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Sample records for accumulation myeloperoxidase activity

  1. (-)-Epicatechin enhances the chlorinating activity of human myeloperoxidase.

    PubMed

    Kirchner, Tina; Flemmig, Jörg; Furtmüller, Paul Georg; Obinger, Christian; Arnhold, Jürgen

    2010-03-01

    The heme-containing enzyme myeloperoxidase (MPO) accumulates at inflammatory sites and is able to catalyse one- and two-electron oxidation reactions. Here it is shown that (-)-epicatechin, which is known to have numerous beneficial health effects, in low micromolar concentration enhances the degradation of monochlorodimedon (MCD) or the chlorination of taurine in a concentration-dependent bell-shaped manner whereas at higher concentrations it sufficiently suppresses the release of hypochlorous acid. Presented reaction mechanisms demonstrate the efficiency of micromolar concentrations of the flavan-3-ol in overcoming the accumulation of compound II that does not participate in the chlorination cycle. In case of MCD the mechanism is more complicated since it also acts as peroxidase substrate with very different reactivity towards compound I (3 x 10(5) M(-1) s(-1)) and compound II (8.8M(-1)s(-1)) at pH 7. By affecting the chlorinating activity of myeloperoxidase (-)-epicatechin may participate in regulation of immune responses at inflammatory sites.

  2. Inhibition of myeloperoxidase and antioxidative activity of Gentiana lutea extracts.

    PubMed

    Nastasijević, Branislav; Lazarević-Pašti, Tamara; Dimitrijević-Branković, Suzana; Pašti, Igor; Vujačić, Ana; Joksić, Gordana; Vasić, Vesna

    2012-07-01

    The aim of this study was to investigate the inhibitory activity of Gentiana lutea extracts on the enzyme myeloperoxidase (MPO), as well as the antioxidant activity of these extracts and their correlation with the total polyphenol content. Extracts were prepared using methanol (100%), water and ethanol aqueous solutions (96, 75, 50 and 25%v/v) as solvents for extraction. Also, isovitexin, amarogentin and gentiopicroside, pharmacologically active constituents of G. lutea were tested as potential inhibitors of MPO. Antioxidant activity of extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging test and also using cyclic voltammetry (CV). Among all extracts, the antioxidant capacity of 50% ethanol aqueous extract was the highest, both when measured using the DPPH test, with IC(50)=20.6 μg/ml, and when using CV. Also, 50% ethanol extract, showed the best inhibition of MPO activity in comparison with other extracts. In the group of the selected G. lutea constituents, gentiopicroside has proved to be the strongest inhibitor of MPO, with IC(50)=0.8 μg/ml. Also, the concentration of G. lutea constituents were determined in all extracts, using Ultra Performance Liquid Chromatography (UPLC).

  3. Cyanate-mediated inhibition of neutrophil myeloperoxidase activity.

    PubMed Central

    Qian, M; Eaton, J W; Wolff, S P

    1997-01-01

    Cyanate (CNO-) forms spontaneously in solutions containing urea, and is present in urine and the body fluids of uraemic patients. We have explored the possibility that CNO- might be one of the unknown substances responsible for the reported impairment, by urine and uraemic plasma, of neutrophil oxidative metabolism (especially as measured by luminol-enhanced chemiluminescence). Luminol-enhanced chemiluminescence generated by human neutrophils derives predominantly from the activity of myeloperoxidase (MPO) which produces hypochlorous acid from H2O2 and Cl-. We hypothesized that CNO- (which resembles the 'pseudohalide' thiocyanate, an alternative substrate for MPO) might somehow interfere with the activity of MPO. In support of this, we find: (i) CNO- inhibits both peroxidative and halogenating activities of MPO and also inhibits the enzyme within intact human neutrophils; (ii) the inhibition is H2O2-dependent, irreversible, accompanied by covalent addition of [14C]CNO- (or a carbon-containing fragment thereof) to the enzyme; (iii) CNO- also inhibits Cl-/H2O2/MPO-mediated bacterial killing. Impairment of this arm of neutrophil bactericidal activity by CNO- formed from urea may be one factor in the risk of urinary-tract infection associated with urinary stasis and perhaps in the generalized increase in susceptibility to infection in uraemic patients. PMID:9337863

  4. Human myeloperoxidase activity is inhibited in vitro by quercetin. Comparison with three related compounds.

    PubMed

    Pincemail, J; Deby, C; Thirion, A; de Bruyn-Dister, M; Goutier, R

    1988-05-15

    Quercetin is an effective inhibitor of human myeloperoxidase (MPO) activity, both with purified enzyme (IC50 = 3.5 microM) and in a system using stimulated human neutrophils. Quercetin is significantly more potent than three other related compounds (rutin, rutin sulfate and troxerutin) and than methimazole, a previously-known myeloperoxidase inhibitor. The inhibitory activity of quercetin is of the competitive type. Moreover, quercetin is directly able to scavenge hypochlorous acid (HOCl), a chlorinated species generated by the MPO/H2O2/Cl- system.

  5. Myeloperoxidase deficiency induces MIP-2 production via ERK activation in zymosan-stimulated mouse neutrophils.

    PubMed

    Tateno, N; Matsumoto, N; Motowaki, T; Suzuki, K; Aratani, Y

    2013-05-01

    Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion. We have previously reported that MPO-deficient (MPO(-/-)) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO(-/-) neutrophils were phosphorylated more strongly than in the wild type when stimulated with zymosan. Exogenous H2O2 treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H2O2 due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan.

  6. Myeloperoxidase deficiency induces MIP-2 production via ERK activation in zymosan-stimulated mouse neutrophils.

    PubMed

    Tateno, N; Matsumoto, N; Motowaki, T; Suzuki, K; Aratani, Y

    2013-05-01

    Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion. We have previously reported that MPO-deficient (MPO(-/-)) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO(-/-) neutrophils were phosphorylated more strongly than in the wild type when stimulated with zymosan. Exogenous H2O2 treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H2O2 due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan. PMID:23438680

  7. Inhibitory effect of curcuminoids and tetrahydrocurcuminoids on equine activated neutrophils and myeloperoxidase activity.

    PubMed

    Franck, T; Kohnen, S; Grulke, S; Neven, P; Goutman, Y; Peters, F; Pirotte, B; Deby-Dupont, G; Serteyn, D

    2008-01-01

    In the horse, the inflammation response to various pathologies (intestinal strangulations, laminitis, etc.) involves an excessive stimulation of the polymorphonuclear neutrophils releasing reactive oxygen species (ROS) and myeloperoxidase (MPO). The aim of the present work was to study the effect of natural polyphenols, curcuminoids and tetrahydrocurcuminoids (THC) on isolated stimulated equine neutrophils and on the activity of purified MPO. The ROS production and the release of MPO by activated neutrophils were measured by chemiluminescence and ELISA techniques, respectively. The activity of purified MPO was measured by studying its nitration, chlorination or oxidation capacity and by using an original method called SIEFED allowing the study of drug interaction with the enzyme without interferences of the medium. Curcuminoids and THC had dose-dependent inhibitory effects on ROS production and MPO release by activated neutrophils and on purified MPO activity. We suggest that the higher efficacy of curcuminoids versus THC could be explained, at least partially, by its chemical structure: the conjugated double bounds and the plane structure of curcuminoids made easier the neutralization of the radical species generated by activated neutrophils and the interaction of the drug with the active site of MPO. These inhibitory effects of curcuminoids on the oxidant activity of equine neutrophils and on MPO activity open therapeutic perspectives in equine pathologies with excessive inflammatory reactions.

  8. Myeloperoxidase Stimulates Neutrophil Degranulation.

    PubMed

    Grigorieva, D V; Gorudko, I V; Sokolov, A V; Kostevich, V A; Vasilyev, V B; Cherenkevich, S N; Panasenko, O M

    2016-08-01

    Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation. PMID:27597056

  9. Characterization and Antioxidant Properties of Six Algerian Propolis Extracts: Ethyl Acetate Extracts Inhibit Myeloperoxidase Activity

    PubMed Central

    Boufadi, Yasmina Mokhtaria; Soubhye, Jalal; Riazi, Ali; Rousseau, Alexandre; Vanhaeverbeek, Michel; Nève, Jean; Boudjeltia, Karim Zouaoui; Van Antwerpen, Pierre

    2014-01-01

    Because propolis contains many types of antioxidant compounds such as polyphenols and flavonoids, it can be useful in preventing oxidative damages. Ethyl acetate extracts of propolis from several Algerian regions show high activity by scavenging free radicals, preventing lipid peroxidation and inhibiting myeloperoxidase (MPO). By fractioning and assaying ethyl acetate extracts, it was observed that both polyphenols and flavonoids contribute to these activities. A correlation was observed between the polyphenol content and the MPO inhibition. However, it seems that kaempferol, a flavonoid, contributes mainly to the MPO inhibition. This molecule is in a high amount in the ethyl acetate extract and demonstrates the best efficiency towards the enzyme with an inhibiting concentration at 50% of 4 ± 2 μM. PMID:24514562

  10. Flavonoids as promoters of the (pseudo-)halogenating activity of lactoperoxidase and myeloperoxidase.

    PubMed

    Gau, Jana; Furtmüller, Paul G; Obinger, Christian; Prévost, Martine; Van Antwerpen, Pierre; Arnhold, Jürgen; Flemmig, Jörg

    2016-08-01

    In this study several flavonoids were tested for their potential to regenerate the (pseudo-)halogenating activity (hypothiocyanite formation) of the heme peroxidases lactoperoxidase (LPO) and myeloperoxidase (MPO) after hydrogen peroxide-mediated enzyme inactivation. Several flavonoid subclasses with varying hydroxylation patterns (especially of the flavonoid B-ring) were examined in order to identify structural properties of efficient enzyme regenerators. Kinetic parameters and second-order rate constants were determined. A 3',4'-dihydroxylated B-ring together with C-ring saturation and hydroxylation were found to be important structural elements, which strongly influence the flavonoid binding and oxidizability by the LPO/MPO redox intermediates Compounds I and II. In combination with docking studies these results allow an understanding of the differences between flavonoids that promote the hypothiocyanite production by LPO and MPO and those that inhibit this enzymatic reaction.

  11. [Myeloperoxidase activity in blood plasma as a criterion of therapy for patients with cardiovascular disease].

    PubMed

    Grigorieva, D V; Gorudko, I V; Kostevich, V A; Sokolov, A V; Buko, I V; Vasilyev, V B; Polonetsky, L Z; Panasenko, O M; Cherenkevich, S N

    2016-03-01

    A significant increase in the myeloperoxidase (MPO) activity has been found in plasma of patients with stable angina and with acute coronary syndrome (ACS) in comparison with the control group. MPO concentration was significantly increased in plasma of ACS patients. Reduced MPO activity in the treated ACS patients correlated with a favorable outcome of the disease. Generally, changes in plasma MPO concentration coincided with changes in lactoferrin concentration thus confirming the role of neutrophil degranulation in the increase of plasma concentrations of these proteins. The increase in MPO activity was obviously determined by modification of the MPO protein caused by reactive oxygen species and halogen in the molar ratio of 1 : 25 and 1 : 50. The decrease in plasma MPO activity may be associated with increased plasma concentrations of the physiological inhibitor of its activity, ceruloplasmin, and also with modification of the MPO protein with reactive oxygen species and halogen at their molar ratio of 1 : 100 and higher. Thus, MPO activity may be used for evaluation of effectiveness of the treatment of cardiovascular diseases. PMID:27420626

  12. A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma.

    PubMed

    Goiffon, Reece J; Martinez, Sara C; Piwnica-Worms, David

    2015-02-10

    Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l(-1) MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders.

  13. A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma

    PubMed Central

    Goiffon, Reece J.; Martinez, Sara C.; Piwnica-Worms, David

    2015-01-01

    Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l−1 MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders. PMID:25666092

  14. Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

    PubMed

    Levine, Adam P; Duchen, Michael R; de Villiers, Simon; Rich, Peter R; Segal, Anthony W

    2015-01-01

    The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

  15. Increased Myeloperoxidase Activity and Protein Nitration Are Indicators of Inflammation in Patients with Chagas' Disease▿

    PubMed Central

    Dhiman, Monisha; Estrada-Franco, Jose Guillermo; Pando, Jasmine M.; Ramirez-Aguilar, Francisco J.; Spratt, Heidi; Vazquez-Corzo, Sara; Perez-Molina, Gladys; Gallegos-Sandoval, Rosa; Moreno, Roberto; Garg, Nisha Jain

    2009-01-01

    In this study, we investigated whether inflammatory responses contribute to oxidative/nitrosative stress in patients with Chagas' disease. We used three tests (enzyme-linked immunosorbent assay, immuno-flow cytometry, and STAT-PAK immunochromatography) to screen human serum samples (n = 1,481) originating from Chiapas, Mexico, for Trypanosoma cruzi-specific antibodies. We identified 121 subjects who were seropositive for T. cruzi-specific antibodies, a finding indicative of an 8.5% seroprevalence in the rural population from Chiapas. Seropositive and seronegative subjects were examined for plasma levels of biomarkers of inflammation, i.e., myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and xanthine oxidase (XOD), as well as for oxidative (advanced oxidation protein products [AOPPs]) and nitrosative (3-nitrotyrosine [3NT]) biomarkers. The seropositive subjects exhibited a significant increase in MPO activity and protein level, the indicator of neutrophil activation. Subsequently, a corresponding increase in AOPP contents, formed by MPO-dependent hypochlorous acid and chloramine formation, was noted in seropositive subjects. The plasma level of 3NT was significantly increased in seropositive subjects, yet we observed no change in XOD activity (O2− source) and nitrate/nitrite contents (denotes iNOS activation and NO production), which implied that direct peroxynitrite formation does not contribute to increased nitrosative damage in chagasic subjects. Instead, a positive correlation between increased MPO activity and protein 3NT formation was observed, which suggested to us that MPO-dependent formation of nitrylchloride that occurs in the presence of physiological NO and O2− concentrations contributes to protein nitration. Overall, our data demonstrate that T. cruzi-induced neutrophil activation is pathological and contributes to MPO-mediated collateral protein oxidative and nitrosative damage in human patients with Chagas' disease. Therapies

  16. Chlorination of guanosine and other nucleosides by hypochlorous acid and myeloperoxidase of activated human neutrophils. Catalysis by nicotine and trimethylamine.

    PubMed

    Masuda, M; Suzuki, T; Friesen, M D; Ravanat, J L; Cadet, J; Pignatelli, B; Nishino, H; Ohshima, H

    2001-11-01

    Activated human neutrophils secrete myeloperoxidase, which generates HOCl from H2O2 and Cl(-). We have found that various (2'-deoxy)nucleosides react with HOCl to form chlorinated (2'-deoxy)nucleosides, including novel 8-chloro(2'-deoxy)guanosine, 5-chloro(2'-deoxy)cytidine, and 8-chloro(2'-deoxy)adenosine formed in yields of 1.6, 1.6, and 0.2%, respectively, when 0.5 mM nucleoside reacted with 0.5 mM HOCl at pH 7.4. The relative chlorination, oxidation, and nitration activities of HOCl, myeloperoxidase, and activated human neutrophils in the presence and absence of nitrite were studied by analyzing 8-chloro-, 8-oxo-7,8-dihydro-, and 8-nitro-guanosine, respectively, using guanosine as a probe. 8-Chloroguanosine was always more easily formed than 8-oxo-7,8-dihydro- or 8-nitro-guanosine. Using electrospray ionization tandem mass spectrometry, we show that several chlorinated nucleosides including 8-chloro(2'-deoxy)guanosine are formed following exposure of isolated DNA or RNA to HOCl. Micromolar concentrations of tertiary amines such as nicotine and trimethylamine dramatically enhanced chlorination of free (2'-deoxy)nucleosides and nucleosides in RNA by HOCl. As the G-463A polymorphism of the MPO gene, which strongly reduces myeloperoxidase mRNA expression, is associated with a reduced risk of lung cancer, chlorination damage of DNA /RNA and nucleosides by myeloperoxidase and its enhancement by nicotine may be important in the pathophysiology of human diseases associated with tobacco habits. PMID:11533049

  17. The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality

    PubMed Central

    Shaeib, Faten; Khan, Sana N.; Thakur, Mili; Kohan-Ghadr, Hamid-Reza; Drewlo, Sascha; Saed, Ghassan M.; Pennathur, Subramaniam; Abu-Soud, Husam M.

    2016-01-01

    Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H2O2) and chloride (Cl-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes. PMID:26982351

  18. MR Imaging of Myeloperoxidase Activity in a Model of the Inflamed Aneurysm Wall

    PubMed Central

    Gounis, M.J.; van der Bom, I.M.J.; Wakhloo, A.K.; Zheng, S.; Chueh, J.-Y.; Kühn, A.L.; Bogdanov, A.A.

    2014-01-01

    Background and Purpose Although myeloperoxidase (MPO) activity in vivo can be visualized using non-invasive imaging, successful clinical translation requires further optimization of the imaging approach. We report a motion-sensitized-driven-equilibrium (MSDE) for the detection of an MPO activity-specific gadolinium (Gd)-containing imaging agent (IA) in experimental aneurysm models that compensates for irregular blood flow enabling vascular wall imaging in the aneurysm. Materials and Methods We deployed a phantom model to optimize a MSDE MR sequence that suppresses complex flow patterns within the aneurysm for detection of an MPO-specific Gd-chelate. The phantom was built from rotational angiography of a rabbit elastase aneurysm model and connected to a cardiac pulse duplicator mimicking rabbit-specific flow conditions. Thereafter, we further refined the MSDE sequence and applied it in vivo to rabbit aneurysm models with and without inflammation in the aneurysmal wall. Under each condition, the aneurysms were imaged before and after intravenous administration of the IA. The signal-to-noise ratio (SNR) of each MR slice through the aneurysm was calculated. Results The MSDE sequence was optimized to reduce flow signal enabling detection of the MPO-IA in the phantom. The optimized imaging protocol in the rabbit model of saccular aneurysms revealed a significant increase in the change of SNR pre- to postcontrast MR signal intensities in the inflamed aneurysms as compared to naïve aneurysms and the adjacent carotid artery (p<0.0001). Conclusion A diagnostic MR protocol was optimized for molecular imaging of an MPO-specific molecular imaging agent in an animal model of brain aneurysms. PMID:25273534

  19. The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality.

    PubMed

    Shaeib, Faten; Khan, Sana N; Thakur, Mili; Kohan-Ghadr, Hamid-Reza; Drewlo, Sascha; Saed, Ghassan M; Pennathur, Subramaniam; Abu-Soud, Husam M

    2016-01-01

    Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H2O2) and chloride (Cl-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes. PMID:26982351

  20. Plasma myeloperoxidase level and polymorphonuclear leukocyte activation in horses suffering from large intestinal obstruction requiring surgery: preliminary results.

    PubMed Central

    Grulke, S; Benbarek, H; Caudron, I; Deby-Dupont, G; Mathy-Hartert, M; Farnir, F; Deby, C; Lamy, M; Serteyn, D

    1999-01-01

    Myeloperoxidase (MPO) is a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Thanks to a radioimmunoassay of equine myeloperoxidase, the authors have observed a significantly higher plasma level of MPO in horses operated for strangulation obstruction of the large intestine (n = 6) than in horses suffering from a non-strangulating displacement of the large intestine (n = 9). For the 2 groups, 3 phases were distinguished: reception (P1), intensive care (P2) and terminal phase (P3). The mean peak values of MPO for these phases were 121.6 ng/mL (P1), 168.6 ng/mL (P2), and 107.0 ng/mL (P3) for the non-strangulating group, and 242.6 ng/mL (P1); 426.0 ng/mL (P2), and 379.5 ng/mL (P3) for the strangulation group. The variations of the mean peak values of plasma MPO were significantly different between the 2 groups and between the different phases. A significant increase of the least square means of MPO was observed between P1 and P2. A significant decrease of the least square means of the number of circulating leukocytes was observed between P1 and P3. Polymorphonuclear neutrophil activation could play a major role in the pathogenesis of acute abdominal disease and endotoxic shock. PMID:10369573

  1. Relationship between the lipophilicity of gallic acid n-alquil esters' derivatives and both myeloperoxidase activity and HOCl scavenging.

    PubMed

    Rosso, Rober; Vieira, Tiago O; Leal, Paulo C; Nunes, Ricardo J; Yunes, Rosendo A; Creczynski-Pasa, Tânia B

    2006-09-15

    The gallic acid and several n-alkyl gallates, with the same number of hydroxyl substituents, varying only in the side carbonic chain length, with respective lipophilicity defined through the C log P, were studied. It evidenced the structure-activity relationship of the myeloperoxidase activity inhibition and the hypochlorous acid scavenger property, as well as its low toxicity in rat hepatic tissue. The gallates with C log P below 3.0 (compounds 2-7) were more active against the enzyme activity, what means that the addition of 1-6 carbons (C log P between 0.92 and 2.92) at the side chain increased approximately 50% the gallic acid effect. However, a relationship between the HOCl scavenging capability and the lipophilicity was not observed. With these results it is possible to suggest that the gallates protect the HOCl targets through two mechanisms: inhibiting its production by the enzyme and scavenging the reactive specie.

  2. Predictive value of serum myeloperoxidase activity for thrombosis of arteriovenous fistulas.

    PubMed

    Stolic, Radojica V; Trajkovic, Goran Z; Kostic, Mirjana; Stolic, Dragica Z; Miric, Dijana J; Kisic, Bojana M; Pajovic, Slavica D; Peric, Vladan M

    2014-07-01

    Myeloperoxidase is a proinflammatory protein that appears as a result of increased oxidative stress. It plays an important role in the promotion and progression of atherosclerosis. The aim of this study was to determine the importance of MPO as a predictive parameter for thrombosis of arteriovenous fistula (AVF). The study involved monitoring patients with AVFs for hemodialysis over a period of 2 years. There were 41 patients, 19 (46%) men and 22 (54%) women, with mean age of 65 ± 12.7 years. Routine laboratory analyses were carried out in all respondents, including determination of MPO concentration. Gender, demographic and anthropometrical characteristics, smoking, alcohol consumption, as well as the presence of diabetic nephropathy, as an etiological factor of kidney disease, were recorded. The group of patients who developed initial thrombosis of the AVFs had significantly different values for leukocytes (8.5 ± 3.8 vs. 7.3 ± 2.1, P = 0.024), erythrocytes (2.8 ± 0.27 vs. 3.2 ± 0.65; P = 0.019), hemoglobin (88.5 ± 81 vs. 99.1 ± 6.02; P = 0.041), and myeloperoxidase (19.3 ± 4.67 vs. 11.1 ± 4.43; P = 0.007) when compared with the group without fistula thrombosis. Diabetic nephropathy (P = 0.02) characterized the group of patients with thrombosis of the fistula. Diabetic nephropathy (B = 2.53, P = 0.049) and MPO (B = 0.03, P = 0.029) were statistically significant predictors of fistula thrombosis. In our study, MPO and diabetic nephropathy were predictors of thrombosis of the AVF.

  3. Polyphenol content and modulatory activities of some tropical dietary plant extracts on the oxidant activities of neutrophils and myeloperoxidase.

    PubMed

    Tsumbu, Cesar N; Deby-Dupont, Ginette; Tits, Monique; Angenot, Luc; Frederich, Michel; Kohnen, Stephane; Mouithys-Mickalad, Ange; Serteyn, Didier; Franck, Thierry

    2012-01-01

    Young leaves of Manihot esculenta Crantz (Euphorbiaceae), Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae) and Pteridium aquilinum (Dennstaedtiaceae) are currently consumed as green vegetables by peoples in sub-Saharan Africa, Latin America, Asia and their migrants living in Western Europe. Sub-Saharan peoples use Manihot, Abelmoschus and Hibiscus also in the folk medicine to alleviate fever and pain, in the treatment of conjunctivitis, rheumatism, hemorrhoid, abscesses, ... The present study investigates the effects of aqueous extracts of those plants on the production of reactive oxygen species (ROS) and the release of myeloperoxidase (MPO) by equine neutrophils activated with phorbol 12-myristate 13-acetate (PMA). The ROS production was measured by lucigenin-enhanced chemiluminescence (CL), and the release of total MPO by an ELISA method. The study also investigates the effect of the extracts on the activity of MPO by studying its nitration activity on tyrosine and by using a new technique called SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection) that allows studying the direct interaction of compounds with the enzyme. In all experiments, the aqueous extracts of the plants developed concentration-dependent inhibitory effects. A moderate heat treatment did not significantly modify the inhibitory capacity of the extracts in comparison to not heated ones. Total polyphenol and flavonoid contents were determined with an HPLC-UV/DAD analysis and a spectroscopic method using Folin-Ciocalteu reagent. Some polyphenols with well-known antioxidant activities (caffeic acid, chlorogenic acid, hyperoside, rosmarinic acid and rutin) were found in the extracts and may partly explain the inhibitory activities observed. The role of those dietary and medicinal plants in the treatment of ROS-dependent inflammatory diseases could have new considerations for health. PMID:22312276

  4. A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests.

    PubMed

    Franck, Thierry; Kohnen, S; Deby-Dupont, G; Grulke, S; Deby, C; Serteyn, D

    2006-07-01

    An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.

  5. A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts.

    PubMed

    Franck, T; Kohnen, S; Boudjeltia, K Zouaoui; Van Antwerpen, P; Bosseloir, A; Niesten, A; Gach, O; Nys, M; Deby-Dupont, G; Serteyn, D

    2009-12-15

    The SIEFED ("Specific Immunological Extraction Followed by Enzymatic Detection") method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H(2)O(2) and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.

  6. Evaluation of the serum catalase and myeloperoxidase activities in chronic arsenic-exposed individuals and concomitant cytogenetic damage.

    PubMed

    Banerjee, Mayukh; Banerjee, Nilanjana; Ghosh, Pritha; Das, Jayanta K; Basu, Santanu; Sarkar, Ajoy K; States, J Christopher; Giri, Ashok K

    2010-11-15

    Chronic arsenic exposure through contaminated drinking water is a major environmental health issue. Chronic arsenic exposure is known to exert its toxic effects by a variety of mechanisms, of which generation of reactive oxygen species (ROS) is one of the most important. A high level of ROS, in turn, leads to DNA damage that might ultimately culminate in cancer. In order to keep the level of ROS in balance, an array of enzymes is present, of which catalase (CAT) and myeloperoxidase (MPO) are important members. Hence, in this study, we determined the activities of these two enzymes in the sera and chromosomal aberrations (CA) in peripheral blood lymphocytes in individuals exposed and unexposed to arsenic in drinking water. Arsenic in drinking water and in urine was used as a measure of exposure. Our results show that individuals chronically exposed to arsenic have significantly higher CAT and MPO activities and higher incidence of CA. We found moderate positive correlations between CAT and MPO activities, induction of CA and arsenic in urine and water. These results indicate that chronic arsenic exposure causes higher CAT and MPO activities in serum that correlates with induction of genetic damage. We conclude that the serum levels of these enzymes might be used as biomarkers of early arsenic exposure induced disease much before the classical dermatological symptoms of arsenicosis begin to appear.

  7. Evaluation of the serum catalase and myeloperoxidase activities in chronic arsenic-exposed individuals and concomitant cytogenetic damage

    SciTech Connect

    Banerjee, Mayukh; Banerjee, Nilanjana; Ghosh, Pritha; Das, Jayanta K.; Basu, Santanu; Sarkar, Ajoy K.; States, J. Christopher; Giri, Ashok K.

    2010-11-15

    Chronic arsenic exposure through contaminated drinking water is a major environmental health issue. Chronic arsenic exposure is known to exert its toxic effects by a variety of mechanisms, of which generation of reactive oxygen species (ROS) is one of the most important. A high level of ROS, in turn, leads to DNA damage that might ultimately culminate in cancer. In order to keep the level of ROS in balance, an array of enzymes is present, of which catalase (CAT) and myeloperoxidase (MPO) are important members. Hence, in this study, we determined the activities of these two enzymes in the sera and chromosomal aberrations (CA) in peripheral blood lymphocytes in individuals exposed and unexposed to arsenic in drinking water. Arsenic in drinking water and in urine was used as a measure of exposure. Our results show that individuals chronically exposed to arsenic have significantly higher CAT and MPO activities and higher incidence of CA. We found moderate positive correlations between CAT and MPO activities, induction of CA and arsenic in urine and water. These results indicate that chronic arsenic exposure causes higher CAT and MPO activities in serum that correlates with induction of genetic damage. We conclude that the serum levels of these enzymes might be used as biomarkers of early arsenic exposure induced disease much before the classical dermatological symptoms of arsenicosis begin to appear.

  8. The effect of indomethacin, myeloperoxidase, and certain steroid hormones on bactericidal activity: an ex vivo and in vivo experimental study

    PubMed Central

    2014-01-01

    Background The role of myeloperoxidase (MPO) is essential in the killing of phagocytosed bacteria. Certain steroid hormones increase MPO plasma concentration. Our aim was to test the effect of MPO, its inhibitor indomethacin, and certain steroid hormones on bactericidal activity. Methods Human polymorphonuclear leukocytes (PMN) were incubated with opsonised Escherichia coli and either MPO, indomethacin, estradiol, or hydrocortisone. Intracellular killing capacity was evaluated with UV microscopy after treatment with fluorescent dye. Next, an in vivo experiment was performed with nine groups of rats: in the first phase of the study indomethacin treatment and Pasteurella multocida infection (Ii), indomethacin treatment without infection (I0), untreated control with infection (Mi) and untreated control without infection (M0); in the second phase of the study rats with infection and testosterone treatment (NT), castration, infection and testosterone treatment (CT), castration, infection and estradiol treatment (CE), non-castrated infected control (N0), and castrated infected control (C0). After treatment bacteria were reisolated from the liver and heart blood on agar plates, and laboratory parameters were analyzed. For the comparison of laboratory results ANOVA or Kruskal-Wallis test and LSD post hoc test was used. Results Indomethacin did not have a remarkable effect on the bacterial killing of PMNs, while the other compounds increased bacterial killing to various degrees. In the animal model indomethacin and infection caused a poor clinical state, a great number of reisolated bacteria, elevated white blood cell (WBC) count, decreased C-reactive protein (CRP) and serum albumin levels. Testosterone treatment resulted in less bacterial colony numbers in group NT, but not in group CT compared to respective controls (N0, C0). Estradiol treatment (CE) decreased colony numbers compared to control (C0). Hormone administration resulted in lower WBC counts, and in group CE, a

  9. Deconstruction of activity-dependent covalent modification of heme in human neutrophil myeloperoxidase by multistage mass spectrometry (MS(4)).

    PubMed

    Geoghegan, Kieran F; Varghese, Alison H; Feng, Xidong; Bessire, Andrew J; Conboy, James J; Ruggeri, Roger B; Ahn, Kay; Spath, Samantha N; Filippov, Sergey V; Conrad, Steven J; Carpino, Philip A; Guimarães, Cristiano R W; Vajdos, Felix F

    2012-03-13

    Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.

  10. p-Hydroxyphenylacetaldehyde is the major product of L-tyrosine oxidation by activated human phagocytes. A chloride-dependent mechanism for the conversion of free amino acids into reactive aldehydes by myeloperoxidase.

    PubMed

    Hazen, S L; Hsu, F F; Heinecke, J W

    1996-01-26

    Reactive aldehydes generated during lipid peroxidation have been implicated in the pathogenesis of atherosclerosis as well as other inflammatory diseases. A potential catalyst for such reactions is myeloperoxidase, a hemeprotein secreted by activated phagocytes. We now report that activated neutrophils utilize the myeloperoxidase-H2O2-chloride system to convert L-tyrosine to p-hydroxyphenylacetaldehyde. Production of p-hydroxyphenylacetaldehyde was nearly quantitative at physiological concentrations of L-tyrosine and chloride. Aldehyde generation required myeloperoxidase, H2O2, L-tyrosine, and chloride ion; it was inhibited by the H2O2 scavenger catalase and by the heme poisons azide and cyanide. Phorbol ester- and calcium ionophore-stimulated human neutrophils likewise generated p-hydroxyphenylacetaldehyde from L-tyrosine by a pathway inhibited by azide, cyanide, and catalase. Aldehyde production accounted for 75% of H2O2 generated by optimally stimulated neutrophils at plasma concentrations of L-tyrosine and chloride. Collectively, these results indicate that activated phagocytes, under physiological conditions, utilize myeloperoxidase to execute the chloride-dependent conversion of L-tyrosine to the lipid-soluble aldehyde, p-hydroxyphenylacetaldehyde, in near quantitative yield. Moreover, like aldehydes derived from lipid peroxidation, amino acid-derived aldehydes may exert potent biological effects in vascular lesions and other sites of inflammation.

  11. Myeloperoxidase-Related Chlorination Activity Is Positively Associated with Circulating Ceruloplasmin in Chronic Heart Failure Patients: Relationship with Neurohormonal, Inflammatory, and Nutritional Parameters

    PubMed Central

    Cabassi, Aderville; Binno, Simone Maurizio; Tedeschi, Stefano; Graiani, Gallia; Galizia, Cinzia; Bianconcini, Michele; Coghi, Pietro; Fellini, Federica; Ruffini, Livia; Govoni, Paolo; Piepoli, Massimo; Perlini, Stefano; Regolisti, Giuseppe; Fiaccadori, Enrico

    2015-01-01

    Rationale. Heart failure (HF) is accompanied by the development of an imbalance between oxygen- and nitric oxide-derived free radical production leading to protein nitration. Both chlorinating and peroxidase cycle of Myeloperoxidase (MPO) contribute to oxidative and nitrosative stress and are involved in tyrosine nitration of protein. Ceruloplasmin (Cp) has antioxidant function through its ferroxidase I (FeOxI) activity and has recently been proposed as a physiological defense mechanism against MPO inappropriate actions. Objective. We investigated the relationship between plasma MPO-related chlorinating activity, Cp and FeOxI, and nitrosative stress, inflammatory, neurohormonal, and nutritional biomarkers in HF patients. Methods and Results. In chronic HF patients (n = 81, 76 ± 9 years, NYHA Class II (26); Class III (29); Class IV (26)) and age-matched controls (n = 17, 75 ± 11 years, CTR), plasma MPO chlorinating activity, Cp, FeOxI, nitrated protein, free Malondialdehyde, BNP, norepinephrine, hsCRP, albumin, and prealbumin were measured. Plasma MPO chlorinating activity, Cp, BNP, norepinephrine, and hsCRP were increased in HF versus CTR. FeOxI, albumin, and prealbumin were decreased in HF. MPO-related chlorinating activity was positively related to Cp (r = 0.363, P < 0.001), nitrated protein, hsCRP, and BNP and inversely to albumin. Conclusions. Plasma MPO chlorinated activity is increased in elderly chronic HF patients and positively associated with Cp, inflammatory, neurohormonal, and nitrosative parameters suggesting a role in HF progression. PMID:26539521

  12. Detailed protocol to assess in vivo and ex vivo myeloperoxidase activity in mouse models of vascular inflammation and disease using hydroethidine.

    PubMed

    Talib, Jihan; Maghzal, Ghassan J; Cheng, David; Stocker, Roland

    2016-08-01

    Myeloperoxidase (MPO) activity contributes to arterial inflammation, vascular dysfunction and disease, including atherosclerosis. Current assessment of MPO activity in biological systems in vivo utilizes 3-chlorotyrosine (3-Cl-Tyr) as a biomarker of hypochlorous acid (HOCl) and other chlorinating species. However, 3-Cl-Tyr is formed in low yield and is subject to further metabolism. Recently, we reported a method to selectively assess MPO-activity in vivo by measuring the conversion of hydroethidine to 2-chloroethidium (2-Cl-E(+)) by liquid chromatography with tandem mass spectrometry (LC-MS/MS) (J. Biol. Chem., 289, 2014, pp. 5580-5595). The hydroethidine-based method has greater sensitivity for MPO activity than measurement of 3-Cl-Tyr. The current methods paper provides a detailed protocol to determine in vivo and ex vivo MPO activity in arteries from mouse models of vascular inflammation and disease by utilizing the conversion of hydroethidine to 2-Cl-E(+). Procedures for the synthesis of standards, preparation of tissue homogenates and the generation of 2-Cl-E(+) are also provided in detail, as are the conditions for LC-MS/MS detection of 2-Cl-E(+). PMID:27184954

  13. A1M/α1-microglobulin is proteolytically activated by myeloperoxidase, binds its heme group and inhibits low density lipoprotein oxidation

    PubMed Central

    Cederlund, Martin; Deronic, Adnan; Pallon, Jan; Sørensen, Ole E.; Åkerström, Bo

    2015-01-01

    α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components. PMID:25698971

  14. Inhibition by polyphenolic phytochemicals and sulfurous compounds of the formation of 8-chloroguanosine mediated by hypochlorous acid, human myeloperoxidase, and activated human neutrophils.

    PubMed

    Nakano, Toshiki; Masuda, Mitsuharu; Suzuki, Toshinori; Ohshima, Hiroshi

    2012-01-01

    Hypochlorous acid (HOCl) produced by myeloperoxidase (MPO) of activated neutrophils can react with nucleic acid bases to form chlorinated nucleosides such as 8-chloroguanosine (Cl-Guo). Chlorination is enhanced by nicotine. We investigated the effects of various natural antioxidants including polyphenolic phytochemicals on the formation of Cl-Guo by HOCl in the presence and the absence of nicotine. Polyphenols, including catechins, curcumin, resveratrol, silibinin, and sulfurous compound α-lipoic acid, were found to inhibit both HOCl- and human MPO-induced Cl-Guo formation dose-dependently. Among the test compounds, (-)-epigallocatechin gallate (EGCG) showed the strongest inhibitory effect. Cl-Guo formation, mediated by activated human neutrophils in the presence of nicotine, was inhibited by EGCG, silibinin, and α-lipoic acid. These results suggest that polyphenols and sulfurous compounds have the potential to inhibit the induction of nucleobase damage mediated by chlorination, with possible application to reducing DNA damage associated with inflammation and cigarette-smoke inhalation. PMID:23221717

  15. Myeloperoxidase levels predict executive function.

    PubMed

    Haslacher, H; Perkmann, T; Lukas, I; Barth, A; Ponocny-Seliger, E; Michlmayr, M; Scheichenberger, V; Wagner, O; Winker, R

    2012-12-01

    The main purpose of the study was to investigate whether baseline myeloperoxidase (MPO) levels are associated with executive cognitive function in individuals with high physical activity. Baseline serum MPO levels of 56 elderly marathon runners and 58 controls were assessed by ELISA. Standardized tests were applied to survey domain-specific cognitive functions. Changes in brain morphology were visualized by magnetic resonance imaging (MRI). High baseline serum MPO levels correlated with worse outcome in tests assessing executive cognitive function in athletes but not in the control group (NAI maze test p<0.05, Trail Making Test ratio p<0.01). In control participants, subcortical white matter hyperintensities were associated with higher scores on the Geriatric Depression Scale (p<0.05), whereas athletes seem to be protected from this effect. During strenuous exercising, MPO as well as its educts may be elevated due to increased oxygen intake and excretion of pro-inflammatory mediators inducing host tissue damage via oxidative stress. This outweighs the potential benefits of physical activity on cognitive function.

  16. Myeloperoxidase levels predict executive function.

    PubMed

    Haslacher, H; Perkmann, T; Lukas, I; Barth, A; Ponocny-Seliger, E; Michlmayr, M; Scheichenberger, V; Wagner, O; Winker, R

    2012-12-01

    The main purpose of the study was to investigate whether baseline myeloperoxidase (MPO) levels are associated with executive cognitive function in individuals with high physical activity. Baseline serum MPO levels of 56 elderly marathon runners and 58 controls were assessed by ELISA. Standardized tests were applied to survey domain-specific cognitive functions. Changes in brain morphology were visualized by magnetic resonance imaging (MRI). High baseline serum MPO levels correlated with worse outcome in tests assessing executive cognitive function in athletes but not in the control group (NAI maze test p<0.05, Trail Making Test ratio p<0.01). In control participants, subcortical white matter hyperintensities were associated with higher scores on the Geriatric Depression Scale (p<0.05), whereas athletes seem to be protected from this effect. During strenuous exercising, MPO as well as its educts may be elevated due to increased oxygen intake and excretion of pro-inflammatory mediators inducing host tissue damage via oxidative stress. This outweighs the potential benefits of physical activity on cognitive function. PMID:22855218

  17. False-positive myeloperoxidase binding activity due to DNA/anti-DNA antibody complexes: a source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic autoantibodies.

    PubMed

    Jethwa, H S; Nachman, P H; Falk, R J; Jennette, J C

    2000-09-01

    Anti-myeloperoxidase antibodies (anti-MPO) are a major type of anti-neutrophil cytoplasmic antibody (ANCA). While evaluating anti-MPO monoclonal antibodies from SCG/Kj mice, we observed several hybridomas that appeared to react with both MPO and DNA. Sera from some patients with systemic lupus erythematosus (SLE) also react with MPO and DNA. We hypothesized that the MPO binding activity is a false-positive result due to the binding of DNA, contained within the antigen binding site of anti-DNA antibodies, to the cationic MPO. Antibodies from tissue culture supernatants from 'dual reactive' hybridomas were purified under high-salt conditions (3 M NaCl) to remove any antigen bound to antibody. The MPO and DNA binding activity were measured by ELISA. The MPO binding activity was completely abrogated while the DNA binding activity remained. The MPO binding activity was restored, in a dose-dependent manner, by the addition of increasing amount of calf-thymus DNA (CT-DNA) to the purified antibody. Sera from six patients with SLE that reacted with both MPO and DNA were treated with DNase and showed a decrease in MPO binding activity compared with untreated samples. MPO binding activity was observed when CT-DNA was added to sera from SLE patients that initially reacted with DNA but not with MPO. These results suggest that the DNA contained within the antigen binding site of anti-DNA antibodies could bind to the highly cationic MPO used as substrate antigen in immunoassays, resulting in a false-positive test.

  18. Superoxide-dependent oxidation of melatonin by myeloperoxidase.

    PubMed

    Ximenes, Valdecir F; Silva, Sueli de O; Rodrigues, Maria R; Catalani, Luiz H; Maghzal, Ghassan J; Kettle, Anthony J; Campa, Ana

    2005-11-18

    Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics. PMID:16148002

  19. A Phytochemical-rich Multivitamin-multimineral Supplement Is Bioavailable and Reduces Serum Oxidized Low-density Lipoprotein, Myeloperoxidase, and Plasminogen Activator Inhibitor-1 in a Four-week Pilot trial of Healthy Individuals

    PubMed Central

    Desai, Anuradha; Lamb, Joseph J.; Chang, Jyh-Lurn; Darland, Gary; Konda, Veera R.

    2014-01-01

    Background: A multivitamin-multimineral supplement combined with a diverse blend of bioactive phytochemicals may provide additional antioxidant capacity and anti-inflammatory property for overall health. This convenient feature may be useful for individuals who want to increase their intake of phytochemicals. Methods: We conducted a pilot study in 15 healthy individuals (8 women and 7 men, mean age 41.7±14.9 years, mean body mass index 28.0±5.6) to investigate the effects of this novel formulation on biomarkers associated with oxidative stress and inflammation. After a 2-week diet that limited intake of fruits and vegetables to 2 servings/day, participants continued with the same restricted diet but began consuming 2 tablets of the study product for the subsequent 4 weeks. Fasting blood samples collected at Week 2 and Week 6 were analyzed and compared using paired t-tests for levels of carotenoids, folate, vitamin B12, homocysteine, oxidized low-density lipoprotein cholesterol (oxLDL), high-sensitivity C-reactive protein (hs-CRP), F2-isoprostane, plasminogen activator inhibitor-1 (PAI-1), and myeloperoxidase. Noninvasive peripheral arterial tonometry (EndoPAT) was also measured. Results: After 4 weeks of supplementation, plasma levels of carotenoids, folate, and vitamin B12, but not homocysteine, were significantly increased (P<.05). Serum levels of oxLDL, PAI-1 and myeloperoxidase were significantly reduced (P<.05), but F2-isoprostane, hs-CRP, and EndoPAT measures were unchanged compared with baseline. The study product was well tolerated. Conclusions: This nutritional supplement is bioavailable as indicated by the significant increase in plasma carotenoids, vitamin B12, and folate levels and may provide health benefits by significantly reducing serum levels of oxLDL, myeloperoxidase, and PAI-1 in healthy individuals. PMID:24808980

  20. Neutrophil myeloperoxidase destruction by ultraviolet irradiation

    SciTech Connect

    Hanker, J.; Giammara, B.; Strauss, G.

    1988-01-01

    The peroxidase activity of enriched leukocyte preparations on coverslips was determined cytochemically with a newly developed method. The techniques utilizes diaminobenzidine medium and cupric nitrate intensification and is suitable for analysis with light microscopy, SEM, and TEM. Blood specimens from control individuals were studied with and without in vitro UV irradiation and compared with those from psoriasis patients exposed therapeutically to various types of UV in phototherapy. All UV irradiated samples showed diminished neutrophil myeloperoxidase (MP) activity although that of the principal eosinophil peroxidase was unaffected. The SEMs supported the contention that decreased neutrophil MP activity might be related to UV induced degranulation. It is believed to be possible, eventually, to equate the observed MP degranulation effect after UV irradiation with diminished ability to fight bacterial infections.

  1. Deconstruction of Activity-Dependent Covalent Modification of Heme in Human Neutrophil Myeloperoxidase by Multistage Mass Spectrometry (MS[superscript 4])

    SciTech Connect

    Geoghegan, Kieran F.; Varghese, Alison H.; Feng, Xidong; Bessire, Andrew J.; Conboy, James J.; Ruggeri, Roger B.; Ahn, Kay; Spath, Samantha N.; Filippov, Sergey V.; Conrad, Steven J.; Carpino, Philip A.; Guimarães, Cristiano R.W.; Vajdos, Felix F.

    2013-03-07

    Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS{sup 4}) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.

  2. Myeloperoxidase in chronic kidney disease.

    PubMed

    Madhusudhana Rao, A; Anand, Usha; Anand, C V

    2011-01-01

    Numerous lines of evidence implicate a role of myeloperoxidase (MPO) in the pathogenesis of cardiovascular disease (CVD). It is a well accepted fact that patients with chronic kidney disease (CKD) are at an increased risk for CVD. MPO is a pro-oxidant enzyme which could be involved in the increased susceptibility of these patients to CVD. Hence, the levels of plasma MPO was determined in healthy controls as well as in patients with CKD [stratified with the level of their kidney failure as CKD stages II-V (end stage renal disease)]. Plasma MPO was assayed by a spectrophotometric method. Serum urea and creatinine were estimated on a clinical chemistry analyzer using standard laboratory procedures. The mean plasma MPO levels were significantly lower with advancing stages of renal failure (P < 0.001). There was a positive correlation between MPO and GFR (r = +0.89, P < 0.001) and a negative correlation with urea (r = -0.85, P < 0.001) and creatinine (r = -0.82, P < 0.001). While an inverse association was observed between plasma MPO and urea in CKD patients, such an association was not observed in control subjects (P = 0.43). In conclusion, the decline in plasma MPO levels may be due to the inhibitory effect of uraemic toxins on the enzyme.

  3. Inactivation of transferrin iron binding capacity by the neutrophil myeloperoxidase system

    SciTech Connect

    Clark, R.A.; Pearson, D.W.

    1989-06-05

    Human serum apotransferrin was exposed to the isolated myeloperoxidase-H2O2-halide system or to phorbol ester-activated human neutrophils. Such treatment resulted in a marked loss in transferrin iron binding capacity as well as concomitant iodination of transferrin. Each component of the cell-free system (myeloperoxidase, H2O2, iodide) or neutrophil system (neutrophils, phorbol ester, iodide) was required in order to observe these changes. In the cell-free system, the H2O2 requirement was fulfilled by either reagent H2O2 or the peroxide-generating system glucose oxidase plus glucose. Both loss of iron binding capacity and transferrin iodination by either the myeloperoxidase system or activated neutrophils were blocked by azide or catalase. The isolated peroxidase system had an acidic pH optimum, whereas the intact cell system was more efficient at neutral pH. The kinetics of changes in iron binding capacity and iodination closely paralleled one another, exhibiting t1/2 values of less than 1 min for the myeloperoxidase-H2O2 system, 3-4 min for the myeloperoxidase-glucose oxidase system, and 8 min for the neutrophil system. That the occupied binding site is protected from the myeloperoxidase system was suggested by (1) a failure to mobilize iron from iron-loaded transferrin, (2) an inverse correlation between initial iron saturation and myeloperoxidase-mediated loss of iron binding capacity, and (3) decreased myeloperoxidase-mediated iodination of iron-loaded versus apotransferrin. Since as little as 1 atom of iodide bound per molecule of transferrin was associated with substantial losses in iron binding capacity, there appears to be a high specificity of myeloperoxidase-catalyzed iodination for residues at or near the iron binding sites. Amino acid analysis of iodinated transferrin (approximately 2 atoms/molecule) demonstrated that iodotyrosine was the predominant iodinated species.

  4. Myeloperoxidase is synthesized as larger phosphorylated precursor.

    PubMed Central

    Hasilik, A; Pohlmann, R; Olsen, R L; von Figura, K

    1984-01-01

    Synthesis and processing of myeloperoxidase were examined in metabolically labeled cells of the human promyelocyte line HL-60 and in an in vitro rabbit reticulocyte lysate system directed with HL-60 mRNA. Radioactivity labeled products were isolated by immunoprecipitation and analyzed by gel electrophoresis and fluorography. In vivo, myeloperoxidase was labeled initially as a 85-K glycosylated polypeptide (75 K after treatment with endo-beta-N-acetylglucosaminidase H). This polypeptide was soon processed to an 81-K intermediate and to smaller mature fragments of 60 K and 13 K within approximately 1 day. A minor portion of the precursor was converted to fragments of 40 K and 43 K. The pattern of labeled polypeptides of mature myeloperoxidase was similar to that of the enzyme purified from human leucocytes. The modifications of the polypeptide and of the oligosaccharide side chains in myeloperoxidase resembled those known to occur during the processing of lysosomal enzymes. In the absence or presence of dog pancreas membranes, myeloperoxidase was synthesized in vitro as a 76-K polypeptide or a 87-K glycosylated polypeptide, respectively. In HL-60 cells [32P]phosphate was incorporated into endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides. The presence of phosphorylated oligosaccharides was inferred from the fact that endocytosis of leucocyte myeloperoxidase in fibroblasts was sensitive to mannose 6-phosphate. It is suggested that myeloperoxidase is synthesized in the rough endoplasmic reticulum as a precursor of larger molecular mass and that the oligosaccharide side chains in the precursor are modified to contain mannose 6-phosphate residues which may be involved in the segregation and transport of the precursor. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6096138

  5. Immunomodulation by neutrophil myeloperoxidase and hydrogen peroxide: differential susceptibility of human lymphocyte functions.

    PubMed

    el-Hag, A; Lipsky, P E; Bennett, M; Clark, R A

    1986-05-01

    The coexistence of activated polymorphonuclear leukocytes and lymphocytes in tumor masses and inflammatory tissues suggests the possibility of interaction between secreted neutrophil products and nearby lymphocytes. To test this hypothesis, we examined the effects of neutrophil myeloperoxidase and H2O2 on lymphocytes. Human peripheral blood mononuclear leukocytes were exposed to myeloperoxidase, an H2O2-generating system (glucose + glucose oxidase), and a halide, and were then tested for functional activities. Natural killer activity against K562 cells, lymphocyte proliferation in response to mitogens, and generation of immunoglobulin-secreting cells were all susceptible to oxidative injury by myeloperoxidase and H2O2. The degree as well as the mechanism of suppression was dependent on the glucose oxidase concentration (i.e., the rate of H2O2 delivery). At low H2O2 flux, myeloperoxidase was essential for induction of lymphocyte suppression; as the rate of H2O2 generation increased, suppression became myeloperoxidase-independent and was mediated by H2O2 alone. Various lymphocyte functions were differentially susceptible to oxidative injury by myeloperoxidase and H2O2. The proliferative response to poke-weed mitogen was the least sensitive, whereas antibody formation was the most sensitive. Proliferative responses to concanavalin A and phytohemagglutinin as well as natural killer activity displayed intermediate degrees of susceptibility. In all assays, lymphocyte viability was greater than 90%. Removal of monocytes from mononuclear leukocytes by adherence to glass increased susceptibility of lymphocytes to oxidative injury. Monocytes in proportions within the range present in peripheral blood mononuclear leukocytes protected lymphocyte functions against oxidative injury by myeloperoxidase and H2O2. This study demonstrates a differential susceptibility of various immune functions to oxidative injury by the neutrophil products myeloperoxidase and H2O2, and shows, in

  6. INACTIVATION OF MYELOPEROXIDASE BY BENZOIC ACID HYDRAZIDE*

    PubMed Central

    Huang, Jiansheng; Smith, Forrest; Panizzi, Jennifer R.; Goodwin, Douglas C.; Panizzi, Peter

    2015-01-01

    Myeloperoxidase (MPO) is expressed by myeloid cells for the purpose of catalyzing the formation of hypochlorous acid, from chloride ions and reaction with a hydrogen peroxide-charged heme covalently bound to the enzyme. Most peroxidase enzymes both plant and mammalian are inhibited by benzoic acid hydrazide (BAH)-containing compounds, but the mechanism underlying MPO inhibition by BAH compounds is largely unknown. Recently, we reported MPO inhibition by BAH and 4-(trifluoromethyl)-BAH was due to hydrolysis of the ester bond between MPO heavy chain glutamate 242 (Glu242) residue and the heme pyrrole A ring, freeing the heme linked light chain MPO subunit from the larger remaining heavy chain portion. Here we probed the structure and function relationship behind this ester bond cleavage using a panel of BAH analogs to gain insight into the constraints imposed by the MPO active site and channel leading to the buried protoporphyrin IX ring. In addition, we show evidence that destruction of the heme ring does not occur by tracking the heme prosthetic group and provide evidence that the mechanism of hydrolysis follows a potential attack of the Glu242 carbonyl leading to a rearrangement causing the release of the vinyl-sulfonium linkage between HC-Met243 and the pyrrole A ring. PMID:25688920

  7. Myeloperoxidase activity and its corresponding mRNA expression as well as gene polymorphism in the population living in the coal-burning endemic fluorosis area in Guizhou of China.

    PubMed

    Zhang, Ting; Shan, Ke-Ren; Tu, Xi; He, Yan; Pei, Jin-Jing; Guan, Zhi-Zhong

    2013-06-01

    The myeloperoxidase (MPO) activity and its corresponding mRNA expression as well as gene polymorphism were investigated in the population who live in the endemic fluorosis area. In the study, 150 people were selected from the coal-burning endemic fluorosis area and 150 normal persons from the non-fluorosis area in Guizhou province of China. The blood samples were collected from these people. The activity of MPO in the plasma was determined by spectrophotometer; the expression of MPO mRNA was measured by employing real-time polymerase chain reaction; DNAs were extracted from the leucocytes in blood and five SNP genotypes of MPO promoter gene detected by a multiplex genotyping method, adapter-ligation-mediated allele-specific amplification. The results showed that the MPO activity and its corresponding mRNA in blood were significantly increased in the population living in the area of fluorosis. The different genotype frequencies of MPO, including -1228G/A, -585T/C, -463G/A, and -163C/T, and the three haplotypes with higher frequencies, including -163C-463G-585T-1228G-1276T, -163C-463G-585T-1228G-1276C, and -163C-463G-585T-1228A-1276T, were significantly associated with fluorosis. The results indicated that the elevated activity of MPO induced by endemic fluorosis may be connected in mechanism to the stimulated expression of MPO mRNA and the changed gene polymorphism.

  8. Myeloperoxidase: a target for new drug development?

    PubMed Central

    Malle, E; Furtmüller, P G; Sattler, W; Obinger, C

    2007-01-01

    Myeloperoxidase (MPO), a member of the haem peroxidase-cyclooxygenase superfamily, is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low- and high-spin ligands is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates, enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme activity of MPO and to ameliorate adverse effects of MPO-derived oxidants will be discussed. Emphasis will be put on mechanism-based inhibitors and high-throughput screening of compounds as well as the discussion of physiologically useful HOCl scavengers. PMID:17592500

  9. An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid, and its application in finding inhibitors which interact directly with the enzyme.

    PubMed

    Franck, T; Minguet, G; Delporte, C; Derochette, S; Zouaoui Boudjeltia, K; Van Antwerpen, P; Gach, O; Deby-Dupont, G; Mouithys-Mickalad, A; Serteyn, D

    2015-06-01

    Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme. The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content. To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation. The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.

  10. Inhibition of oxidation activity of myeloperoxidase (MPO) by propylthiouracil (PTU) and anti-MPO antibodies from patients with PTU-induced vasculitis.

    PubMed

    Zhang, Ai-Hua; Chen, Min; Gao, Ying; Zhao, Ming-Hui; Wang, Hai-Yan

    2007-02-01

    Propylthiouracil (PTU) could induce antineutrophil cytoplasmic antibody (ANCA) associated vasculitis. This study aimed to investigate the inhibitory effects on MPO oxidation activity by PTU and MPO-ANCA from patients with primary microscopic polyangiitis (MPA) and PTU-induced vasculitis. IgG preparations were purified from MPO-ANCA-positive sera from seven patients with PTU-induced vasculitis and ten patients with primary MPA. The oxidation activity of MPO was measured in the presence of PTU and MPO-ANCA-positive IgG preparations from patients with PTU-induced vasculitis and primary MPA respectively. PTU could competitively inhibit the oxidation activity of MPO dose dependently. MPO-ANCA-positive IgG preparations from 6/7 patients with PTU-induced vasculitis and only 3/10 from patients with primary MPA could inhibit the MPO activity in a dose-dependent manner. In conclusions, the oxidation activity of MPO could be inhibited by PTU and PTU-induced MPO-ANCA in a dose-dependent manner, which might be involved in the pathogenesis PTU-induced vasculitis.

  11. Low-Density Lipoprotein Modified by Myeloperoxidase in Inflammatory Pathways and Clinical Studies

    PubMed Central

    Vanhamme, Luc; Roumeguère, Thierry; Zouaoui Boudjeltia, Karim

    2013-01-01

    Oxidation of low-density lipoprotein (LDL) has a key role in atherogenesis. Among the different models of oxidation that have been studied, the one using myeloperoxidase (MPO) is thought to be more physiopathologically relevant. Apolipoprotein B-100 is the unique protein of LDL and is the major target of MPO. Furthermore, MPO rapidly adsorbs at the surface of LDL, promoting oxidation of amino acid residues and formation of oxidized lipoproteins that are commonly named Mox-LDL. The latter is not recognized by the LDL receptor and is accumulated by macrophages. In the context of atherogenesis, Mox-LDL accumulates in macrophages leading to foam cell formation. Furthermore, Mox-LDL seems to have specific effects and triggers inflammation. Indeed, those oxidized lipoproteins activate endothelial cells and monocytes/macrophages and induce proinflammatory molecules such as TNFα and IL-8. Mox-LDL may also inhibit fibrinolysis mediated via endothelial cells and consecutively increase the risk of thrombus formation. Finally, Mox-LDL has been involved in the physiopathology of several diseases linked to atherosclerosis such as kidney failure and consequent hemodialysis therapy, erectile dysfunction, and sleep restriction. All these issues show that the investigations of MPO-dependent LDL oxidation are of importance to better understand the inflammatory context of atherosclerosis. PMID:23983406

  12. Active transport and accumulation of bicarbonate by a unicellular cyanobacterium.

    PubMed

    Miller, A G; Colman, B

    1980-09-01

    The rates of inorganic carbon accumulation and carbon fixation in light by the unicellular cyanobacterim Coccohloris peniocystis have been determined. Cells incubated in the light in medium containing H14CO3- were rapidly separated from the medium by centrifugation through silicone oil into a strongly basic terminating solution. Samples of these inactivated cells were assayed to determine total 14C accumulation, and acid-treated samples were assayed to determine 14C fixation. The rate of transport of inorganic into illuminated cells was faster than the rate of CO2 production in the medium from HCO3- dehydration. This evidence for HCO3- transport in these cells is in agreement with our previous results based upon measurements of photosynthetic O2 evolution. A substantial pool of inorganic carbon was bulit up within the cells presumably as HCO3- before the onset of the maximum rate of photosynthesis. Large accumulation ratios were observed, greater than 1,000 times the external HCO3- concentration. Accumulation did not occur in the dark and was greatly suppressed by the photosynthesis inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 3-chloro-carbonylcyanide phenylhydrazone. These results indicate that the accumulation of inorganic carbon in these cells involves a light-dependent active transport process. PMID:6773925

  13. Plasminogen activator inhibitor-1 antisense oligodeoxynucleotides abrogate mesangial fibronectin accumulation.

    PubMed

    Park, Jehyun; Seo, Ji Yeon; Ha, Hunjoo

    2010-12-01

    Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 µM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

  14. Myeloperoxidase-produced Genomic DNA-centered Radicals and Protection by Resveratrol

    EPA Science Inventory

    Myeloperoxidase (MPO) released by activated neutrophils, production of hypochlorous acid (HOCI) and oxidation of the genomic DNA in epithelial cells is thought to initiate and promote carcinogenesis. In this study we applied the 5,5-dimethyl-l-pyrroline N-oxide (DMPO)-based i;nmu...

  15. Enantioselective epoxidation and carbon-carbon bond cleavage catalyzed by Coprinus cinereus peroxidase and myeloperoxidase.

    PubMed

    Tuynman, A; Spelberg, J L; Kooter, I M; Schoemaker, H E; Wever, R

    2000-02-01

    We demonstrate that myeloperoxidase (MPO) and Coprinus cinereus peroxidase (CiP) catalyze the enantioselective epoxidation of styrene and a number of substituted derivatives with a reasonable enantiomeric excess (up to 80%) and in a moderate yield. Three major differences with respect to the chloroperoxidase from Caldariomyces fumago (CPO) are observed in the reactivity of MPO and CiP toward styrene derivatives. First, in contrast to CPO, MPO and CiP produced the (S)-isomers of the epoxides in enantiomeric excess. Second, for MPO and CiP the H(2)O(2) had to be added very slowly (10 eq in 16 h) to prevent accumulation of catalytically inactive enzyme intermediates. Under these conditions, CPO hardly showed any epoxidizing activity; only with a high influx of H(2)O(2) (300 eq in 1.6 h) was epoxidation observed. Third, both MPO and CiP formed significant amounts of (substituted) benzaldehydes as side products as a consequence of C-alpha-C-beta bond cleavage of the styrene derivatives, whereas for CPO and cytochrome c peroxidase this activity is not observed. C-alpha-C-beta cleavage was the most prominent reaction catalyzed by CiP, whereas with MPO the relative amount of epoxide formed was higher. This is the first report of peroxidases catalyzing both epoxidation reactions and carbon-carbon bond cleavage. The results are discussed in terms of mechanisms involving ferryl oxygen transfer and electron transfer, respectively.

  16. Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

    PubMed

    Bian, Zhi-Ping; Li, Xiong-Zhi; Wu, Heng-Fang; Xu, Jin-Dan; Gu, Chun-Rong; Chen, Xiang-Jian; Yang, Di

    2016-04-01

    Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

  17. PARP activation promotes nuclear AID accumulation in lymphoma cells.

    PubMed

    Tepper, Sandra; Jeschke, Julia; Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-03-15

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma.

  18. PARP activation promotes nuclear AID accumulation in lymphoma cells

    PubMed Central

    Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-01-01

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma. PMID:26921193

  19. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    PubMed

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  20. Specific Sequence Motifs Direct the Oxygenation and Chlorination of Tryptophan by Myeloperoxidase

    PubMed Central

    Fu, Xiaoyun; Wang, Yi; Kao, Jeffery; Irwin, Angela; d’Avignon, André; Mecham, Robert P.; Parks, William C.; Heinecke, Jay W.

    2008-01-01

    Most studies of protein oxidation have typically focused on the reactivity of single amino acid side chains while ignoring the potential importance of adjacent sequences in directing the reaction pathway. We previously showed that hypochlorous acid (HOCl), a specific product of myeloperoxidase, inactivates matrilysin by modifying adjacent tryptophan and glycine (WG) residues in the catalytic domain. Here, we use model peptides that mimic the region of matrilysin involved in this reaction, VVWGTA, VVWATA and the library VVWXTA, to determine whether specific sequence motifs are targeted for chlorination or oxygenation by myeloperoxidase. Our results demonstrate that HOCl generated by myeloperoxidase or activated neutrophils converts the peptide VVWGTA to a chlorinated product, WG+32(Cl). Tandem mass spectrometry in concert with high resolution 1H and two-dimensional NMR analysis revealed that the modification required cross-linking of the tryptophan to the amide of glycine followed by chlorination of the indole ring of tryptophan. In contrast, when glycine in the peptide was replaced with alanine, the major products were mono- and di-oxygenated tryptophan residues. When the peptide library VVWXTA (where X represents all 20 common amino acids) was exposed to HOCl, only WG produced a high yield of the chloroindolenine derivative. However, when glycine was replaced by other amino acids, oxygenated tryptophan derivatives were the major products. Our observations indicate that WG may represent a specific sequence motif in proteins that is targeted for chlorination by myeloperoxidase. PMID:16548523

  1. Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates*

    PubMed Central

    Forbes, Louisa V.; Sjögren, Tove; Auchère, Françoise; Jenkins, David W.; Thong, Bob; Laughton, David; Hemsley, Paul; Pairaudeau, Garry; Turner, Rufus; Eriksson, Håkan; Unitt, John F.; Kettle, Anthony J.

    2013-01-01

    The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation. PMID:24194519

  2. Insights into myeloperoxidase biosynthesis from its inherited deficiency.

    PubMed

    Nauseef, W M

    1998-09-01

    Myeloperoxidase (MPO) is a heme protein present in the granules of neutrophils and monocytes. The activated neutrophil releases MPO into the phagolysosome or into the extracellular space in response to a variety of agonists. During concomitant activation of the NADPH-dependent oxidase, the stimulated neutrophil also generates hydrogen peroxide, and in this way the MPO-hydrogen peroxide-halide system exerts its potent microbicidal activity. Recent interest in MPO has extended well beyond the domain of innate host defense against infection and includes generalized inflammatory diseases, atherosclerosis, and degenerative neurologic diseases. Search of the various data banks using the cDNA sequence for MPO has uncovered previously unsuspected relationships among peroxidatively active proteins in widely different species. In addition, application of the analytical tools of cell and molecular biology has allowed definition of specific genotypes underlying MPO deficiency and the impact of particular mutations on the fate of MPO precursors along the biosynthetic pathway. In parallel, such studies have allowed significant advances in understanding of the normal steps in MPO biosynthesis and intracellular targeting.

  3. Payload drug vs. nanocarrier biodegradation by myeloperoxidase- and peroxynitrite-mediated oxidations: pharmacokinetic implications

    NASA Astrophysics Data System (ADS)

    Seo, Wanji; Kapralov, Alexandr A.; Shurin, Galina V.; Shurin, Michael R.; Kagan, Valerian E.; Star, Alexander

    2015-05-01

    With the advancement of nanocarriers for drug delivery into biomedical practice, assessments of drug susceptibility to oxidative degradation by enzymatic mechanisms of inflammatory cells become important. Here, we investigate oxidative degradation of a carbon nanotube-based drug carrier loaded with Doxorubicin. We employed myeloperoxidase-catalysed and peroxynitrite-mediated oxidative conditions to mimic the respiratory burst of neutrophils and macrophages, respectively. In addition, we revealed that the cytostatic and cytotoxic effects of free Doxorubicin, but not nanotube-carried drug, on melanoma and lung carcinoma cell lines were abolished in the presence of tumor-activated myeloid regulatory cells that create unique myeloperoxidase- and peroxynitrite-induced oxidative conditions. Both ex vivo and in vitro studies demonstrate that the nanocarrier protects the drug against oxidative biodegradation.With the advancement of nanocarriers for drug delivery into biomedical practice, assessments of drug susceptibility to oxidative degradation by enzymatic mechanisms of inflammatory cells become important. Here, we investigate oxidative degradation of a carbon nanotube-based drug carrier loaded with Doxorubicin. We employed myeloperoxidase-catalysed and peroxynitrite-mediated oxidative conditions to mimic the respiratory burst of neutrophils and macrophages, respectively. In addition, we revealed that the cytostatic and cytotoxic effects of free Doxorubicin, but not nanotube-carried drug, on melanoma and lung carcinoma cell lines were abolished in the presence of tumor-activated myeloid regulatory cells that create unique myeloperoxidase- and peroxynitrite-induced oxidative conditions. Both ex vivo and in vitro studies demonstrate that the nanocarrier protects the drug against oxidative biodegradation. Electronic supplementary information (ESI) available: Experimental details and data from characterization of materials synthesis and degradation studies. See DOI: 10

  4. Kinetic evidence for rapid oxidation of (-)-epicatechin by human myeloperoxidase

    SciTech Connect

    Spalteholz, Holger; Furtmueller, Paul Georg; Jakopitsch, Christa; Obinger, Christian; Schewe, Tankred; Sies, Helmut; Arnhold, Juergen

    2008-07-11

    Apocynin has been reported to require dimerization by myeloperoxidase (MPO) to inhibit leukocyte NADPH oxidase. (-)-Epicatechin, a dietary flavan-3-ol, has been identified as a 'prodrug' of apocynin-like metabolites that inhibit endothelial NADPH oxidase activity and elevate the cellular level of nitric oxide. Since (-)-epicatechin has tentatively been identified as substrate of MPO, we studied the one-electron oxidation of (-)-epicatechin by MPO. By using multi-mixing stopped-flow technique, we demonstrate that (-)-epicatechin is one of the most efficient electron donors for heme peroxidases investigated so far. Second order rate constants for the (-)-epicatechin-mediated conversion of MPO-compound I to compound II and compound II to resting enzyme were estimated to be 1.9 x 10{sup 7} and 4.5 x 10{sup 6} M{sup -1} s{sup -1}, respectively (pH 7, 25 deg. C). The data indicate that (-)-epicatechin is capable of undergoing fast MPO-mediated one-electron oxidation.

  5. The molecular mechanism for interaction of ceruloplasmin and myeloperoxidase

    NASA Astrophysics Data System (ADS)

    Bakhautdin, Bakytzhan; Bakhautdin, Esen Göksöy

    2016-04-01

    Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change.

  6. Evaluation of Antiradical and Anti-Inflammatory Activities of Ethyl Acetate and Butanolic Subfractions of Agelanthus dodoneifolius (DC.) Polhill & Wiens (Loranthaceae) Using Equine Myeloperoxidase and Both PMA-Activated Neutrophils and HL-60 Cells

    PubMed Central

    Boly, Rainatou; Franck, Thierry; Kohnen, Stephan; Lompo, Marius; Guissou, Innocent Pierre; Dubois, Jacques; Serteyn, Didier; Mouithys-Mickalad, Ange

    2015-01-01

    The ethyl acetate and n-butanolic subfractions of Agelanthus dodoneifolius were investigated for their antioxidant and antimyeloperoxidase (MPO) activities. The reactive oxygen species (ROS) generation was assessed by lucigenin-enhanced chemiluminescence (CL) and dichlorofluorescein- (DCF-) induced fluorescence techniques from phorbol myristate acetate- (PMA-) stimulated equine neutrophils and human myeloid cell line HL-60, respectively. In parallel, the effects of the tested subfractions were evaluated on the total MPO release by stimulated neutrophils and on the specific MPO activity by means of immunological assays. The results showed the potent activity of the butanolic subfraction, at least in respect of the chemiluminescence test (IC50 = 0.3 ± 0.1 µg/mL) and the ELISA and SIEFED assays (IC50 = 2.8 ± 1.2 µg/mL and 1.3 ± 1.0 µg/mL), respectively. However, the ethyl acetate subfraction was found to be the most potent in the DCF assay as at the highest concentration, DCF fluorescence intensity decreases of about 50%. Moreover, we demonstrated that the ethyl acetate subfraction was rich in catechin (16.51%) while it was not easy to identify the main compounds in the butanolic subfraction using the UPLC-MS/MS technique. Nevertheless, taken together, our results provide evidence that Agelanthus dodoneifolius subfractions may represent potential sources of natural antioxidants and of antimyeloperoxidase compounds. PMID:25821497

  7. EFFECT OF VAPOR-PHASE BIOREACTOR OPERATION ON BIOMASS ACCUMULATION, DISTRIBUTION, AND ACTIVITY. (R826168)

    EPA Science Inventory

    Excess biomass accumulation and activity loss in vapor-phase bioreactors (VPBs) can lead to unreliable long-term operation. In this study, temporal and spatial variations in biomass accumulation, distribution and activity in VPBs treating toluene-contaminated air were monitored o...

  8. Myeloperoxidase genotype, fruit and vegetable consumption, and breast cancer risk.

    PubMed

    Ahn, Jiyoung; Gammon, Marilie D; Santella, Regina M; Gaudet, Mia M; Britton, Julie A; Teitelbaum, Susan L; Terry, Mary Beth; Neugut, Alfred I; Josephy, P David; Ambrosone, Christine B

    2004-10-15

    Myeloperoxidase (MPO), an antimicrobial enzyme in the breast, generates reactive oxygen species (ROS) endogenously. An MPO G463A polymorphism exists in the promoter region, with the variant A allele conferring lower transcription activity than the common G allele. Because oxidative stress may play a role in breast carcinogenesis, we evaluated MPO genotypes in relation to breast cancer risk among 1,011 cases and 1,067 controls from the Long Island Breast Cancer Study Project (1996-1997). We also assessed the potential modifying effects of dietary antioxidants and hormonally related risk factors on these relationships. Women over 20 years with incident breast cancer who were residents of Nassau and Suffolk Counties, NY, were identified as potential cases. Population-based controls were frequency matched by 5-year age groups. Genotyping was performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) technology, and suspected breast cancer risk factors and usual dietary intake were assessed during an in-person interview. Unconditional logistic regression was used to estimate odds ratios and 95% confidence intervals. Having at least one A allele was associated with an overall 13% reduction in breast cancer risk. When consumption of fruits and vegetables and specific dietary antioxidants were dichotomized at the median, inverse associations with either GA or AA genotypes were most pronounced among women who consumed higher amounts of total fruits and vegetables (odds ratio, 0.75; 95% confidence interval, 0.58-0.97); this association was not noted among the low-consumption group (P for interaction = 0.04). Relationships were strongest among premenopausal women. Results from this first study of MPO genotypes and breast cancer risk indicate that MPO variants, related to reduced generation of ROS, are associated with decreased breast cancer risk, and emphasize the importance of fruit and vegetable consumption in reduction of breast

  9. Myeloperoxidase deficiency ameliorates progression of chronic kidney disease in mice.

    PubMed

    Lehners, Alexander; Lange, Sascha; Niemann, Gianina; Rosendahl, Alva; Meyer-Schwesinger, Catherine; Oh, Jun; Stahl, Rolf; Ehmke, Heimo; Benndorf, Ralf; Klinke, Anna; Baldus, Stephan; Wenzel, Ulrich Otto

    2014-08-15

    Myeloperoxidase (MPO) is an enzyme expressed in neutrophils and monocytes/macrophages. Beside its well-defined role in innate immune defence, it may also be responsible for tissue damage. To identify the role of MPO in the progression of chronic kidney disease (CKD), we investigated CKD in a model of renal ablation in MPO knockout and wild-type mice. CKD was induced by 5/6 nephrectomy. Mice were followed for 10 wk to evaluate the impact of MPO deficiency on renal morbidity. Renal ablation induced CKD in wild-type mice with increased plasma levels of MPO compared with controls. No difference was found between MPO-deficient and wild-type mice regarding albuminuria 1 wk after renal ablation, indicating similar acute responses to renal ablation. Over the next 10 wk, however, MPO-deficient mice developed significantly less albuminuria and glomerular injury than wild-type mice. This was accompanied by a significantly lower renal mRNA expression of the fibrosis marker genes plasminogen activator inhibitor-I, collagen type III, and collagen type IV as well as matrix metalloproteinase-2 and matrix metalloproteinase-9. MPO-deficient mice also developed less renal inflammation after renal ablation, as indicated by a lower infiltration of CD3-positive T cells and F4/80-positive monocytes/macrophages compared with wild-type mice. In vitro chemotaxis of monocyte/macrophages isolated from MPO-deficient mice was impaired compared with wild-type mice. No significant differences were observed for mortality and blood pressure after renal ablation. In conclusion, these results demonstrate that MPO deficiency ameliorates renal injury in the renal ablation model of CKD in mice.

  10. Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

    PubMed

    Shaeib, Faten; Khan, Sana N; Ali, Iyad; Najafi, Tohid; Maitra, Dhiman; Abdulhamid, Ibrahim; Saed, Ghassan M; Pennathur, Subramaniam; Abu-Soud, Husam M

    2015-01-01

    Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation. PMID:25835505

  11. An aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung inflammation in mice.

    PubMed

    Bachoual, Rafik; Talmoudi, Wifak; Boussetta, Tarek; Braut, Françoise; El-Benna, Jamel

    2011-06-01

    Punica granatum peel aqueous extract (PGE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. In this study we investigated the effect of PGE on human neutrophil reactive oxygen species (ROS) production in vitro and on LPS-induced lung inflammation in vivo in mice. Neutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H(2)O(2) was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethyl benzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. PGE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLF)- or phorbol myristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. PGE had no effect on superoxide anion generation, suggesting that it does not directly inhibit NADPH oxidase activity or activation pathways, or scavenge superoxide anions. PGE did not scavenge H(2)O(2) but directly inhibited myeloperoxidase activity in vitro. In vivo studies showed that PGE also attenuated LPS-induced lung inflammation in mice. So this study reveals that PGE inhibits neutrophil MPO activity and attenuates LPS-induced lung inflammation in mice. Inhibition of MPO activity by PGE could explain its anti-inflammatory action. PMID:21376769

  12. An aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung inflammation in mice.

    PubMed

    Bachoual, Rafik; Talmoudi, Wifak; Boussetta, Tarek; Braut, Françoise; El-Benna, Jamel

    2011-06-01

    Punica granatum peel aqueous extract (PGE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. In this study we investigated the effect of PGE on human neutrophil reactive oxygen species (ROS) production in vitro and on LPS-induced lung inflammation in vivo in mice. Neutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H(2)O(2) was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethyl benzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. PGE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLF)- or phorbol myristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. PGE had no effect on superoxide anion generation, suggesting that it does not directly inhibit NADPH oxidase activity or activation pathways, or scavenge superoxide anions. PGE did not scavenge H(2)O(2) but directly inhibited myeloperoxidase activity in vitro. In vivo studies showed that PGE also attenuated LPS-induced lung inflammation in mice. So this study reveals that PGE inhibits neutrophil MPO activity and attenuates LPS-induced lung inflammation in mice. Inhibition of MPO activity by PGE could explain its anti-inflammatory action.

  13. Comparative Ni tolerance and accumulation potentials between Mesembryanthemum crystallinum (halophyte) and Brassica juncea: Metal accumulation, nutrient status and photosynthetic activity.

    PubMed

    Amari, Taoufik; Ghnaya, Tahar; Debez, Ahmed; Taamali, Manel; Ben Youssef, Nabil; Lucchini, Giorgio; Sacchi, Gian Attilio; Abdelly, Chedly

    2014-11-01

    Saline soils often constitute sites of accumulation of industrial and urban wastes contaminated by heavy metals. Halophytes, i.e. native salt-tolerant species, could be more suitable for heavy metal phytoextraction from saline areas than glycophytes, most frequently used so far. In the framework of this approach, we assess here the Ni phytoextraction potential in the halophyte Mesembryanthemum crystallinum compared with the model species Brassica juncea. Plants were hydroponically maintained for 21 days at 0, 25, 50, and 100μM NiCl2. Nickel addition significantly restricted the growth activity of both species, and to a higher extent in M. crystallinum, which did not, however, show Ni-related toxicity symptoms on leaves. Interestingly, photosynthesis activity, chlorophyll content and photosystem II integrity assessed by chlorophyll fluorescence were less impacted in Ni-treated M. crystallinum as compared to B. juncea. The plant mineral nutrition was differently affected by NiCl2 exposure depending on the element, the species investigated and even the organ. In both species, roots were the preferential sites of Ni(2+) accumulation, but the fraction translocated to shoots was higher in B. juncea than in M. crystallinum. The relatively good tolerance of M. crystallinum to Ni suggests that this halophyte species could be used in the phytoextraction of moderately polluted saline soils. PMID:25171515

  14. Luminol-dependent photoemission from single neutrophil stimulated by phorbol ester and calcium ionophore--role of degranulation and myeloperoxidase

    SciTech Connect

    Suematsu, M.; Oshio, C.; Miura, S.; Suzuki, M.; Houzawa, S.; Tsuchiya, M.

    1988-08-30

    Luminol-dependent photonic burst from phorbol ester-treated single neutrophil was visually investigated by using an ultrasensitive photonic image intensifier microscope. Neutrophils stimulated by phorbol myristate acetate (0.1 microgram/ml) alone produced a negligible level of photonic activities in the presence of luminol (10 micrograms/ml). The additional application of 0.1 microM Ca2+ ionophore A23187 induced explosive changes of photonic burst corresponding to the distribution of neutrophils, and these photonic activities were gradually spread to extracellular space. Sodium azide, which prevents myeloperoxidase activity, inhibited Ca2+ ionophore-induced photonic burst from phorbol ester-treated neutrophil. These findings suggest a prerequisite role of degranulation and myeloperoxidase release in luminol-dependent photoemission from stimulated neutrophils.

  15. Myeloperoxidase-dependent Lipid Peroxidation Promotes the Oxidative Modification of Cytosolic Proteins in Phagocytic Neutrophils*

    PubMed Central

    Wilkie-Grantham, Rachel P.; Magon, Nicholas J.; Harwood, D. Tim; Kettle, Anthony J.; Vissers, Margreet C.; Winterbourn, Christine C.; Hampton, Mark B.

    2015-01-01

    Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation. PMID:25697357

  16. Gold enrichment in active geothermal systems by accumulating colloidal suspensions

    NASA Astrophysics Data System (ADS)

    Hannington, Mark; Harðardóttir, Vigdis; Garbe-Schönberg, Dieter; Brown, Kevin L.

    2016-04-01

    The origins of high-grade hydrothermal ore deposits are debated, but active geothermal systems provide important clues to their formation. The highest concentrations of gold are found in geothermal systems with direct links to island arc magmatism. Yet, similar concentrations have also been found in the absence of any input from arc magmas, for example, in the Reykjanes geothermal field, Iceland. Here we analyse brine samples taken from deep wells at Reykjanes and find that gold concentrations in the reservoir zone have increased over the past seven years from an average of 3 ppb to 14 ppb. The metal concentrations greatly exceed the maximum solubility of gold in the reservoir under saturated conditions and are now nearly two orders of magnitude higher than in mid-ocean ridge black smoker fluids--the direct analogues of Reykjanes deep liquids. We suggest that ongoing extraction of brine, the resulting pressure drop, and increased boiling have caused gold to drop out of solution and become trapped in the reservoir as a colloidal suspension. This process may explain how the stock of metal in the reservoirs of fossil geothermal systems could have increased over time and thus become available for the formation of gold-rich ore deposits.

  17. Mechanism of interferon-gamma production by monocytes stimulated with myeloperoxidase and neutrophil extracellular traps.

    PubMed

    Yamaguchi, Rui; Kawata, Jin; Yamamoto, Toshitaka; Ishimaru, Yasuji; Sakamoto, Arisa; Ono, Tomomichi; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-08-01

    Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell-cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFN-gamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signal-regulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase.

  18. Myeloperoxidase-dependent effect of amines on functions of isolated neutrophils.

    PubMed Central

    Thomas, E L; Grisham, M B; Jefferson, M M

    1983-01-01

    Isolated neutrophilic leukocytes were incubated with primary amines and related nitrogenous compounds. Stimulation of neutrophil oxygen (O2) metabolism with phorbol myristate acetate or opsonized zymosan resulted in production of hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (C1-) to hypochlorous acid (HOC1), and the reaction of HOC1 with the added compounds to yield nitrogen-chlorine (N-C1) derivatives. Formation of N-C1 derivatives of low lipid solubility resulted in accumulation of the derivatives in the extracellular medium. These oxidizing agents were identified and measured on the basis of their absorption spectra and their ability to oxidize 5-thio-2-nitrobenzoic acid to the disulfide form. The yield of N-Cl derivatives was in the order: taurine greater than Tris greater than spermidine greater than spermine greater than glucosamine greater than putrescine greater than guanidinoacetate. Accumulation of N-C1 derivatives was also observed in the absence of added amines, owing to the reaction of HOC1 with endogenous taurine and other amines that were released from the cells into the medium. In the presence of compounds that yield lipophilic N-C1 derivatives, little or no accumulation of oxidizing agents was observed. Instead, these compounds inhibited the accumulation of N-C1 derivatives that was obtained with taurine, and their effect was competitive with taurine. Inhibition was in the order: methylamine greater than ethanolamine greater than phenylethylamine greater than p-toluenesulfonamide greater than ammonia greater than guanidine. Formation of lipophilic N-C1 derivatives also resulted in inhibition of O2 uptake and glucose metabolism. Inhibition was prevented by adding catalase to eliminate H2O2, dapsone to inhibit myeloperoxidase, taurine to compete for reaction with HOC1, or compounds that are rapidly oxidized by HOC1 or N-C1 derivatives, to reduce these oxidizing agents. The results indicate that: (a) formation of N-C1

  19. Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells.

    PubMed

    Phung, Thuy Thi Bich; Sugamata, Ryuichi; Uno, Kazuko; Aratani, Yasuaki; Ozato, Keiko; Kawachi, Shoji; Thanh Nguyen, Liem; Nakayama, Toshinori; Suzuki, Kazuo

    2011-12-01

    Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.

  20. Myeloperoxidase-Oxidized LDLs Enhance an Anti-Inflammatory M2 and Antioxidant Phenotype in Murine Macrophages

    PubMed Central

    Sauvage, Aude; Van Steenbrugge, Martine

    2016-01-01

    Macrophages and oxidized LDLs play a key role in atherogenesis but their heterogeneity has been neglected up to now. Macrophages are prone to polarization and subsets of polarized macrophages have been described in atheromas. LDLs can be oxidized not only chemically by copper (Ox-LDLs) but also enzymatically by myeloperoxidase (MpOx-LDLs) resulting in oxidized LDLs poor in lipid peroxides. The effects of physiologically relevant myeloperoxidase-oxidized LDLs on macrophage polarization or on polarized macrophages remain largely unknown. In this study, the effects of LDLs on macrophage polarization were investigated by monitoring the expression of M1 and M2 genes following stimulation with native LDLs, Ox-LDLs, or MpOx-LDLs in RAW 264.7 cells. Except for MRC1, which is induced only by Ox-LDLs, MpOx-LDLs induced an overexpression of most of the selected marker genes at the mRNA level. MpOx-LDLs also modulate marker gene expression in polarized macrophages favoring notably anti-inflammatory Arg1 expression in M2 cells and also in the other phenotypes. Noteworthy, MpOx-LDLs were the most efficient to accumulate lipids intracellularly in (un)polarized macrophages whatever the phenotype. These data were largely confirmed in murine bone marrow-derived macrophages. Our data suggest that MpOx-LDLs were the most efficient to accumulate within cells and to enhance an anti-inflammatory and antioxidant phenotype in M2 cells and also in the other macrophage phenotypes.

  1. Potential Role of Plasma Myeloperoxidase Level in Predicting Long-Term Outcome of Acute Myocardial Infarction

    PubMed Central

    Kaya, Mehmet Gungor; Yalcin, Ridvan; Okyay, Kaan; Poyraz, Fatih; Bayraktar, Nilufer; Pasaoglu, Hatice; Boyaci, Bulent; Cengel, Atiye

    2012-01-01

    We investigated the prognostic importance of plasma myeloperoxidase levels in patients with ST-elevation myocardial infarction (STEMI) at long-term follow-up, and we analyzed the correlations between plasma myeloperoxidase levels and other biochemical values. We evaluated 73 consecutive patients (56 men; mean age, 56 ±11 yr) diagnosed with acute STEMI and 46 age- and sex-matched healthy control participants. Patients were divided into 2 groups according to the median myeloperoxidase level (Group 1: plasma myeloperoxidase ≤68 ng/mL; and Group 2: plasma myeloperoxidase >68 ng/mL). Patients were monitored for the occurrence of major adverse cardiovascular events (MACE), which were defined as cardiac death; reinfarction; new hospital admission for angina; heart failure; and revascularization procedures. The mean follow-up period was 25 ± 16 months. Plasma myeloperoxidase levels were higher in STEMI patients than in control participants (82 ± 34 vs 20 ±12 ng/mL; P=0.001). Composite MACE occurred in 12 patients with high myeloperoxidase levels (33%) and in 4 patients with low myeloperoxidase levels (11%) (P=0.02). The incidences of nonfatal recurrent myocardial infarction and verified cardiac death were higher in the high-mye-loperoxidase group. In multivariate analysis, high plasma myeloperoxidase levels were independent predictors of MACE (odds ratio = 3.843; <95% confidence interval, 1.625–6.563; P=0.003). High plasma myeloperoxidase levels identify patients with a worse prognosis after acute STEMI at 2-year follow-up. Evaluation of plasma myeloperoxidase levels might be useful in determining patients at high risk of death and MACE who can benefit from further aggressive treatment and closer follow-up. PMID:22949765

  2. Polyol accumulation by Aspergillus oryzae at low water activity in solid-state fermentation.

    PubMed

    Ruijter, George J G; Visser, Jaap; Rinzema, Arjen

    2004-04-01

    Polyol accumulation and metabolism were examined in Aspergillus oryzae cultured on whole wheat grains or on wheat dough as a model for solid-state culture. In solid-state fermentation (SSF), water activity (a(w)) is typically low resulting in osmotic stress. In addition to a high level of mannitol, which is always present in the cells, A. oryzae accumulated high concentrations of glycerol, erythritol and arabitol at relatively low a(w) (0.96-0.97) in SSF. Accumulation of such a mixture of polyols is rather unusual and might be typical for SSF. A. oryzae mycelium accumulating various polyols at low a(w) contained at least four distinct polyol dehydrogenases with highest activities toward glycerol, erythritol, D-arabitol and mannitol. NADP(+)-dependent glycerol dehydrogenase activity correlated very well with glycerol accumulation. A similar correlation was observed for erythritol and NADP(+)-erythritol dehydrogenase suggesting that NADP(+)-dependent glycerol and erythritol dehydrogenases are involved in biosynthesis of glycerol and erythritol, respectively, and that these enzymes are induced by osmotic stress.

  3. Betanin inhibits the myeloperoxidase/nitrite-induced oxidation of human low-density lipoproteins.

    PubMed

    Allegra, Mario; Tesoriere, Luisa; Livrea, Maria A

    2007-03-01

    Production of nitrogen dioxide by the activity of myeloperoxidase (MPO) in the presence of nitrite is now considered a key step in the pathophysiology of low-density lipoprotein (LDL) oxidation. This study shows that betanin, a phytochemical of the betalain class, inhibits the production of lipid hydroperoxides in human LDL submitted to a MPO/nitrite-induced oxidation. Kinetic measurements including time-course of particle oxidation and betanin consumption, either in the presence or in the absence of nitrite, suggest that the antioxidant effect is possibly the result of various actions. Betanin scavenges the initiator radical nitrogen dioxide and can also act as a lipoperoxyl radical-scavenger. In addition, unidentified oxidation product(s) of betanin by MPO/nitrite inhibit(s) the MPO/nitrite-induced LDL oxidation as effectively as the parent compound. In the light of betanin bioavailability and post-absorbtion distribution in humans, present findings may suggest favourable in vivo activity of this phytochemical.

  4. ANTIMICROBIAL ACTIVITY OF COPPER AND ZINC ACCUMULATED BY EASTERN OYSTER AMEBOCYTES

    EPA Science Inventory

    Fisher, William S. Submitted. Antimicrobial Activity of Copper and Zinc Accumulated by Eastern Oyster Amebocytes. J. Shellfish Res. 54 p. (ERL,GB 1196).

    The distribution of eastern oysters Crassostrea virginica near terrestrial watersheds has led to a general impression t...

  5. Role of Myeloperoxidase in Patients with Chronic Kidney Disease

    PubMed Central

    Kisic, Bojana; Miric, Dijana; Dragojevic, Ilija; Rasic, Julijana; Popovic, Ljiljana

    2016-01-01

    Chronic kidney disease (CKD) is a worldwide public health problem. Patients with CKD have a number of disorders in the organism, and the presence of oxidative stress and systemic inflammation in these patients is the subject of numerous studies. Chronic inflammation joined with oxidative stress contributes to the development of numerous complications: accelerated atherosclerosis process and cardiovascular disease, emergence of Type 2 diabetes mellitus, development of malnutrition, anaemia, hyperparathyroidism, and so forth, affecting the prognosis and quality of life of patients with CKD. In this review we presented the potential role of the myeloperoxidase enzyme in the production of reactive/chlorinating intermediates and their role in oxidative damage to biomolecules in the body of patients with chronic kidney disease and end-stage renal disease. In addition, we discussed the role of modified lipoprotein particles under the influence of prooxidant MPO intermediates in the development of endothelial changes and cardiovascular complications in renal failure. PMID:27127544

  6. Myeloperoxidase-antineutrophil cytoplasmic antibody-associated sensorineural hearing loss.

    PubMed

    Maguchi, S; Fukuda, S; Chida, E; Terayama, Y

    2001-05-01

    A 36-year-old female with hyperthyroidism that had been treated with propilthiouracil (PTU) complained of tinnitus and hearing loss in both ears. She was treated with steroid administration by an otolaryngologist; however, hearing continued to fluctuate when the steroids were tapered. Laboratory evaluation revealed a decreased complement level and elevated levels of immunoglobulin M (IgM) and myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA). With the withdrawal of PTU and high-dose methylprednisolone, she had excellent return of right-sided hearing. In recent years, there have been many reports about MPO-ANCA-associated small vessel vasculitis. Although any organ may be affected by this disease, there are no reports about MPO-ANCA-associated progressive hearing loss without any other organ involvement. The present case suggests the possibility that inner ear blood flow impairment due to ANCA-associated small vessel vasculitis induces the so-called autoimmune sensorineural hearing loss.

  7. Phospho-dependent Accumulation of GABABRs at Presynaptic Terminals after NMDAR Activation.

    PubMed

    Hannan, Saad; Gerrow, Kim; Triller, Antoine; Smart, Trevor G

    2016-08-16

    Here, we uncover a mechanism for regulating the number of active presynaptic GABAB receptors (GABABRs) at nerve terminals, an important determinant of neurotransmitter release. We find that GABABRs gain access to axon terminals by lateral diffusion in the membrane. Their relative accumulation is dependent upon agonist activation and the presence of the two distinct sushi domains that are found only in alternatively spliced GABABR1a subunits. Following brief activation of NMDA receptors (NMDARs) using glutamate, GABABR diffusion is reduced, causing accumulation at presynaptic terminals in a Ca(2+)-dependent manner that involves phosphorylation of GABABR2 subunits at Ser783. This signaling cascade indicates how synaptically released glutamate can initiate, via a feedback mechanism, increased levels of presynaptic GABABRs that limit further glutamate release and excitotoxicity.

  8. Phospho-dependent Accumulation of GABABRs at Presynaptic Terminals after NMDAR Activation.

    PubMed

    Hannan, Saad; Gerrow, Kim; Triller, Antoine; Smart, Trevor G

    2016-08-16

    Here, we uncover a mechanism for regulating the number of active presynaptic GABAB receptors (GABABRs) at nerve terminals, an important determinant of neurotransmitter release. We find that GABABRs gain access to axon terminals by lateral diffusion in the membrane. Their relative accumulation is dependent upon agonist activation and the presence of the two distinct sushi domains that are found only in alternatively spliced GABABR1a subunits. Following brief activation of NMDA receptors (NMDARs) using glutamate, GABABR diffusion is reduced, causing accumulation at presynaptic terminals in a Ca(2+)-dependent manner that involves phosphorylation of GABABR2 subunits at Ser783. This signaling cascade indicates how synaptically released glutamate can initiate, via a feedback mechanism, increased levels of presynaptic GABABRs that limit further glutamate release and excitotoxicity. PMID:27498877

  9. Ethnic differences in pre-adipocyte intracellular lipid accumulation and alkaline phosphatase activity.

    PubMed

    Ali, Aus T; Chirambo, George; Penny, Clement; Paiker, Janice E; Ikram, Faisel; Psaras, George; Crowther, Nigel J

    2015-01-01

    Alkaline phosphatase (ALP) increases lipid accumulation in human pre-adipocytes. This study was performed to assess whether ethnic differences in the prevalence of obesity in African and European females are related to differences in pre-adipocyte lipid accretion and ALP activity. Pre-adipocytes were isolated from 13 black and 14 white females. Adipogenesis was quantified using the lipid dye, Oil red O, whilst ALP activity was assayed in cell extracts on day zero and 12days after initiating adipogenesis. Lipid levels (OD units/mg protein) were lower in pre-adipocytes from white than black females on day 0 (0.36±0.05 versus 0.44±0.03, respectively; p<0.0005) and day 12 (1.18±0.14 versus 1.80±0.22, respectively; p<0.0005), as was ALP activity (mU/mg protein) on day zero (36.5±5.8 versus 136.4±10.9, respectively; p<0.0005) and day 12 (127±16 versus 278±27, respectively; p<0.0005). Treatment of pre-adipocytes with histidine, an ALP inhibitor, blocked lipid accumulation. Thus, lipid uptake is higher in pre-adipocytes isolated from black compared to white females which parallels the obesity prevalence rates in these population groups. The reason for higher fat accumulation in pre-adipocytes isolated from black females may be related to higher ALP activity.

  10. AMPK over-activation leads to accumulation of α-synuclein oligomers and decrease of neurites

    PubMed Central

    Jiang, Peizhou; Gan, Ming; Ebrahim, Abdul Shukkur; Castanedes-Casey, Monica; Dickson, Dennis W.; Yen, Shu-Hui C.

    2012-01-01

    Neuronal inclusions of α-synuclein (α-syn), termed Lewy bodies, are a hallmark of Parkinson disease (PD). Increased α-syn levels can occur in brains of aging human and neurotoxin treated mice. Since previous studies have shown increased brain lactate levels in aging brains, in PD affected subjects when compared to age-matched controls, and in mice treated with MPTP, we tested the effects of lactate exposure on α-syn in a cell based-study. We demonstrated that (i) lactate treatment led to α-syn accumulation and oligomerization in a time- and concentration-dependent manner, (ii) such alterations were mediated via adenosine-monophosphate activated protein kinase (AMPK) and associated with increasing cytoplasmic phosphorylated AMPK levels, (iii) AMPK activation facilitated α-syn accumulation and phosphorylation, (iv) lactate treatment or overexpression of active form of AMPK decreased α-syn turnover and neurite outgrowth and (v) Lewy body-bearing neurons displayed abnormal cytoplasmic distribution of phosphorylated AMPK, which normally is located in nuclei. Together, our results suggest that chronic neuronal accumulation of α-syn induced by lactate-triggered AMPK activation in aging brains may be a novel mechanism underlying α-synucleionpathies in PD and related disorders. PMID:23200460

  11. Accumulated Bending Energy Elicits Neutral Sphingomyelinase Activity in Human Red Blood Cells

    PubMed Central

    López, David J.; Egido-Gabas, Meritxell; López-Montero, Iván; Busto, Jon V.; Casas, Josefina; Garnier, Marie; Monroy, Francisco; Larijani, Banafshé; Goñi, Félix M.; Alonso, Alicia

    2012-01-01

    We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A2. The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypo-and hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A2 activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stress-induced ceramide synthesis and apoptosis. PMID:22824271

  12. Tomato responds to green peach aphid infestation with the activation of trehalose metabolism and starch accumulation.

    PubMed

    Singh, Vijay; Shah, Jyoti

    2012-06-01

    The disaccharide trehalose and trehalose-6-phosphate that are present in trace amounts are suggested to have a signaling function in plants. Recently, it was demonstrated that trehalose metabolism contributes to Arabidopsis thaliana defense against the green peach aphid (GPA; Myzus persicae Sülzer), an important insect pest of a large variety of plants. TPS11 (TREHALOSE PHOSPHATE SYNTHASE11)-dependent trehalose metabolism was shown to curtail GPA infestation by promoting starch accumulation and expression of the PAD4 (PHYTOALEXIN-DEFICIENT4) gene, which has important roles in regulating antibiosis and antixenosis against GPA. Here we show that trehalose metabolism is similarly activated in leaves of GPA-infested tomato (Solanum lycopersicum) plants and likely contributes to tomato defense against GPA. GPA-infested leaves of tomato accumulated trehalose, which was accompanied by the transient upregulation of SlTPS11, a homolog of the Arabidopsis TPS11. GPA-infestation was also accompanied by starch accumulation and the upregulation of SlPAD4, the tomato homolog of Arabidopsis PAD4. Furthermore, trehalose application induced SlPAD4 expression and starch accumulation, and curtailed GPA infestation, suggesting that like in Arabidopsis trehalose contributes to tomato defense against GPA.

  13. Distinctive role of activated tumor-associated macrophages in photosensitizer accumulation

    NASA Astrophysics Data System (ADS)

    Korbelik, Mladen; Krosl, Gorazd

    1995-05-01

    Cells dissociated from tumors (carcinomas and sarcomas) growing subcutaneously in mice that have been administered Photofrin or other photosensitizers were analyzed by flow cytometry. Monoclonal antibodies were used for identification of major cellular populations contained in these tumors. The results demonstrate that a subpopulation of tumor-associated macrophages (TAMs) is unique among tumor cell populations in that it excels in the accumulation of very high levels of photosensitizers. These macrophages showed an increased expression of interleukin 2 receptor, which is indicative of their activated state. since macrophages were reported to concentrate in the periphery of human neoplasms, it is suggested that activates TAMs are the determinants of tumor-localized photosensitizer fluorescence.

  14. Proteinase 3-ANCA Vasculitis versus Myeloperoxidase-ANCA Vasculitis

    PubMed Central

    Hilhorst, Marc; van Paassen, Pieter

    2015-01-01

    In patients with GN or vasculitis, ANCAs are directed against proteinase 3 (PR3) or myeloperoxidase (MPO). The differences between PR3-ANCA-associated vasculitis (AAV) and MPO-AAV described in the past have been supplemented during the last decade. In this review, we discuss the differences between these two small-vessel vasculitides, focusing especially on possible etiologic and pathophysiologic differences. PR3-AAV is more common in northern parts of the world, whereas MPO-AAV is more common in southern regions of Europe, Asia, and the Pacific, with the exception of New Zealand and Australia. A genetic contribution has been extensively studied, and there is a high prevalence of the HLA-DPB1*04:01 allele in patients with PR3-AAV as opposed to patients with MPO-AAV and/or healthy controls. Histologically, MPO-AAV and PR3-AAV are similar but show qualitative differences when analyzed carefully. Clinically, both serotypes are difficult to distinguish, but quantitative differences are present. More organs are affected in PR3-AAV, whereas renal limited vasculitis occurs more often in patients with MPO-AAV. For future clinical trials, we advocate classifying patients by ANCA serotype as opposed to the traditional disease type classification. PMID:25956510

  15. Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization.

    PubMed

    Okada, Sabrina Sayori; de Oliveira, Edson Mendes; de Araújo, Tomaz Henrique; Rodrigues, Maria Rita; Albuquerque, Renata Chaves; Mortara, Renato Arruda; Taniwaki, Noemi Nosomi; Nakaya, Helder Imoto; Campa, Ana; Moreno, Ana Carolina Ramos

    2016-02-01

    Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes. PMID:26632272

  16. Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses.

    PubMed

    Minguet, Grégory; Franck, Thierry; Joris, Jean; Ceusters, Justine; Mouithys-Mickalad, Ange; Serteyn, Didier; Sandersen, Charlotte

    2015-05-15

    Volatile anaesthestics have shown to modulate the oxidative response of polymorphonuclear neutrophils (PMNs). We investigated the effects of isoflurane and sevoflurane on the degranulation of total and active myeloperoxidase (MPO) from horse PMNs and their direct interaction with MPO activity. Whole blood from horse was incubated in 1 and 2 minimal alveolar concentrations (MAC) of isoflurane or sevoflurane for 1h and PMNs were stimulated with cytochalasin B (CB) plus N-formyl-méthionyl-leucyl-phenylalanine (fMLP). After stimulation, the plasma was collected to measure total and active MPO by enzyme-linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. The effects of 1 and 2 MAC of isoflurane and sevoflurane on the peroxidase and chlorination activity of pure MPO were assessed by fluorescence using Amplex red and 3'-(p-aminophenyl) fluorescein (APF) respectively and in parallel with a SIEFED assay to estimate the potential interaction of the anaesthetics with the enzyme. Although isoflurane and sevoflurane had inconsistent effects on total MPO release, both volatile agents reduced active MPO release and showed a direct inhibition on the peroxidase and the chlorination activity of the enzyme. A persistent interaction between MPO and anaesthetics was evidenced with isoflurane but not with sevoflurane.

  17. Effect of acetic acid on lipid accumulation by glucose-fed activated sludge cultures

    SciTech Connect

    Mondala, Andro; Hernandez, Rafael; French, Todd; McFarland, Linda; Sparks, Darrell; Holmes, William; Haque, Monica

    2012-01-01

    The effect of acetic acid, a lignocellulose hydrolysis by-product, on lipid accumulation by activated sludge cultures grown on glucose was investigated. This was done to assess the possible application of lignocellulose as low-cost and renewable fermentation substrates for biofuel feedstock production. Results: Biomass yield was reduced by around 54% at a 2 g L -1 acetic acid dosage but was increased by around 18% at 10 g L -1 acetic acid dosage relative to the control run. The final gravimetric lipid contents at 2 and 10 g L -1 acetic acid levels were 12.5 + 0.7% and 8.8 + 3.2% w/w, respectively, which were lower than the control (17.8 + 2.8% w/w). However, biodiesel yields from activated sludge grown with acetic acid (5.6 + 0.6% w/w for 2 g L -1 acetic acid and 4.2 + 3.0% w/w for 10 g L -1 acetic acid) were higher than in raw activated sludge (1-2% w/w). The fatty acid profiles of the accumulated lipids were similar with conventional plant oil biodiesel feedstocks. Conclusions: Acetic acid enhanced biomass production by activated sludge at high levels but reduced lipid production. Further studies are needed to enhance acetic acid utilization by activated sludge microorganisms for lipid biosynthesis.

  18. Plasma membrane overgrowth causes fibrotic collagen accumulation and immune activation in Drosophila adipocytes

    PubMed Central

    Zang, Yiran; Wan, Ming; Liu, Min; Ke, Hongmei; Ma, Shuangchun; Liu, Lu-Ping; Ni, Jian-Quan; Carlos Pastor-Pareja, José

    2015-01-01

    Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. Little is known about the factors that determine preferential onset of fibrosis in particular tissues. Here we show that plasma membrane (PM) overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. We found that loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of PM is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion. Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response. Our work indicates that traffic imbalances and PM topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases. DOI: http://dx.doi.org/10.7554/eLife.07187.001 PMID:26090908

  19. Activity and Accumulation of Cell Division-Promoting Phenolics in Tobacco Tissue Cultures 1

    PubMed Central

    Teutonico, Rita A.; Dudley, Matthew W.; Orr, John D.; Lynn, David G.; Binns, Andrew N.

    1991-01-01

    Dehydrodiconiferyl alcohol glucosides (DCGs) are derivatives of the phenylpropanoid pathway that have been isolated from Catharansus roseus L. (Vinca rosea) crown gall tumors. Fractions containing purified DCGs have been shown previously to promote the growth of cytokinin-requiring tissues of tobacco in the absence of exogenous cytokinins. In this study, we utilized synthetic DCG isomers to confirm the cell division-promoting activity of DCG isomers A and B and show that they neither promote shoot meristem initiation on Nicotiana tabacum L., cv Havana 425, leaf explants nor induce betacyanin synthesis in amaranth seedlings. Analysis of cultured tobacco pith tissue demonstrated that DCG accumulation was stimulated by cytokinin treatment and correlated with cytokinin-induced cell division. Thus, the accumulation of metabolites that could replace cytokinin in cell division bioassays is stimulated by cytokinins. These data support the model that DCGs are a component of a cytokinin-mediated regulatory circuit controlling cell division. ImagesFigure 2 PMID:16668384

  20. Salivary Myeloperoxidase, Assessed by 3,3'-Diaminobenzidine Colorimetry, Can Differentiate Periodontal Patients from Nonperiodontal Subjects.

    PubMed

    Klangprapan, Supaporn; Chaiyarit, Ponlatham; Hormdee, Doosadee; Kampichai, Amonrujee; Khampitak, Tueanjit; Daduang, Jureerut; Tavichakorntrakool, Ratree; Panijpan, Bhinyo; Boonsiri, Patcharee

    2016-01-01

    Periodontal diseases, which result from inflammation of tooth supporting tissues, are highly prevalent worldwide. Myeloperoxidase (MPO), from certain white blood cells in saliva, is a biomarker for inflammation. We report our study on the salivary MPO activity and its association with severity of periodontal diseases among Thai patients. Periodontally healthy subjects (n = 11) and gingivitis (n = 32) and periodontitis patients (n = 19) were enrolled. Assessments of clinically periodontal parameters were reported as percentages for gingival bleeding index (GI) and bleeding on probing (BOP), whereas pocket depth (PD) and clinical attachment loss (CAL) were measured in millimeters and then made to index scores. Salivary MPO activity was measured by colorimetry using 3,3'-diaminobenzidine as substrate. The results showed that salivary MPO activity in periodontitis patients was significantly higher than in healthy subjects (p = 0.003) and higher than in gingivitis patients (p = 0.059). No difference was found between gingivitis and healthy groups (p = 0.181). Significant correlations were observed (p < 0.01) between salivary MPO activity and GI (r = 0.632, p < 0.001), BOP (r = 0.599, p < 0.001), PD (r = 0.179, p = 0.164), and CAL (r = 0.357, p = 0.004) index scores. Sensitivity (94.12%), specificity (54.55%), and positive (90.57%) and negative (66.67%) predictive values indicate that salivary MPO activity has potential use as a screening marker for oral health of the Thai community. PMID:27274868

  1. Myeloperoxidase Inactivates TIMP-1 by Oxidizing Its N-terminal Cysteine Residue

    PubMed Central

    Wang, Yi; Rosen, Henry; Madtes, David K.; Shao, Baohai; Martin, Thomas R.; Heinecke, Jay W.; Fu, Xiaoyun

    2016-01-01

    An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation. PMID:17726014

  2. Activation of PPARα by Fatty Acid Accumulation Enhances Fatty Acid Degradation and Sulfatide Synthesis.

    PubMed

    Yang, Yang; Feng, Yuyao; Zhang, Xiaowei; Nakajima, Takero; Tanaka, Naoki; Sugiyama, Eiko; Kamijo, Yuji; Aoyama, Toshifumi

    2016-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first reaction in the mitochondrial fatty acid β-oxidation pathway. VLCAD deficiency is associated with the accumulation of fat in multiple organs and tissues, which results in specific clinical features including cardiomyopathy, cardiomegaly, muscle weakness, and hepatic dysfunction in infants. We speculated that the abnormal fatty acid metabolism in VLCAD-deficient individuals might cause cell necrosis by fatty acid toxicity. The accumulation of fatty acids may activate peroxisome proliferator-activated receptor (PPAR), a master regulator of fatty acid metabolism and a potent nuclear receptor for free fatty acids. We examined six skin fibroblast lines, derived from VLCAD-deficient patients and identified fatty acid accumulation and PPARα activation in these cell lines. We then found that the expression levels of three enzymes involved in fatty acid degradation, including long-chain acyl-CoA synthetase (LACS), were increased in a PPARα-dependent manner. This increased expression of LACS might enhance the fatty acyl-CoA supply to fatty acid degradation and sulfatide synthesis pathways. In fact, the first and last reactions in the sulfatide synthesis pathway are regulated by PPARα. Therefore, we also measured the expression levels of enzymes involved in sulfatide metabolism and the regulation of cellular sulfatide content. The levels of these enzymes and cellular sulfatide content both increased in a PPARα-dependent manner. These results indicate that PPARα activation plays defensive and compensative roles by reducing cellular toxicity associated with fatty acids and sulfuric acid. PMID:27644403

  3. RIM Promotes Calcium Channel Accumulation at Active Zones of the Drosophila Neuromuscular Junction

    PubMed Central

    Graf, Ethan R.; Valakh, Vera; Wright, Christina M.; Wu, Chunlai; Liu, Zhihua; Zhang, Yong Q.; DiAntonio, Aaron

    2012-01-01

    Summary Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca2+ channels and proteins that influence Ca2+ channel accumulation at release sites. Here we identify Drosophila RIM and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission, due to a significant reduction in both the clustering of Ca2+ channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Since RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca2+ channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca2+ channel accumulation but is not a Rab3 effector for release machinery distribution across release sites. PMID:23175814

  4. Myeloperoxidase-Oxidized LDLs Enhance an Anti-Inflammatory M2 and Antioxidant Phenotype in Murine Macrophages

    PubMed Central

    Sauvage, Aude; Van Steenbrugge, Martine

    2016-01-01

    Macrophages and oxidized LDLs play a key role in atherogenesis but their heterogeneity has been neglected up to now. Macrophages are prone to polarization and subsets of polarized macrophages have been described in atheromas. LDLs can be oxidized not only chemically by copper (Ox-LDLs) but also enzymatically by myeloperoxidase (MpOx-LDLs) resulting in oxidized LDLs poor in lipid peroxides. The effects of physiologically relevant myeloperoxidase-oxidized LDLs on macrophage polarization or on polarized macrophages remain largely unknown. In this study, the effects of LDLs on macrophage polarization were investigated by monitoring the expression of M1 and M2 genes following stimulation with native LDLs, Ox-LDLs, or MpOx-LDLs in RAW 264.7 cells. Except for MRC1, which is induced only by Ox-LDLs, MpOx-LDLs induced an overexpression of most of the selected marker genes at the mRNA level. MpOx-LDLs also modulate marker gene expression in polarized macrophages favoring notably anti-inflammatory Arg1 expression in M2 cells and also in the other phenotypes. Noteworthy, MpOx-LDLs were the most efficient to accumulate lipids intracellularly in (un)polarized macrophages whatever the phenotype. These data were largely confirmed in murine bone marrow-derived macrophages. Our data suggest that MpOx-LDLs were the most efficient to accumulate within cells and to enhance an anti-inflammatory and antioxidant phenotype in M2 cells and also in the other macrophage phenotypes. PMID:27656049

  5. Myeloperoxidase-dependent Inactivation of Surfactant Protein D in Vitro and in Vivo*

    PubMed Central

    Crouch, Erika C.; Hirche, Tim O.; Shao, Baohai; Boxio, Rachel; Wartelle, Julien; Benabid, Rym; McDonald, Barbara; Heinecke, Jay; Matalon, Sadis; Belaaouaj, Azzaq

    2010-01-01

    Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H2O2-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity. PMID:20228064

  6. Myeloperoxidase deletion prevents high-fat diet-induced obesity and insulin resistance.

    PubMed

    Wang, Qilong; Xie, Zhonglin; Zhang, Wencheng; Zhou, Jun; Wu, Yue; Zhang, Miao; Zhu, Huaiping; Zou, Ming-Hui

    2014-12-01

    Activation of myeloperoxidase (MPO), a heme protein primarily expressed in granules of neutrophils, is associated with the development of obesity. However, whether MPO mediates high-fat diet (HFD)-induced obesity and obesity-associated insulin resistance remains to be determined. Here, we found that consumption of an HFD resulted in neutrophil infiltration and enhanced MPO expression and activity in epididymal white adipose tissue, with an increase in body weight gain and impaired insulin signaling. MPO knockout (MPO(-/-)) mice were protected from HFD-enhanced body weight gain and insulin resistance. The MPO inhibitor 4-aminobenzoic acid hydrazide reduced peroxidase activity of neutrophils and prevented HFD-enhanced insulin resistance. MPO deficiency caused high body temperature via upregulation of uncoupling protein-1 and mitochondrial oxygen consumption in brown adipose tissue. Lack of MPO also attenuated HFD-induced macrophage infiltration and expression of proinflammatory cytokines. We conclude that activation of MPO in adipose tissue contributes to the development of obesity and obesity-associated insulin resistance. Inhibition of MPO may be a potential strategy for prevention and treatment of obesity and insulin resistance.

  7. UV-B-Induced PR-1 Accumulation Is Mediated by Active Oxygen Species.

    PubMed

    Green, R.; Fluhr, R.

    1995-02-01

    Depletion of the stratospheric ozone layer may result in an increase in the levels of potentially harmful UV-B radiation reaching the surface of the earth. We have found that UV-B is a potent inducer of the plant pathogenesis-related protein PR-1 in tobacco leaves. UV-B fluences required for PR-1 accumulation are similar to those of other UV-B-induced responses. The UV-B-induced PR-1 accumulation was confined precisely to the irradiated area of the leaf but displayed no leaf tissue specificity. A study of some of the possible components of the signal transduction pathway between UV-B and PR-1 induction showed that photosynthetic processes are not essential, and photoreversible DNA damage is not involved. Antioxidants and cycloheximide were able to block the induction of PR-1 by UV-B, and treatment of leaves with a generator of reactive oxygen resulted in the accumulation of PR-1 protein. These results demonstrate an absolute requirement for active oxygen species and protein synthesis in this UV-B signal transduction pathway. In contrast, we also show that other elicitors, notably salicylic acid, are able to elicit PR-1 via nonreactive oxygen species-requiring pathways.

  8. Temporal accumulation of salicylic acid activates the defense response against Colletotrichum in strawberry.

    PubMed

    Grellet-Bournonville, Carlos F; Martinez-Zamora, Martín G; Castagnaro, Atilio P; Díaz-Ricci, Juan Carlos

    2012-05-01

    Many authors have reported interactions between strawberry cultivars and pathogenic microorganisms, yet little is known about the mechanisms triggered in the plant. In this paper we examine the participation of the salicylic acid (SA) signaling pathway involved in the response of Fragaria x ananassa cv. Pájaro plants to pathogens. Strawberry plants were challenged with the virulent strain M11 of Colletotrichum acutatum, or with the avirulent strain M23 of Colletotrichum fragariae which confers resistance to the former. Our study showed that the isolate M23 induced a temporal SA accumulation that was accompanied with the induction of PR-1 gene expression in strawberry plants. Such events occured after the oxidative burst, evaluated as the accumulation of hydrogen peroxide and superoxide anion, and many hours before the protection could be detected. Similar results were obtained with exogenously applied SA. Results obtained supports the hypothesis that strawberry plants activate a SA mediated defense mechanisms that is effective against a causal agent of anthracnose. In contrast, plants inoculated with M11 did not show oxidative burst, SA accumulation or PR1 gene induction. This is the first report about a defense response signaling pathway studied in strawberry plants. PMID:22366637

  9. Polygonum cuspidatum inhibits pancreatic lipase activity and adipogenesis via attenuation of lipid accumulation

    PubMed Central

    2013-01-01

    Background Obesity causes metabolic disease and is a serious health problem around the world. Polygonum cuspidatum (POCU1b) has been used clinically for the treatment of constipation, gallstones, hepatitis, and inflammation in East Asian countries. The principal aim of this study was to investigate for the first time whether the extract of Polygonum cuspidatum (POCU) biologically affects adipogenesis in 3 T3-L1 preadipocytes. Methods Fractions (n-hexan, ethyl acetate, n-butanol, and water) of POCU ethanol extract were evaluated in vitro for their inhibitory activities on pancreatic lipase. Of the fractions, the n-butanol of POCU ethanol extract (POCU1b) was examined anti-obesity activity in 3 T3-L1 preadipocytes. To examine the inhibitory effect of POCU1b on adipogenesis, 3 T3-L1 preadipocytes were treated every the other day with POCU1b at various concentrations (0 ~ 25 μg/mL) for twelve days. Oil-red O staining and triglyceride content assay were performed to determine the lipid accumulation. The expression of mRNA and proteins associated lipid accumulation was measured using RT-PCR and Western blotting analysis. We also examined the effect of POCU1b on level of phosphorylated AMP-activated protein kinase (pAMPK) in 3 T3-L1 preadipocytes with POCU1b at various concentrations during adipocyte differentiation. Results POCU1b exhibited the most pronounced inhibitory effects on pancreatic lipase activity. We found that POCU1b inhibited adipocyte differentiation in 3 T3-L1 preadipocytes in a dose-dependent manner, as evidenced by the reduced formation of lipid droplets and decreased glycerol-3-phosphate dehydrogenase (GPDH) activity. We also showed that the expression levels of adipocyte differentiation-related protein (ADRP) and perilipin (a protein that coats lipid droplets in adipocytes) were both reduced after POCU1b treatment. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and CCAAT/enhancer-binding protein-alpha (C/EBP-α) proteins, both

  10. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation1[OPEN

    PubMed Central

    Okumura, Masaki; Inoue, Shin-ichiro; Kuwata, Keiko

    2016-01-01

    Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  11. Thiocyanate supplementation decreases atherosclerotic plaque in mice expressing human myeloperoxidase.

    PubMed

    Morgan, P E; Laura, R P; Maki, R A; Reynolds, W F; Davies, M J

    2015-06-01

    Elevated levels of the heme enzyme myeloperoxidase (MPO) are associated with adverse cardiovascular outcomes. MPO predominantly catalyzes formation of the oxidants hypochlorous acid (HOCl) from Cl(-), and hypothiocyanous acid (HOSCN) from SCN(-), with these anions acting as competitive substrates. HOSCN is a less powerful and more specific oxidant than HOCl, and selectively targets thiols; such damage is largely reversible, unlike much HOCl-induced damage. We hypothesized that increased plasma SCN(-), and hence HOSCN formation instead of HOCl, may decrease artery wall damage. This was examined using high-fat fed atherosclerosis-prone LDLR(-/-) mice transgenic for human MPO, with and without SCN(-) (10 mM) added to drinking water. Serum samples, collected fortnightly, were analyzed for cholesterol, triglycerides, thiols, MPO, and SCN(-); study-long exposure was calculated by area under the curve (AUC). Mean serum SCN(-) concentrations were elevated in the supplemented mice (200-320 μM) relative to controls (< 120 μM). Normalized aortic root plaque areas at sacrifice were 26% lower in the SCN(-)-supplemented mice compared with controls (P = 0.0417), but plaque morphology was not appreciably altered. Serum MPO levels steadily increased in mice on the high-fat diet, however, comparison of SCN(-)-supplemented versus control mice showed no significant changes in MPO protein, cholesterol, or triglyceride levels; thiol levels were decreased in supplemented mice at one time-point. Plaque areas increased with higher cholesterol AUC (r = 0.4742; P = 0.0468), and decreased with increasing SCN(-) AUC (r = - 0.5693; P = 0.0134). These data suggest that increased serum SCN(-) levels, which can be achieved in humans by dietary manipulation, may decrease atherosclerosis burden.

  12. Thiocyanate supplementation decreases atherosclerotic plaque in mice expressing human myeloperoxidase

    PubMed Central

    Morgan, P. E.; Laura, R. P.; Maki, R. A.; Reynolds, W. F.; Davies, M. J.

    2015-01-01

    Elevated levels of the heme enzyme myeloperoxidase (MPO) are associated with adverse cardiovascular outcomes. MPO predominantly catalyzes formation of the oxidants hypochlorous acid (HOCl) from Cl−, and hypothiocyanous acid (HOSCN) from SCN−, with these anions acting as competitive substrates. HOSCN is a less powerful and more specific oxidant than HOCl, and selectively targets thiols; such damage is largely reversible, unlike much HOCl-induced damage. We hypothesized that increased plasma SCN−, and hence HOSCN formation instead of HOCl, may decrease artery wall damage. This was examined using high-fat fed atherosclerosis-prone LDLR−/− mice transgenic for human MPO, with and without SCN− (10 mM) added to drinking water. Serum samples, collected fortnightly, were analyzed for cholesterol, triglycerides, thiols, MPO and SCN−; study-long exposure was calculated by area under the curve (AUC). Mean serum SCN− concentrations were elevated in the supplemented mice (200-320 μM) relative to controls (<120 μM). Normalized aortic root plaque areas at sacrifice were 26% lower in the SCN−-supplemented mice compared to controls (P=0.0417), but plaque morphology was not appreciably altered. Serum MPO levels steadily increased in mice on the high-fat diet, however, comparison of SCN−- supplemented vs. control mice showed no significant changes in MPO protein, cholesterol or triglyceride levels; thiol levels were decreased in supplemented mice at one time-point. Plaque areas increased with higher cholesterol AUC (r=0.4742; P=0.0468), and decreased with increasing SCN− AUC (r=−0.5693; P=0.0134). These data suggest that increased serum SCN− levels, which can be achieved in humans by dietary manipulation, may decrease atherosclerosis burden. PMID:25812586

  13. Bioactivation of myelotoxic xenobiotics by human neutrophil myeloperoxidase

    SciTech Connect

    Roy, R.R.

    1989-01-01

    Many environmental pollutants and drugs are toxic to the bone marrow. Some of these xenobiotics may initiate toxicity after undergoing bioactivation to free radicals and/or other reactive electrophiles. Peroxidases are a group of enzymes that catalyze the one-electron oxidative bioactivation of a variety of xenobiotics in vitro. Myeloperoxidase (MPO) is a peroxidative enzyme found in very high concentration in the neutrophils of human bone marrow. In this study, human MPO was evaluated to determine its ability to catalyze the in vitro bioactivation of known bone marrow toxicants that contain the aromatic hydroxyl (Ar-OH), aromatic amine (Ar-N-R{sub 2}), or heterocyclic tertiary amine ({double bond}N-R) moieties. The formation of free radical metabolites during the MPO-catalyzed bioactivation of hydroquinone and catechol (benzene metabolites), mitoxantrone and ametantrone (antitumor drugs), and chlorpromazine and promazine (antipsychotic drugs) was demonstrated by EPR spectroscopy. The reactivity of the products formed during the MPO catalyzed bioactivation of ({sup 14}C)hydroquinone and ({sup 14}C)catechol was shown by their covalent binding to protein and DNA in vitro. The covalently binding metabolite in each case is postulated to be the quinone form of the xenobiotic. In addition, both GSH and NADH were oxidized by the reactive intermediate(s) formed during the MPO-catalyzed bioactivation of many of the bone marrow toxicants tested. It was also shown that p,p-biphenol stimulated the MPO catalyzed bioactivation of both hydroquinone and catechol, while p-cresol stimulated the MPO-catalyzed bioactivation of catechol.

  14. PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes

    PubMed Central

    Haddock, Christopher J.; Blomenkamp, Keith; Gautam, Madhav; James, Jared; Mielcarska, Joanna; Gogol, Edward; Teckman, Jeffrey; Skowyra, Dorota

    2014-01-01

    Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome

  15. PiZ mouse liver accumulates polyubiquitin conjugates that associate with catalytically active 26S proteasomes.

    PubMed

    Haddock, Christopher J; Blomenkamp, Keith; Gautam, Madhav; James, Jared; Mielcarska, Joanna; Gogol, Edward; Teckman, Jeffrey; Skowyra, Dorota

    2014-01-01

    Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome

  16. Endogenous nitric oxide accumulation is involved in the antifungal activity of Shikonin against Candida albicans

    PubMed Central

    Liao, Zebin; Yan, Yu; Dong, Huaihuai; Zhu, Zhenyu; Jiang, Yuanying; Cao, Yingying

    2016-01-01

    The aim of the present study was to investigate the role of nitric oxide (NO) in the antifungal activity of Shikonin (SK) against Candida albicans (C. albicans) and to clarify the underlying mechanism. The results showed that the NO donors S-nitrosoglutathione (GSNO) and L-arginine could enhance the antifungal activity of SK, whereas the NO production inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) attenuated antifungal action. Using the fluorescent dye 3-amino,4-aminomethyl-2′, 7-difluorescein, diacetate (DAF-FM DA), we found that the accumulation of NO in C. albicans was increased markedly by SK in a time- and dose-dependent manner. In addition, the results of real-time reverse transcription-PCR (RT-PCR) demonstrated that the transcription level of YHB1 in C. albicans was greatly increased upon incubation of SK. Consistently, the YHB1-null mutant (yhb1Δ/Δ) exhibited a higher susceptibility to SK than wild-type cells. In addition, although the transcription level of CTA4 in C. albicans was not significantly changed when exposed to SK, the CTA4-null mutant (cta4Δ/Δ) was more susceptible to SK. Collectively, SK is the agent found to execute its antifungal activity directly via endogenous NO accumulation, and NO-mediated damage is related to the suppression of YHB1 and the function of CTA4. PMID:27530748

  17. Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

    PubMed Central

    Nguyen, Hien Thi Thu; Kristiansen, Rikke; Vestergaard, Mette; Wimmer, Reinhard

    2015-01-01

    Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. 13C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using 3H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions. PMID:25956769

  18. Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions.

    PubMed

    Nguyen, Hien Thi Thu; Kristiansen, Rikke; Vestergaard, Mette; Wimmer, Reinhard; Nielsen, Per Halkjær

    2015-07-01

    Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. (13)C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using (3)H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions. PMID:25956769

  19. Localization of hydrogen peroxide accumulation and diamine oxidase activity in pea root nodules under aluminum stress.

    PubMed

    Sujkowska-Rybkowska, Marzena; Borucki, Wojciech

    2014-02-01

    Aluminum (Al) is one of the environmental stressors that induces formation of reactive oxygen species (ROS) in plants. Hydrogen peroxide (H2O2) and H2O2-generated apoplast diamine oxidase (DAO) activity were detected cytochemically via transmission electron microscopy (TEM), in pea (Pisum sativum L.) root nodules exposed to high (50 μM AlCl3, for 2 and 24h) Al stress. The nodules were shown to respond to Al stress by disturbances in infection thread (IT) growth, bacteria endocytosis, premature degeneration of bacteroidal tissue and generation of H2O2 in nodule apoplast. Large amounts of peroxide were found at the same sites as high DAO activity under Al stress, suggesting that DAO is a major source of Al-induced peroxide accumulation in the nodules. Peroxide distribution and DAO activity in the nodules of both control plants and Al-treated ones were typically found in the plant cell walls, intercellular spaces and infection threads. However, 2 h Al treatment increased DAO activity and peroxide accumulation in the nodule apoplast and bacteria within threads. A prolonged Al treatment (24 h) increased the H2O2 content and DAO activity in the nodule apoplast, especially in the thread walls, matrix and bacteria within infection threads. In addition to ITs, prematurely degenerated bacteroids, which occurred in response to Al, were associated with intense staining for H2O2 and DAO activity. These results suggest the involvement of DAO in the production of a large amount of H2O2 in the nodule apoplast under Al stress. The role of reactive oxygen species in pea-Rhizobium symbiosis under Al stress is discussed. PMID:24246127

  20. Sulfite-mediated oxidation of myeloperoxidase to a free radical: immuno-spin trapping detection in human neutrophils.

    PubMed

    Ranguelova, Kalina; Rice, Annette B; Lardinois, Olivier M; Triquigneaux, Mathilde; Steinckwich, Natacha; Deterding, Leesa J; Garantziotis, Stavros; Mason, Ronald P

    2013-07-01

    Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of the reactive sulfur trioxide ((•)SO3(-)), peroxymonosulfate ((-)O3SOO(•)), and sulfate (SO4(•-)) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions, and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of this study was to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage, possibly leading to (bi)sulfite-exacerbated allergic reactions.

  1. Nitric oxide fumigation stimulates flavonoid and phenolic accumulation and enhances antioxidant activity of mushroom.

    PubMed

    Dong, Jufang; Zhang, Ming; Lu, Li; Sun, Lina; Xu, Maojun

    2012-12-01

    The effects of nitric oxide (NO) on antioxidant activity and contents of phenolics and flavonoids in mushroom Russula griseocarnosa were investigated. Freshly harvested mushrooms were fumigated with 0, 10, 20 and 30μLL(-1) NO at 20°C for 2h and then taken to examine the antioxidant activities using assays of reducing power, chelating effect on ferrous ions, scavenging effect on hydroxyl free radicals, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. The results showed that the antioxidant activities of the mushrooms fumigated with NO were significantly increased when compared to the controls. Moreover, NO fumigation significantly enhanced phenolic and flavonoid contents and stimulated the activities of phenylalanine ammonia-lyase and chalcone synthase. The results indicated that NO fumigation might have potential application for enhancing the bioactive compounds and improving antioxidant activities in the mushrooms. Furthermore, the data suggested that the NO-induced phenolic and flavonoid accumulation was due to the activation of the biosynthetic pathways in the mushrooms.

  2. Modification of High Density Lipoprotein by Myeloperoxidase Generates a Pro-inflammatory Particle*

    PubMed Central

    Undurti, Arundhati; Huang, Ying; Lupica, Joseph A.; Smith, Jonathan D.; DiDonato, Joseph A.; Hazen, Stanley L.

    2009-01-01

    High density lipoprotein (HDL) is the major atheroprotective particle in plasma. Recent studies demonstrate that myeloperoxidase (MPO) binds to HDL in vivo, selectively targeting apolipoprotein A1 (apoA1) of HDL for oxidative modification and concurrent loss in cholesterol efflux and lecithin cholesterol acyl transferase activating activities, generating a “dysfunctional HDL” particle. We now show that (patho)physiologically relevant levels of MPO-catalyzed oxidation result in loss of non-cholesterol efflux activities of HDL including anti-apoptotic and anti-inflammatory functions. One mechanism responsible is shown to involve the loss of modified HDL binding to the HDL receptor, scavenger receptor B1, and concurrent acquisition of saturable and specific binding to a novel unknown receptor independent of scavenger receptors CD36 and SR-A1. HDL modification by MPO is further shown to confer pro-inflammatory gain of function activities as monitored by NF-κB activation and surface vascular cell adhesion molecule levels on aortic endothelial cells exposed to MPO-oxidized HDL. The loss of non-cholesterol efflux activities and the gain of pro-inflammatory functions requires modification of the entire particle and can be recapitulated by oxidation of reconstituted HDL particles comprised of apoA1 and nonoxidizable phosphatidylcholine species. Multiple site-directed mutagenesis studies of apoA1 suggest that the pro-inflammatory activity of MPO-modified HDL does not involve methionine, tyrosine, or tryptophan, oxidant-sensitive residues previously mapped as sites of apoA1 oxidation within human atheroma. Thus, MPO-catalyzed oxidation of HDL results not only in the loss of classic atheroprotective reverse cholesterol transport activities of the lipoprotein but also both the loss of non-cholesterol efflux related activities and the gain of pro-inflammatory functions. PMID:19726691

  3. Interplay between enterobactin, myeloperoxidase and lipocalin 2 regulates E. coli survival in the inflamed gut.

    PubMed

    Singh, Vishal; Yeoh, Beng San; Xiao, Xia; Kumar, Manish; Bachman, Michael; Borregaard, Niels; Joe, Bina; Vijay-Kumar, Matam

    2015-01-01

    During an inflammatory response in the gut, some commensal bacteria such as E. coli can thrive and contribute to disease. Here we demonstrate that enterobactin (Ent), a catecholate siderophore released by E. coli, is a potent inhibitor of myeloperoxidase (MPO), a bactericidal enzyme of the host. Glycosylated Ent (salmochelin) and non-catecholate siderophores (yersiniabactin and ferrichrome) fail to inhibit MPO activity. An E. coli mutant (ΔfepA) that overproduces Ent, but not an Ent-deficient double mutant (ΔaroB/ΔfepA), inhibits MPO activity and exhibits enhanced survival in inflamed guts. This survival advantage is counter-regulated by lipocalin 2, a siderophore-binding host protein, which rescues MPO from Ent-mediated inhibition. Spectral analysis reveals that Ent interferes with compound I [oxoiron, Fe(IV)=O] and reverts the enzyme back to its native ferric [Fe(III)] state. These findings define a fundamental mechanism by which E. coli surpasses the host innate immune responses during inflammatory gut diseases and gains a distinct survival advantage. PMID:25964185

  4. The myeloperoxidase product hypochlorous acid generates irreversible high-density lipoprotein receptor inhibitors

    PubMed Central

    Binder, Veronika; Ljubojevic, Senka; Haybaeck, Johannes; Holzer, Michael; El-Gamal, Dalia; Schicho, Rudolf; Pieske, Burkert; Heinemann, Akos; Marsche, Gunther

    2014-01-01

    Objective Elevated levels of advanced oxidation protein products (AOPPs) have been described in several chronic inflammatory diseases, like chronic renal insufficiency, rheumatoid arthritis and atherosclerosis. Recent findings revealed that AOPPs are inhibitors of the major high-density lipoprotein (HDL) receptor, scavenger receptor class B, type 1 (SR-BI). Here we investigated what oxidation induced structural alterations convert plasma albumin into an HDL-receptor inhibitor. Approach and Results Exposure of albumin to the physiological oxidant, hypochlorous acid, generated high affinity SR-BI ligands. Protection of albumin lysine-residues prior exposure to hypochlorous acid as well as regeneration of N-chloramines after oxidation of albumin completely prevented binding of oxidized albumin to SR-BI, indicating that modification of albumin lysine-residues is required to generate SR-BI ligands. Of particular interest, N-chloramines within oxidized albumin promoted irreversible binding to SR-BI, resulting in permanent receptor blockade. We observed that the SR-BI inhibitory activity of albumin isolated from chronic kidney disease patients correlated with the content of the myeloperoxidase-specific oxidation product 3-chlorotyrosine and was associated with alterations in the composition of HDL. Conclusion Given that several potential atheroprotective activities of HDL are mediated by SR-BI, the present results raise the possibility that oxidized plasma albumin, through permanent SR-BI blockade, contributes to the pathophysiology of cardiovascular disease. PMID:23493288

  5. Myeloperoxidase scavenges peroxynitrite: A novel anti-inflammatory action of the heme enzyme

    PubMed Central

    Koyani, Chintan N.; Flemmig, Joerg; Malle, Ernst; Arnhold, Juergen

    2015-01-01

    Peroxynitrite, a potent pro-inflammatory and cytotoxic species, interacts with a variety of heme containing proteins. We addressed the question whether (i) the interaction of myeloperoxidase (MPO, an enzyme generating hypochlorous acid from hydrogen peroxide and chloride ions) with peroxynitrite affects the clearance of peroxynitrite, and (ii) if peroxynitrite could modulate the chlorinating activity of MPO. Our results show that this interaction promotes the decomposition of the highly reactive pro-inflammatory oxidant, whereby MPO Compound II (but not Compound I) is formed. The efficiency of MPO to remove peroxynitrite was enhanced by l-tyrosine, nitrite and (−)-epicatechin, substances known to reduce Compound II with high reaction rate. Next, peroxynitrite (added as reagent) diminished the chlorinating activity of MPO in the presence of hydrogen peroxide. Alternatively, SIN-1, a peroxynitrite donor, reduced hypochlorous acid formation by MPO, as measured by aminophenyl fluorescein oxidation (time kinetics) and taurine chloramine formation (end point measurement). At inflammatory loci, scavenging of peroxynitrite by MPO may overcome the uncontrolled peroxynitrite decomposition and formation of reactive species, which lead to cell/tissue damage. PMID:25731855

  6. Wasting our time? Allocated versus accumulated physical activity in afterschool programs

    PubMed Central

    Brazendale, Keith; Beets, Michael W.; Weaver, Robert G.; Huberty, Jennifer L.; Pate, Russell R.; Beighle, Aaron

    2015-01-01

    Background Afterschool programs (ASPs) can provide opportunities for children to accumulate moderate-to-vigorous physical activity (MVPA). The optimal amount of time ASPs should allocate for physical activity (PA) on a daily basis to ensure children achieve policy-stated PA recommendations remains unknown. Methods Children (n = 1248, 5–12 years) attending 20 ASPs wore accelerometers up to 4 non-consecutive week days for the duration of the ASPs during spring 2013 (February-April). Daily schedules were obtained from each ASP. Results Across 20 ASPs, three programs allocated ≤30 min, five approximately 45 min, four 60 min, four 75 min, and four ≥105 min for PA opportunities daily (min.d−1). Children accumulated the highest levels of MVPA in ASPs that allocated ≥60 min.d−1 for PA opportunities (24.8–25.1 min.d−1 for boys and 17.1–19.4 min.d−1 for girls) versus ASPs allocating ≤45 min.d−1 for PA opportunities (19.7 min.d−1 and 15.6 min.d−1 for boys and girls, respectively). There were no differences in the amount of MVPA accumulated by children among ASPs that allocated 60 min.d−1 (24.8 min.d−1 for boys and 17.1 min.d−1 for girls), 75 min.d−1 (25.1 min.d−1 for boys and 19.4 min.d−1 for girls) or ≥105 min.d−1 (23.8 min.d−1 for boys and 17.8 min.d−1 for girls). Across ASPs, 26% of children (31% for boys and 14% for girls) met the recommended 30 minutes of MVPA. Conclusions Allocating more than one hour of PA opportunities is not associated with an increase in MVPA during ASPs. Allocating 60 min.d−1, in conjunction with enhancing PA opportunities, can potentially serve to maximize children’s accumulation of MVPA during ASPs. PMID:25271393

  7. Epigallocatechin-3-Gallate Inhibition of Myeloperoxidase and Its Counter-Regulation by Dietary Iron and Lipocalin 2 in Murine Model of Gut Inflammation.

    PubMed

    Yeoh, Beng San; Aguilera Olvera, Rodrigo; Singh, Vishal; Xiao, Xia; Kennett, Mary J; Joe, Bina; Lambert, Joshua D; Vijay-Kumar, Matam

    2016-04-01

    Green tea-derived polyphenol (-)-epigallocatechin-3-gallate (EGCG) has been extensively studied for its antioxidant and anti-inflammatory properties in models of inflammatory bowel disease, yet the underlying molecular mechanism is not completely understood. Herein, we demonstrate that EGCG can potently inhibit the proinflammatory enzyme myeloperoxidase in vitro in a dose-dependent manner over a range of physiologic temperatures and pH values. The ability of EGCG to mediate its inhibitory activity is counter-regulated by the presence of iron and lipocalin 2. Spectral analysis indicated that EGCG prevents the peroxidase-catalyzed reaction by reverting the reactive peroxidase heme (compound I:oxoiron) back to its native inactive ferric state, possibly via the exchange of electrons. Further, administration of EGCG to dextran sodium sulfate-induced colitic mice significantly reduced the colonic myeloperoxidase activity and alleviated proinflammatory mediators associated with gut inflammation. However, the efficacy of EGCG against gut inflammation is diminished when orally coadministered with iron. These findings indicate that the ability of EGCG to inhibit myeloperoxidase activity is one of the mechanisms by which it exerts mucoprotective effects and that counter-regulatory factors such as dietary iron and luminal lipocalin 2 should be taken into consideration for optimizing clinical management strategies for inflammatory bowel disease with the use of EGCG treatment. PMID:26968114

  8. A modification to the Activated Sludge Model No. 2 based on the competition between phosphorus-accumulating organisms and glycogen-accumulating organisms.

    PubMed

    Manga, J; Ferrer, J; Garcia-Usach, F; Seco, A

    2001-01-01

    A modification to the ASM2 is proposed which permits representation of the competition between phosphorus accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) in a nutrient removal activated sludge system. Some important aspects, which are not considered in ASM2, are discussed. The proposed modification includes denitrification by PAOs, PAO glycogen storage capability and GAO metabolism model. It is shown that the proposed modification is capable of describing pilot plant data using a single set of stoichiometric and kinetic parameters over three different sludge ages (16, 14 and 12 days). The modified ASM2 may be applicable to a wide range of situations where PAOs and GAOs can compete. This modification may well provide a better understanding about GAO behaviour.

  9. The role of the sulfonium linkage in the stabilization of the ferrous form of myeloperoxidase: a comparison with lactoperoxidase.

    PubMed

    Brogioni, Silvia; Stampler, Johanna; Furtmüller, Paul G; Feis, Alessandro; Obinger, Christian; Smulevich, Giulietta

    2008-05-01

    In all mammalian peroxidases, the heme is covalently attached to the protein via two ester linkages between conserved aspartate (Asp94) and glutamate residues (Glu242) and modified methyl groups on pyrrole rings A and C. Only myeloperoxidase has an additional sulfonium ion linkage between the sulfur atom of the conserved methionine 243 and the beta-carbon of the vinyl group on pyrrole ring A. Upon reduction from Fe(III) to Fe(II), lactoperoxidase (LPO) but not myeloperoxidase (MPO) is shown to adopt three distinct active site conformations which depend on pH and time. Comparative spectroscopic analysis (UV-Vis absorption and resonance Raman) of the ferrous forms of LPO, wild-type MPO and the variants Asp94Val, Glu242Gln, Met243Thr and Met243Val clearly demonstrate that a single, stable ferrous form of MPO is present only in those proteins which retain an intact sulfonium linkage. By contrast, both ferrous Met243Thr and Met243Val can assume two conformations. They resemble ferrous LPO, being five-coordinated high-spin species that are distinguished by the strength of the proximal Fe-histidine bond. This bond weakens with time or decreasing pH, as indicated by the Fe-histidine stretching bands.

  10. Along-strike Variations in Active Strain Accumulation in the Northwest Himalaya

    NASA Astrophysics Data System (ADS)

    Morell, K. D.; Ahmad, T.; Sandiford, M.; Codilean, A. T.; Fulop, R. H.

    2015-12-01

    The spatial distribution of channel steepness, erosion rate, and physiographic data highlight pronounced along-strike changes in active strain accumulation in the northwest Himalaya. In particular, the data suggest that the mid-crustal ramp of the Main Himalayan Thrust could merge along-strike with an active portion of the Main Boundary Thrust near longitude ~77º E. This along-strike change in active fault geometry also coincides with the lateral termination of both lesser and greater Himalayan sequences, a significant reduction in total shortening within the wedge, and pronounced variations in regional seismicity. Recent activity along extensional structures in the high Himalaya of this same region appears to have led to significant reorganization, modification and capture of the Sutlej River basin, one of the largest Himalayan river systems. Given the recent 2015 Gorkha earthquake along a comparable section ~500-km along strike, these new constraints on active fault architecture could have regional implications for how strain is partitioned along seismogenic faults in the northwest Himalaya.

  11. A review on soil carbon accumulation due to the management change of major Brazilian agricultural activities.

    PubMed

    La Scala jr, N; De Figueiredo, E B; Panosso, A R

    2012-08-01

    Agricultural areas deal with enormous CO2 intake fluxes offering an opportunity for greenhouse effect mitigation. In this work we studied the potential of soil carbon sequestration due to the management conversion in major agricultural activities in Brazil. Data from several studies indicate that in soybean/maize, and related rotation systems, a significant soil carbon sequestration was observed over the year of conversion from conventional to no-till practices, with a mean rate of 0.41 Mg C ha(-1) year(-1). The same effect was observed in sugarcane fields, but with a much higher accumulation of carbon in soil stocks, when sugarcane fields are converted from burned to mechanised based harvest, where large amounts of sugarcane residues remain on the soil surface (1.8 Mg C ha(-1) year(-1)). The higher sequestration potential of sugarcane crops, when compared to the others, has a direct relation to the primary production of this crop. Nevertheless, much of this mitigation potential of soil carbon accumulation in sugarcane fields is lost once areas are reformed, or intensive tillage is applied. Pasture lands have shown soil carbon depletion once natural areas are converted to livestock use, while integration of those areas with agriculture use has shown an improvement in soil carbon stocks. Those works have shown that the main crop systems of Brazil have a huge mitigation potential, especially in soil carbon form, being an opportunity for future mitigation strategies.

  12. Irrigation with industrial wastewater activates antioxidant system and osmoprotectant accumulation in lettuce, turnip and tomato plants.

    PubMed

    Hashem, H A; Hassanein, R A; El-Deep, M H; Shouman, A I

    2013-09-01

    We focused on the impact of industrial wastes on the water quality of the El-Amia drain in Egypt and the effect of irrigation with industrial wastewater on the growth, cell membranes, photosynthetic pigment content, the antioxidant system and selected osmoprotectants (proline, total amino nitrogen and soluble sugars) in three crop plants: turnip, tomato and lettuce. Furthermore, the present work focused on the analysis of the heavy metal content and its accumulation in the studied plants. For this purpose, water samples were collected 1, 10 and 19 km from the beginning of the drain and used for irrigation, with fresh water as a control. We found that industrial wastewater contained significant amounts of heavy metals (Cd, Ni and Co) warranted a pollution problem as their amounts exceed the maximum recommended concentrations according to FAO guidelines for trace metals in irrigation water. The three crop plants accumulate significant amounts of heavy metals in their shoots and roots and showed a significant decrease in leaf area, fresh weight and dry weight of shoots and roots, accompanied by a marked reduction in photosynthetic pigment content and damage to cell membranes, as indicated by increased electrolyte leakage and a lower membrane stability index. Significant increases in the activities of antioxidant enzymes and in the glutathione, proline, soluble sugar and total amino nitrogen content in response to irrigation with wastewater may be defense mechanisms induced in response to heavy metal stress.

  13. Irrigation with industrial wastewater activates antioxidant system and osmoprotectant accumulation in lettuce, turnip and tomato plants.

    PubMed

    Hashem, H A; Hassanein, R A; El-Deep, M H; Shouman, A I

    2013-09-01

    We focused on the impact of industrial wastes on the water quality of the El-Amia drain in Egypt and the effect of irrigation with industrial wastewater on the growth, cell membranes, photosynthetic pigment content, the antioxidant system and selected osmoprotectants (proline, total amino nitrogen and soluble sugars) in three crop plants: turnip, tomato and lettuce. Furthermore, the present work focused on the analysis of the heavy metal content and its accumulation in the studied plants. For this purpose, water samples were collected 1, 10 and 19 km from the beginning of the drain and used for irrigation, with fresh water as a control. We found that industrial wastewater contained significant amounts of heavy metals (Cd, Ni and Co) warranted a pollution problem as their amounts exceed the maximum recommended concentrations according to FAO guidelines for trace metals in irrigation water. The three crop plants accumulate significant amounts of heavy metals in their shoots and roots and showed a significant decrease in leaf area, fresh weight and dry weight of shoots and roots, accompanied by a marked reduction in photosynthetic pigment content and damage to cell membranes, as indicated by increased electrolyte leakage and a lower membrane stability index. Significant increases in the activities of antioxidant enzymes and in the glutathione, proline, soluble sugar and total amino nitrogen content in response to irrigation with wastewater may be defense mechanisms induced in response to heavy metal stress. PMID:23790475

  14. WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis

    PubMed Central

    Broun, Pierre; Poindexter, Patricia; Osborne, Erin; Jiang, Cai-Zhong; Riechmann, José Luis

    2004-01-01

    Epicuticular wax forms a layer of hydrophobic material on plant aerial organs, which constitutes a protective barrier between the plant and its environment. We report here the identification of WIN1, an Arabidopsis thaliana ethylene response factor-type transcription factor, which can activate wax deposition in overexpressing plants. We constitutively expressed WIN1 in transgenic Arabidopsis plants, and found that leaf epidermal wax accumulation was up to 4.5-fold higher in these plants than in control plants. A significant increase was also found in stems. Interestingly, ≈50% of the additional wax could only be released by complete lipid extractions, suggesting that not all of the wax is superficial. Gene expression analysis indicated that a number of genes, such as CER1, KCS1, and CER2, which are known to be involved in wax biosynthesis, were induced in WIN1 overexpressors. This observation indicates that induction of wax accumulation in transgenic plants is probably mediated through an increase in the expression of genes encoding enzymes of the wax biosynthesis pathway. PMID:15070782

  15. Neutrophil myeloperoxidase and its substrates: formation of specific markers and reactive compounds during inflammation

    PubMed Central

    Kato, Yoji

    2016-01-01

    Myeloperoxidase is an inflammatory enzyme that generates reactive hypochlorous acid in the presence of hydrogen peroxide and chloride ion. However, this enzyme also uses bromide ion or thiocyanate as a substrate to form hypobromous or hypothiocyanous acid, respectively. These species play important roles in host defense against the invasion of microorganisms. In contrast, these enzyme products modify biomolecules in hosts during excess inflammation, indicating that the action of myeloperoxidase is both beneficial and harmful. Myeloperoxidase uses other endogenous compounds, such as serotonin, urate, and l-tyrosine, as substrates. This broad-range specificity may have some biological implications. Target molecules of this enzyme and its products vary, including low-molecular weight thiols, proteins, nucleic acids, and lipids. The modified products represent biomarkers of myeloperoxidase action. Moderate inhibition of this enzyme might be critical for the prevention/modulation of excess, uncontrolled inflammatory events. Some phytochemicals inhibit myeloperoxidase, which might explain the reductive effect caused by the intake of vegetables and fruits on cardiovascular diseases. PMID:27013775

  16. Flavonoids induce HIF-1alpha but impair its nuclear accumulation and activity.

    PubMed

    Triantafyllou, Anastasia; Mylonis, Ilias; Simos, George; Bonanou, Sophia; Tsakalof, Andreas

    2008-02-15

    Hypoxia-inducible factor-1alpha (HIF-1alpha) is the regulatory subunit of the transcription factor HIF-1, which is highly involved in the pathology of diseases associated with tissue hypoxia. In this study we investigated the ability of plant flavonoids to induce HIF-1alpha and regulate HIF-1 transcriptional activity in HeLa cells. We demonstrate for the first time that the flavonoids baicalein, luteolin and fisetin, as well as the previously investigated quercetin, induce HIF-1alpha under normal oxygen pressure, whereas kaempferol, taxifolin, and rutin are inactive. We further reveal that the capability of flavonoids to bind efficiently intracellular iron and their lipophilicity are essential for HIF-1alpha induction. Despite the ability of flavonoids to stabilize HIF-1alpha, the transcriptional activity of HIF-1 induced by flavonoids was significantly lower than that observed with the iron chelator and known HIF-1 inducer, desferrioxamine (DFO). Furthermore, when cells in which HIF-1 had been induced by DFO were also treated with flavonoids, the transcriptional activity of HIF-1 was strongly impaired without simultaneous reduction in HIF-1alpha protein levels. Localization of HIF-1alpha by immuno- and direct fluorescence microscopy and in vitro phosphorylation assays suggest that flavonoids inhibit HIF-1 activity by impairing the MAPK-dependent phosphorylation of HIF-1alpha, thereby decreasing its nuclear accumulation.

  17. Accumulated source imaging of brain activity with both low and high-frequency neuromagnetic signals

    PubMed Central

    Xiang, Jing; Luo, Qian; Kotecha, Rupesh; Korman, Abraham; Zhang, Fawen; Luo, Huan; Fujiwara, Hisako; Hemasilpin, Nat; Rose, Douglas F.

    2014-01-01

    Recent studies have revealed the importance of high-frequency brain signals (>70 Hz). One challenge of high-frequency signal analysis is that the size of time-frequency representation of high-frequency brain signals could be larger than 1 terabytes (TB), which is beyond the upper limits of a typical computer workstation's memory (<196 GB). The aim of the present study is to develop a new method to provide greater sensitivity in detecting high-frequency magnetoencephalography (MEG) signals in a single automated and versatile interface, rather than the more traditional, time-intensive visual inspection methods, which may take up to several days. To address the aim, we developed a new method, accumulated source imaging, defined as the volumetric summation of source activity over a period of time. This method analyzes signals in both low- (1~70 Hz) and high-frequency (70~200 Hz) ranges at source levels. To extract meaningful information from MEG signals at sensor space, the signals were decomposed to channel-cross-channel matrix (CxC) representing the spatiotemporal patterns of every possible sensor-pair. A new algorithm was developed and tested by calculating the optimal CxC and source location-orientation weights for volumetric source imaging, thereby minimizing multi-source interference and reducing computational cost. The new method was implemented in C/C++ and tested with MEG data recorded from clinical epilepsy patients. The results of experimental data demonstrated that accumulated source imaging could effectively summarize and visualize MEG recordings within 12.7 h by using approximately 10 GB of computer memory. In contrast to the conventional method of visually identifying multi-frequency epileptic activities that traditionally took 2–3 days and used 1–2 TB storage, the new approach can quantify epileptic abnormalities in both low- and high-frequency ranges at source levels, using much less time and computer memory. PMID:24904402

  18. Activity blockade and GABAA receptor blockade produce synaptic scaling through chloride accumulation in embryonic spinal motoneurons and interneurons.

    PubMed

    Lindsly, Casie; Gonzalez-Islas, Carlos; Wenner, Peter

    2014-01-01

    Synaptic scaling represents a process whereby the distribution of a cell's synaptic strengths are altered by a multiplicative scaling factor. Scaling is thought to be a compensatory response that homeostatically controls spiking activity levels in the cell or network. Previously, we observed GABAergic synaptic scaling in embryonic spinal motoneurons following in vivo blockade of either spiking activity or GABAA receptors (GABAARs). We had determined that activity blockade triggered upward GABAergic scaling through chloride accumulation, thus increasing the driving force for these currents. To determine whether chloride accumulation also underlies GABAergic scaling following GABAAR blockade we have developed a new technique. We expressed a genetically encoded chloride-indicator, Clomeleon, in the embryonic chick spinal cord, which provides a non-invasive fast measure of intracellular chloride. Using this technique we now show that chloride accumulation underlies GABAergic scaling following blockade of either spiking activity or the GABAAR. The finding that GABAAR blockade and activity blockade trigger scaling via a common mechanism supports our hypothesis that activity blockade reduces GABAAR activation, which triggers synaptic scaling. In addition, Clomeleon imaging demonstrated the time course and widespread nature of GABAergic scaling through chloride accumulation, as it was also observed in spinal interneurons. This suggests that homeostatic scaling via chloride accumulation is a common feature in many neuronal classes within the embryonic spinal cord and opens the possibility that this process may occur throughout the nervous system at early stages of development.

  19. Quantification of Interictal Neuromagnetic Activity in Absence Epilepsy with Accumulated Source Imaging.

    PubMed

    Xiang, Jing; Tenney, Jeffrey R; Korman, Abraham M; Leiken, Kimberly; Rose, Douglas F; Harris, Elana; Yuan, Weihong; Horn, Paul S; Holland, Katherine; Loring, David W; Glauser, Tracy A

    2015-11-01

    Aberrant brain activity in childhood absence epilepsy (CAE) during seizures has been well recognized as synchronous 3 Hz spike-and-wave discharges on electroencephalography. However, brain activity from low- to very high-frequency ranges in subjects with CAE between seizures (interictal) has rarely been studied. Using a high-sampling rate magnetoencephalography (MEG) system, we studied ten subjects with clinically diagnosed but untreated CAE in comparison with age- and gender-matched controls. MEG data were recorded from all subjects during the resting state. MEG sources were assessed with accumulated source imaging, a new method optimized for localizing and quantifying spontaneous brain activity. MEG data were analyzed in nine frequency bands: delta (1-4 Hz), theta (4-8 Hz), alpha (8-12 Hz), beta (12-30 Hz), low-gamma (30-55 Hz), high-gamma (65-90 Hz), ripple (90-200 Hz), high-frequency oscillation (HFO, 200-1,000 Hz), and very high-frequency oscillation (VHFO, 1,000-2,000 Hz). MEG source imaging revealed that subjects with CAE had higher odds of interictal brain activity in 200-1,000 and 1,000-2,000 Hz in the parieto-occipito-temporal junction and the medial frontal cortices as compared with controls. The strength of the interictal brain activity in these regions was significantly elevated in the frequency bands of 90-200, 200-1,000 and 1,000-2,000 Hz for subjects with CAE as compared with controls. The results indicate that CAE has significantly aberrant brain activity between seizures that can be noninvasively detected. The measurements of high-frequency neuromagnetic oscillations may open a new window for investigating the cerebral mechanisms of interictal abnormalities in CAE. PMID:25359158

  20. Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages

    PubMed Central

    Namatame, Ichiji; Tomoda, Hiroshi; Ishibashi, Shun; Ōmura, Satoshi

    2004-01-01

    Beauveriolides I and III, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and III strongly inhibited the cholesteryl ester (CE) synthesis with IC50 values of 0.78 and 0.41 μM, respectively, without showing significant effects on the triacylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and III with IC50 values of 6.0 and 5.5 μM, respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and III exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveriolides I and III are the first microbial cyclodepsipeptides having an in vivo antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents. PMID:14718664

  1. Experimental evaluation of decrease in the activities of polyphosphate/glycogen-accumulating organisms due to cell death and activity decay in activated sludge.

    PubMed

    Hao, Xiaodi; Wang, Qilin; Cao, Yali; van Loosdrecht, Mark C M

    2010-06-15

    Decrease in bacterial activity (biomass decay) in activated sludge can result from cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The goal of this study was to experimentally differentiate between cell death and activity decay as the cause of decrease in bacterial activity. By means of measuring maximal anaerobic phosphate release rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in situ hybridization (FISH), the decay rates and death rates of polyphosphate-accumulating organisms (PAOs) in a biological nutrient removal (BNR) system and a laboratory phosphate removing sequencing batch reactor (SBR) system were determined, respectively, under famine conditions. In addition, the decay rate and death rate of glycogen-accumulating organisms (GAOs) in a SBR system with an enrichment culture of GAOs were also measured under famine conditions. Hereto the maximal anaerobic volatile fatty acid uptake rates, live/dead staining, and FISH were used. The experiments revealed that in the BNR and enriched PAO-SBR systems, activity decay contributed 58% and 80% to the decreased activities of PAOs, and that cell death was responsible for 42% and 20% of decreases in their respective activities. In the enriched GAOs system, activity decay constituted a proportion of 74% of the decreased activity of GAOs, and cell death only accounted for 26% of the decrease of their activity.

  2. Utilizing G2/M retention effect to enhance tumor accumulation of active targeting nanoparticles

    PubMed Central

    Hu, Guanlian; Cun, Xingli; Ruan, Shaobo; Shi, Kairong; Wang, Yang; Kuang, Qifang; Hu, Chuan; Xiao, Wei; He, Qin; Gao, Huile

    2016-01-01

    In recent years, active targeting strategies by ligand modification have emerged to enhance tumor accumulation of NP, but their clinical application was strictly restricted due to the complex preparation procedures, poor stability and serious toxicity. An effective and clinical translational strategy is required to satisfy the current problems. Interestingly, the internalization of NP is intimately related with cell cycle and the expression of receptors is not only related with cancer types but also cell cycle progression. So the cellular uptake of ligand modified NP may be related with cell cycle. However, few investigations were reported about the relationship between cell cycle and the internalization of ligand modified NP. Herein, cellular uptake of folic acid (FA) modified NP after utilizing chemotherapeutic to retain the tumor cells in G2/M phase was studied and a novel strategy was designed to enhance the active targeting effect. In our study, docetaxel (DTX) notably synchronized cells in G2/M phase and pretreatment with DTX highly improved in vitro and in vivo tumor cell targeting effect of FA decorated NP (FANP). Since FA was a most common used tumor active targeting ligand, we believe that this strategy possesses broader prospects in clinical application for its simplicity and effectiveness. PMID:27273770

  3. Excess beta-catenin promotes accumulation of transcriptionally active p53.

    PubMed Central

    Damalas, A; Ben-Ze'ev, A; Simcha, I; Shtutman, M; Leal, J F; Zhurinsky, J; Geiger, B; Oren, M

    1999-01-01

    beta-catenin is a multifunctional protein, acting both as a structural component of the cell adhesion machinery and as a transducer of extracellular signals. Deregulated beta-catenin protein expression, due to mutations in the beta-catenin gene itself or in its upstream regulator, the adenomatous polyposis coli (APC) gene, is prevalent in colorectal cancer and in several other tumor types, and attests to the potential oncogenic activity of this protein. Increased expression of beta-catenin is an early event in colorectal carcinogenesis, and is usually followed by a later mutational inactivation of the p53 tumor suppressor. To examine whether these two key steps in carcinogenesis are interrelated, we studied the effect of excess beta-catenin on p53. We report here that overexpression of beta-catenin results in accumulation of p53, apparently through interference with its proteolytic degradation. This effect involves both Mdm2-dependent and -independent p53 degradation pathways, and is accompanied by augmented transcriptional activity of p53 in the affected cells. Increased p53 activity may provide a safeguard against oncogenic deregulation of beta-catenin, and thus impose a pressure for mutational inactivation of p53 during the later stages of tumor progression. PMID:10357817

  4. Myeloperoxidase-derived oxidants rapidly oxidize and disrupt zinc-cysteine/histidine clusters in proteins.

    PubMed

    Cook, Naomi L; Pattison, David I; Davies, Michael J

    2012-12-01

    Zinc is an abundant cellular transition metal ion, which binds avidly to protein cysteine (Cys) and histidine (His) residues to form zinc-Cys/His clusters; these play a key role in the function of many proteins (e.g., DNA binding and repair enzymes, transcription factors, nitric oxide synthase). Leukocyte-derived myeloperoxidase generates powerful oxidants including hypochlorous (HOCl), hypobromous (HOBr), and hypothiocyanous (HOSCN) acids from H(2)O(2) and (pseudo)halide ions. Excessive or misplaced formation of these species is associated with cellular dysfunction, apoptosis and necrosis, and multiple inflammatory diseases. HOCl and HOBr react rapidly with sulfur-containing compounds, and HOSCN reacts specifically with thiols. Consequently, we hypothesized that zinc-Cys/His clusters would be targets for these oxidants, and the activity of such enzymes would be perturbed. This hypothesis has been tested using yeast alcohol dehydrogenase (YADH), which contains a well-characterized Zn(1)Cys(2)His(1) cluster. Incubation of YADH with pathologically relevant concentrations of HOSCN, HOCl, and HOBr resulted in rapid oxidation of the protein (rate constants, determined by competition kinetics, for reaction of HOCl and HOSCN with YADH being (3.3±0.9)×10(8) and (2.9±0.4)×10(4) M(-1) s(-1) per YADH monomer, respectively), loss of enzyme activity, Zn(2+) release, changes in protein structure (particularly formation of disulfide cross-links), and oxidation of Cys residues. The loss of enzyme activity correlated with Zn(2+) release, loss of thiols, and changes in protein structure. We conclude that exposure of zinc-Cys/His clusters to inflammatory oxidants can result in impaired protein activity, thiol oxidation, and Zn(2+) release. These reactions may contribute to inflammation-induced tissue damage.

  5. Myeloperoxidase-derived oxidants rapidly oxidize and disrupt zinc-cysteine/histidine clusters in proteins.

    PubMed

    Cook, Naomi L; Pattison, David I; Davies, Michael J

    2012-12-01

    Zinc is an abundant cellular transition metal ion, which binds avidly to protein cysteine (Cys) and histidine (His) residues to form zinc-Cys/His clusters; these play a key role in the function of many proteins (e.g., DNA binding and repair enzymes, transcription factors, nitric oxide synthase). Leukocyte-derived myeloperoxidase generates powerful oxidants including hypochlorous (HOCl), hypobromous (HOBr), and hypothiocyanous (HOSCN) acids from H(2)O(2) and (pseudo)halide ions. Excessive or misplaced formation of these species is associated with cellular dysfunction, apoptosis and necrosis, and multiple inflammatory diseases. HOCl and HOBr react rapidly with sulfur-containing compounds, and HOSCN reacts specifically with thiols. Consequently, we hypothesized that zinc-Cys/His clusters would be targets for these oxidants, and the activity of such enzymes would be perturbed. This hypothesis has been tested using yeast alcohol dehydrogenase (YADH), which contains a well-characterized Zn(1)Cys(2)His(1) cluster. Incubation of YADH with pathologically relevant concentrations of HOSCN, HOCl, and HOBr resulted in rapid oxidation of the protein (rate constants, determined by competition kinetics, for reaction of HOCl and HOSCN with YADH being (3.3±0.9)×10(8) and (2.9±0.4)×10(4) M(-1) s(-1) per YADH monomer, respectively), loss of enzyme activity, Zn(2+) release, changes in protein structure (particularly formation of disulfide cross-links), and oxidation of Cys residues. The loss of enzyme activity correlated with Zn(2+) release, loss of thiols, and changes in protein structure. We conclude that exposure of zinc-Cys/His clusters to inflammatory oxidants can result in impaired protein activity, thiol oxidation, and Zn(2+) release. These reactions may contribute to inflammation-induced tissue damage. PMID:23032100

  6. Myeloperoxidase and crystalline bodies in the granules of DMBA-induced rat chloroma cells.

    PubMed

    Ioachim, H L; Keller, S; Sabbath, M; Andersson, B; Dorsett, B; Essner, E

    1972-01-01

    Chloroma (chloroleukemia) was induced in a splenectomized rat by repeatedly administering dimethylbenz(a)anthracene (DMBA) and was serially transplanted thereafter. Composed of immature myeloid cells, the tumor imparted a green discoloration to the tissues that it infiltrated extensively. Chloroma cells fluoresced red in ultraviolet light, produced a characteristic curve in spectrophotometry, and contained large amounts of myeloperoxidase. They included numerous intracytoplasmic granules of both types A and B, which contained occasional crystalline bars. Permanent lines of chloroma cells were established in tissue culture. These cells, while maintaining their initial morphology, ceased producing myeloperoxidase and subsequently induced white tumors when they were isotransplanted.

  7. Mercury modulates selenium activity via altering its accumulation and speciation in garlic (Allium sativum).

    PubMed

    Zhao, Jiating; Hu, Yi; Gao, Yuxi; Li, Yufeng; Li, Bai; Dong, Yuanxing; Chai, Zhifang

    2013-06-01

    Combined pollution of selenium (Se) and mercury (Hg) has been known in Wanshan district (Guizhou Province, China). A better understanding of how Se and Hg interact in plants and the phytotoxicity thereof will provide clues about how to avoid or mitigate adverse effects of Se/Hg on local agriculture. In this study, the biological activity of Se has been investigated in garlic with or without Hg exposure. Se alone can promote garlic growth at low levels (<0.1 mg L(-1)), whereas it inhibits garlic growth at high levels (>1 mg L(-1)). The promotive effect of Se in garlic can be enhanced by low Hg exposure (<0.1 mg L(-1)). When both Se and Hg are at high levels, there is a general antagonistic effect between these two elements in terms of phytotoxicity. Inductively coupled plasma mass spectrometry (ICP-MS) data suggest that Se is mainly concentrated in garlic roots, compared to the leaves and the bulbs. Se uptake by garlic in low Se medium (<0.1 mg L(-1)) can be significantly enhanced as Hg exposure levels increase (P < 0.05), while it can be inhibited by Hg when Se exposure levels exceed 1 mg L(-1). The synchrotron radiation X-ray fluorescence (SRXRF) mapping further shows that Se is mainly concentrated in the stele of the roots, bulbs and the veins of the leaves, and Se accumulation in garlic can be reduced by Hg. The X-ray absorption near edge structure (XANES) study indicates that Se is mainly formed in C-Se-C form in garlic. Hg can decrease the content of inorganic Se mainly in SeO3(2-) form in garlic while increasing the content of organic Se mainly in C-Se-C form (MeSeCys and its derivatives). Hg-mediated changes in Se species along with reduced Se accumulation in garlic may account for the protective effect of Hg against Se phytotoxicity.

  8. Virucidal efficacy of treatment with photodynamically activated curcumin on murine norovirus bio-accumulated in oysters.

    PubMed

    Wu, Juan; Hou, Wei; Cao, Binbin; Zuo, Tao; Xue, Changhu; Leung, Albert Wingnang; Xu, Chuanshan; Tang, Qing-Juan

    2015-09-01

    Norovirus (NoV) is one of the most important seafood- and water-borne viruses, and is a major cause of acute gastroenteritis outbreaks. In the present study we investigated the effect of curcumin as a sensitizer to photodynamic treatment both in buffer and in oysters against murine norovirus 1 (MNV-1), a surrogate of NoV. MNV-1 cultured in buffer and MNV-1 bio-accumulated in oysters were irradiated with a novel LED light source with a wavelength of 470nm and an energy of 3.6J/cm(2). Inactivation of MNV-1 was investigated by plaque assays. After virus was extracted from the gut of oysters treated over a range of curcumin concentrations, the ultrastructural morphology of the virus was observed using electron microscopy, and the integrity of viral nucleic acids and stability of viral capsid proteins were also determined. Results showed that the infectivity of MNV-1 was significantly inhibited by 1-3logPFU/ml, with significant damage to viral nucleic acids in a curcumin dose-dependent manner after photodynamic activation. Virus morphology was altered after the photodynamic treatment with curcumin, presumably due to the change of the viral capsid protein structures. The data suggest that treatment of oysters with photodynamic activation of curcumin is a potentially efficacious and cost-effective method to inactivate food-borne NoV. Further studies are necessary to evaluate the toxicology of this approach in detail and perform sensory evaluation of the treated product.

  9. Empagliflozin Protects against Diet-Induced NLRP-3 Inflammasome Activation and Lipid Accumulation.

    PubMed

    Benetti, Elisa; Mastrocola, Raffaella; Vitarelli, Giovanna; Cutrin, Juan Carlos; Nigro, Debora; Chiazza, Fausto; Mayoux, Eric; Collino, Massimo; Fantozzi, Roberto

    2016-10-01

    The aim of this study was to evaluate the effects of chronic treatment with empagliflozin, a potent and selective sodium glucose cotransporter-2 inhibitor, in a murine model of diet-induced obesity and insulin resistance, focusing on drug effects on body weight reduction and nucleotide-binding domain, leucine-rich repeat containing protein (NLRP)-3 inflammasome activation, which have never been investigated to date. Male C57BL/6 mice were fed control or a high fat-high sugar (HFHS) diet for 4 months. Over the last 2 months, subsets of animals were treated with empagliflozin (1-10 mg/kg) added to the diet. Empagliflozin evoked body weight reduction (P < 0.001 for the highest dose) and positive effects on fasting glycemia and homeostasis model assessment of insulin resistance. In addition, the drug was able to reduce renal tubular damage and liver triglycerides level in a dose-dependent manner. Interestingly, empagliflozin also decreased cardiac lipid accumulation. Moreover, diet-induced activation of NLRP-3 in kidney and liver (not observed in the heart) was dose-dependently attenuated by empagliflozin. Our results clearly demonstrate the ability of empagliflozin to counteract the deleterious effects evoked by chronic exposure to HFHS diet. Most notably, empagliflozin treatment was associated with NLRP-3 inflammasome signaling modulation, suggesting that this inhibition may contribute to the drug therapeutic effects. PMID:27440421

  10. Platelet Activation Due to Hemodynamic Shear Stresses: Damage Accumulation Model and Comparison to In Vitro Measurements

    PubMed Central

    Nobili, Matteo; Sheriff, Jawaad; Morbiducci, Umberto; Redaelli, Alberto; Bluestein, Danny

    2009-01-01

    The need to optimize the thrombogenic performance of blood recirculating cardiovascular devices, e.g., prosthetic heart valves (PHV) and ventricular assist devices (VAD), is accentuated by the fact that most of them require lifelong anticoagulation therapy that does not eliminate the risk of thromboembolic complications. The formation of thromboemboli in the flow field of these devices is potentiated by contact with foreign surfaces and regional flow phenomena that stimulate blood clotting, especially platelets. With the lack of appropriate methodology, device manufacturers do not specifically optimize for thrombogenic performance. Such optimization can be facilitated by formulating a robust numerical methodology with predictive capabilities of flow-induced platelet activation. In this study, a phenomenological model for platelet cumulative damage, identified by means of genetic algorithms (GAs), was correlated with in vitro experiments conducted in a Hemodynamic Shearing Device (HSD). Platelets were uniformly exposed to flow shear representing the lower end of the stress levels encountered in devices, and platelet activity state (PAS) was measured in response to six dynamic shear stress waveforms representing repeated passages through a device, and correlated to the predictions of the damage accumulation model. Experimental results demonstrated an increase in PAS with a decrease in “relaxation” time between pulses. The model predictions were in very good agreement with the experimental results. PMID:18204318

  11. Demystifying "free will": the role of contextual information and evidence accumulation for predictive brain activity.

    PubMed

    Bode, Stefan; Murawski, Carsten; Soon, Chun Siong; Bode, Philipp; Stahl, Jutta; Smith, Philip L

    2014-11-01

    Novel multivariate pattern classification analyses have enabled the prediction of decision outcomes from brain activity prior to decision-makers' reported awareness. These findings are often discussed in relation to the philosophical concept of "free will". We argue that these studies demonstrate the role of unconscious processes in simple free choices, but they do not inform the philosophical debate. Moreover, these findings are difficult to relate to cognitive decision-making models, due to misleading assumptions about random choices. We review evidence suggesting that sequential-sampling models, which assume accumulation of evidence towards a decision threshold, can also be applied to free decisions. If external evidence is eliminated by the task instructions, decision-makers might use alternative, subtle contextual information as evidence, such as their choice history, that is not consciously monitored and usually concealed by the experimental design. We conclude that the investigation of neural activity patterns associated with free decisions should aim to investigate how decisions are jointly a function of internal and external contexts, rather than to resolve the philosophical "free will" debate.

  12. Lysyl oxidase: Influence of dietary copper on accumulation land functional activity in rat skin

    SciTech Connect

    Romero-Chapman, N.; Tinker, D.; Uriu-Hare, J.; Keen C.L.; Gacheru, S.; Rucker, R.B. )

    1991-03-11

    Lysyl oxidase (Lys. Ox.) functions extracellularly and catalyzes the oxidative deamination of peptidyl lysine in collagen and elastin. Lys. Ox. was purified 150- to 175-fold from urea extracts of rat skin and uteri. Both {sup {minus}}40 and {sup {minus}}32 kDa polypeptide chains could be isolated from rat skin with apparent Lys. Ox. activity. Antibodies raised in chickens against the {sup {minus}}40 kDa form of Lys. Ox. detected the {sup {minus}}32 kDa form in immunoblots. Consequently, it is inferred that the {sup {minus}}32 kDa form of Lys. Ox. is processed from the {sup {minus}}40 kDa form of the enzyme. Antibodies were also used to prepare antirat Lys. Ox. affinity columns to separate Lys. Ox. from other proteins. Sixteen hours after an oral dose of Cu-67, about 6-8% of the Cu-67 was incorporated into rat skin was found in associated with Lys. Ox. The Lys. Ox. concentration in rat skin was 2.5 to 7.5 nmoles (determined by enzyme liked immunosorption assays). Changing the Cu status by feeding a diet low in Cu did not influence Lys. Ox. accumulation or the % age of Cu-67 in skin as Lys. Ox. However, Lys. Ox. function activity was one-third normal values in rats deprived of Cu.

  13. Transportation and Accumulation of Redox Active Species at the Buried Interfaces of Plasticized Membrane Electrodes.

    PubMed

    Sohail, Manzar; De Marco, Roland; Jarolímová, Zdeňka; Pawlak, Marcin; Bakker, Eric; He, Ning; Latonen, Rose-Marie; Lindfors, Tom; Bobacka, Johan

    2015-09-29

    The transportation and accumulation of redox active species at the buried interface between glassy carbon electrodes and plasticized polymeric membranes have been studied using synchrotron radiation X-ray photoelectron spectroscopy (SR-XPS), near edge X-ray absorption fine structure (NEXAFS), in situ electrochemical Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy, cyclic voltammetry (CV), chronoamperometry (CA), and electrochemical impedance spectroscopy (EIS). Ferrocene tagged poly(vinyl chloride) [FcPVC], ferrocene (Fc), and its derivatives together with tetracyanoquinodimethane (TCNQ) doped plasticized polymeric membrane electrodes have been investigated, so as to extend the study of the mechanism of this reaction chemistry to different time scales (both small and large molecules with variable diffusion coefficients) using a range of complementary electrochemical and surface analysis techniques. This study also provides direct spectroscopic evidence for the transportation and electrochemical reactivity of redox active species, regardless of the size of the electrochemically reactive molecule, at the buried interface of the substrate electrode. With all redox dopants, when CA electrolysis was performed, redox active species were undetectable (<1 wt % of signature elements or below the detection limit of SR-XPS and NEXAFS) in the outermost surface layers of the membrane, while a high concentration of redox species was located at the electrode substrate as a consequence of the deposition of the reaction product (Fc(+)-anion complex) at the buried interface between the electrode and the membrane. This reaction chemistry for redox active species within plasticized polymeric membranes may be useful in the fashioning of multilayered polymeric devices (e.g., chemical sensors, organic electronic devices, protective laminates, etc.) based on an electrochemical tunable deposition of redox molecules at the buried substrate electrode beneath

  14. Myeloperoxidase Serves as a Redox Switch that Regulates Apoptosis in Epithelial Ovarian Cancer

    PubMed Central

    Saed, Ghassan M.; Ali-Fehmi, Rouba; Jiang, Zhong L.; Fletcher, Nicole M.; Diamond, Michael P.; Abu-Soud, Husam M.; Munkarah, Adnan R.

    2009-01-01

    Objectives Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO+)-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO+. We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. Methods MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT-PCR, ELISA, and TUNEL assays. Results MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. Conclusion MPO and iNOS are co-localized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions. PMID:19962178

  15. Essential role of CD11a in CD8+ T-cell accumulation and activation in adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T-cells, particularly CD8+ T-cells, are major participants in obesity-linked adipose tissue inflammation. We examined the mechanisms of CD8+ T-cell accumulation and activation in adipose tissue and the role of CD11a, a beta2 integrin. CD8+ T-cells in adipose tissue of obese mice showed activated phe...

  16. Induction of Mast Cell Accumulation by Tryptase via a Protease Activated Receptor-2 and ICAM-1 Dependent Mechanism

    PubMed Central

    Liu, Xin; Wang, Junling; Zhang, Huiyun; Zhan, Mengmeng; Chen, Hanqiu; Fang, Zeman; Xu, Chiyan; Chen, Huifang; He, Shaoheng

    2016-01-01

    Mast cells are primary effector cells of allergy, and recruitment of mast cells in involved tissue is one of the key events in allergic inflammation. Tryptase is the most abundant secretory product of mast cells, but little is known of its influence on mast cell accumulation. Using mouse peritoneal model, cell migration assay, and flow cytometry analysis, we investigated role of tryptase in recruiting mast cells. The results showed that tryptase induced up to 6.7-fold increase in mast cell numbers in mouse peritoneum following injection. Inhibitors of tryptase, an antagonist of PAR-2 FSLLRY-NH2, and pretreatment of mice with anti-ICAM-1, anti-CD11a, and anti-CD18 antibodies dramatically diminished tryptase induced mast cell accumulation. On the other hand, PAR-2 agonist peptides SLIGRL-NH2 and tc-LIGRLO-NH2 provoked mast cell accumulation following injection. These implicate that tryptase induced mast cell accumulation is dependent on its enzymatic activity, activation of PAR-2, and interaction between ICAM-1 and LFA-1. Moreover, induction of trans-endothelium migration of mast cells in vitro indicates that tryptase acts as a chemoattractant. In conclusion, provocation of mast cell accumulation by mast cell tryptase suggests a novel self-amplification mechanism of mast cell accumulation. Mast cell stabilizers as well as PAR-2 antagonist agents may be useful for treatment of allergic reactions. PMID:27378825

  17. Effect of Inoculation with Glomus versiforme on Cadmium Accumulation, Antioxidant Activities and Phytochelatins of Solanum photeinocarpum

    PubMed Central

    Tan, Shi-Yun; Jiang, Qiu-Yun; Zhuo, Feng; Liu, Hui; Wang, Yu-Tao; Li, Shao-Shan; Ye, Zhi-Hong; Jing, Yuan-Xiao

    2015-01-01

    The plant growth, phosphate acquisition, Cd translocation, phytochelatins (PCs) production and antioxidant parameters [superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione (GSH), ascorbate (ASA) and malonaldehyde (MDA)] were investigated in Cd-hyperaccumulator Solanum photeinocarpum inoculated with Glomus versiforme BGC GD01C (Gv) in Cd-added soils (0, 5, 10, 20, 40 mg Cd kg-1 soil). Mycorrhizal colonization rates were generally high (from 77% to 94%), and hardly affected by Cd. Gv colonization significantly enhanced P acquisition, growth and total Cd uptakes in both shoots and roots of S. photeinocarpum at all Cd levels. Meanwhile, Gv symbiosis significantly increased Cd concentration in the roots, and decreased Cd concentration in the shoots at all Cd levels, which indicates that Gv could promote phytostabilization by enhancing Cd accumulation in the roots to inhibit its translocation to shoots and the “dilution effects” linked to an increase in plant dry matter yield and a reduced Cd partitioning to shoots. Moreover, the improvement of CAT, POD and APX activities in the leaves of mycorrhizal plants infers that Gv symbiosis helped S. photeinocarpum to relieve oxidative damage to biomolecules in Cd-contaminated soil. The evident decline of MDA content in the leaves of mycorrhizal plants indicates that Gv symbiosis evidently improved antioxidant activities, and the enhancement of PCs production in the leaves of mycorrhizal plants suggests that Gv-inoculated plant may be more efficient to relieve Cd phytotoxicity. Therefore, the possible mechanisms of Cd phytotoxicity alleviation by Gv can be concluded as the decline of Cd concentration in the shoots and the improvement of P acquisition, PCs production and activities of CAT, POD, APX in mycorrhizal plants. PMID:26176959

  18. Effect of Inoculation with Glomus versiforme on Cadmium Accumulation, Antioxidant Activities and Phytochelatins of Solanum photeinocarpum.

    PubMed

    Tan, Shi-Yun; Jiang, Qiu-Yun; Zhuo, Feng; Liu, Hui; Wang, Yu-Tao; Li, Shao-Shan; Ye, Zhi-Hong; Jing, Yuan-Xiao

    2015-01-01

    The plant growth, phosphate acquisition, Cd translocation, phytochelatins (PCs) production and antioxidant parameters [superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione (GSH), ascorbate (ASA) and malonaldehyde (MDA)] were investigated in Cd-hyperaccumulator Solanum photeinocarpum inoculated with Glomus versiforme BGC GD01C (Gv) in Cd-added soils (0, 5, 10, 20, 40 mg Cd kg-1 soil). Mycorrhizal colonization rates were generally high (from 77% to 94%), and hardly affected by Cd. Gv colonization significantly enhanced P acquisition, growth and total Cd uptakes in both shoots and roots of S. photeinocarpum at all Cd levels. Meanwhile, Gv symbiosis significantly increased Cd concentration in the roots, and decreased Cd concentration in the shoots at all Cd levels, which indicates that Gv could promote phytostabilization by enhancing Cd accumulation in the roots to inhibit its translocation to shoots and the "dilution effects" linked to an increase in plant dry matter yield and a reduced Cd partitioning to shoots. Moreover, the improvement of CAT, POD and APX activities in the leaves of mycorrhizal plants infers that Gv symbiosis helped S. photeinocarpum to relieve oxidative damage to biomolecules in Cd-contaminated soil. The evident decline of MDA content in the leaves of mycorrhizal plants indicates that Gv symbiosis evidently improved antioxidant activities, and the enhancement of PCs production in the leaves of mycorrhizal plants suggests that Gv-inoculated plant may be more efficient to relieve Cd phytotoxicity. Therefore, the possible mechanisms of Cd phytotoxicity alleviation by Gv can be concluded as the decline of Cd concentration in the shoots and the improvement of P acquisition, PCs production and activities of CAT, POD, APX in mycorrhizal plants. PMID:26176959

  19. Effect of Inoculation with Glomus versiforme on Cadmium Accumulation, Antioxidant Activities and Phytochelatins of Solanum photeinocarpum.

    PubMed

    Tan, Shi-Yun; Jiang, Qiu-Yun; Zhuo, Feng; Liu, Hui; Wang, Yu-Tao; Li, Shao-Shan; Ye, Zhi-Hong; Jing, Yuan-Xiao

    2015-01-01

    The plant growth, phosphate acquisition, Cd translocation, phytochelatins (PCs) production and antioxidant parameters [superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione (GSH), ascorbate (ASA) and malonaldehyde (MDA)] were investigated in Cd-hyperaccumulator Solanum photeinocarpum inoculated with Glomus versiforme BGC GD01C (Gv) in Cd-added soils (0, 5, 10, 20, 40 mg Cd kg-1 soil). Mycorrhizal colonization rates were generally high (from 77% to 94%), and hardly affected by Cd. Gv colonization significantly enhanced P acquisition, growth and total Cd uptakes in both shoots and roots of S. photeinocarpum at all Cd levels. Meanwhile, Gv symbiosis significantly increased Cd concentration in the roots, and decreased Cd concentration in the shoots at all Cd levels, which indicates that Gv could promote phytostabilization by enhancing Cd accumulation in the roots to inhibit its translocation to shoots and the "dilution effects" linked to an increase in plant dry matter yield and a reduced Cd partitioning to shoots. Moreover, the improvement of CAT, POD and APX activities in the leaves of mycorrhizal plants infers that Gv symbiosis helped S. photeinocarpum to relieve oxidative damage to biomolecules in Cd-contaminated soil. The evident decline of MDA content in the leaves of mycorrhizal plants indicates that Gv symbiosis evidently improved antioxidant activities, and the enhancement of PCs production in the leaves of mycorrhizal plants suggests that Gv-inoculated plant may be more efficient to relieve Cd phytotoxicity. Therefore, the possible mechanisms of Cd phytotoxicity alleviation by Gv can be concluded as the decline of Cd concentration in the shoots and the improvement of P acquisition, PCs production and activities of CAT, POD, APX in mycorrhizal plants.

  20. Epitope Analysis of Anti-Myeloperoxidase Antibodies in Patients with ANCA-Associated Vasculitis

    PubMed Central

    Gou, Shen-Ju; Xu, Peng-Cheng; Chen, Min; Zhao, Ming-Hui

    2013-01-01

    Objective Increasing evidences have suggested the pathogenic role of anti-neutrophil cytoplasmic antibodies (ANCA) directing myeloperoxidase (MPO) in ANCA-associated vasculitis (AAV). The current study aimed to analyze the association between the linear epitopes of MPO-ANCA and clinicopathological features of patients with AAV. Methods Six recombinant linear fragments, covering the whole length amino acid sequence of a single chain of MPO, were produced from E.coli. Sera from 77 patients with AAV were collected at presentation. 13 out of the 77 patients had co-existence of serum anti-GBM antibodies. Ten patients also had sequential sera during follow up. The epitope specificities were detected by enzyme-linked immunosorbent assay using the recombinant fragments as solid phase ligands. Results Sera from 45 of the 77 (58.4%) patients with AAV showed a positive reaction to one or more linear fragments of the MPO chain. The Birmingham Vasculitis Activity Scores and the sera creatinine were significantly higher in patients with positive binding to the light chain fragment than that in patients without the binding. The epitopes recognized by MPO-ANCA from patients with co-existence of serum anti-GBM antibodies were mainly located in the N-terminus of the heavy chain. In 5 out of the 6 patients, whose sera in relapse recognize linear fragments, the reactivity to linear fragments in relapse was similar to that of initial onset. Conclusion The epitope specificities of MPO-ANCA were associated with disease activity and some clinicopathological features in patients with ANCA-associated vasculitis. PMID:23577119

  1. Effect of nonsurgical periodontal therapy on salivary myeloperoxidase levels: A biochemical study

    PubMed Central

    Dagar, Mona; Deepa, Dhruv Kumar; Molly, Madan; Sharma, Anamika; Khattak, Braham Prakash

    2015-01-01

    Background: Myeloperoxidase (MPO), the most abundant protein in neutrophils, is the focus of inflammatory pathologies. MPO could participate in the initiation and progression of periodontal disease. Materials and Methods: A total of 60 patients with healthy gingiva, gingivitis, periodontitis between age group of 20–55 years were selected. Group I - 20 Patients with healthy gingiva, Group II - 20 Patients with generalized gingivitis, Group III - 20 Patients with generalized chronic periodontitis, Group IV - 20 Patients of Group III after 1-month of scaling and root planning. The following parameters were recorded: Gingival index, plaque index, bleeding on probing index, probing pocket depth, clinical attachment level, salivary MPO levels. All the parameters were then statistically analyzed. Results: The mean MPO levels in Group I recorded was - 0.320 + 0.06, Group II was - 0.183 + 0.04, Group III was - 0.814 + 0.08 and Group IV was - 0.386 + 0.08 respectively. All these values were statistically significant when compared between the four groups (P < 0.05). A significantly elevated salivary MPO levels were found in subjects with chronic periodontitis as compared to the gingivitis group and the healthy group (P < 0.05). However, moderate but statistically significant increase in the MPO levels were observed in the gingivitis group as compared to the healthy group (P < 0.05). Furthermore, significant reduction in MPO levels were observed in Group IV after 1-month of nonsurgical periodontal therapy. Conclusion: The activities of MPO enzyme were significantly increased in the saliva of patients with periodontal disease in comparison to healthy individuals. Furthermore, nonsurgical periodontal therapy was found to be effective in improving clinical parameters and in reducing MPO levels. Salivary enzymes like MPO could be considered as a biochemical marker of periodontal disease activity. PMID:26644720

  2. Functional Consequence of Positive Selection Revealed through Rational Mutagenesis of Human Myeloperoxidase

    PubMed Central

    Loughran, Noeleen B.; Hinde, Sara; McCormick-Hill, Sally; Leidal, Kevin G.; Bloomberg, Sarah; Loughran, Sinéad T.; O’Connor, Brendan; Ó'Fágáin, Ciarán; Nauseef, William M.; O’Connell, Mary J.

    2012-01-01

    Myeloperoxidase (MPO) is a member of the mammalian heme peroxidase (MHP) multigene family. Whereas all MHPs oxidize specific halides to generate the corresponding hypohalous acid, MPO is unique in its capacity to oxidize chloride at physiologic pH to produce hypochlorous acid (HOCl), a potent microbicide that contributes to neutrophil-mediated host defense against infection. We have previously resolved the evolutionary relationships in this functionally diverse multigene family and predicted in silico that positive Darwinian selection played a major role in the observed functional diversities (Loughran NB, O'Connor B, O'Fagain C, O'Connell MJ. 2008. The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions. BMC Evol Biol. 8:101). In this work, we have replaced positively selected residues asparagine 496 (N496), tyrosine 500 (Y500), and leucine 504 (L504) with the amino acids present in the ancestral MHP and have examined the effects on the structure, biosynthesis, and activity of MPO. Analysis in silico predicted that N496F, Y500F, or L504T would perturb hydrogen bonding in the heme pocket of MPO and thus disrupt the structural integrity of the enzyme. Biosynthesis of the mutants stably expressed in human embryonic kidney 293 cells yielded apoproMPO, the heme-free, enzymatically inactive precursor of MPO, that failed to undergo normal maturation or proteolytic processing. As a consequence of the maturational arrest at the apoproMPO stage of development, cells expressing MPO with mutations N496F, Y500F, L504T, individually or in combination, lacked normal peroxidase or chlorinating activity. Taken together, our data provide further support for the in silico predictions of positive selection and highlight the correlation between positive selection and functional divergence. Our data demonstrate that directly probing the functional importance of positive selection can provide important insights into understanding protein evolution

  3. Propylthiouracil (PTU)-induced vasculitis associated with antineutrophil antibody against myeloperoxidase (MPO-ANCA).

    PubMed

    Nakamori, Yoshitaka; Tominaga, Takayuki; Inoue, Yasushi; Shinohara, Kenji

    2003-06-01

    A 54-year-old woman had been administered propylthiouracil (PTU) for Graves' disease for 4 years. Recently, she complained of hemoptysis due to pulmonary alveolar hemorrhage causing anemia, and also had microhematuria. Antineutrophil cytoplasmic antibody against myeloperoxidase (MPO-ANCA) was positive, and she was diagnosed with PTU-induced vasculitis. Cessation of PTU and the administration of corticosteroids ameliorated these manifestations.

  4. Accumulation of fossil fuels and metallic minerals in active and ancient rift lakes

    USGS Publications Warehouse

    Robbins, E.I.

    1983-01-01

    A study of active and ancient rift systems around the world suggests that accumulations of fossil fuels and metallic minerals are related to the interactions of processes that form rift valleys with those that take place in and around rift lakes. The deposition of the precursors of petroleum, gas, oil shale, coal, phosphate, barite, Cu-Pb-Zn sulfides, and uranium begins with erosion of uplifted areas, and the consequent input of abundant nutrients and solute loads into swamps and tectonic lakes. Hot springs and volcanism add other nutrients and solutes. The resulting high biological productivity creates oxidized/reduced interfaces, and anoxic and H2S-rich bottom waters which preserves metal-bearing organic tissues and horizons. In the depositional phases, the fine-grained lake deposits are in contact with coarse-grained beach, delta, river, talus, and alluvial fan deposits. Earthquake-induced turbidites also are common coarse-grained deposits of rift lakes. Postdepositional processes in rifts include high heat flow and a resulting concentration of the organic and metallic components that were dispersed throughout the lakebeds. Postdepositional faulting brings organic- and metal-rich sourcebeds in contact with coarse-grained host and reservoir rocks. A suite of potentially economic deposits is therefore a characteristic of rift valleys. ?? 1983.

  5. Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice

    PubMed Central

    Saito, Mariko; Wu, Gusheng; Hui, Maria; Masiello, Kurt; Dobrenis, Kostantin; Ledeen, Robert W.; Saito, Mitsuo

    2015-01-01

    Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration. PMID:26063460

  6. Accumulate repeat accumulate codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

  7. An Accumulated Activity Effective Index for Promoting Physical Activity: A Design and Development Study in a Mobile and Pervasive Health Context

    PubMed Central

    Liu, Chung-Tse

    2015-01-01

    Background Increased physical activity has become a principal personal health goal worldwide because sufficient physical activity can reduce the risk of many adverse conditions. Motivating individuals to increase their levels of physical activity can increase life expectancy and contribute to a healthy life. Sharing and comparison of physical activity information by using the Internet, with fewer privacy concerns, might also help encourage people to promote and maintain sufficient physical activity. To promote and manage physical activity, an accumulated activity effective index (AAEI) is proposed in this paper. Objective The purpose of the AAEI design is to maintain and promote physical activity. The public can easily accept a clear indicator that reveals the current status of physical activity. The AAEI is not only an assessment and tracking tool for personal physical activity, but is also useful for goal setting and for sharing content with the Internet community. Methods The AAEI is derived from input in the form of accumulated physical activity, and evaluates the status of physical activities and days spent exercising. The term AAEI(t1,t2) is an index of the accumulated physical activity in the time interval (t1,t2), where the base unit of time is the day. The AAEI is determined according to accumulated physical activity and is adjusted using the previous status of physical activity. The previous status of physical activity is estimated according to the number of days spent exercising and the accumulated physical activity that has been performed. An analysis of the AAEI performance was conducted using a simulation model and a real-world trial with 2 participants. Results The AAEI increased as the physical activity and days spent exercising accumulated. Conversely, the AAEI decreased with lack of physical activity and increased resting days. In simulation, the shape of the AAEI line indicated different types of exercise. The moving average AAEI represented long

  8. Role of high-avidity binding of human neutrophil myeloperoxidase in the killing of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Miyasaki, K T; Zambon, J J; Jones, C A; Wilson, M E

    1987-01-01

    The binding of the neutrophil enzyme myeloperoxidase (MPO) to microbial surfaces is believed to be the first step in its microbicidal activity. The MPO-H2O2-Cl- system is responsible for most oxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. There appear to be three forms of MPO (MPO I, II, and III), all of which can kill this organism in the presence of H2O2 and chloride. In this study, we characterized the binding of native human neutrophil MPO to A. actinomycetemcomitans by an elution procedure dependent on the cationic detergent cetyltrimethylammonium bromide. Binding of native MPO was rapid and reached apparent equilibrium within 1 min. A proportion of binding under equilibrium conditions was saturable and highly avid, with a capacity of 4,500 sites per cell and a dissociation constant of 7.9 X 10(-10) M. At equal protein concentrations, more MPO III bound than MPO II, and more MPO II bound than MPO I. The high-avidity interaction was inhibitable with yeast mannan and with the serotype-defining mannan of A. actinomycetemcomitans. Binding was also partially reversible with yeast mannan. MPO bound to the high-avidity sites did not oxidize guaiacol but oxidized chloride, as detected by the chlorination of taurine. MPO bound to the high-avidity sites was incapable of killing A. actinomycetemcomitans alone in the presence of H2O2 and Cl-, but potentiated killing when sufficient additional MPO was provided. The killing of A. actinomycetemcomitans by the MPO-H2O2-Cl- system was inhibited by yeast mannan and a serotype-defining mannan of A. actinomycetemcomitans. We conclude that high-avidity binding of MPO to the surface of A. actinomycetemcomitans is a mannan-specific interaction and that MPO bound to the high-avidity sites is essential but not alone sufficient to kill A. actinomycetemcomitans. PMID:3032796

  9. Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I.

    PubMed

    Chan, Gary K L; Witkowski, Andrzej; Gantz, Donald L; Zhang, Tianqi O; Zanni, Martin T; Jayaraman, Shobini; Cavigiolio, Giorgio

    2015-04-24

    High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo.

  10. Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I*

    PubMed Central

    Chan, Gary K. L.; Witkowski, Andrzej; Gantz, Donald L.; Zhang, Tianqi O.; Zanni, Martin T.; Jayaraman, Shobini; Cavigiolio, Giorgio

    2015-01-01

    High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo. PMID:25759391

  11. Roles of myeloperoxidase and GAPDH in interferon-gamma production of GM-CSF-dependent macrophages.

    PubMed

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-02-01

    Interferon (IFN)-gamma is highly expressed in atherosclerotic lesions and may have an important role in atherogenesis. Myeloperoxidase (MPO), the most abundant protein in neutrophils, is a marker of plaque vulnerability and a possible bridge between inflammation and cardiovascular disease. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has also been implicated in the pathogenesis of atherosclerosis. The present study investigated the role of neutrophil activation in atherosclerosis. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IFN-gamma protein by GM-CSF-dependent-macrophages was investigated by enzyme-linked immunosorbent assay after stimulation with MPO. GM-CSF enhanced macrophage expression of the mannose receptor (CD206), which is involved in MPO uptake. MPO increased IFN-gamma production by GM-CSF-dependent macrophages in a concentration-dependent manner. Pretreatment of macrophages with small interfering RNA (siRNA) for CD206 or extracellular signal-regulated kinase (ERK)-2 attenuated IFN-gamma production, while siRNA for ERK-1 did not. GAPDH is known to bind to adenylate/uridylate (AU)-rich elements of RNA and may influence IFN-gamma protein expression by binding to the AU-rich element of IFN-gamma mRNA. Interestingly, pretreatment with siRNA for GAPDH significantly reduced IFN-gamma production by macrophages, while it did not affect TF protein expression. In conclusion, MPO upregulates IFN-gamma production by GM-CSF-dependent-macrophages via the CD206/ERK-2 signaling pathway, while silencing GAPDH reduces IFN-gamma production. PMID:27441256

  12. Ammonia activates pacC and patulin accumulation in an acidic environment during apple colonization by Penicillium expansum.

    PubMed

    Barad, Shiri; Espeso, Eduardo A; Sherman, Amir; Prusky, Dov

    2016-06-01

    Penicillium expansum, the causal agent of blue mould rot, causes severe post-harvest fruit maceration simultaneously with the secretion of d-gluconic acid (GLA) and the mycotoxin patulin in colonized tissue. The factor(s) inducing patulin biosynthesis during colonization of the host acidic environment is unclear. During the colonization of apple fruit in vivo and growth in culture, P. expansum secretes pH-modulating GLA and ammonia. Although patulin and its possible opportunistic precursor GLA accumulate together during fungal development, ammonia is detected on the colonized tissue's leading edge and after extended culture, close to patulin accumulation. Here, we demonstrate ammonia-induced transcript activation of the global pH modulator PacC and patulin accumulation in the presence of GLA by: (i) direct exogenous treatment of P. expansum growing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the patulin biosynthesis cluster. Ammonia induced patulin accumulation concurrently with the transcript activation of pacC and patulin biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacC transcript expression under acidic conditions. Electrophoretic mobility shift assays using P. expansum PacC and antibodies to the different cleaved proteins showed that PacC is not protected against proteolytic signalling at pH 4.5 relative to pH 7.0, but NH4 addition did not further enhance its proteolytic cleavage. Ammonia enhanced the activation of palF transcript in the Pal pathway under acidic conditions. Ammonia accumulation in the host environment by the pathogen under acidic pH may be a regulatory cue for pacC activation, towards the accumulation of secondary metabolites, such as patulin.

  13. Ammonia activates pacC and patulin accumulation in an acidic environment during apple colonization by Penicillium expansum.

    PubMed

    Barad, Shiri; Espeso, Eduardo A; Sherman, Amir; Prusky, Dov

    2016-06-01

    Penicillium expansum, the causal agent of blue mould rot, causes severe post-harvest fruit maceration simultaneously with the secretion of d-gluconic acid (GLA) and the mycotoxin patulin in colonized tissue. The factor(s) inducing patulin biosynthesis during colonization of the host acidic environment is unclear. During the colonization of apple fruit in vivo and growth in culture, P. expansum secretes pH-modulating GLA and ammonia. Although patulin and its possible opportunistic precursor GLA accumulate together during fungal development, ammonia is detected on the colonized tissue's leading edge and after extended culture, close to patulin accumulation. Here, we demonstrate ammonia-induced transcript activation of the global pH modulator PacC and patulin accumulation in the presence of GLA by: (i) direct exogenous treatment of P. expansum growing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the patulin biosynthesis cluster. Ammonia induced patulin accumulation concurrently with the transcript activation of pacC and patulin biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacC transcript expression under acidic conditions. Electrophoretic mobility shift assays using P. expansum PacC and antibodies to the different cleaved proteins showed that PacC is not protected against proteolytic signalling at pH 4.5 relative to pH 7.0, but NH4 addition did not further enhance its proteolytic cleavage. Ammonia enhanced the activation of palF transcript in the Pal pathway under acidic conditions. Ammonia accumulation in the host environment by the pathogen under acidic pH may be a regulatory cue for pacC activation, towards the accumulation of secondary metabolites, such as patulin. PMID:26420024

  14. Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2

    PubMed Central

    Yin, Yaling; Wang, Yali; Gao, Di; Ye, Jinwang; Wang, Xin; Fang, Lin; Wu, Dongqin; Pi, Guilin; Lu, Chengbiao; Zhou, Xin-Wen; Yang, Ying; Wang, Jian-Zhi

    2016-01-01

    Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer’s disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments. PMID:27277673

  15. Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2.

    PubMed

    Yin, Yaling; Wang, Yali; Gao, Di; Ye, Jinwang; Wang, Xin; Fang, Lin; Wu, Dongqin; Pi, Guilin; Lu, Chengbiao; Zhou, Xin-Wen; Yang, Ying; Wang, Jian-Zhi

    2016-01-01

    Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer's disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments. PMID:27277673

  16. Active hydrocarbon biosynthesis and accumulation in a green alga, Botryococcus braunii (race A).

    PubMed

    Hirose, Mana; Mukaida, Fukiko; Okada, Sigeru; Noguchi, Tetsuko

    2013-08-01

    Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [(14)C]acetate. Incorporation of (14)C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii, and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.

  17. Intracellular mechanisms coupled to NPY Y2 and Y5 receptor activation and lipid accumulation in murine adipocytes.

    PubMed

    Rosmaninho-Salgado, Joana; Cortez, Vera; Estrada, Marta; Santana, Magda M; Gonçalves, Alexandra; Marques, Ana Patrícia; Cavadas, Cláudia

    2012-12-01

    The formation of adipose tissue is a process that includes the pre-adipocyte proliferation and differentiation to adipocytes that are cells specialized in lipid accumulation. The adipocyte differentiation is a process driven by the coordinated expression of various transcription factors, such as peroxisome proliferator-activated receptor (PPAR-γ). Neuropeptide Y (NPY) induces adipocyte proliferation and differentiation but the NPY receptors and the intracellular pathways involved in these processes are still not clear. In the present work we studied the role of NPY receptors and the intracellular pathways involved in the stimulatory effect of NPY on lipid accumulation. The murine pre-adipocyte cell line, 3T3-L1, was used as a cell model. Adipogenesis was evaluated by quantifying lipid accumulation by Oil red-O assay and by analyzing PPAR-γ expression using the Western blotting assay. Adipocytes were incubated with NPY (100nM) and a decrease on lipid accumulation and PPAR-γ expression was observed in the presence of NPY Y(2) receptor antagonist (BIIE0246, 1μM) or NPY Y(5) antagonist. Furthermore, NPY Y(2) (NPY(3-36), 100nM) or NPY Y(5) (NPY(19-23)(GLY(1), Ser(3), Gln(4), Thr(6), Ala(31), Aib(32), Gln(34)) PP, 100nM) receptor agonists increased lipid accumulation and PPAR-γ expression. We further investigate the intracellular pathways associated with NPY Y(2) and NPY Y(5) receptor activation. Our results show NPY induces PPAR-γ expression and lipid accumulation through NPY Y(2) and NPY Y(5) receptors activation. PKC and PLC inhibitors inhibit lipid accumulation induced by NPY Y(5) receptor agonist. Moreover, our results suggest that lipid accumulation induced by NPY Y(2) receptor activation occurs through PKA, MAPK and PI3K pathways. In conclusion, this study contributes to a step forward on the knowledge of intracellular mechanisms associated with NPY receptors activation on adipocytes and contributes to a better understanding and the development of new

  18. Phosphatase production and activity in Citrobacter freundii and a naturally occurring, heavy-metal-accumulating Citrobacter sp.

    PubMed

    Montgomery, D M; Dean, A C; Wiffen, P; Macaskie, L E

    1995-10-01

    The ability of a naturally occurring Citrobacter sp. to accumulate cadmium has been attributed to cellular precipitation of CdHPO4, utilizing HPO4(2-) liberated via the activity of an overproduced, Cd-resistant acid-type phosphatase. Phosphatase production and heavy metal accumulation by batch cultures of this strain (N14) and a phosphatase-deficient mutant were compared with two reference strains of Citrobacter freundii. Only strain N14 expressed a high level of acid phosphatase and accumulated lanthanum and uranyl ion enzymically. Acid phosphatase is regulated via carbon-starvation; although the C. freundii strains overexpressed phosphatase activity in carbon-limiting continuous culture, this was approximately 20-fold less than the activity of strain N14 grown similarly. Citrobacter strain N14 was originally isolated from a metal-contaminated soil environment; phosphatase overproduction and metal accumulation were postulated as a detoxification mechanism. However, application of Cd-stress, and enrichment for Cd-resistant C. freundii ('training'), reduced the phosphatase activity of this organism by about 50% as compared to Cd-unstressed cultures. The acid phosphatase of C. freundii and Citrobacter N14 had a similar pattern of resistance to some diagnostic reagents. The enzyme of the latter is similar to the PhoN acid phosphatase of Salmonella typhimurium described by other workers; the results are discussed with respect to the known phosphatases of the enterobacteria.

  19. Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: structural and functional effects.

    PubMed

    De Cristofaro, R; Landolfi, R

    2000-02-01

    The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin. PMID:10739383

  20. Infection-induced type I interferons activate CD11b on B-1 cells for subsequent lymph node accumulation

    PubMed Central

    Waffarn, Elizabeth E.; Hastey, Christine J.; Dixit, Neha; Choi, Youn Soo; Cherry, Simon; Kalinke, Ulrich; Simon, Scott I.; Baumgarth, Nicole

    2016-01-01

    Innate-like B-1a lymphocytes rapidly redistribute to regional mediastinal lymph nodes (MedLN) during influenza infection to generate protective IgM. Here we demonstrate that influenza infection-induced type I interferons directly stimulate body cavity B-1 cells and are a necessary signal required for B-1 cell accumulation in MedLN. Vascular mimetic flow chamber studies show that type I interferons increase ligand-mediated B-1 cell adhesion under shear stress by inducing high-affinity conformation shifts of surface-expressed integrins. In vivo trafficking experiments identify CD11b as the non-redundant, interferon-activated integrin required for B-1 cell accumulation in MedLN. Thus CD11b on B-1 cells senses infection-induced innate signals and facilitates their rapid sequester into secondary lymphoid tissues, thereby regulating the accumulation of polyreactive IgM producers at sites of infection. PMID:26612263

  1. Dietary fructose feeding increases adipose methylglyoxal accumulation in rats in association with low expression and activity of glyoxalase-2.

    PubMed

    Masterjohn, Christopher; Park, Youngki; Lee, Jiyoung; Noh, Sang K; Koo, Sung I; Bruno, Richard S

    2013-08-21

    Methylglyoxal is a precursor to advanced glycation endproducts that may contribute to diabetes and its cardiovascular-related complications. Methylglyoxal is successively catabolized to D-lactate by glyoxalase-1 and glyoxalase-2. The objective of this study was to determine whether dietary fructose and green tea extract (GTE) differentially regulate methylglyoxal accumulation in liver and adipose, mediated by tissue-specific differences in the glyoxalase system. We fed six week old male Sprague-Dawley rats a low-fructose diet (10% w/w) or a high-fructose diet (60% w/w) containing no GTE or GTE at 0.5% or 1.0% for nine weeks. Fructose-fed rats had higher (P < 0.05) adipose methylglyoxal, but GTE had no effect. Plasma and hepatic methylglyoxal were unaffected by fructose and GTE. Fructose and GTE also had no effect on the expression or activity of glyoxalase-1 and glyoxalase-2 at liver or adipose. Regardless of diet, adipose glyoxalase-2 activity was 10.8-times lower (P < 0.05) than adipose glyoxalase-1 activity and 5.9-times lower than liver glyoxalase-2 activity. Adipose glyoxalase-2 activity was also inversely related to adipose methylglyoxal (r = -0.61; P < 0.05). These findings suggest that fructose-mediated adipose methylglyoxal accumulation is independent of GTE supplementation and that its preferential accumulation in adipose compared to liver is due to low constitutive expression of glyoxalase-2.

  2. Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization.

    PubMed

    Carter, Russell E; Seidel, Jessica L; Lindquist, Britta E; Sheline, Christian T; Shuttleworth, C William

    2013-06-01

    Spreading depolarization (SD) is a feed-forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn(2+) release and intracellular accumulation occurs with SD, and elevated intracellular Zn(2+) ([Zn(2+) ]i ) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn(2+) ]i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn(2+) ]i loading prior to SD, by transient ZnCl2 application with the Zn(2+) ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn(2+) ]i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn(2+) release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn(2+) ]i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn(2+) ]i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn(2+) ]i accumulation in a range of brain injury models. PMID:23495967

  3. Accumulated oxygen deficit and shuttle run performance in physically active men and women.

    PubMed

    Ramsbottom, R; Nevill, M E; Nevill, A M; Hazeldine, R

    1997-04-01

    The aim of this study was to establish the validity of using shuttle run performance over 20 m to predict accumulated oxygen deficit. A new high-intensity shuttle run test (HIST) was devised, during which subjects ran to exhaustion at a speed equivalent to 120% of their performance attained during a progressive shuttle run test. The reliability of the new test was examined and found to be acceptable for 18 subjects who performed the test twice on separate days (r = 0.84, P < 0.01, study I). The discriminating ability of the new test was examined by comparing the distance covered by eight sprint- and eight endurance-trained athletes at 120% of their respective progressive shuttle run performances (615 +/- 111 vs 273 +/- 84 m, P < 0.01, study II). The strongest predictor of accumulated oxygen deficit for 27 subjects was found to be the geometric mean of the performances on the new test and on the progressive shuttle run test (r = 0.74, study III). The regression equation for this relationship was then used to estimate the accumulated oxygen deficit for a second group of 16 subjects (study IV). The correlation between the estimated and measured accumulated oxygen deficits was significant (r = 0.79, P < 0.01). The results from studies III and IV were therefore combined with the data from six new subjects to give a regression equation for predictive purposes based on 49 subjects.

  4. Accumulation of duct cells with activated YAP parallels fibrosis progression in NonAlcoholic Fatty Liver Disease

    PubMed Central

    Machado, Mariana Verdelho; Michelotti, Gregory Alexander; de Almeida, Thiago Pereira; Xie, Guanhua; Premont, Richard; Cortez-Pinto, Helena; Diehl, Anna Mae

    2015-01-01

    Background & Aims Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem-cell associated factor, controls liver growth in healthy adult mice. Increasing nuclear localization of YAP triggers accumulation of reactive-appearing ductular cells (YAP+RDC) with liver progenitor capabilities. The significance of YAP activation, and mechanisms involved, are unknown in diseased livers. We evaluated the hypothesis that YAP is more activated in injured livers that are scarring than in those that are regenerating effectively. Methods Immunohistochemistry and qRT-PCR analysis were used to localize and quantify changes in YAP and RDC in 52 patients with nonalcoholic fatty liver disease (NAFLD) and two mouse models of diet-induced nonalcoholic steatohepatitis (NASH). Results were correlated with liver disease severity, metabolic risk factors, and factors proven to control NAFLD progression. Results YAP increased in NAFLD where it mainly localized in nuclei of RDC that expressed progenitor markers. Accumulation of YAP+RDC paralleled the severity of hepatocyte injury and accumulation of Sonic hedgehog (Shh), but not steatosis or metabolic risk factors. YAP+RDC expressed osteopontin, a Shh-regulated fibrogenic factor. Myofibroblast accumulation, fibrosis, and numbers of YAP+RDC strongly correlated. In murine NASH models, atrophic fibrotic livers contained significantly more YAP+RDC than livers with less severe NASH. Conclusion YAP+RDC promote scarring, rather than effective regeneration, during NASH. PMID:26070409

  5. Rosehip Extract Inhibits Lipid Accumulation in White Adipose Tissue by Suppressing the Expression of Peroxisome Proliferator-activated Receptor Gamma.

    PubMed

    Nagatomo, Akifumi; Nishida, Norihisa; Matsuura, Yoichi; Shibata, Nobuhito

    2013-06-01

    Recent studies have shown that Rosa canina L. and tiliroside, the principal constituent of its seeds, exhibit anti-obesity and anti-diabetic activities via enhancement of fatty acid oxidation in the liver and skeletal muscle. However, the effects of rosehip, the fruit of this plant, extract (RHE), or tiliroside on lipid accumulation in adipocytes have not been analyzed. We investigated the effects of RHE and tiliroside on lipid accumulation and protein expression of key transcription factors in both in vitro and in vivo models. RHE and tiliroside inhibited lipid accumulation in a dose-dependent manner in 3T3-L1 cells. We also analyzed the inhibitory effect of RHE on white adipose tissue (WAT) in high-fat diet (HFD)-induced obesity mice model. Male C57BL/6J mice were fed HFD or HFD supplemented with 1% RHE (HFDRH) for 8 weeks. The HFDRH-fed group gained less body weight and had less visceral fat than the HFD-fed group. Liver weight was significantly lower in the HFDRH-fed group and total hepatic lipid and triglyceride (TG) content was also reduced. A significant reduction in the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was observed in epididymal fat in the HFDRH-fed group, in comparison with controls, through Western blotting. These results suggest that downregulation of PPARγ expression is involved, at least in part, in the suppressive effect of RHE on lipid accumulation in WAT.

  6. Rosehip Extract Inhibits Lipid Accumulation in White Adipose Tissue by Suppressing the Expression of Peroxisome Proliferator-activated Receptor Gamma

    PubMed Central

    Nagatomo, Akifumi; Nishida, Norihisa; Matsuura, Yoichi; Shibata, Nobuhito

    2013-01-01

    Recent studies have shown that Rosa canina L. and tiliroside, the principal constituent of its seeds, exhibit anti-obesity and anti-diabetic activities via enhancement of fatty acid oxidation in the liver and skeletal muscle. However, the effects of rosehip, the fruit of this plant, extract (RHE), or tiliroside on lipid accumulation in adipocytes have not been analyzed. We investigated the effects of RHE and tiliroside on lipid accumulation and protein expression of key transcription factors in both in vitro and in vivo models. RHE and tiliroside inhibited lipid accumulation in a dose-dependent manner in 3T3-L1 cells. We also analyzed the inhibitory effect of RHE on white adipose tissue (WAT) in high-fat diet (HFD)-induced obesity mice model. Male C57BL/6J mice were fed HFD or HFD supplemented with 1% RHE (HFDRH) for 8 weeks. The HFDRH-fed group gained less body weight and had less visceral fat than the HFD-fed group. Liver weight was significantly lower in the HFDRH-fed group and total hepatic lipid and triglyceride (TG) content was also reduced. A significant reduction in the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was observed in epididymal fat in the HFDRH-fed group, in comparison with controls, through Western blotting. These results suggest that downregulation of PPARγ expression is involved, at least in part, in the suppressive effect of RHE on lipid accumulation in WAT. PMID:24471115

  7. Activity-dependent synaptic GRIP1 accumulation drives synaptic scaling up in response to action potential blockade

    PubMed Central

    Gainey, Melanie A.; Tatavarty, Vedakumar; Nahmani, Marc; Lin, Heather; Turrigiano, Gina G.

    2015-01-01

    Synaptic scaling is a form of homeostatic plasticity that stabilizes neuronal firing in response to changes in synapse number and strength. Scaling up in response to action-potential blockade is accomplished through increased synaptic accumulation of GluA2-containing AMPA receptors (AMPAR), but the receptor trafficking steps that drive this process remain largely obscure. Here, we show that the AMPAR-binding protein glutamate receptor-interacting protein-1 (GRIP1) is essential for regulated synaptic AMPAR accumulation during scaling up. Synaptic abundance of GRIP1 was enhanced by activity deprivation, directly increasing synaptic GRIP1 abundance through overexpression increased the amplitude of AMPA miniature excitatory postsynaptic currents (mEPSCs), and shRNA-mediated GRIP1 knockdown prevented scaling up of AMPA mEPSCs. Furthermore, knockdown and replace experiments targeting either GRIP1 or GluA2 revealed that scaling up requires the interaction between GRIP1 and GluA2. Finally, GRIP1 synaptic accumulation during scaling up did not require GluA2 binding. Taken together, our data support a model in which activity-dependent trafficking of GRIP1 to synaptic sites drives the forward trafficking and enhanced synaptic accumulation of GluA2-containing AMPAR during synaptic scaling up. PMID:26109571

  8. Expression of cadR Enhances its Specific Activity for Cd Detoxification and Accumulation in Arabidopsis.

    PubMed

    Li, Jingrui; Wei, Xuezhi; Yu, Pengli; Deng, Xin; Xu, Wenxiu; Ma, Mi; Zhang, Haiyan

    2016-08-01

    Cadmium (Cd) is a transition metal that is highly toxic in biological systems. Anthropogenic emissions of Cd have increased biogeochemical cycling and the amount of Cd in the biosphere. Here we studied the utility of a bacterial Cd-binding protein, CadR, for the remediation of Cd contamination. CadR was successfully targeted to chloroplasts using a constitutive Cauliflower mosaic virus (CaMV) 35S promoter or a shoot-specific Chl a/b-binding protein 2 gene (CAB2) promoter and an RbcS (small subunit of the Rubisco complex) transit peptide. Under short-term (2 d) exposure to Cd, the cadR transgenic plants showed up to a 2.9-fold Cd accumulation in roots compared with untransformed plants. Under medium term (7 d) exposure to Cd, the concentrations of Cd in leaves began to increase but there were no differences between the wild type and the cadR transgenic plants. Under long-term (16 d) exposure to Cd, the cadR transgenic plants accumulated greater amounts of Cd in leaves than the untransformed plants. Total Cd accumulation (µg per plant) in shoots and roots of the plants expressing cadR were significantly higher (up to 3.5-fold in shoots and 5.2-fold in roots) than those of the untransformed plants. We also found that targeting CadR to chloroplasts facilitated chloroplastic metal homeostasis and Chl b accumulation. Our results demonstrate that manipulating chelating capacity in chloroplasts or in the cytoplasm may be effective in modifying both the accumulation of and resistance to Cd.

  9. Expression of cadR Enhances its Specific Activity for Cd Detoxification and Accumulation in Arabidopsis.

    PubMed

    Li, Jingrui; Wei, Xuezhi; Yu, Pengli; Deng, Xin; Xu, Wenxiu; Ma, Mi; Zhang, Haiyan

    2016-08-01

    Cadmium (Cd) is a transition metal that is highly toxic in biological systems. Anthropogenic emissions of Cd have increased biogeochemical cycling and the amount of Cd in the biosphere. Here we studied the utility of a bacterial Cd-binding protein, CadR, for the remediation of Cd contamination. CadR was successfully targeted to chloroplasts using a constitutive Cauliflower mosaic virus (CaMV) 35S promoter or a shoot-specific Chl a/b-binding protein 2 gene (CAB2) promoter and an RbcS (small subunit of the Rubisco complex) transit peptide. Under short-term (2 d) exposure to Cd, the cadR transgenic plants showed up to a 2.9-fold Cd accumulation in roots compared with untransformed plants. Under medium term (7 d) exposure to Cd, the concentrations of Cd in leaves began to increase but there were no differences between the wild type and the cadR transgenic plants. Under long-term (16 d) exposure to Cd, the cadR transgenic plants accumulated greater amounts of Cd in leaves than the untransformed plants. Total Cd accumulation (µg per plant) in shoots and roots of the plants expressing cadR were significantly higher (up to 3.5-fold in shoots and 5.2-fold in roots) than those of the untransformed plants. We also found that targeting CadR to chloroplasts facilitated chloroplastic metal homeostasis and Chl b accumulation. Our results demonstrate that manipulating chelating capacity in chloroplasts or in the cytoplasm may be effective in modifying both the accumulation of and resistance to Cd. PMID:27382127

  10. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation

    PubMed Central

    Twu, Yuh-Ching; Lee, Tzong-Shyuan; Lin, Yun-Lian; Hsu, Shih-Ming; Wang, Yuan-Hsi; Liao, Chia-Yu; Wang, Chung-Kwe; Liang, Yu-Chih; Liao, Yi-Jen

    2016-01-01

    In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis. PMID:27420058

  11. High concentrations of myeloperoxidase in the equine uterus as an indicator of endometritis.

    PubMed

    Parrilla-Hernandez, Sonia; Ponthier, Jérôme; Franck, Thierry Y; Serteyn, Didier D; Deleuze, Stéfan C

    2014-04-15

    Intraluminal fluid and excessive abnormal hyperedema are regularly used for the diagnosis of endometritis in the mare, which is routinely confirmed by the presence of neutrophils on endometrial smears. Studies show a relation between neutrophils and myeloperoxidase (MPO), an enzyme contained in and released by neutrophils during degranulation or after cell lysis. This enzyme has been found in many fluids and tissues, and associated with different inflammatory pathologies in the horse. The aims of this study were to assess the presence and concentration of MPO in the equine uterus, and to investigate its relation with neutrophils, and other clinical signs of endometritis. Mares (n = 51) were evaluated for the presence of intraluminal fluid and excessive endometrial edema before breeding, and a small volume lavage and cytology samples were obtained. From 69 cycles, supernatant of the uterine flushes was analyzed with a specific equine MPO ELISA assay to measure MPO concentration. Cytology samples were used for the diagnosis of endometritis. Myeloperoxidase was present in the uterus of all estrus mares in highly variable concentrations. Myeloperoxidase concentrations were significantly (P < 0.05) higher in samples with positive cytologies and in the presence of intraluminal fluid. Occasionally, some samples with negative cytologies showed high MPO concentration, but the opposite was never observed. Cycles presenting hyperedema weren't associated with high concentration of MPO, intraluminal fluid, or positive cytology, making it a poor diagnostic tool of endometritis. PMID:24565475

  12. Accumulation fatty acids of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense

    NASA Astrophysics Data System (ADS)

    Leyva, Luis A.; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E.

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  13. Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoAcarboxylase, temperature, and co-immobilization with Azospirillum brasilense [corrected].

    PubMed

    Leyva, Luis A; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  14. Novel phosphatidylethanolamine derivatives accumulate in circulation in hyperlipidemic ApoE−/− mice and activate platelets via TLR2

    PubMed Central

    Biswas, Sudipta; Xin, Liang; Panigrahi, Soumya; Zimman, Alejandro; Wang, Hua; Yakubenko, Valentin P.; Byzova, Tatiana V.; Salomon, Robert G.

    2016-01-01

    A prothrombotic state and increased platelet reactivity are common in dyslipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products, including hydroxy-ω-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid, can also be modified by hydroxy-ω-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PEs) are present in the plasma of hyperlipidemic ApoE−/− mice. CAP-PEs directly bind to TLR2 and induces platelet integrin αIIbβ3 activation and P-selectin expression in a Toll-like receptor 2 (TLR2)-dependent manner. Platelet activation by CAP-PEs includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of tumor necrosis factor receptor–associated factor 6. This in turn activates the Src family kinases, spleen tyrosine kinase and PLCγ2, and platelet integrins. Murine intravital thrombosis studies demonstrated that CAP-PEs accelerate thrombosis in TLR2-dependent manner and that TLR2 contributes to accelerate thrombosis in mice in the settings of hyperlipidemia. Our study identified the novel end-products of lipid peroxidation, accumulating in circulation in hyperlipidemia and inducing platelet activation by promoting cross-talk between innate immunity and integrin activation signaling pathways. PMID:27015965

  15. Augmentation of Human Macrophage Candidacidal Capacity by Recombinant Human Myeloperoxidase and GranulocyteMacrophage Colony-Stimulating Factor

    PubMed Central

    Maródi, László; Tournay, Christophe; Káposzta, Rita; Johnston, Richard B.; Moguilevsky, Nicole

    1998-01-01

    Phagocyte myeloperoxidase (MPO) is believed to be particularly important in defense against candida infection. We reported earlier that monocytes, rich in MPO, killed Candida albicans at a significantly higher rate and extent than did monocyte-derived macrophages, known to lack MPO, and that C. albicans is less resistant to MPO-dependent oxidants than less pathogenic Candida species. We hypothesized, therefore, that the capacity of macrophages to kill C. albicans might be improved in the presence of MPO. In this study, we evaluated the ability of recombinant human MPO (rhMPO) to augment the killing of C. albicans by resident macrophages and macrophages activated by recombinant human granulocyte-macrophage colony-stimulating factor. Addition of rhMPO (concentration range, 0.8 to 6.4 U/ml) to suspensions of resident and activated macrophages and opsonized C. albicans resulted in concentration-dependent and significant increases in candida killing. This enhancement was particularly pronounced with activated macrophages, whether C. albicans was opsonized or unopsonized and ingested through the macrophage mannose receptor. rhMPO did not affect the killing of C. albicans by monocytes, nor did it affect phagocytosis of opsonized or unopsonized C. albicans. These results indicate that exogenous rhMPO can augment the candidacidal capacity of both resident and activated macrophages, with a more profound effect on activated cells. We suggest that rhMPO may be effective in the treatment of invasive candidiasis. PMID:9596743

  16. Salivary Myeloperoxidase, Assessed by 3,3′-Diaminobenzidine Colorimetry, Can Differentiate Periodontal Patients from Nonperiodontal Subjects

    PubMed Central

    Klangprapan, Supaporn; Chaiyarit, Ponlatham; Hormdee, Doosadee; Kampichai, Amonrujee; Khampitak, Tueanjit; Daduang, Jureerut; Tavichakorntrakool, Ratree; Panijpan, Bhinyo; Boonsiri, Patcharee

    2016-01-01

    Periodontal diseases, which result from inflammation of tooth supporting tissues, are highly prevalent worldwide. Myeloperoxidase (MPO), from certain white blood cells in saliva, is a biomarker for inflammation. We report our study on the salivary MPO activity and its association with severity of periodontal diseases among Thai patients. Periodontally healthy subjects (n = 11) and gingivitis (n = 32) and periodontitis patients (n = 19) were enrolled. Assessments of clinically periodontal parameters were reported as percentages for gingival bleeding index (GI) and bleeding on probing (BOP), whereas pocket depth (PD) and clinical attachment loss (CAL) were measured in millimeters and then made to index scores. Salivary MPO activity was measured by colorimetry using 3,3′-diaminobenzidine as substrate. The results showed that salivary MPO activity in periodontitis patients was significantly higher than in healthy subjects (p = 0.003) and higher than in gingivitis patients (p = 0.059). No difference was found between gingivitis and healthy groups (p = 0.181). Significant correlations were observed (p < 0.01) between salivary MPO activity and GI (r = 0.632, p < 0.001), BOP (r = 0.599, p < 0.001), PD (r = 0.179, p = 0.164), and CAL (r = 0.357, p = 0.004) index scores. Sensitivity (94.12%), specificity (54.55%), and positive (90.57%) and negative (66.67%) predictive values indicate that salivary MPO activity has potential use as a screening marker for oral health of the Thai community. PMID:27274868

  17. Relationship between in vitro binding activity and in vivo tumor accumulation of radiolabeled monoclonal antibodies

    SciTech Connect

    Sakahara, H.; Endo, K.; Koizumi, M.; Nakashima, T.; Kunimatsu, M.; Watanabe, Y.; Kawamura, Y.; Nakamura, T.; Tanaka, H.; Kotoura, Y.

    1988-02-01

    The relationship between in vitro cell binding and in vivo tumor accumulation of radiolabeled antibodies was studied using /sup 125/I- and /sup 111/In-labeled monoclonal antibodies to human osteosarcoma, and a human osteosarcoma xenograft (KT005) in nude mice. Three monoclonal antibodies--OST6, OST7, and OST15--raised against human osteosarcoma recognize the same antigen molecule. Although the binding of both /sup 125/I- and /sup 111/In-labeled OST6 to KT005 cells was higher than that of radiolabeled OST7 in vitro, /sup 125/I-labeled OST6 showed a faster clearance from the circulation and a lower accumulation in the transplanted tumor than /sup 125/I-labeled OST7. In contrast to the radioiodinated antibodies, the in vivo tumor accumulation of /sup 111/In-labeled OST6 was higher, although not significantly, than that of /sup 111/In-labeled OST7. OST15 showed the lowest binding in vitro, and its in vivo tumor localization was also lower than the others. The discrepancy in tumor uptake between OST6 and OST7 labeled with either /sup 125/I or /sup 111/In may have been a result of differing blood clearance. These results suggest that binding studies can be used to exclude from in vivo use those antibodies which show very poor binding in vitro, while in vivo serum clearance may be a better test for choosing antibodies with similar binding.

  18. Eucommia ulmoides Oliver Extract, Aucubin, and Geniposide Enhance Lysosomal Activity to Regulate ER Stress and Hepatic Lipid Accumulation

    PubMed Central

    Lee, Hwa-Young; Lee, Geum-Hwa; Lee, Mi-Rin; Kim, Hye-Kyung; Kim, Nan-young; Kim, Seung-Hyun; Lee, Yong-Chul; Kim, Hyung-Ryong; Chae, Han-Jung

    2013-01-01

    Eucommia ulmoides Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of Eucommia ulmoides Oliver extracts (EUE) on hepatic dyslipidemia and its related mechanisms by in vitro and in vivo studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitate-induced endoplasmic reticulum (ER) stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia. PMID:24349058

  19. Neu-164 and Neu-107, two novel antioxidant and anti-myeloperoxidase compounds, inhibit acute cigarette smoke-induced lung inflammation.

    PubMed

    Thatcher, Thomas H; Hsiao, Hsi-Min; Pinner, Elhanan; Laudon, Moshe; Pollock, Stephen J; Sime, Patricia J; Phipps, Richard P

    2013-07-15

    Cigarette smoke is a profound proinflammatory stimulus that causes acute lung inflammation and chronic lung disease, including chronic obstructive pulmonary disease (COPD, emphysema, and chronic bronchitis), via a variety of mechanisms, including oxidative stress. Cigarette smoke contains high levels of free radicals, whereas inflammatory cells, including macrophages and neutrophils, express enzymes, including NADPH oxidase, nitric oxide synthase, and myeloperoxidase, that generate reactive oxygen species in situ and contribute to inflammation and tissue damage. Neu-164 and Neu-107 are small-molecule inhibitors of myeloperoxidase, as well as potent antioxidants. We hypothesized that Neu-164 and Neu-107 would inhibit acute cigarette smoke-induced inflammation. Adult C57BL/6J mice were exposed to mainstream cigarette smoke for 3 days to induce acute inflammation and were treated daily by inhalation with Neu-164, Neu-107, or dexamethasone as a control. Inflammatory cells and cytokines were assessed by bronchoalveolar lavage and histology. mRNA levels of endogenous antioxidant genes heme oxygenase-1 and glutamate-cysteine ligase modifier subunit were determined by qPCR. Cigarette smoke exposure induced acute lung inflammation with accumulation of neutrophils and upregulation of proinflammatory cytokines, including IL-6, macrophage inflammatory protein-2, and keratinocyte-derived cytokine. Both Neu-164 and Neu-107 significantly reduced the accumulation of inflammatory cells and the expression of inflammatory cytokines as effectively as dexamethasone. Upregulation of endogenous antioxidant genes was dampened. Neu-164 and Neu-107 inhibit acute cigarette smoke-induced inflammation by scavenging reactive oxygen species in cigarette smoke and by inhibiting further oxidative stress caused by inflammatory cells. These compounds may have promise in preventing or treating lung disease associated with chronic smoke exposure, including COPD.

  20. CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner

    SciTech Connect

    Dosemeci, Ayse; Thein, Soe; Yang, Yijung; Reese, Thomas S.; Tao-Cheng, Jung-Hwa

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer CYLD is a deubiquitinase specific for lysine63-linked polyubiquitins. Black-Right-Pointing-Pointer Presence of CYLD in PSDs is established by biochemistry and immunoEM. Black-Right-Pointing-Pointer CYLD accumulates on PSDs upon depolarization of neurons. Black-Right-Pointing-Pointer Accumulation of CYLD at PSDs may regulate trafficking/degradation of synaptic proteins. -- Abstract: Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

  1. Galactinol synthase enzyme activity influences raffinose family oligosaccharides (RFO) accumulation in developing chickpea (Cicer arietinum L.) seeds.

    PubMed

    Gangola, Manu P; Jaiswal, Sarita; Kannan, Udhaya; Gaur, Pooran M; Båga, Monica; Chibbar, Ravindra N

    2016-05-01

    To understand raffinose family oligosaccharides (RFO) metabolism in chickpea (Cicer arietinum L.) seeds, RFO accumulation and corresponding biosynthetic enzymes activities were determined during seed development of chickpea genotypes with contrasting RFO concentrations. RFO concentration in mature seeds was found as a facilitator rather than a regulating step of seed germination. In mature seeds, raffinose concentrations ranged from 0.38 to 0.68 and 0.75 to 0.99 g/100 g, whereas stachyose concentrations varied from 0.79 to 1.26 and 1.70 to 1.87 g/100 g indicating significant differences between low and high RFO genotypes, respectively. Chickpea genotypes with high RFO concentration accumulated higher concentrations of myo-inositol and sucrose during early seed developmental stages suggesting that initial substrate concentrations may influence RFO concentration in mature seeds. High RFO genotypes showed about two to three-fold higher activity for all RFO biosynthetic enzymes compared to those with low RFO concentrations. RFO biosynthetic enzymes activities correspond with accumulation of individual RFO during seed development.

  2. Galactinol synthase enzyme activity influences raffinose family oligosaccharides (RFO) accumulation in developing chickpea (Cicer arietinum L.) seeds.

    PubMed

    Gangola, Manu P; Jaiswal, Sarita; Kannan, Udhaya; Gaur, Pooran M; Båga, Monica; Chibbar, Ravindra N

    2016-05-01

    To understand raffinose family oligosaccharides (RFO) metabolism in chickpea (Cicer arietinum L.) seeds, RFO accumulation and corresponding biosynthetic enzymes activities were determined during seed development of chickpea genotypes with contrasting RFO concentrations. RFO concentration in mature seeds was found as a facilitator rather than a regulating step of seed germination. In mature seeds, raffinose concentrations ranged from 0.38 to 0.68 and 0.75 to 0.99 g/100 g, whereas stachyose concentrations varied from 0.79 to 1.26 and 1.70 to 1.87 g/100 g indicating significant differences between low and high RFO genotypes, respectively. Chickpea genotypes with high RFO concentration accumulated higher concentrations of myo-inositol and sucrose during early seed developmental stages suggesting that initial substrate concentrations may influence RFO concentration in mature seeds. High RFO genotypes showed about two to three-fold higher activity for all RFO biosynthetic enzymes compared to those with low RFO concentrations. RFO biosynthetic enzymes activities correspond with accumulation of individual RFO during seed development. PMID:26953100

  3. Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase.

    PubMed

    Victor, Victor M; Rovira-Llopis, Susana; Bañuls, Celia; Diaz-Morales, Noelia; Martinez de Marañon, Arantxa; Rios-Navarro, Cesar; Alvarez, Angeles; Gomez, Marcelino; Rocha, Milagros; Hernández-Mijares, Antonio

    2016-01-01

    Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.

  4. Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

    PubMed Central

    Victor, Victor M.; Rovira-Llopis, Susana; Bañuls, Celia; Diaz-Morales, Noelia; Martinez de Marañon, Arantxa; Rios-Navarro, Cesar; Alvarez, Angeles; Gomez, Marcelino; Rocha, Milagros; Hernández-Mijares, Antonio

    2016-01-01

    Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR. PMID:27007571

  5. Accumulation of wildtype and ALS-linked mutated VAPB impairs activity of the proteasome.

    PubMed

    Moumen, Anice; Virard, Isabelle; Raoul, Cédric

    2011-01-01

    Cellular homeostasis relies on a tight control of protein synthesis, folding and degradation, in which the endoplasmic reticulum (ER) quality control and the ubiquitin proteasome system (UPS) have an instrumental function. ER stress and aberrant accumulation of misfolded proteins represent a pathological signature of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder caused by the selective degeneration of motoneurons in the brain and spinal cord. Mutations in the ER-resident protein VAPB have been associated with familial forms of the disease. ALS-linked mutations cause VAPB to form cytoplasmic aggregates. We previously demonstrated that viral-mediated expression of both wildtype and mutant human VAPB (hVAPB) leads to an ER stress response that contributes to the selective death of motoneurons. However, the mechanisms behind ER stress, defective UPS and hVAPB-associated motoneuron degeneration remain elusive. Here, we show that the overexpression of wildtype and mutated hVAPB, which is found to be less stable than the wildtype protein, leads to the abnormal accumulation of ubiquitin and ubiquitin-like protein conjugates in non-human primate cells. We observed that overexpression of both forms of hVAPB elicited an ER stress response. Treatment of wildtype and mutated hVAPB expressing cells with the ER stress inhibitor salubrinal diminished the burden of ubiquitinated proteins, suggesting that ER stress contributes to the impairment of proteasome function. We also found that both wildtype and mutated hVAPB can associate with the 20S proteasome, which was found to accumulate at the ER with wildtype hVAPB or in mutant hVAPB aggregates. Our results suggest that ER stress and corruption of the proteasome function might contribute to the aberrant protein homeostasis associated with hVAPB.

  6. Sulforaphane, an activator of Nrf2, suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes.

    PubMed

    Shinkai, Yasuhiro; Sumi, Daigo; Fukami, Ikuo; Ishii, Tetsuro; Kumagai, Yoshito

    2006-03-20

    Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role in metabolism and excretion of xenobiotics. Exposure of primary mouse hepatocytes to SFN resulted in activation of Nrf2 and significant elevation of protein expressions responsible for excretion of arsenic into extracellular space. Pretreatment with SFN 24 h prior to arsenite exposure reduced not only arsenic accumulation in the cells but also cellular toxicity of this metalloid. Therefore, our findings indicate a potential function of SFN in reducing cellular arsenic levels, thereby diminishing arsenic toxicity. PMID:16516206

  7. Sulforaphane, an activator of Nrf2, suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes.

    PubMed

    Shinkai, Yasuhiro; Sumi, Daigo; Fukami, Ikuo; Ishii, Tetsuro; Kumagai, Yoshito

    2006-03-20

    Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role in metabolism and excretion of xenobiotics. Exposure of primary mouse hepatocytes to SFN resulted in activation of Nrf2 and significant elevation of protein expressions responsible for excretion of arsenic into extracellular space. Pretreatment with SFN 24 h prior to arsenite exposure reduced not only arsenic accumulation in the cells but also cellular toxicity of this metalloid. Therefore, our findings indicate a potential function of SFN in reducing cellular arsenic levels, thereby diminishing arsenic toxicity.

  8. Accumulation of biologically active furanocoumarins in Ruta graveolens ssp. divaricata (Tenore) Gams in vitro culture.

    PubMed

    Ekiert, H; Abou-Mandour, A A; Czygan, F Ch

    2005-01-01

    The study was designed to investigate dynamics of accumulation of five linear furanocoumarins and umbelliferone in stationary liquid cultures of Ruta graveolens ssp. divaricata (Tenore) Gams during 6-week growth cycles. The contents of individual metabolites in biomass increased 1.8-3.5 times while their total content rose 2.3 times. Maximum contents of xanthotoxin, bergapten and isopimpinellin (112.3, 76.2 and 84.0mg/100g d.w., respectively) and maximum total content of all metabolites (283.4 mg/100 g d.w.), obtained on 35th culture day, are interesting from practical point of view.

  9. Present-day Block Motions and Strain Accumulation on Active Faults in the Caribbean

    NASA Astrophysics Data System (ADS)

    Symithe, S. J.; Calais, E.; Freed, A. M.

    2014-12-01

    The quasi-frontal subduction of the north and south American plates under the Lesser Antilles and the left and right lateral strike-slip along the northern and southern margins of the Caribbean plate offer the opportunity to study the transition from subduction to strike-slip between major plates. In addition, the segmentation and degree of interplate coupling at the Lesser Antilles subduction is key to our understanding of the earthquake potential of a subduction whose length is similar to the rupture area of the Mw9.0, 2011, Tohoku earthquake in Japan. We used the block modeling approach described in Meade and Loveless (2009) to test the optimal block geometry for the northern, eastern and southern boundaries of the Caribbean plate. We solved for angular velocities for each block/plate and strain accumulation rates for all major faults in the region. Then we calculated the variations in interplate coupling along the subduction plate boundaries using the accumulated strain rates. We tested 11 different block geometries; they are all based on geological evidences unless they are suggested by discrepancies within the GPS and seismological data or by previously published results. We confirm the existence of the micro Gonave plate. The boundary between the Micro-Gonave plate and the Hispaniola crustal block is better suited along the Haitian-Thrust-Belt instead of the Neiba-Matheux fault. The interseismic GPS velocities do not show evidence for a distinct North Lesser Antilles block. We found a totally uncoupled section of the subduction starting from the Puerto-Rico trench to the end of the Lesser Antilles section. All the relative motion of the Caribbean block is lost aseismically along the boundary of that portion of the subduction. While we found strong coupling along the northern Hispaniola section, most of the deformation on this region is being accumulated along intrablock faults with very low strain (~2mm/yr) along the intraplate subduction interface. We also

  10. Active accumulation of gold nanorods in tumor in response to near-infrared laser irradiation.

    PubMed

    Shiotani, Atsushi; Akiyama, Yasuyuki; Kawano, Takahito; Niidome, Yasuro; Mori, Takeshi; Katayama, Yoshiki; Niidome, Takuro

    2010-11-17

    Gold nanorods, rod-shaped gold nanoparticles, have strong absorbance in the near-infrared region, and the absorbed light energy can be converted to heat, the so-called photothermal effect. The gold nanorods were coated with thermoresponsive polymers, which have different phase transition temperatures that were controlled by adding comonomers, N,N-dimethylacrylamide (DMAA) or acrylamide (AAm) to N-isopropylacrylamide (NIPAM). The phase transition temperatures of poly(NIPAM-DMAA) and poly(NIPAM-AAm)-coated gold nanorods were 38 and 41 °C, respectively, while polyNIPAM-coated gold nanorods showed phase transition at 34 °C. Irradiation of the coated gold nanorods using the near-infrared laser induced a decrease in their sizes due to a phase transition of the polymer layers. Poly(NIPAM-AAm)-coated gold nanorods stably circulated in the blood flow without a phase transition after intravenous injection. Irradiation of near-infrared light at a tumor after the injection resulted in the gold specifically accumulating in the tumor. This novel accumulation technique which combines a thermoresponsive polymer and the photothermal effect of the gold nanorods should be a powerful tool for targeted delivery in response to light irradiation.

  11. Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts.

    PubMed

    Nitta, Yoko; Yasukata, Fumiko; Kitamoto, Noritoshi; Ito, Mikiko; Sakaue, Motoyoshi; Kikuzaki, Hiroe; Ueno, Hiroshi

    2016-03-01

    Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 μM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

  12. A Fuzzy Logic Prompting Mechanism Based on Pattern Recognition and Accumulated Activity Effective Index Using a Smartphone Embedded Sensor.

    PubMed

    Liu, Chung-Tse; Chan, Chia-Tai

    2016-01-01

    Sufficient physical activity can reduce many adverse conditions and contribute to a healthy life. Nevertheless, inactivity is prevalent on an international scale. Improving physical activity is an essential concern for public health. Reminders that help people change their health behaviors are widely applied in health care services. However, timed-based reminders deliver periodic prompts suffer from flexibility and dependency issues which may decrease prompt effectiveness. We propose a fuzzy logic prompting mechanism, Accumulated Activity Effective Index Reminder (AAEIReminder), based on pattern recognition and activity effective analysis to manage physical activity. AAEIReminder recognizes activity levels using a smartphone-embedded sensor for pattern recognition and analyzing the amount of physical activity in activity effective analysis. AAEIReminder can infer activity situations such as the amount of physical activity and days spent exercising through fuzzy logic, and decides whether a prompt should be delivered to a user. This prompting system was implemented in smartphones and was used in a short-term real-world trial by seventeenth participants for validation. The results demonstrated that the AAEIReminder is feasible. The fuzzy logic prompting mechanism can deliver prompts automatically based on pattern recognition and activity effective analysis. AAEIReminder provides flexibility which may increase the prompts' efficiency. PMID:27548184

  13. A Fuzzy Logic Prompting Mechanism Based on Pattern Recognition and Accumulated Activity Effective Index Using a Smartphone Embedded Sensor.

    PubMed

    Liu, Chung-Tse; Chan, Chia-Tai

    2016-08-19

    Sufficient physical activity can reduce many adverse conditions and contribute to a healthy life. Nevertheless, inactivity is prevalent on an international scale. Improving physical activity is an essential concern for public health. Reminders that help people change their health behaviors are widely applied in health care services. However, timed-based reminders deliver periodic prompts suffer from flexibility and dependency issues which may decrease prompt effectiveness. We propose a fuzzy logic prompting mechanism, Accumulated Activity Effective Index Reminder (AAEIReminder), based on pattern recognition and activity effective analysis to manage physical activity. AAEIReminder recognizes activity levels using a smartphone-embedded sensor for pattern recognition and analyzing the amount of physical activity in activity effective analysis. AAEIReminder can infer activity situations such as the amount of physical activity and days spent exercising through fuzzy logic, and decides whether a prompt should be delivered to a user. This prompting system was implemented in smartphones and was used in a short-term real-world trial by seventeenth participants for validation. The results demonstrated that the AAEIReminder is feasible. The fuzzy logic prompting mechanism can deliver prompts automatically based on pattern recognition and activity effective analysis. AAEIReminder provides flexibility which may increase the prompts' efficiency.

  14. A Fuzzy Logic Prompting Mechanism Based on Pattern Recognition and Accumulated Activity Effective Index Using a Smartphone Embedded Sensor

    PubMed Central

    Liu, Chung-Tse; Chan, Chia-Tai

    2016-01-01

    Sufficient physical activity can reduce many adverse conditions and contribute to a healthy life. Nevertheless, inactivity is prevalent on an international scale. Improving physical activity is an essential concern for public health. Reminders that help people change their health behaviors are widely applied in health care services. However, timed-based reminders deliver periodic prompts suffer from flexibility and dependency issues which may decrease prompt effectiveness. We propose a fuzzy logic prompting mechanism, Accumulated Activity Effective Index Reminder (AAEIReminder), based on pattern recognition and activity effective analysis to manage physical activity. AAEIReminder recognizes activity levels using a smartphone-embedded sensor for pattern recognition and analyzing the amount of physical activity in activity effective analysis. AAEIReminder can infer activity situations such as the amount of physical activity and days spent exercising through fuzzy logic, and decides whether a prompt should be delivered to a user. This prompting system was implemented in smartphones and was used in a short-term real-world trial by seventeenth participants for validation. The results demonstrated that the AAEIReminder is feasible. The fuzzy logic prompting mechanism can deliver prompts automatically based on pattern recognition and activity effective analysis. AAEIReminder provides flexibility which may increase the prompts’ efficiency. PMID:27548184

  15. Down-regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii.

    PubMed

    Michelet, Laure; Roach, Thomas; Fischer, Beat B; Bedhomme, Mariette; Lemaire, Stéphane D; Krieger-Liszkay, Anja

    2013-06-01

    In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H2 O2 in the 10 μm range. This concentration range seems to be necessary to activate H2 O2 -dependent signalling pathways stimulating the expression of H2 O2 responsive genes, such as the heat shock protein HSP22C. Catalase knock-down mutants had lost the transient accumulation of H2 O2 , suggesting that a decrease in catalase activity was the key element for establishing a transient H2 O2 burst. Catalase was inactivated by a one-electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H2 O2 , inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast-directed protection enzymes.

  16. EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA.

    PubMed

    GREENAWALT, J W; ROSSI, C S; LEHNINGER, A L

    1964-10-01

    Rat liver mitochondria allowed to accumulate maximal amounts of Ca(++) and HPO(4) (=) ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca(++) and HPO(4) (=) from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca(++)-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca(++)-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca(++)-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca(++) and phosphate from the mitochondria into the medium.

  17. Reactive Oxygen Species-Induced TXNIP Drives Fructose-Mediated Hepatic Inflammation and Lipid Accumulation Through NLRP3 Inflammasome Activation

    PubMed Central

    Zhang, Xian; Zhang, Jian-Hua; Chen, Xu-Yang; Hu, Qing-Hua; Wang, Ming-Xing; Jin, Rui; Zhang, Qing-Yu; Wang, Wei; Wang, Rong; Kang, Lin-Lin; Li, Jin-Sheng; Li, Meng

    2015-01-01

    Abstract Aims: Increased fructose consumption predisposes the liver to nonalcoholic fatty liver disease (NAFLD), but the mechanisms are elusive. Thioredoxin-interacting protein (TXNIP) links oxidative stress to NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and this signaling axis may be involved in fructose-induced NAFLD. Here, we explore the role of reactive oxygen species (ROS)-induced TXNIP overexpression in fructose-mediated hepatic NLRP3 inflammasome activation, inflammation, and lipid accumulation. Results: Rats were fed a 10% fructose diet for 8 weeks and treated with allopurinol and quercetin during the last 4 weeks. Five millimolars of fructose-exposed hepatocytes (primary rat hepatocytes, rat hepatic parenchymal cells [RHPCs], HLO2, HepG2) were co-incubated with antioxidants or caspase-1 inhibitor or subjected to TXNIP or NLRP3 siRNA interference. Fructose induced NLRP3 inflammasome activation and pro-inflammatory cytokine secretion, janus-activated kinase 2/signal transducers and activators of transcription 3-mediated inflammatory signaling, and expression alteration of lipid metabolism-related genes in cultured hepatocytes and rat livers. NLRP3 silencing and caspase-1 suppression blocked these effects in primary rat hepatocytes and RHPCs, confirming that inflammasome activation alters hepatocyte lipid metabolism. Hepatocellular ROS and TXNIP were increased in animal and cell models. TXNIP silencing blocked NLRP3 inflammasome activation, inflammation, and lipid metabolism perturbations but not ROS induction in fructose-exposed hepatocytes, whereas antioxidants addition abrogated TXNIP induction and diminished the detrimental effects in fructose-exposed hepatocytes and rat livers. Innovation and Conclusions: This study provides a novel mechanism for fructose-induced NAFLD pathogenesis by which the ROS-TXNIP pathway mediates hepatocellular NLRP3 inflammasome activation, inflammation and lipid accumulation. Antioxidant

  18. Accumulate-Repeat-Accumulate-Accumulate-Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Sam; Thorpe, Jeremy

    2004-01-01

    Inspired by recently proposed Accumulate-Repeat-Accumulate (ARA) codes [15], in this paper we propose a channel coding scheme called Accumulate-Repeat-Accumulate-Accumulate (ARAA) codes. These codes can be seen as serial turbo-like codes or as a subclass of Low Density Parity Check (LDPC) codes, and they have a projected graph or protograph representation; this allows for a high-speed iterative decoder implementation using belief propagation. An ARAA code can be viewed as a precoded Repeat-and-Accumulate (RA) code with puncturing in concatenation with another accumulator, where simply an accumulator is chosen as the precoder; thus ARAA codes have a very fast encoder structure. Using density evolution on their associated protographs, we find examples of rate-lJ2 ARAA codes with maximum variable node degree 4 for which a minimum bit-SNR as low as 0.21 dB from the channel capacity limit can be achieved as the block size goes to infinity. Such a low threshold cannot be achieved by RA or Irregular RA (IRA) or unstructured irregular LDPC codes with the same constraint on the maximum variable node degree. Furthermore by puncturing the accumulators we can construct families of higher rate ARAA codes with thresholds that stay close to their respective channel capacity thresholds uniformly. Iterative decoding simulation results show comparable performance with the best-known LDPC codes but with very low error floor even at moderate block sizes.

  19. ORM Promotes Skeletal Muscle Glycogen Accumulation via CCR5-Activated AMPK Pathway in Mice

    PubMed Central

    Qin, Zhen; Wan, Jing-Jing; Sun, Yang; Wang, Peng-Yuan; Su, Ding-Feng; Lei, Hong; Liu, Xia

    2016-01-01

    We found previously that acute phase protein orosomucoid reacts to fatigue and activates C-C chemokine receptor type 5 to increase muscle glycogen storage and enhance muscle endurance (Lei et al., 2016). To explore the underlying molecular mechanisms, we investigated the role of AMP-activated protein kinase, a critical fuel sensor in skeletal muscle, in C-C chemokine receptor type 5-mediated orosomucoid action. It was found orosomucoid increased skeletal muscle AMP-activated protein kinase activation in a time- and dose- dependent manner, which was largely prevented by pharmacological blocking or knockout of C-C chemokine receptor type 5. Administration of orosomucoid also significantly increased the de-phosphorylation and activity of muscle glycogen synthase, the rate-limiting enzyme for glycogen synthesis. The effect was largely absent in mice deficient in C-C chemokine receptor type 5−/− or AMP-activated protein kinase α2−/−, the predominant isoform in skeletal muscle. Moreover, deletion of AMP-activated protein kinase α2 abolished the effect of orosomucoid on fatigue and muscle glycogen. These findings indicate that orosomucoid may promote glycogen storage and enhance muscle function through C-C chemokine receptor type 5-mdiated activation of AMP-activated protein kinase, which in turn activates glycogen synthase and increases muscle glycogen. PMID:27679573

  20. ORM Promotes Skeletal Muscle Glycogen Accumulation via CCR5-Activated AMPK Pathway in Mice.

    PubMed

    Qin, Zhen; Wan, Jing-Jing; Sun, Yang; Wang, Peng-Yuan; Su, Ding-Feng; Lei, Hong; Liu, Xia

    2016-01-01

    We found previously that acute phase protein orosomucoid reacts to fatigue and activates C-C chemokine receptor type 5 to increase muscle glycogen storage and enhance muscle endurance (Lei et al., 2016). To explore the underlying molecular mechanisms, we investigated the role of AMP-activated protein kinase, a critical fuel sensor in skeletal muscle, in C-C chemokine receptor type 5-mediated orosomucoid action. It was found orosomucoid increased skeletal muscle AMP-activated protein kinase activation in a time- and dose- dependent manner, which was largely prevented by pharmacological blocking or knockout of C-C chemokine receptor type 5. Administration of orosomucoid also significantly increased the de-phosphorylation and activity of muscle glycogen synthase, the rate-limiting enzyme for glycogen synthesis. The effect was largely absent in mice deficient in C-C chemokine receptor type 5(-/-) or AMP-activated protein kinase α2(-/-), the predominant isoform in skeletal muscle. Moreover, deletion of AMP-activated protein kinase α2 abolished the effect of orosomucoid on fatigue and muscle glycogen. These findings indicate that orosomucoid may promote glycogen storage and enhance muscle function through C-C chemokine receptor type 5-mdiated activation of AMP-activated protein kinase, which in turn activates glycogen synthase and increases muscle glycogen. PMID:27679573

  1. ORM Promotes Skeletal Muscle Glycogen Accumulation via CCR5-Activated AMPK Pathway in Mice

    PubMed Central

    Qin, Zhen; Wan, Jing-Jing; Sun, Yang; Wang, Peng-Yuan; Su, Ding-Feng; Lei, Hong; Liu, Xia

    2016-01-01

    We found previously that acute phase protein orosomucoid reacts to fatigue and activates C-C chemokine receptor type 5 to increase muscle glycogen storage and enhance muscle endurance (Lei et al., 2016). To explore the underlying molecular mechanisms, we investigated the role of AMP-activated protein kinase, a critical fuel sensor in skeletal muscle, in C-C chemokine receptor type 5-mediated orosomucoid action. It was found orosomucoid increased skeletal muscle AMP-activated protein kinase activation in a time- and dose- dependent manner, which was largely prevented by pharmacological blocking or knockout of C-C chemokine receptor type 5. Administration of orosomucoid also significantly increased the de-phosphorylation and activity of muscle glycogen synthase, the rate-limiting enzyme for glycogen synthesis. The effect was largely absent in mice deficient in C-C chemokine receptor type 5−/− or AMP-activated protein kinase α2−/−, the predominant isoform in skeletal muscle. Moreover, deletion of AMP-activated protein kinase α2 abolished the effect of orosomucoid on fatigue and muscle glycogen. These findings indicate that orosomucoid may promote glycogen storage and enhance muscle function through C-C chemokine receptor type 5-mdiated activation of AMP-activated protein kinase, which in turn activates glycogen synthase and increases muscle glycogen.

  2. [Pigment accumulation and functional activity of chloroplasts in common Pisum sativum L. mutants with low chlorophyll level (chlorotica)].

    PubMed

    Ladygin, V G

    2003-01-01

    Pea mutants chlorotica 2004 and 2014 with a low content of chlorophyll were studied. The mutant 2004 has light green leaves and stem, and the mutant 2014 has yellow green leaves and stem. They accumulate approximately 80 and 50% chlorophylls of the parent form of pea Torsdag cv. The content of carotene in carotenoids of the mutant 2004 was much lower, and the accumulation of lutein and violaxanthine was increased. The accumulation of all carotenoids in the mutant 2014 decreased almost proportionally to a decrease in the chlorophyll content. The rate of CO2 evolution in mutant chlorotica 2004 and 2014 was established to be lower. The quantum efficiency of photosynthesis in the mutants was 29-30% lower as compared to the control, and in hybrid plants it was 1.5-2-fold higher. It is assumed that the increase in the activity of the night-time respiration in gas exchange of chlorotica mutants and the drop of photosynthesis lead to a decrease in biomass increment. The results obtained allow us to conclude that the mutation of chlorotica 2004 and 2014 affects the genes controlling the formation and functioning of different components of the photosynthetic apparatus.

  3. Effect of TCDD on ACARAT activity and vitamin A accumulation in the kidney of male Sprague-Dawley rats

    SciTech Connect

    Jurek, M.A.; Powers, R.H.; Gilbert, L.C.; Aust, S.D.

    1987-05-01

    Previous studies have shown that rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit symptoms of vitamin A deficiency, including hypophagia, failure of normal growth, loss of hepatic vitamin A and accumulation of vitamin A in the kidney. They observed that male Sprague-Dawley rats treated with a single dose of TCDD gained less weight than control rats over a 12 day period. Treated rats showed a progressive loss of hepatic retinyl esters to levels 55% that of control rats. Treated rats accumulated renal vitamin A, with retinyl palmitate levels reaching 5.2x that of control animals, while retinol levels were elevated to 1.5x that of control rats. The ratio of retinyl palmitate to retinol was significantly greater in treated rats than in the control rats. Acyl CoA:Retinol Acyl Transferase (ACARART) activity was 2x greater in kidneys from treated rats, and positively correlated with retinyl palmitate concentrations. They suggest that accumulation of retinyl esters in the kidney occurs as a result of retinol esterification, in response to the TCDD-induced vitamin A deficiency.

  4. Neutrophil-Mediated Regulation of Innate and Adaptive Immunity: The Role of Myeloperoxidase

    PubMed Central

    Odobasic, Dragana; Kitching, A. Richard; Holdsworth, Stephen R.

    2016-01-01

    Neutrophils are no longer seen as leukocytes with a sole function of being the essential first responders in the removal of pathogens at sites of infection. Being armed with numerous pro- and anti-inflammatory mediators, these phagocytes can also contribute to the development of various autoimmune diseases and can positively or negatively regulate the generation of adaptive immune responses. In this review, we will discuss how myeloperoxidase, the most abundant neutrophil granule protein, plays a key role in the various functions of neutrophils in innate and adaptive immunity. PMID:26904693

  5. A new hexacyclic triterpene acid from the roots of Euscaphis japonica and its inhibitory activity on triglyceride accumulation.

    PubMed

    Li, Yan-Ci; Tian, Ke; Sun, Li-Juan; Long, Hui; Li, Lu-Jun; Wu, Zheng-Zhi

    2016-03-01

    A new taraxerene-type hexacyclic triterpene acid named (12R,13S)-3-methoxy-12,13-cyclo-taraxerene-2,14-diene-1-one-28-oic acid (1), together with a known compound 3,7-dihydroxy-5-octanolide (2), was isolated from the roots of Euscaphis japonica. The structure of new compound 1 was elucidated on the basis of NMR, HR-ESIMS and X-ray diffraction analysis. It showed promising inhibitory activity on oleic acid induced triglyceride accumulation on HepG2 cells. PMID:26828452

  6. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    PubMed Central

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  7. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

  8. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  9. Accumulate-Repeat-Accumulate-Accumulate Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Samuel; Thorpe, Jeremy

    2007-01-01

    Accumulate-repeat-accumulate-accumulate (ARAA) codes have been proposed, inspired by the recently proposed accumulate-repeat-accumulate (ARA) codes. These are error-correcting codes suitable for use in a variety of wireless data-communication systems that include noisy channels. ARAA codes can be regarded as serial turbolike codes or as a subclass of low-density parity-check (LDPC) codes, and, like ARA codes they have projected graph or protograph representations; these characteristics make it possible to design high-speed iterative decoders that utilize belief-propagation algorithms. The objective in proposing ARAA codes as a subclass of ARA codes was to enhance the error-floor performance of ARA codes while maintaining simple encoding structures and low maximum variable node degree.

  10. Design and synthesis of a series of piperazine-1-carboxamidine derivatives with antifungal activity resulting from accumulation of endogenous reactive oxygen species.

    PubMed

    François, Isabelle E; Thevissen, Karin; Pellens, Klaartje; Meert, Els M; Heeres, Jan; Freyne, Eddy; Coesemans, Erwin; Viellevoye, Marcel; Deroose, Frederik; Martinez Gonzalez, Sonia; Pastor, Joaquin; Corens, David; Meerpoel, Lieven; Borgers, Marcel; Ausma, Jannie; Dispersyn, Gerrit D; Cammue, Bruno P

    2009-10-01

    In this study, we screened a library of 500 compounds for fungicidal activity via induction of endogenous reactive oxygen species (ROS) accumulation. Structure-activity relationship studies showed that piperazine-1-carboxamidine analogues with large atoms or large side chains substituted on the phenyl group at the R(3) and R(5) positions are characterized by a high ROS accumulation capacity in Candida albicans and a high fungicidal activity. Moreover, we could link the fungicidal mode of action of the piperazine-1-carboxamidine derivatives to the accumulation of endogenous ROS. PMID:19705386

  11. Activity-dependent accumulation of calcium in Purkinje cell dendritic spines

    SciTech Connect

    Andrews, S.B.; Leapman, R.D.; Landis, D.M.; Reese, T.S.

    1988-03-01

    The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 +/- 0.6 mmol/kg of dry weight +/- SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 +/- 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCl resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.

  12. Activation of accumulated nitrite reduction by immobilized Pseudomonas stutzeri T13 during aerobic denitrification.

    PubMed

    Ma, Fang; Sun, Yilu; Li, Ang; Zhang, Xuening; Yang, Jixian

    2015-01-01

    The excellent removal efficiency of nitrate by the aerobic denitrifier, Pseudomonas stutzeri T13, was achieved in free cells system. However, poor nitrite reduction prevents efficient aerobic denitrification because of the nitrite accumulation. This problem could be conquered by immobilizing the cells on supports. In this study, strain T13 was immobilized by mycelial pellets (MPs), polyurethane foam cubes (PFCs) and sodium alginate beads (SABs). Higher removal percentages of TN in MP (43.78%), PFC (42.31%) and SAB (57.25%) systems were achieved compared with the free cell system (29.7%). Furthermore, the optimal condition for immobilized cell systems was as follows: 30°C, 100rpm shaking speed and pH 7. The shock-resistance of SAB system was relatively poor, which could collapse under either alkaline (pH=9) or high rotating (200rpm) conditions. The recycling experiments demonstrated that the high steady TN removal rate could be maintained for seven cycles in both MP and PFC systems. PMID:25827250

  13. Floc-forming properties of polyphosphate accumulating organisms in activated sludge.

    PubMed

    Larsen, P; Eriksen, P S; Lou, M A; Thomsen, T R; Kong, Y H; Nielsen, J L; Nielsen, P H

    2006-01-01

    The physico-chemical characteristics of polyphosphate-accumulating organisms (PAO) involved in enhanced biological phosphorus removal (EBPR) was investigated in order to find a novel method for phosphorus recovery. If the physico-chemical characteristics of PAO are different from those of other main floc components, it may be possible to enrich PAO in bulk water or in the floc material for improved recovery of phosphorus. A combination of shear tests, chemical manipulation, and quantification of PAO by fluorescence in situ hybridization was applied. The microcolony strength of both Rhodocyclus-related PAO and Actinobacteria-related PAO was generally high as no treatment could break up more than 20% of all PAO in microcolonies. In contrast, it was possible to remove 20-40% of the organic matter and other bacterial cells by applying a high pH value or adding EDTA. With that a selective enrichment of PAO in the remaining floc material was possible. The feasibility of applying this selective PAO enrichment in flocs remains to be evaluated in full-scale plants for P-recovery. PMID:16898159

  14. Adenylyl cyclase activation underlies intracellular cyclic AMP accumulation, cyclic AMP transport, and extracellular adenosine accumulation evoked by beta-adrenergic receptor stimulation in mixed cultures of neurons and astrocytes derived from rat cerebral cortex.

    PubMed

    Rosenberg, P A; Li, Y

    1995-09-18

    We have previously shown that stimulation of cortical cultures containing both neurons and astrocytes with the beta-adrenergic agonist isoproterenol (ISO) results in transport of cAMP from astrocytes followed by extracellular hydrolysis to adenosine [Rosenberg et al. J. Neurosci. 14 (1994) 2953-2965]. In this study we found that the endogenous catecholamines epinephrine (EPI) and norepinephrine (NE), but not dopamine, serotonin, or histamine, all at 10 microM, significantly stimulated intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation in cortical cultures. Detailed dose-response experiments were performed for NE and EPI, as well as ISO. For each catecholamine, the potencies in evoking intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation were similar. These data provide additional evidence that a single common mechanism, namely beta-adrenergic mediated activation of adenylyl cyclase, underlies intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation. It appears that regulation of extracellular adenosine levels via cAMP transport and extracellular hydrolysis to adenosine may be a final common pathway of neuromodulation in cerebral cortex for catecholamines, and, indeed, any substance whose receptors are coupled to adenylyl cyclase.

  15. Influence of myeloperoxidase on colon tumor occurrence in inflamed versus non-inflamed colons of ApcMin/+ mice☆

    PubMed Central

    Al-Salihi, Mazin; Reichert, Ethan; Fitzpatrick, F.A.

    2015-01-01

    Control of colorectal cancer needs to be tailored to its etiology. Tumor promotion mechanisms in colitis-associated colon cancer differ somewhat from the mechanisms involved in hereditary and sporadic colorectal cancer. Unlike sporadic or inherited tumors, some experimental models show that colitis-associated colon tumors do not require cyclooxygenase (COX) expression for progression, and non-steroidal anti-inflammatory drugs (NSAIDs) which prevent sporadic or inherited colon cancer do not prevent colitis-associated colon cancer. We report that myeloperoxidase (MPO), an ancestor of the COX isoenzymes, is a determinant of colitis-associated colon tumors in ApcMin/+ mice. During experimentally induced colitis, inhibition of MPO by resorcinol dampened colon tumor development. Conversely, in the bowels of ApcMin/+ mice without colitis, resorcinol administration or ‘knockout’ of MPO gene coincided with a slight, but discernible increase in colon tumor incidence. Acrolein, a by-product of MPO catalysis, formed a covalent adduct with the phosphatase tensin homolog (PTEN) tumor suppressor and enhanced the activity of the Akt kinase proto-oncogene in vitro and in vivo. Thus, MPO may be an important determinant of diet and inflammation on colon cancer risk via its effect on endogenous exposure to oxidants and acrolein. We propose a hypothetical model to explain an apparent dichotomy between colon tumor occurrence and MPO inhibition in inflamed versus non-inflamed colons. PMID:26262998

  16. An extraovarian protein accumulated in mosquito oocytes is a carboxypeptidase activated in embryos

    SciTech Connect

    Wenlong Cho; Deitsch, K.W.; Raikhel, A.S. )

    1991-12-01

    The authors report a phenomenon previously unknown for oviparous animals; in Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized extraovarially and then internalized by oocytes. The cDNA encoding mosquito vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP cDNA hybridizes to a 1.5-kilobase mRNA present only in the fat body of vitellogenic females. The deduced amino acid sequence of VCP shares significant homology with members of the serine carboxypeptidase family. Binding assays using a serine protease inhibitor, ({sup 3}H)diisopropyl fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic development. Activation of VCP is associated with the reduction in its size from 53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48-kDa, form of VCP is maximally present at the middle of embryonic development and disappears by the end.

  17. Development of tuberous roots and sugar accumulation as related to invertase activity and mineral nutrition.

    PubMed

    Ricardo, C P; Sovia, D

    1974-03-01

    Sucrose storage in tuberous roots was not observed when the tissues had very high activities of acid invertase. High activities of the enzyme were always present in the roots at early stages of their development. In species where the activity of the enzyme decreased during root development, sucrose was stored. Thus, acid invertase was undetectable in mature roots of carrots (Daucus carota L.) where sucrose formed almost 80% of the dry matter. Conversely, radish (Raphanus sativus L.) and turnip (Brassica rapa L.) roots, in which the activity of the enzyme remained high until maturity, did not store appreciable amounts of sucrose (2% and 9%, respectively, of the dry matter in the mature roots), reducing sugars being the main reserve (more than 80% of the dry matter in mature turnips). The correlation between sucrose content and acid invertase activity was furthermore evident in both sucrose- and hexose-storing roots when the activity of this enzyme was affected by changes in the mineral nutrition. Deficiencies of nitrogen and sulphur reduced the activity of acid and alkaline invertases and led to increase in sucrose content and decrease in reducing sugars. However, the decline of alkaline invertase activity in tissues low in acid invertase had no clear effect on sugar content. Sodium chloride (10(-1)M) affected acid invertase and sugars in a manner similar to that of the two deficiencies, but had practically no effect on alkaline invertase. The changes in sugar content produced by the variations in mineral nutrition were small in hexose-storing roots in relation to those of sucrose-storing roots. It is possible that this result is related to the different levels of acid invertase in the two types of roots.

  18. Active but inoperable thrombin is accumulated in a plasma protein layer surrounding Streptococcus pyogenes.

    PubMed

    Naudin, Clément; Hurley, Sinead M; Malmström, Erik; Plug, Tom; Shannon, Oonagh; Meijers, Joost C M; Mörgelin, Matthias; Björck, Lars; Herwald, Heiko

    2015-10-01

    Activation of thrombin is a critical determinant in many physiological and pathological processes including haemostasis and inflammation. Under physiological conditions many of these functions are involved in wound healing or eradication of an invading pathogen. However, when activated systemically, thrombin can contribute to severe and life-threatening conditions by causing complications such as multiple multi-organ failure and disseminated intravascular coagulation. In the present study we investigated how the activity of thrombin is modulated when it is bound to the surface of Streptococcus pyogenes. Our data show that S. pyogenes bacteria become covered with a proteinaceous layer when incubated with human plasma, and that thrombin is a constituent of this layer. Though the coagulation factor is found attached to the bacteria with a functional active site, thrombin has lost its capacity to interact with its natural substrates and inhibitors. Thus, the interaction of bacteria with human plasma renders thrombin completely inoperable at the streptococcal surface. This could represent a host defense mechanism to avoid systemic activation of coagulation which could be otherwise induced when bacteria enter the circulation and cause systemic infection.

  19. [Combined effects of copper and simulated acid rain on copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa].

    PubMed

    He, Shan-Ying; Gao, Yong-Jie; Shentu, Jia-Li; Chen, Kun-Bai

    2011-02-01

    A pot experiment was conducted to study the combined effects of Cu (0-1500 mg x kg(-1)) and simulated acid rain (pH 2.5-5.6) on the copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa. With the increasing concentration of soil Cu, the Cu accumulation in R. acetosa increased, being higher in root than in stem and leaf. The exposure to low pH acid rain promoted the Cu uptake by R. acetosa. With the increase of soil Cu concentration and/or of acid rain acidity, the biomass of R. acetosa decreased, leaf and root MDA contents increased and had good correlation with soil Cu concentration, and the SOD and POD activities in leaf and root displayed a decreasing trend after an initial increase. This study showed that R. acetosa had a strong adaptive ability to Cu and acid rain stress, exhibiting a high application potential in the remediation of Cu-contaminated soil in acid rain areas.

  20. [Combined effects of copper and simulated acid rain on copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa].

    PubMed

    He, Shan-Ying; Gao, Yong-Jie; Shentu, Jia-Li; Chen, Kun-Bai

    2011-02-01

    A pot experiment was conducted to study the combined effects of Cu (0-1500 mg x kg(-1)) and simulated acid rain (pH 2.5-5.6) on the copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa. With the increasing concentration of soil Cu, the Cu accumulation in R. acetosa increased, being higher in root than in stem and leaf. The exposure to low pH acid rain promoted the Cu uptake by R. acetosa. With the increase of soil Cu concentration and/or of acid rain acidity, the biomass of R. acetosa decreased, leaf and root MDA contents increased and had good correlation with soil Cu concentration, and the SOD and POD activities in leaf and root displayed a decreasing trend after an initial increase. This study showed that R. acetosa had a strong adaptive ability to Cu and acid rain stress, exhibiting a high application potential in the remediation of Cu-contaminated soil in acid rain areas. PMID:21608265

  1. A practical method for quantification of phosphorus- and glycogen-accumulating organism populations in activated sludge systems.

    PubMed

    López-Vázquez, Carlos M; Hooijmans, Christine M; Brdjanovic, Damir; Gijzen, Huub J; van Loosdrecht, Mark C M

    2007-12-01

    Enhanced biological phosphorus removal (EBPR) from wastewater relies on the enrichment of activated sludge with phosphorus-accumulating organisms (PAOs). The presence and proliferation of glycogen-accumulating organisms (GAOs), which compete for substrate with PAOs, may be detrimental for EBPR systems, leading to deterioration and, in extreme cases, failure of the process. Therefore, from both process evaluation and modeling perspectives, the estimation of PAO and GAO populations in activated sludge systems is a relevant issue. A simple method for the quantification of PAO and GAO population fractions in activated sludge systems is presented in this paper. To develop such a method, the activity observed in anaerobic batch tests executed with different PAO/GAO ratios, by mixing highly enriched PAO and GAO cultures, was studied. Strong correlations between PAO/GAO population ratios and biomass activity were observed (R2 > 0.97). This served as a basis for the proposal of a simple and practical method to quantify the PAO and GAO populations in activated sludge systems, based on commonly measured and reliable analytical parameters (i.e., mixed liquor suspended solids, acetate, and orthophosphate) without requiring molecular techniques. This method relies on the estimation of the total active biomass population under anaerobic conditions (PAO plus GAO populations), by measuring the maximum acetate uptake rate in the presence of excess acetate. Later, the PAO and GAO populations present in the activated sludge system can be estimated, by taking into account the PAO/GAO ratio calculated on the basis of the anaerobic phosphorus release-to-acetate consumed ratio. The proposed method was evaluated using activated sludge from municipal wastewater treatment plants. The results from the quantification performed following the proposed method were compared with direct population estimations carried out with fluorescence in situ hybridization analysis (determining Candidatus

  2. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus

    PubMed Central

    Wang, Xi; Pan, Ya-Jie; Chang, Bo-Wen; Hu, Yan-Bo; Guo, Xiao-Rui; Tang, Zhong-Hua

    2016-01-01

    We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. PMID:27314017

  3. Effects of Age and Experience on Physical Activity Accumulation during Kin-Ball

    ERIC Educational Resources Information Center

    Hastie, Peter A.; Langevin, Francois; Wadsworth, Danielle

    2011-01-01

    With a specific agenda of creating a fun activity that emphasized teamwork, cooperation, and sportsmanship, Mario Demers, a Canadian physical education professor, created Kin-Ball in the mid 1980s. The game involves three teams of four players each in which a large ball (4 feet diameter and 2.2 pounds weight (1.22 m and 1 kg, respectively) is sent…

  4. Protection by Nitric Oxide Donors of Isolated Rat Hearts Is Associated with Activation of Redox Metabolism and Ferritin Accumulation

    PubMed Central

    Grievink, Hilbert; Zeltcer, Galina; Drenger, Benjamin; Berenshtein, Eduard; Chevion, Mordechai

    2016-01-01

    Preconditioning (PC) procedures (ischemic or pharmacological) are powerful procedures used for attaining protection against prolonged ischemia and reperfusion (I/R) injury, in a variety of organs, including the heart. The detailed molecular mechanisms underlying the protection by PC are however, complex and only partially understood. Recently, an ‘iron-based mechanism’ (IBM), that includes de novo ferritin synthesis and accumulation, was proposed to explain the specific steps in cardioprotection generated by IPC. The current study investigated whether nitric oxide (NO), generated by exogenous NO-donors, could play a role in the observed IBM of cardioprotection by IPC. Therefore, three distinct NO-donors were investigated at different concentrations (1–10 μM): sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). Isolated rat hearts were retrogradely perfused using the Langendorff configuration and subjected to prolonged ischemia and reperfusion with or without pretreatment by NO-donors. Hemodynamic parameters, infarct sizes and proteins of the methionine-centered redox cycle (MCRC) were analyzed, as well as cytosolic aconitase (CA) activity and ferritin protein levels. All NO-donors had significant effects on proteins involved in the MCRC system. Nonetheless, pretreatment with 10 μM SNAP was found to evoke the strongest effects on Msr activity, thioredoxin and thioredoxin reductase protein levels. These effects were accompanied with a significant reduction in infarct size, increased CA activity, and ferritin accumulation. Conversely, pretreatment with 2 μM SIN-1 increased infarct size and was associated with slightly lower ferritin protein levels. In conclusion, the abovementioned findings indicate that NO, depending on its bio-active redox form, can regulate iron metabolism and plays a role in the IBM of cardioprotection against reperfusion injury. PMID:27447933

  5. Protection by Nitric Oxide Donors of Isolated Rat Hearts Is Associated with Activation of Redox Metabolism and Ferritin Accumulation.

    PubMed

    Grievink, Hilbert; Zeltcer, Galina; Drenger, Benjamin; Berenshtein, Eduard; Chevion, Mordechai

    2016-01-01

    Preconditioning (PC) procedures (ischemic or pharmacological) are powerful procedures used for attaining protection against prolonged ischemia and reperfusion (I/R) injury, in a variety of organs, including the heart. The detailed molecular mechanisms underlying the protection by PC are however, complex and only partially understood. Recently, an 'iron-based mechanism' (IBM), that includes de novo ferritin synthesis and accumulation, was proposed to explain the specific steps in cardioprotection generated by IPC. The current study investigated whether nitric oxide (NO), generated by exogenous NO-donors, could play a role in the observed IBM of cardioprotection by IPC. Therefore, three distinct NO-donors were investigated at different concentrations (1-10 μM): sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). Isolated rat hearts were retrogradely perfused using the Langendorff configuration and subjected to prolonged ischemia and reperfusion with or without pretreatment by NO-donors. Hemodynamic parameters, infarct sizes and proteins of the methionine-centered redox cycle (MCRC) were analyzed, as well as cytosolic aconitase (CA) activity and ferritin protein levels. All NO-donors had significant effects on proteins involved in the MCRC system. Nonetheless, pretreatment with 10 μM SNAP was found to evoke the strongest effects on Msr activity, thioredoxin and thioredoxin reductase protein levels. These effects were accompanied with a significant reduction in infarct size, increased CA activity, and ferritin accumulation. Conversely, pretreatment with 2 μM SIN-1 increased infarct size and was associated with slightly lower ferritin protein levels. In conclusion, the abovementioned findings indicate that NO, depending on its bio-active redox form, can regulate iron metabolism and plays a role in the IBM of cardioprotection against reperfusion injury. PMID:27447933

  6. In vitro evaluation of Bacopa monniera on anti-Helicobacter pylori activity and accumulation of prostaglandins.

    PubMed

    Goel, R K; Sairam, K; Babu, M Dora; Tavares, I A; Raman, A

    2003-01-01

    Bacopa monniera is an Indian tratidional medicine widely used to improve intellectual functions. Earlier, we had reported the prophylactic and curative effects of standardized extract of Bacopa monniera (BME) in various gastric ulcer models. The effect was due to augmentation of the defensive mucosal factors like increase in mucin secretion, life span of mucosal cells and gastric antioxidant effect rather than on the offensive acid-pepsin secretion. The present study includes evaluation of standardized BME (bacoside A content--35.5 +/- 0.9) on other contributing factors towards ulcerogenesis. BME in the dose of 1000 microg/ml showed anti-Helicobacter pylori activity in vitrol and in the dose of 10 microg/ml increased in vitro of prostanoids (PGE and PGI2) in human colonic mucosal incubates. It may be concluded that these factors may contribute to antiulcerogenic activity of BME.

  7. [Changed accumulation of active ingredient in different localities and growth period of Hemsleya zhejiangensis (Cucurbitaceae)].

    PubMed

    Yang, Wang-Wei; Lei, Zu-Pei; Wang, Wei-Min; Liang, Wei-qing; Zhou, Wei-Qing; Jin, Xiao-Feng

    2014-08-01

    In this paper, the content of moisture, ethanol-soluble extractives, total saponins and polysaccharide of different tuber samples of Hemsleya zhejiangensis, from different localities, years and seasons, were detected based upon Chinese Pharmacopoeia 2010 version. The samples of roots, stems and leaves in summer were detected as well. The results are mainly as follows. (1)With tuber quality increasing, the content of total saponins increased and then decreased. The individual quality of tubers getting 594.06 g, the content of total saponins reached the peak. (2) The content of active ingredients in different localities was significantly different, and the population of Wuyanling had the maximum content of total saponins and polysaccharide. (3) The content of active ingredients revealed stability between the years 2012 and 2013, but the content of polysaccharide was significantly different. The content in 2012 was higher than that of 2013. (4) The content of active ingredients reached the peak in autumn, which was the best harvest season. (5) Among different component content detection of nutritional organs, tubers had the maximum content of ethanol-soluble extractives, total saponins and polysaccharide. Leaves also contained higher content of ethanol-soluble extractives and total saponins than roots and stems. All of these provide theoretical basis for plant, harvest and production of H. zhejiangensis, which is an endemic, rare, and endangered medicinal plants.

  8. Kinetic Modeling of the Arabidopsis Cryptochrome Photocycle: FADH(o) Accumulation Correlates with Biological Activity.

    PubMed

    Procopio, Maria; Link, Justin; Engle, Dorothy; Witczak, Jacques; Ritz, Thorsten; Ahmad, Margaret

    2016-01-01

    Cryptochromes are flavoprotein photoreceptors with multiple signaling roles during plant de-etiolation and development. Arabidopsis cryptochromes (cry1 and cry2) absorb light through an oxidized flavin (FADox) cofactor which undergoes reduction to both FADH° and FADH(-) redox states. Since the FADH° redox state has been linked to biological activity, it is important to estimate its concentration formed upon illumination in vivo. Here we model the photocycle of isolated cry1 and cry2 proteins with a three-state kinetic model. Our model fits the experimental data for flavin photoconversion in vitro for both cry1 and cry2, providing calculated quantum yields which are significantly lower in cry1 than for cry2. The model was applied to the cryptochrome photocycle in vivo using biological activity in plants as a readout for FADH° concentration. The fit to the in vivo data provided quantum yields for cry1 and cry2 flavin reduction similar to those obtained in vitro, with decreased cry1 quantum yield as compared to cry2. These results validate our assumption that FADH° concentration correlates with biological activity. This is the first reported attempt at kinetic modeling of the cryptochrome photocycle in relation to macroscopic signaling events in vivo, and thereby provides a theoretical framework to the components of the photocycle that are necessary for cryptochrome response to environmental signals. PMID:27446119

  9. Kinetic Modeling of the Arabidopsis Cryptochrome Photocycle: FADHo Accumulation Correlates with Biological Activity

    PubMed Central

    Procopio, Maria; Link, Justin; Engle, Dorothy; Witczak, Jacques; Ritz, Thorsten; Ahmad, Margaret

    2016-01-01

    Cryptochromes are flavoprotein photoreceptors with multiple signaling roles during plant de-etiolation and development. Arabidopsis cryptochromes (cry1 and cry2) absorb light through an oxidized flavin (FADox) cofactor which undergoes reduction to both FADH° and FADH− redox states. Since the FADH° redox state has been linked to biological activity, it is important to estimate its concentration formed upon illumination in vivo. Here we model the photocycle of isolated cry1 and cry2 proteins with a three-state kinetic model. Our model fits the experimental data for flavin photoconversion in vitro for both cry1 and cry2, providing calculated quantum yields which are significantly lower in cry1 than for cry2. The model was applied to the cryptochrome photocycle in vivo using biological activity in plants as a readout for FADH° concentration. The fit to the in vivo data provided quantum yields for cry1 and cry2 flavin reduction similar to those obtained in vitro, with decreased cry1 quantum yield as compared to cry2. These results validate our assumption that FADH° concentration correlates with biological activity. This is the first reported attempt at kinetic modeling of the cryptochrome photocycle in relation to macroscopic signaling events in vivo, and thereby provides a theoretical framework to the components of the photocycle that are necessary for cryptochrome response to environmental signals. PMID:27446119

  10. Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

    PubMed

    Li, Wei; Shan, Xiao-Yu; Wang, Zhi-Yao; Lin, Xiao-Yu; Li, Chen-Xu; Cai, Chao-Yang; Abbas, Ghulam; Zhang, Meng; Shen, Li-Dong; Hu, Zhi-Qiang; Zhao, He-Ping; Zheng, Ping

    2016-01-01

    The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater. PMID:26595097

  11. Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

    PubMed

    Li, Wei; Shan, Xiao-Yu; Wang, Zhi-Yao; Lin, Xiao-Yu; Li, Chen-Xu; Cai, Chao-Yang; Abbas, Ghulam; Zhang, Meng; Shen, Li-Dong; Hu, Zhi-Qiang; Zhao, He-Ping; Zheng, Ping

    2016-01-01

    The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater.

  12. meso-Dihydroguaiaretic acid inhibits hepatic lipid accumulation by activating AMP-activated protein kinase in human HepG2 cells.

    PubMed

    Lee, Myoung-Su; Kim, Kyung Jin; Kim, Daeyoung; Lee, Kyung-Eun; Hwang, Jae-Kwan

    2011-01-01

    Hepatic lipid accumulation is a major risk factor for dyslipidemia, nonalcoholic fatty liver disease, and insulin resistance. The present study was conducted to evaluate hypolipidemic effects of meso-dihydroguaiaretic acid (MDA), anti-oxidative and anti-inflammatory compound isolated from the Myristica fragrans HOUTT., by oil red O staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot. MDA significantly inhibited insulin-induced hepatic lipid accumulation in a dose-dependent manner. The lipid-lowering effect of MDA was accompanied by increased expression of proteins involved in fatty acid oxidation and decreased expression of lipid synthetic proteins. In addition, MDA activated AMP-activated protein kinase (AMPK) as determined by phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK. The effects of MDA on lipogenic protein expression were suppressed by pretreatment with compound C, an AMPK inhibitor. Taken together, these findings show that MDA inhibits insulin-induced lipid accumulation in human HepG2 cells by suppressing expression of lipogenic proteins through AMPK signaling, suggesting a potent lipid-lowering agent. PMID:21963507

  13. Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation

    PubMed Central

    Alleaume-Butaux, Aurélie; Nicot, Simon; Pietri, Mathéa; Baudry, Anne; Dakowski, Caroline; Tixador, Philippe; Ardila-Osorio, Hector; Haeberlé, Anne-Marie; Bailly, Yannick; Peyrin, Jean-Michel; Launay, Jean-Marie; Kellermann, Odile; Schneider, Benoit

    2015-01-01

    In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases. PMID:26241960

  14. Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation.

    PubMed

    Alleaume-Butaux, Aurélie; Nicot, Simon; Pietri, Mathéa; Baudry, Anne; Dakowski, Caroline; Tixador, Philippe; Ardila-Osorio, Hector; Haeberlé, Anne-Marie; Bailly, Yannick; Peyrin, Jean-Michel; Launay, Jean-Marie; Kellermann, Odile; Schneider, Benoit

    2015-08-01

    In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.

  15. Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation.

    PubMed

    Alleaume-Butaux, Aurélie; Nicot, Simon; Pietri, Mathéa; Baudry, Anne; Dakowski, Caroline; Tixador, Philippe; Ardila-Osorio, Hector; Haeberlé, Anne-Marie; Bailly, Yannick; Peyrin, Jean-Michel; Launay, Jean-Marie; Kellermann, Odile; Schneider, Benoit

    2015-08-01

    In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases. PMID:26241960

  16. Development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of barley and its virescens mutant.

    PubMed

    Kyle, D J; Zalik, S

    1982-06-01

    The development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of greening wild-type barley (Hordeum vulgare L. cv. Gateway) and its virescens mutant were studied. The rate of chlorophyll accumulation per plastid was faster in the wild-type than in the mutant seedlings upon illumination after 6 days of etiolation, but was not different after 8 days. Although the protein content per plastid did not vary during greening, there was a change in the sodium dodecyl sulfate-polyacrylamide gel polypeptide profiles. High molecular weight proteins of 96,000 and 66,000 decreased whereas those at 34,000, 27,000 and 22,000 increased in relative quantity as a function of greening. The fully greened mutant seedlings were not deficient in the light-harvesting chlorophyll protein complex (LHC) or the reaction centers of photosystem I and photosystem II. Photosystem I-associated photochemical activities appeared within the first hour of plastid development and photosystem II associated activities and O(2) evolution within the next 6 hours. In all cases, the developmental rates per unit protein were slower in the mutant following 6 days of etiolation, but no differences between the two genotypes could be seen after 8 days due to a decrease in the developmental rate of the wild-type chloroplasts. An increase in photosynthetic unit size associated with plastid morphogenesis was faster in the wild-type seedlings after 6 days, but again the difference was negligible after 8 days. It was concluded that no single measured photochemical parameter is affected by this mutation, but rather, all aspects of chloroplast development are affected similarly by an overall reduction in the rate of chloroplast morphogenesis. This mutant, therefore, undergoes the normal pattern of proplastid to chloroplast development, but at a markedly reduced rate.

  17. Adsorption and Catalytic Activity of Glucose Oxidase Accumulated on OTCE upon the Application of External Potential

    PubMed Central

    Benavidez, Tomás E.; Torrente, Daniel; Marucho, Marcelo; Garcia, Carlos D.

    2014-01-01

    This article describes the adsorption of glucose oxidase (GOx) onto optically transparent carbon electrodes (OTCE) under the effect of applied potential and the analysis of the enzymatic activity of the resulting GOx/OTCE substrates. In order to avoid electrochemical interferences with the enzyme redox center, control electrochemical experiments were performed using flavin adenine dinucleotide (FAD) and GOx/OTCE substrates. Then, the enzyme adsorption experiments were carried out as a function of the potential applied (ranged from the open circuit potential to +950 mV), the pH solution, the concentration of enzyme, and the ionic strength on the environment. The experimental results demonstrated that an increase in the adsorbed amount of GOx on the OTCE can be achieved when the potential was applied. Although the increase in the adsorbed amount was examined as a function of the potential, a maximum enzymatic activity was observed in the GOx/OTCE substrate achieved at +800 mV. These experiments suggest that although an increase in the amount of enzyme adsorbed can be obtained by the application of an external potential to the electrode, the magnitude of such potential can produce detrimental effects in the conformation of the adsorbed protein and should be carefully considered. As such, the article describes a simple and rational approach to increase the amount of enzyme adsorbed on a surface and can be applied to improve the sensitivity of a variety of biosensors. PMID:25261840

  18. Acidobacteria dominate the active bacterial communities of Arctic tundra with widely divergent winter-time snow accumulation and soil temperatures.

    PubMed

    Männistö, Minna K; Kurhela, Emilia; Tiirola, Marja; Häggblom, Max M

    2013-04-01

    The timing and extent of snow cover is a major controller of soil temperature and hence winter-time microbial activity and plant diversity in Arctic tundra ecosystems. To understand how snow dynamics shape the bacterial communities, we analyzed the bacterial community composition of windswept and snow-accumulating shrub-dominated tundra heaths of northern Finland using DNA- and RNA-based 16S rRNA gene community fingerprinting (terminal restriction fragment polymorphism) and clone library analysis. Members of the Acidobacteria and Proteobacteria dominated the bacterial communities of both windswept and snow-accumulating habitats with the most abundant phylotypes corresponding to subdivision (SD) 1 and 2 Acidobacteria in both the DNA- and RNA-derived community profiles. However, different phylotypes within Acidobacteria were found to dominate at different sampling dates and in the DNA- vs. RNA-based community profiles. The results suggest that different species within SD1 and SD2 Acidobacteria respond to environmental conditions differently and highlight the wide functional diversity of these organisms even within the SD level. The acidic tundra soils dominated by ericoid shrubs appear to select for diverse stress-tolerant Acidobacteria that are able to compete in the nutrient poor, phenolic-rich soils. Overall, these communities seem stable and relatively insensitive to the predicted changes in the winter-time snow cover.

  19. Regulatory T cells with multiple suppressive and potentially pro-tumor activities accumulate in human colorectal cancer.

    PubMed

    Timperi, Eleonora; Pacella, Ilenia; Schinzari, Valeria; Focaccetti, Chiara; Sacco, Luca; Farelli, Francesco; Caronna, Roberto; Del Bene, Gabriella; Longo, Flavia; Ciardi, Antonio; Morelli, Sergio; Vestri, Anna Rita; Chirletti, Piero; Barnaba, Vincenzo; Piconese, Silvia

    2016-07-01

    Tregs can contribute to tumor progression by suppressing antitumor immunity. Exceptionally, in human colorectal cancer (CRC), Tregs are thought to exert beneficial roles in controlling pro-tumor chronic inflammation. The goal of our study was to characterize CRC-infiltrating Tregs at multiple levels, by phenotypical, molecular and functional evaluation of Tregs from the tumor site, compared to non-tumoral mucosa and peripheral blood of CRC patients. The frequency of Tregs was higher in mucosa than in blood, and further significantly increased in tumor. Ex vivo, those Tregs suppressed the proliferation of tumor-infiltrating CD8(+) and CD4(+) T cells. A differential compartmentalization was detected between Helios(high) and Helios(low) Treg subsets (thymus-derived versus peripherally induced): while Helios(low) Tregs were enriched in both sites, only Helios(high) Tregs accumulated significantly and specifically in tumors, displayed a highly demethylated TSDR region and contained high proportions of cells expressing CD39 and OX40, markers of activation and suppression. Besides the suppression of T cells, Tregs may contribute to CRC progression also through releasing IL-17, or differentiating into Tfr cells that potentially antagonize a protective Tfh response, events that were both detected in tumor-associated Tregs. Overall, our data indicate that Treg accumulation may contribute through multiple mechanisms to CRC establishment and progression. PMID:27622025

  20. Ultraviolet Radiation-Elicited Enhancement of Isoflavonoid Accumulation, Biosynthetic Gene Expression, and Antioxidant Activity in Astragalus membranaceus Hairy Root Cultures.

    PubMed

    Jiao, Jiao; Gai, Qing-Yan; Wang, Wei; Luo, Meng; Gu, Cheng-Bo; Fu, Yu-Jie; Ma, Wei

    2015-09-23

    In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting isoflavonoid accumulation. The optimum enhancement for isoflavonoid production was achieved in 34-day-old AMHRCs elicited by 86.4 kJ/m(2) of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g dry weight (DW), which was 2.29-fold higher relative to control (232.93 ± 3.08 μg/g DW). UV-B up-regulated the transcriptional expressions of all investigated genes involved in isoflavonoid biosynthetic pathway. PAL and C4H were found to be two potential key genes that controlled isoflavonoid biosynthesis. Moreover, a significant increase was noted in antioxidant activity of extracts from UV-B-elicited AMHRCs (IC50 values = 0.85 and 1.08 mg/mL) in comparison with control (1.38 and 1.71 mg/mL). Overall, this study offered a feasible elicitation strategy to enhance isoflavonoid accumulation in AMHRCs and also provided a basis for metabolic engineering of isoflavonoid biosynthesis in the future.

  1. The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets.

    PubMed

    Arena, Ellen T; Auweter, Sigrid D; Antunes, L Caetano M; Vogl, A Wayne; Han, Jun; Guttman, Julian A; Croxen, Matthew A; Menendez, Alfredo; Covey, Scott D; Borchers, Christoph H; Finlay, B Brett

    2011-11-01

    To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.

  2. Ultraviolet Radiation-Elicited Enhancement of Isoflavonoid Accumulation, Biosynthetic Gene Expression, and Antioxidant Activity in Astragalus membranaceus Hairy Root Cultures.

    PubMed

    Jiao, Jiao; Gai, Qing-Yan; Wang, Wei; Luo, Meng; Gu, Cheng-Bo; Fu, Yu-Jie; Ma, Wei

    2015-09-23

    In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting isoflavonoid accumulation. The optimum enhancement for isoflavonoid production was achieved in 34-day-old AMHRCs elicited by 86.4 kJ/m(2) of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g dry weight (DW), which was 2.29-fold higher relative to control (232.93 ± 3.08 μg/g DW). UV-B up-regulated the transcriptional expressions of all investigated genes involved in isoflavonoid biosynthetic pathway. PAL and C4H were found to be two potential key genes that controlled isoflavonoid biosynthesis. Moreover, a significant increase was noted in antioxidant activity of extracts from UV-B-elicited AMHRCs (IC50 values = 0.85 and 1.08 mg/mL) in comparison with control (1.38 and 1.71 mg/mL). Overall, this study offered a feasible elicitation strategy to enhance isoflavonoid accumulation in AMHRCs and also provided a basis for metabolic engineering of isoflavonoid biosynthesis in the future. PMID:26370303

  3. Hesperetin Induces Apoptosis in Breast Carcinoma by Triggering Accumulation of ROS and Activation of ASK1/JNK Pathway.

    PubMed

    Palit, Shreyasi; Kar, Susanta; Sharma, Gunjan; Das, Pijush K

    2015-08-01

    Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF-7 cells in a concentration- and time-dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF-10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl-serine externalization, DNA fragmentation, caspase-7 activation, and PARP cleavage. Apoptosis was associated with caspase-9 activation, mitochondrial membrane potential loss, release of cytochrome c, and increase in Bax:Bcl-2 ratio. Pre-treatment with caspase-9 specific inhibitor (Z-LEHD-fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow-cytometric analysis revealed triggering of ROS in a time-dependent manner. Pre-treatment with ROS scavenger N-acetylcysteine (NAC) and glutathione markedly abrogated hesperetin-mediated apoptosis whereas carbonyl cyanide m-chlorophenylhydrazone (CCCP) pretreatment along with DHR123-based flow-cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pre-treatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetin-mediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin-mediated apoptosis suggesting that hesperetin-mediated apoptosis of MCF-7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA-MB-231 cells via intrinsic pathway via activation of caspase -9 and -3 and increase in Bax:Bcl-2 ratio.

  4. Accumulation of phenanthrene by roots of intact wheat (Triticum acstivnm L.) seedlings: passive or active uptake?

    PubMed Central

    2010-01-01

    Background Polycyclic aromatic hydrocarbons (PAHs) are of particular concern due to their hydrophobic, recalcitrant, persistent, potentially carcinogenic, mutagenic and toxic properties, and their ubiquitous occurrence in the environment. Most of the PAHs in the environment are present in surface soil. Plants grown in PAH-contaminated soils or water can become contaminated with PAHs because of their uptake. Therefore, they may threaten human and animal health. However, the mechanism for PAHs uptake by crop roots is little understood. It is important to understand exactly how PAHs are transported into the plant root system and into the human food chain, since it is beneficial in governing crop contamination by PAHs, remedying soils or waters polluted by PAHs with plants, and modeling potential uptake for risk assessment. Results The possibility that plant roots may take up phenanthrene (PHE), a representative of PAHs, via active process was investigated using intact wheat (Triticum acstivnm L.) seedlings in a series of hydroponic experiments. The time course for PHE uptake into wheat roots grown in Hoagland solution containing 5.62 μM PHE for 36 h could be separated into two periods: a fast uptake process during the initial 2 h and a slow uptake component thereafter. Concentration-dependent PHE uptake was characterized by a smooth, saturable curve with an apparent Km of 23.7 μM and a Vmax of 208 nmol g-1 fresh weight h-1, suggesting a carrier-mediated uptake system. Competition between PHE and naphthalene for their uptake by the roots further supported the carrier-mediated uptake system. Low temperature and 2,4-dinitrophenol (DNP) could inhibit PHE uptake equally, indicating that metabolism plays a role in PHE uptake. The inhibitions by low temperature and DNP were strengthened with increasing concentration of PHE in external solution within PHE water solubility (7.3 μM). The contribution of active uptake to total absorption was almost 40% within PHE water

  5. X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH.

    PubMed

    Yue, K T; Taylor, K L; Kinkade, J M; Sinclair, R B; Powers, L S

    1997-04-01

    Myeloperoxidase (MPO), an important enzyme in the oxygen-dependent host defense system of human polymorphonuclear leukocytes, utilizes hydrogen peroxide to catalyze the production of hypochlorous acid, an oxidizing bactericidal agent. While MPO shows significant sequence homology with other peroxidases and this homology is particularly striking among the active-site residues, MPO exhibits unusual spectral features and the unique ability to catalyze the oxidation of chloride ions. We have investigated the MPO active-site with X-ray absorption (XAS) and resonance Raman (RRS) spectroscopies at neutral pH and also at the physiological acidic pH (pH approximately 3) and have compared these results with those of horseradish peroxidase (HRP). At pH 7.5, XAS results show that the iron heme active site is 6-coordinate where the distal ligand is likely nitrogen or oxygen, but not sulfur. The heme is distorted compared to HRP, other peroxidases, and heme compounds, but at pH approximately 3, the distal ligand is lost and the heme is less distorted. RRS results under identical pH conditions show that the skeletal core-size sensitive modes and v3 are shifted to higher frequency at pH approximately 3 indicating a 6- to 5-coordination change of high spin ferric heme. In addition, a new band at 270 cm(-1) is observed at pH approximately 3 which is consistent with the loss of the sixth ligand. The higher symmetry of the heme at pH approximately 3 is reflected by a single v4 mode in the (RRS) spectrum. HRP also loses its loosely associated distal water at this pH, but little change in heme distortion is observed. This change suggests that loss of the distal ligand in MPO releases stress on the heme which may facilitate binding of chloride ion.

  6. Lumbricus terrestris L. activity increases the availability of metals and their accumulation in maize and barley.

    PubMed

    Ruiz, E; Alonso-Azcárate, J; Rodríguez, L

    2011-03-01

    The effect of the earthworm Lumbricus terrestris L. on metal availability in two mining soils was assessed by means of chemical extraction methods and a pot experiment using crop plants. Results from single and sequential extractions showed that L. terrestris had a slight effect on metal fractionation in the studied soils: only metals bound to the soil organic matter were significantly increased in some cases. However, we found that L. terrestris significantly increased root, shoot and total Pb and Zn concentrations in maize and barley for the soil with the highest concentrations of total and available metals. Specifically, shoot Pb concentration was increased by a factor of 7.5 and 3.9 for maize and barley, respectively, while shoot Zn concentration was increased by a factor of 3.7 and 1.7 for maize and barley, respectively. Our results demonstrated that earthworm activity increases the bioavailability of metals in soils. PMID:21190761

  7. Exploring Effective Strategies for Increasing the Amount of Moderate-to-Vigorous Physical Activity Children Accumulate during Recess: A Quasi-Experimental Intervention Study

    ERIC Educational Resources Information Center

    Efrat, Merav W.

    2013-01-01

    Background: Less than half of elementary children meet the physical activity recommendations of 30 to 60 minutes of moderate-to-vigorous physical activity (MVPA) on a daily basis. Recess provides the single biggest opportunity for children to accumulate MVPA. This study explored whether a teacher's social prompting to be active during recess…

  8. Reduced photosynthetic activity is directly correlated with 2-(3H)-benzoxazolinone accumulation in lettuce leaves.

    PubMed

    Sánchez-Moreiras, Adela M; Oliveros-Bastidas, Alberto; Reigosa, Manuel J

    2010-02-01

    2-(3H)-benzoxazolinone (BOA) is a secondary plant metabolite previously found to inhibit plant growth and development. The phytotoxic activity of BOA has been extensively demonstrated over the last years. However, the relation of BOA phytotoxicity with BOA accumulation in plant leaves has not been thoroughly investigated. In this work, BOA phytotoxicity on photosynthesis (PhiPSII and Pn) of lettuce (Lactuca sativa L. cv. Great Lakes) was studied, and these results were correlated with BOA quantities in the leaves. BOA-treated plants showed reduced photosynthesis rate 6 h after the beginning of the treatment, and the efficiency of photosystem II started to be affected 10 h after treatment. These results were correlated with an increasing concentration of BOA in leaves that starts 6 h after treatment and shows a maximum at 96 h.

  9. Accumulation of health promoting phytochemicals in wild relatives of tomato and their contribution to in vitro antioxidant activity.

    PubMed

    Meléndez-Martínez, Antonio J; Fraser, Paul D; Bramley, Peter M

    2010-07-01

    Harnessing natural variation is an important aspect of modern marker assisted breeding. Traditionally breeding programmes have focused on increased yield and resistance to biotic and abiotic pressures. However, consumer demands for improved quality have lead to increased effort into the breeding of nutritional quality traits in crop plants. In the present study, health-related phytochemicals (carotenoids, tocopherols and phenolics) present in green, yellow and red wild relatives of tomato have been analyzed during fruit development and ripening. This study shows that the differences in the final colour of the fruits were due to a distinct accumulation of carotenoids mainly related to the expression of the phytoene synthase-1 gene (Psy-1). In ripe red-fruited tomatoes, the different deposition of pigments gave rise in some cases to colour differences visually discernible by the consumer. Important quantitative differences between and across taxa were noticed for the in vitro antioxidant activity (AA) of the samples. PMID:20457456

  10. Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins

    PubMed Central

    Poletto, Mattia; Lirussi, Lisa; Wilson, David M.; Tell, Gianluca

    2014-01-01

    Nucleophosmin (NPM1) is a multifunctional protein that controls cell growth and genome stability via a mechanism that involves nucleolar–cytoplasmic shuttling. It is clear that NPM1 also contributes to the DNA damage response, yet its exact function is poorly understood. We recently linked NPM1 expression to the functional activation of the major abasic endonuclease in mammalian base excision repair (BER), apurinic/apyrimidinic endonuclease 1 (APE1). Here we unveil a novel role for NPM1 as a modulator of the whole BER pathway by 1) controlling BER protein levels, 2) regulating total BER capacity, and 3) modulating the nucleolar localization of several BER enzymes. We find that cell treatment with the genotoxin cisplatin leads to concurrent relocalization of NPM1 and BER components from nucleoli to the nucleoplasm, and cellular experiments targeting APE1 suggest a role for the redistribution of nucleolar BER factors in determining cisplatin toxicity. Finally, based on the use of APE1 as a representative protein of the BER pathway, our data suggest a function for BER proteins in the regulation of ribogenesis. PMID:24648491

  11. Synthesis of fructooligosaccharides in banana 'prata' and its relation to invertase activity and sucrose accumulation.

    PubMed

    Der Agopian, Roberta Ghedini; Purgatto, Eduardo; Cordenunsi, Beatriz Rosana; Lajolo, Franco Maria

    2009-11-25

    Levels of sucrose and total fructooligosaccharides (FOS) were quantified in different phases of banana 'Prata' ripening during storage at ambient (approximately 19 degrees C) and low (approximately 10 degrees C) temperature. Total FOS levels were detected in the first days after harvest, whereas 1-kestose remained undetectable until the sucrose levels reached approximately 200 mg/g (dry weight) in both groups. Sucrose levels increased slowly but constantly at low temperature, but they elevated rapidly when the temperature was raised to 19 degrees C. Total FOS and sucrose levels were higher in bananas stored at low temperature than in the control group. In both samples, total FOS levels were higher than those of 1-kestose. The carbohydrate profiles obtained by HPLC and TLC suggest the presence of neokestose, 6-kestose, and bifurcose. The enzymes putatively involved in banana fructosyltransferase activity were also evaluated. Results obtained indicate that the banana enzyme responsible for the synthesis of FOS by transfructosylation is an invertase rather than a sucrose-sucrosyl transferase-like enzyme. PMID:19860446

  12. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling.

    PubMed

    Böhme, Mathias A; Beis, Christina; Reddy-Alla, Suneel; Reynolds, Eric; Mampell, Malou M; Grasskamp, Andreas T; Lützkendorf, Janine; Bergeron, Dominique Dufour; Driller, Jan H; Babikir, Husam; Göttfert, Fabian; Robinson, Iain M; O'Kane, Cahir J; Hell, Stefan W; Wahl, Markus C; Stelzl, Ulrich; Loll, Bernhard; Walter, Alexander M; Sigrist, Stephan J

    2016-10-01

    Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

  13. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling.

    PubMed

    Böhme, Mathias A; Beis, Christina; Reddy-Alla, Suneel; Reynolds, Eric; Mampell, Malou M; Grasskamp, Andreas T; Lützkendorf, Janine; Bergeron, Dominique Dufour; Driller, Jan H; Babikir, Husam; Göttfert, Fabian; Robinson, Iain M; O'Kane, Cahir J; Hell, Stefan W; Wahl, Markus C; Stelzl, Ulrich; Loll, Bernhard; Walter, Alexander M; Sigrist, Stephan J

    2016-10-01

    Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission. PMID:27526206

  14. Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

    PubMed Central

    Barak, Yoram; van Rijn, Jaap

    2000-01-01

    Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H+-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed. PMID

  15. Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes

    SciTech Connect

    Seo, Min Suk; Kim, Jung Hwan; Kim, Hye Jung; Chang, Ki Churl; Park, Sang Won

    2015-04-15

    Honokiol is a bioactive neolignan compound isolated from the species of Magnolia. This study was designed to elucidate the cellular mechanism by which honokiol alleviates the development of non-alcoholic steatosis. HepG2 cells were treated with honokiol for 1 h, and then exposed to 1 mM free fatty acid (FFA) for 24 h to simulate non-alcoholic steatosis in vitro. C57BL/6 mice were fed with a high-fat diet for 28 days, and honokiol (10 mg/kg/day) was daily treated. Honokiol concentration-dependently attenuated intracellular fat overloading and triglyceride (TG) accumulation in FFA-exposed HepG2 cells. These effects were blocked by pretreatment with an AMP-activated protein kinase (AMPK) inhibitor. Honokiol significantly inhibited sterol regulatory element-binding protein-1c (SREBP-1c) maturation and the induction of lipogenic proteins, stearoyl-CoA desaturase-1 (SCD-1) and fatty acid synthase (FAS) in FFA-exposed HepG2 cells, but these effects were blocked by pretreatment of an AMPK inhibitor. Honokiol induced AMPK phosphorylation and subsequent acetyl-CoA carboxylase (ACC) phosphorylation, which were inhibited by genetic deletion of liver kinase B1 (LKB1). Honokiol stimulated LKB1 phosphorylation, and genetic deletion of LKB1 blocked the effect of honokiol on SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-exposed HepG2 cells. Honokiol attenuated the increases in hepatic TG and lipogenic protein levels and fat accumulation in the mice fed with high-fat diet, while significantly induced LKB1 and AMPK phosphorylation. Taken together, our findings suggest that honokiol has an anti-lipogenic effect in hepatocytes, and this effect may be mediated by the LKB1–AMPK signaling pathway, which induces ACC phosphorylation and inhibits SREBP-1c maturation in hepatocytes. - Highlights: • Honokiol attenuates lipid accumulation induced by free fatty acid in hepatocyte. • Honokiol inhibits the increase in lipogenic enzyme levels induced by free fatty

  16. {delta}-ALAD activity variations in red blood cells in response to lead accumulation in rock doves (Columba livia)

    SciTech Connect

    Gonzalez, M.; Tejedor, M.C.

    1992-10-01

    The enzyme {delta}-aminolevulinic acid dehydratase ({delta}-ALAD, E.C. 4.2.1.24), catalyses the second step of the haeme biosynthetic pathway and is required to maintain the haemoglobin and cytochrome content in red cells. {delta}-ALAD is not only found in bone marrow cells, the major site of haeme synthesis, but also in circulating erythrocytes and other tissues. An inverse correlation was found between {delta}-ALAD activity in red blood cells and lead concentration in the blood. The degree of {delta}-ALAD inhibition in erythrocytes has been widely accepted as a standard bioassay to detect acute and chronic lead exposure in humans and in avians. The value of this parameter as an indicator for environmental lead has been often reported in doves and Scanlon. In lead-treated rats, an increase in {delta}-ALAD activity in bone marrow cells and in blood samples was shown by radioimmunoassay at 5 and 9 days after the treatment. Similarly, the amount of {delta}-ALAD seems to be more sensitive to lead in avian species than in mammals, the usefulness of blood {delta}-ALAD activity as an index of lead exposure has already been questioned by Hutton in the pigeon and by Jaffe et al. in humans. The present investigation studied the toxic effects of lead on rock dove red blood cell {delta}-ALAD activity in two situations: in doves treated with lead acetate in the laboratory and in doves exposed to the environment of Alcala de Henares. The final lead blood concentrations were lower in the environmental than in the laboratory doves. {delta}-ALAD activity in bone marrow cells and the relationships between lead accumulation and enzyme activity in red cells, are examined. 20 refs., 5 figs., 1 tab.

  17. Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway.

    PubMed

    Maitra, Dhiman; Shaeib, Faten; Abdulhamid, Ibrahim; Abdulridha, Rasha M; Saed, Ghassan M; Diamond, Michael P; Pennathur, Subramaniam; Abu-Soud, Husam M

    2013-10-01

    Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl(-)) and hydrogen peroxide (H₂O₂). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV-visible spectra and H₂O₂-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H₂O₂ (10 µM), precluding the enzyme from functioning at maximum activity (80-90% inhibition). MPO heme destruction occurred only in the presence of Cl(-). Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO-Compound I-Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.

  18. [Use of the granulocytic myeloperoxidase release reaction to diagnose food additive allergies].

    PubMed

    Titova, N D

    2011-03-01

    Adverse reactions to food additives are difficult to diagnose due to the diversity of mechanisms involved in their realization and to the absence of reasonably reliable methods for their determination. Eighty-three patients with allergic diseases were examined using the granulocytic myeloperoxidase release reaction (MRR) to diagnose intolerance reactions to food additives (E102, E122, E124, E132, E110, E2111). MRR revealed leukocyte hypersensitivity to tartrazine in 10.8%, sunset yellow in 4.8%, ponceau in 13.2%, indigo carmine in 8.4%, carmoisine and benzoate in 9.6%. The findings were correlated with history data and the levels of IgE antibodies to these dyes. The practical use of the proposed MRR method makes it possible to enhance the accuracy of diagnosis of allergy to food additives. PMID:21584968

  19. A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection

    PubMed Central

    Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth

    2015-01-01

    Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease. PMID:26116672

  20. A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection.

    PubMed

    Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth

    2015-09-01

    Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease. PMID:26116672

  1. Decreased myeloperoxidase expressing cells in the aged rat brain after excitotoxic damage.

    PubMed

    Campuzano, Oscar; Castillo-Ruiz, Maria del Mar; Acarin, Laia; Gonzalez, Berta; Castellano, Bernardo

    2011-09-01

    Brain aging is associated to several morphological and functional alterations that influence the evolution and outcome of CNS damage. Acute brain injury such as an excitotoxic insult induces initial tissue damage followed by associated inflammation and oxidative stress, partly attributed to neutrophil recruitment and the expression of oxidative enzymes such as myeloperoxidase (MPO), among others. However, to date, very few studies have focused on how age can influence neutrophil infiltration after acute brain damage. Therefore, to evaluate the age-dependent pattern of neutrophil cell infiltration following an excitotoxic injury, intrastriatal injection of N-methyl-d-aspartate was performed in young and aged male Wistar rats. Animals were sacrificed at different times between 12h post-lesion (hpl) to 14 days post-lesion (dpl). Cryostat sections were processed for myeloperoxidase (MPO) immunohistochemistry, and double labeling for either neuronal cells (NeuN), astrocytes (GFAP), perivascular macrophages (ED-2), or microglia/macrophages (tomato lectin histochemistry). Our observations showed that MPO + cells were observed in the injured striatum from 12 hpl (when maximum values were found) until 7 dpl, when cell density was strongly diminished. However, at all survival times analyzed, the overall density of MPO + cells was lower in the aged versus the adult injured striatum. MPO + cells were mainly identified as neutrophils (especially at 12 hpl and 1 dpl), but it should be noted that MPO + neurons and microglia/macrophages were also found. MPO + neurons were most commonly observed at 12 hpl and reduced in the aged. MPO + microglia/macrophages were the main population expressing MPO from 3 dpl, when density was also reduced in aged subjects. These results point to neutrophil infiltration as another important factor contributing to the different responses of the adult and aged brain to damage, highlighting the need of using aged animals for the study of acute age

  2. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    PubMed

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV. PMID:26436122

  3. Essential role of SBP-1 activation in oxygen deprivation induced lipid accumulation and increase in body width/length ratio in Caenorhabditis elegans.

    PubMed

    Taghibiglou, Changiz; Martin, Henry G S; Rose, Jacqueline K; Ivanova, Nadia; Lin, Conny H C; Lau, H Lee; Rai, Susan; Wang, Yu Tian; Rankin, Catharine H

    2009-02-18

    Epidemiological evidence suggests a link between chronic oxygen starvation and fat accumulation/obesity, however the underlying mechanism remains unclear. Using Caenorhabditis elegans we found extended oxygen deprivation resulted in activation of SBP-1, the worm homologue of SREBP1, a transcription factor important in maintaining lipid homeostasis. SBP-1 knockdown prevented hypoxia-induced fat accumulation and the associated increase in worm width/length ratio, demonstrating that SBP-1/SREBP1 plays an essential role in hypoxia-induced lipid accumulation and body shape alteration. This study provides the first evidence suggesting that activation of SREBP1 may be a critical pathogenic factor contributing to chronic hypoxia associated excessive fat accumulation/obesity in humans.

  4. Interseismic strain accumulation across an active thrust system: an InSAR case study in the Himalaya

    NASA Astrophysics Data System (ADS)

    Grandin, R.; Doin, M.; Ducret, G.; Bollinger, L.; Pinel-Puysségur, B.; Lasserre, C.; Jolivet, R.

    2011-12-01

    The major active thrust system underlying the Himalayan range produces recurrent large earthquakes, posing a significant threat to the densely populated Indo-Gangetic basin. Measuring the interseismic deformation associated with this fault system could provide important constraints on the geometry of the locked faults that are bound to rupture in future great earthquakes. This has so far been considered out of reach of InSAR techniques, due to decorrelation, prominent topographic features, and unfavourable climatic conditions. However, preliminary tests carried out with the archived ASAR data provided by ESA's ENVISAT satellite since 2002 have shown that recent advances in InSAR processing may now allow geodesists to tackle most of these perturbations. In this context, applying these advanced techniques to the case of the Himalayas is both a challenging and necessary task. We will present the methodology and the first results of an InSAR study of interseismic strain accumulation across the Himalayas. Small-baseline processing of ENVISAT data using a combination of ROI_PAC software, NSBAS processing chain and MulSAR technique yields a sufficient number of coherent interferograms to compute a preliminary average velocity map of interseismic uplift. Time-space variations of stratified tropospheric delay observed in these interferogrames are mitigated using a prediction deduced from outputs of the ECMWF global meteorological reanalysis ERA-Interim. Finally, a correction of DEM errors from the wrapped InSAR data set further improves the coherence of interferograms with a large perpendicular baseline. Comparison of the InSAR LOS velocity maps with microseismic activity detected near the transition zone at the base of the seismogenic portion of the Main Himalayan Thrust is expected to provide constraints on the process of elastic strain accumulation during the interseismic period. This will help in understanding the interaction between the construction of topography

  5. Certain forms of matrix metalloproteinase-9 accumulate in the extracellular space after microdialysis probe implantation and middle cerebral artery occlusion/reperfusion.

    PubMed

    Planas, Anna M; Justicia, Carles; Solé, Sònia; Friguls, Bibiana; Cervera, Alvaro; Adell, Albert; Chamorro, Angel

    2002-08-01

    Matrix metalloproteinases (MMPs) are activated in focal cerebral ischemia. The activation of MMP-9 is involved in blood-brain barrier breakdown and tissue remodeling. The MMPs are released to the extracellular space, but the form and fate of secreted enzymes in brain are unknown. Using microdialysis in vivo, the authors studied whether ischemia-induced MMP-9 in brain tissue was related to free MMP-9 in the extracellular fluid. A microdialysis probe was placed into the right striatum and microdialysis was initiated 24 hours later in controls (n = 7). One hour prior to microdialysis, a group of rats (n = 7) was subjected to 1-hour occlusion of the right middle cerebral artery, followed by reperfusion. Dialysates were collected at discrete time points up to 24 hours, and subjected to zymography and Western blot analysis. The MMP-9 was released after ischemia and accumulated in the extracellular space at 24 hours (P < 0.05). Free MMP-9 forms include mainly the 95-kd proform, and, to a lesser extent, dimers and cleaved active forms (70 kd), but not the 88-kd form found in tissue. Probe implantation and microdialysis increased free MMP-9 in the dialysate. This increase was concomitant with neutrophil infiltration after the mechanical lesion, as myeloperoxidase was found by means of Western blot analysis in the brain hemisphere subjected to microdialysis (P < 0.005), and immunohistochemistry revealed the presence of myeloperoxidase stain surrounding the site of probe implantation. The results suggest that certain forms of MMP-9 are released and accumulate in the extracellular space after brain injury, and that vascular alterations and neutrophil recruitment elicit MMP-9 activation in the brain after focal ischemia and trauma.

  6. Antioxidant Effects of Vitamins C and E on the Low-Density Lipoprotein Oxidation Mediated by Myeloperoxidase

    PubMed Central

    Samsam Shariat, Seyed Ziyae Aldin; Mostafavi, Sayed Abolfazl; Khakpour, Farzad

    2013-01-01

    Background: Oxidative modification of low-density lipoprotein (LDL) appears to be an early step in the pathogenesis of atherosclerosis. Meanwhile, myeloperoxidase (MPO)-catalyzed reaction is one of the potent pathways for LDL oxidation in vivo. The aim of this study was to evaluate in vitro antioxidant effects of vitamins C and E on LDL oxidation mediated by MPO. Methods: MPO was isolated from fresh plasma by sequential centrifugation using density ultracentrifugation. It was incubated with LDL and the LDL oxidation level was determined spectrophotometrically by measuring conjugated diene absorbance at 234 nm. Furthermore, vitamin C (50-200 mM) and vitamin E (10-40 mM) were added and the LDL oxidation level was determined. Results: The purity index of MPO and its enzymatic activity were 0.69 and 1127 U/mg protein, respectively. It was demonstrated that vitamin C in vitro inhibited LDL oxidation mediated by MPO; however, vitamin E was unable to act in the same way. The protection by vitamin C was concentration dependent and maximum protective effect of vitamin C was observed at 150 mM, where about 64% of the LDL oxidation was inhibited. Vitamin C increased lag time of LDL oxidation mediated by MPO up to 2.4 times. Conclusion: It can be concluded from our results that vitamin C is able to improve LDL resistance to oxidative modification in vitro. In addition, vitamin C might be effective in LDL oxidation mediated by MPO in vivo, resulting in reduction of atherosclerosis process rate. PMID:23279831

  7. Micropruina glycogenica gen. nov., sp. nov., a new Gram-positive glycogen-accumulating bacterium isolated from activated sludge.

    PubMed

    Shintani, T; Liu, W T; Hanada, S; Kamagata, Y; Miyaoka, S; Suzuki, T; Nakamura, K

    2000-01-01

    A new Gram-positive non-spore-forming bacterium, strain Lg2T, was isolated from an activated sludge reactor showing enhanced biological phosphorus removal activity. The new isolate was a slowly growing organism and was capable of accumulating large amounts of intracellular glycogen from substrate taken up. Both oxidase and catalase were produced. The new isolate contained meso-diaminopimelic acid (DAP) in the cell wall. Complex fatty acid patterns with iso-C14:0, anteiso-C15:0, C16:0, iso-C16:0 and four other minor saturated or unsaturated straight-chain fatty acids were detected. The isolate contained a high genomic G+C content (70.5 mol%). Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate in the high G+C Gram-positive group with Microlunatus phosphovorus and Friedmanniella antarctica as the closest relatives (sequence similarities are 93 and 92 %, respectively). These three organisms shared common features in morphology, but strain Lg2T could be differentiated from the other species by its peptidoglycan type (meso-DAP), fatty acid composition, carbon source utilization profile and G+C content. On the basis of these findings, it is proposed that a new genus and species, Micropruina glycogenica, should be created for the new isolate; the type strain is strain Lg2T (= JCM 10248T).

  8. PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

    PubMed

    Xu, Haiqing; Kim, Sooah; Sorek, Hagit; Lee, Youngsuk; Jeong, Deokyeol; Kim, Jungyeon; Oh, Eun Joong; Yun, Eun Ju; Wemmer, David E; Kim, Kyoung Heon; Kim, Soo Rin; Jin, Yong-Su

    2016-03-01

    The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

  9. Activity in Inferior Parietal and Medial Prefrontal Cortex Signals the Accumulation of Evidence in a Probability Learning Task

    PubMed Central

    d'Acremont, Mathieu; Fornari, Eleonora; Bossaerts, Peter

    2013-01-01

    In an uncertain environment, probabilities are key to predicting future events and making adaptive choices. However, little is known about how humans learn such probabilities and where and how they are encoded in the brain, especially when they concern more than two outcomes. During functional magnetic resonance imaging (fMRI), young adults learned the probabilities of uncertain stimuli through repetitive sampling. Stimuli represented payoffs and participants had to predict their occurrence to maximize their earnings. Choices indicated loss and risk aversion but unbiased estimation of probabilities. BOLD response in medial prefrontal cortex and angular gyri increased linearly with the probability of the currently observed stimulus, untainted by its value. Connectivity analyses during rest and task revealed that these regions belonged to the default mode network. The activation of past outcomes in memory is evoked as a possible mechanism to explain the engagement of the default mode network in probability learning. A BOLD response relating to value was detected only at decision time, mainly in striatum. It is concluded that activity in inferior parietal and medial prefrontal cortex reflects the amount of evidence accumulated in favor of competing and uncertain outcomes. PMID:23401673

  10. Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

    PubMed Central

    Krebs, Melanie; Beyhl, Diana; Görlich, Esther; Al-Rasheid, Khaled A. S.; Marten, Irene; Stierhof, York-Dieter; Hedrich, Rainer; Schumacher, Karin

    2010-01-01

    The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H+-ATPase (V-ATPase) and the vacuolar H+-pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn2+/H+-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na+/H+-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vha-a2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth. PMID:20133698

  11. Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation.

    PubMed

    ElKeeb, A M; Collier, M E W; Maraveyas, A; Ettelaie, C

    2015-08-01

    We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial cells. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release, respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA-mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cells, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53. PMID:25903973

  12. Energy release, beam attenuation radiation damage, gas production and accumulation of long-lived activity in Pb, Pb-Bi and Hg targets

    SciTech Connect

    Shubin, Yu.N.

    1996-06-01

    The calculation and analysis of the nuclei concentrations and long-lived residual radioactivity accumulated in Pb, Pb-Bi and Hg targets irradiated by 800 MeV, 30 mA proton beam have been performed. The dominating components to the total radioactivity of radionuclides resulting from fission and spallation reactions and radiative capture by both target nuclei and accumulated radioactive nuclei for various irradiation and cooling times were analyzed. The estimations of spectral component contributions of neutron and proton fluxes to the accumulated activity were carried out. The contributions of fission products to the targets activity and partial activities of main long-lived fission products to the targets activity and partial activities of main long-lived fission products were evaluated. The accumulation of Po isotopes due to reactions induced by secondary alpha-particles were found to be important for the Pb target as compared with two-step radiative capture. The production of Tritium in the targets and its contribution to the total targets activity was considered in detail. It is found that total activities of both targets are close to one another.

  13. Accumulation and washout of 99mTc-sestamibi in osteoarthritic subchondral bone may indicate increased osteoclastic activity accompanying microfractures: a case study.

    PubMed

    Tanaka, Ryo; Ebine, Masato

    2016-01-01

    We show for the first time that imaging of osteoarthritic subchondral bone with 99mTc-sestamibi (MIBI) may be a valuable diagnostic tool to assess the severity of subchondral sclerosis. This is a report on a patient who had osteoarthritis of both knees and where MIBI was accumulated in the lesion sites. Due to the accumulation of MIBI in the bone, we believe that excessive stress had been applied to the bone and microfractures had activated bone remodeling. Typically, MIBI accumulation is not observed in the bone, but MIBI is believed to exhibit high accumulation where there is an increased presence of osteoblasts and osteoclasts. Osteoclasts absorb calcium, consequently causing a depolarization of the mitochondrial membrane potential, which may decrease the ability to retain MIBI and enhance washout. Pathological examination of the tissues of this case confirmed an abundance of osteoclasts. This suggests that using MIBI may be useful for determining the increased presence of osteoblasts and osteoclasts.

  14. Cadmium accumulation retard activity of functional components of photo assimilation and growth of rice cultivars amended with vermicompost.

    PubMed

    Sebastian, Abin; Prasad, M N V

    2013-01-01

    Cadmium (Cd) uptake mediated alterations in functional components of photo assimilation during conversion of cow dung and poultry cast to vermicompost were studied in two Indian rice cultivars; MO 16 and MTU 7029. It was found that higher amount of Cd accumulate in plants grown in soil amended with vermicompost which in turn damaged functional components in photo assimilation. Enhancement of root growth was recognized as reason for Cd accumulation. Metabolic alterations noticed among plants were not taken place during application of raw materials used for vermicomposting such as cow dung and poultry cast amendment. Rice varieties accumulated Cd differentially where MTU 7029 accumulated more Cd compare to MO 16. It was also noticed that existence of negative correlation between zinc status of the plant and Cd accumulation.

  15. Group 3 innate lymphoid cells accumulate and exhibit disease-induced activation in the meninges in EAE.

    PubMed

    Hatfield, Julianne K; Brown, Melissa A

    2015-10-01

    Innate lymphoid cells are immune cells that reside in tissues that interface with the external environment and contribute to the first line defense against pathogens. However, they also have roles in promoting chronic inflammation. Here we demonstrate that group 3 ILCs, (ILC3s - CD45+Lin-IL-7Rα+RORγt+), are normal residents of the meninges and exhibit disease-induced accumulation and activation in EAE. In addition to production of the pro-inflammatory cytokines IL-17 and GM-CSF, ILC3s constitutively express CD30L and OX40L, molecules required for memory T cell survival. We show that disease-induced trafficking of transferred wild type T cells to the meninges is impaired in ILC3-deficient Rorc-/- mice. Furthermore, lymphoid tissue inducer cells, a c-kit+ ILC3 subset that promotes ectopic lymphoid follicle development, a hallmark of many autoimmune diseases, are reduced in the meninges of EAE-resistant c-kit mutant Kit(W/Wv) mice. We propose that ILC3s sustain neuroinflammation by supporting T cell survival and reactivation in the meninges.

  16. Age‐related remodeling of small arteries is accompanied by increased sphingomyelinase activity and accumulation of long‐chain ceramides

    PubMed Central

    Ohanian, Jacqueline; Liao, Aiyin; Forman, Simon P.; Ohanian, Vasken

    2014-01-01

    Abstract The structure and function of large arteries alters with age leading to increased risk of cardiovascular disease. Age‐related large artery remodeling and arteriosclerosis is associated with increased collagen deposition, inflammation, and endothelial dysfunction. Bioactive sphingolipids are known to regulate these processes, and are also involved in aging and cellular senescence. However, less is known about age‐associated alterations in small artery morphology and function or whether changes in arterial sphingolipids occur in aging. We show that mesenteric small arteries from old sheep have increased lumen diameter and media thickness without a change in media to lumen ratio, indicative of outward hypertrophic remodeling. This remodeling occurred without overt changes in blood pressure or pulse pressure indicating it was a consequence of aging per se. There was no age‐associated change in mechanical properties of the arteries despite an increase in total collagen content and deposition of collagen in a thickened intima layer in arteries from old animals. Analysis of the sphingolipid profile showed an increase in long‐chain ceramide (C14–C20), but no change in the levels of sphingosine or sphingosine‐1‐phosphate in arteries from old compared to young animals. This was accompanied by a parallel increase in acid and neutral sphingomyelinase activity in old arteries compared to young. This study demonstrates remodeling of small arteries during aging that is accompanied by accumulation of long‐chain ceramides. This suggests that sphingolipids may be important mediators of vascular aging. PMID:24872355

  17. Canthin-6-one displays antiproliferative activity and causes accumulation of cancer cells in the G2/M phase.

    PubMed

    Dejos, Camille; Voisin, Pierre; Bernard, Marianne; Régnacq, Matthieu; Bergès, Thierry

    2014-11-26

    Canthinones are natural substances with a wide range of biological activities, including antipyretic, antiparasitic, and antimicrobial. Antiproliferative and/or cytotoxic effects of canthinones on cancer cells have also been described, although their mechanism of action remains ill defined. To gain better insight into this mechanism, the antiproliferative effect of a commercially available canthin-6-one (1) was examined dose-dependently on six cancer cell lines (human prostate, PC-3; human colon, HT-29; human lymphocyte, Jurkat; human cervix, HeLa; rat glioma, C6; and mouse embryonic fibroblasts, NIH-3T3). Cytotoxic effects of 1 were investigated on the same cancer cell lines by procaspase-3 cleavage and on normal human skin fibroblasts. Strong antiproliferative effects of the compound were observed in all cell lines, whereas cytotoxic effects were very dependent on cell type. A better definition of the mechanism of action of 1 was obtained on PC-3 cells, by showing that it decreases BrdU incorporation into DNA by 60% to 80% and mitotic spindle formation by 70% and that it causes a 2-fold accumulation of cells in the G2/M phase of the cell cycle. Together, the data suggest that the primary effect of canthin-6-one (1) is antiproliferative, possibly by interfering with the G2/M transition. Proapoptotic effects might result from this disturbance of the cell cycle. PMID:25379743

  18. Effect of a microbiota activator on accumulated ammonium and microbial community structure in a pilot-scale membrane bioreactor.

    PubMed

    Sato, Yuya; Hori, Tomoyuki; Navarro, Ronald R; Ronald, Navarro R; Habe, Hiroshi; Ogata, Atsushi

    2015-01-01

    Microbiota activators (MAs) have been used to improve the reactor performances of biological wastewater treatment processes. In this study, to remove ammonium (NH4(+)) accumulated during the pre-operation of a pilot-scale membrane bioreactor (MBR) under high-organic-loading conditions, an MA was added to the MBR system and the resulting changes in reactor performances and microbial communities were monitored for 12 days. The NH4(+) concentrations in the sludge and effluent decreased (from 427 to 246 mg/L in the sludge (days 1-9)), and mixed liquor suspended solid increased (from 6,793 to 11,283 mg/L (days 1-12)) after the addition of MA. High-throughput Illumina sequencing of 16S rRNA genes revealed that the microbial community structure changed along with the NH4(+) removal resulting from the MA addition. In particular, the relative abundance of an Acidovorax-related operational taxonomic unit (OTU) increased significantly, accounting for approximately 50% of the total microbial population at day 11. In contrast, the ammonia-oxidizing bacteria and archaea showed low abundances (<0.05%), and no anaerobic ammonia oxidizers were detected. These results suggested that the Acidovorax-related OTU was mainly involved in the NH4(+) removal in the MBR, probably due to its ammonia-assimilating metabolism. PMID:26377133

  19. Houttuynia cordata attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells.

    PubMed

    Kang, Hyun; Koppula, Sushruta

    2014-01-01

    Houttuynia cordata (H. cordata) from the family Saururaceae is a perennial herb native to Southeast Asia. It possesses a range of medicinal properties to treat several disease symptoms including allergic inflammation and anaphylaxis. In the present investigation, we provided the molecular mechanisms underlying the role of H. cordata extract (HCE) in the prevention of high glucose-induced lipid accumulation in human HepG2 hepatocytes. HepG2 cells were pre-treated with various concentrations of HCE (0, 10, 20, 40, and 80 μg/mL) and treated with serum-free medium with normal glucose (5 mM) for 1 h, followed by exposure to high glucose (25 mM D-glucose) for 24 h. HCE significantly and dose-dependently attenuated lipid accumulation in human HepG2 hepatocytes when exposed to high glucose (25 mM D-glucose) (p < 0.05, p < 0.01 and p < 0.001 at 20, 40, and 80 μg/mL concentrations, respectively). Further, HCE attenuated the expression of fatty acid synthase (FAS), sterol regulatory element-binding protein-1 and glycerol 3-phosphate acyltransferases (GPATs). The adenosine monophosphate-activated protein kinase (AMPK) was also activated by HCE treatment when exposed to high glucose (25 mM D-glucose) in human HepG2 hepatocytes. This study suggests the hypolipidemic effects of HCE by the inhibition of lipid biosynthesis mediated through AMPK signaling, which may play an active role and can be developed as an anti-obesity agent. PMID:24871657

  20. Adenoviral overexpression and small interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated collagen accumulation in normal and keloid fibroblasts.

    PubMed

    Tuan, Tai-Lan; Hwu, Paul; Ho, Wendy; Yiu, Peter; Chang, Richard; Wysocki, Annette; Benya, Paul D

    2008-11-01

    Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.

  1. Myeloperoxidase Oxidized LDL Interferes with Endothelial Cell Motility through miR-22 and Heme Oxygenase 1 Induction: Possible Involvement in Reendothelialization of Vascular Injuries

    PubMed Central

    Daher, Jalil; Martin, Maud; Rousseau, Alexandre; Nuyens, Vincent; Fayyad-Kazan, Hussein; Van Antwerpen, Pierre; Courbebaisse, Guy; Martiat, Philippe; Badran, Bassam; Dequiedt, Frank

    2014-01-01

    Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque. PMID:25530680

  2. Oenothera paradoxa defatted seeds extract and its bioactive component penta-O-galloyl-β-D-glucose decreased production of reactive oxygen species and inhibited release of leukotriene B4, interleukin-8, elastase, and myeloperoxidase in human neutrophils.

    PubMed

    Kiss, Anna K; Filipek, Agnieszka; Czerwińska, Monika; Naruszewicz, Marek

    2010-09-22

    In this study, we analyzed ex vivo the effect of an aqueous extract of Oenothera paradoxa defatted seeds on the formation of neutrophil-derived oxidants. For defining active compounds, we also tested lypophilic extract constituents such as gallic acid, (+)-catechin, ellagic acid, and penta-O-galloyl-β-D-glucose and a hydrophilic fraction containing polymeric procyanidins. The anti-inflammatory potential of the extract and compounds was tested by determining the release from activated neutrophils of elastase, myeloperoxidase, interleukin-8 (IL-8), and leukotriene B4 (LTB4), which are considered relevant for the pathogenesis of cardiovascular diseases. The extract of O. paradoxa defatted seeds displays potent antioxidant effects against both 4β-phorbol-12β-myristate-α13-acetate- and formyl-met-leu-phenylalanine-induced reactive oxygen species production in neutrophils with IC50 values around 0.2 μg/mL. All types of polyphenolics present in the extract contributed to the extract antioxidant activity. According to their IC50 values, penta-O-galloyl-β-D-glucose was the more potent constituent of the extract. In cell-free assays, we demonstrated that this effect is partially due to the scavenging of O2- and H2O2 oxygen species. The extract and especially penta-O-galloyl-β-D-glucose significantly inhibit elastase, myeloperoxidase IL-8, and LTB4 release with an IC50 for penta-O-galloyl-β-D-glucose of 17±1, 15±1, 6.5±2.5, and around 20 μM, respectively. The inhibition of penta-O-galloyl-β-D-glucose on reactive oxygen species and especially on O2- production, myeloperoxidase, and chemoattractant release may reduce the interaction of polymorphonuclear leukocyte with the vascular endothelium and by that potentially diminish the risk of progression of atherosclerosis development.

  3. Susceptibility to arsenic-induced hyperkeratosis and oxidative stress genes myeloperoxidase and catalase.

    PubMed

    Ahsan, Habibul; Chen, Yu; Kibriya, Muhammad G; Islam, Mohammad N; Slavkovich, Vesna N; Graziano, Joseph H; Santella, Regina M

    2003-11-10

    Chronic exposure to inorganic arsenic is known to cause non-melanocytic skin and internal cancers in humans. We examined whether genetic susceptibility, as determined by single nucleotide polymorphisms -463G-->A and -262C-->T in the oxidative stress genes myeloperoxidase (MPO) and catalase (CAT), respectively, are associated with the risk of arsenic-induced hyperkeratotic skin lesions-precursors of skin cancer-in a case-control study in Bangladesh. Carriers of the susceptible MPO and CAT genotypes were at elevated risk (OR 2.1 and 95% CI 0.7-6.2 for MPO; OR 1.9 and 95% CI 0.8-4.7 for CAT) of hyperkeratosis after adjustment for arsenic exposure and other covariates. Subjects carrying the high-risk MPO genotype and with high arsenic exposure were at almost six times (OR 5.8; 95% CI 1.1-30.1) elevated risk of developing hyperkeratosis as compared to those carrying the low-risk genotype and with low arsenic exposure. Similarly, highly exposed subjects carrying the high-risk CAT genotype were at more than four times (OR 4.6; 95% CI 1.4-15.6) elevated risk of developing hyperkeratosis as compared to those carrying the low-risk genotype and with low arsenic exposure. Our findings, although based on small numbers, suggest that the oxidative stress genes MPO and CAT may influence the risk of arsenic-induced premalignant hyperkeratotic skin lesions.

  4. Changes in the viscosity of hyaluronic acid after exposure to a myeloperoxidase-derived oxidant

    SciTech Connect

    Baker, M.S.; Green, S.P.; Lowther, D.A.

    1989-04-01

    Both purified hyaluronic acid (HA) and bovine synovial fluid react with OCI-, the major oxidant produced by the myeloperoxidase (MPO)/H/sub 2/O/sub 2//CI- system, resulting in a decrease in their specific viscosity. This reaction is inhibited in the presence of excess methionine. H/sub 2/O/sub 2/ alone decreases the viscosity of HA, presumably by the Fenton reaction, in the absence (but not in the presence) of the iron chelator, diethyltriaminepentacetic acid (DETAPAC). In the presence of DETAPAC, incubation of HA with the complete MPO/H/sub 2/O/sub 2//CI- system lowered the viscosity of HA. Analysis of 3H-HA exposed to OCI- by gel filtration chromatography indicated that cleavage of HA occurred only at higher OCI- concentrations. We suggest that the reduction in viscosity of HA by the MPO/H/sub 2/O/sub 2//CI- system may be due to a combination of oxidative cleavage and changes in the conformation of the molecule. We speculate that the changes in the molecular size of rheumatoid synovial fluid HA may be due to the action of the neutrophil MPO/H/sub 2/O/sub 2//CI- system.

  5. Immunohistochemical Expression of Myeloperoxidase in Placental Samples of Systematic Lupus Erythematosus Pregnancies

    PubMed Central

    Heidari, Zahra; Mahmoudzadeh Sagheb, Hamidreza; Sheibak, Nadia

    2016-01-01

    Objective: Reports of increased level of Myeloperoxidase (MPO) in plasma and placental extracts of Systematic Lupus Erythematosus (SLE) has been proposed that MPO may have an important role in this pregnancy complication. In present study immunohistochemical MPO expression was investigated in placental samples of SLE women compared with normal controls. Materials and methods: Ten patients with SLE were recruited as case group. Control group was selected from mothers with normal uncomplicated pregnancies. A monoclonal antibody specific for MPO was used for immunohistochemical staining and then the staining was quantified and differences between groups were compared using Mann-Whitney U test. Results: There were significant differences in the expression levels of MPO in the syncytiotrophoblast cells and the extravillous trophoblast cells between the control and SLE groups (p < 0.05). There were no significant differences in the expression level of MPO in the vascular endothelium and the relative number of the MPO-positive leukocytes in placental tissue between SLE and control groups (p > 0.05). Conclusion: The present study showed that MPO expression is increased in syncytiotrophoblast cells and the extravillous trophoblast cells of SLE placentas compared to healthy subjects. It seems that these changes are able to impress structure and function of placenta and survival of the fetus. PMID:27648095

  6. Immunohistochemical Expression of Myeloperoxidase in Placental Samples of Systematic Lupus Erythematosus Pregnancies

    PubMed Central

    Heidari, Zahra; Mahmoudzadeh Sagheb, Hamidreza; Sheibak, Nadia

    2016-01-01

    Objective: Reports of increased level of Myeloperoxidase (MPO) in plasma and placental extracts of Systematic Lupus Erythematosus (SLE) has been proposed that MPO may have an important role in this pregnancy complication. In present study immunohistochemical MPO expression was investigated in placental samples of SLE women compared with normal controls. Materials and methods: Ten patients with SLE were recruited as case group. Control group was selected from mothers with normal uncomplicated pregnancies. A monoclonal antibody specific for MPO was used for immunohistochemical staining and then the staining was quantified and differences between groups were compared using Mann-Whitney U test. Results: There were significant differences in the expression levels of MPO in the syncytiotrophoblast cells and the extravillous trophoblast cells between the control and SLE groups (p < 0.05). There were no significant differences in the expression level of MPO in the vascular endothelium and the relative number of the MPO-positive leukocytes in placental tissue between SLE and control groups (p > 0.05). Conclusion: The present study showed that MPO expression is increased in syncytiotrophoblast cells and the extravillous trophoblast cells of SLE placentas compared to healthy subjects. It seems that these changes are able to impress structure and function of placenta and survival of the fetus.

  7. Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant

    PubMed Central

    Guo, Ling-Xia; Shi, Cai-Yun; Liu, Xiao; Ning, Dong-Yuan; Jing, Long-Fei; Yang, Huan; Liu, Yong-Zhong

    2016-01-01

    ‘Hong Anliu’ (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of ‘Anliu’ (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between ‘HAL’ and ‘AL’. Compared with ‘AL’, the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in ‘HAL’. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H+-ATPase, vacuolar H+-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in ‘HAL’. These results implied that ‘HAL’ has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with ‘AL’. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in ‘HAL’. PMID:27385485

  8. Endocrine activity of persistent organic pollutants accumulated in human silicone implants--Dosing in vitro assays by partitioning from silicone.

    PubMed

    Gilbert, Dorothea; Mayer, Philipp; Pedersen, Mikael; Vinggaard, Anne Marie

    2015-11-01

    Persistent organic pollutants (POPs) accumulated in human tissues may pose a risk for human health by interfering with the endocrine system. This study establishes a new link between actual human internal POP levels and the endocrine active dose in vitro, applying partitioning-controlled dosing from silicone to the H295R steroidogenesis assay: (1) Measured concentrations of POPs in silicone breast implants were taken from a recent study and silicone disks were loaded according to these measurements. (2) Silicone disks were transferred into H295R cell culture plates in order to control exposure of the adrenal cells by equilibrium partitioning. (3) Hormone production of the adrenal cells was measured as toxicity endpoint. 4-Nonylphenol was used for method development, and the new dosing method was compared to conventional solvent-dosing. The two dosing modes yielded similar dose-dependent hormonal responses of H295R cells. However, with the partitioning-controlled freely dissolved concentrations (Cfree) as dose metrics, dose-response curves were left-shifted by two orders of magnitude relative to spiked concentrations. Partitioning-controlled dosing of POPs resulted in up to 2-fold increases in progestagen and corticosteroid levels at Cfree of individual POPs in or below the femtomolar range. Silicone acted not only as source of the POPs but also as a sorption sink for lipophilic hormones, stimulating the cellular hormone production. Methodologically, the study showed that silicone can be used as reference partitioning phase to transfer in vivo exposure in humans (silicone implants) to in vitro assays (partition-controlled dosing). The main finding was that POPs at the levels at which they are found in humans can interfere with steroidogenesis in a human adrenocortical cell line. PMID:26264162

  9. [Effects of ryegrass and arbuscular mycorrhiza on activities of antioxidant enzymes, accumulation and chemical forms of cadmium in different varieties of tomato].

    PubMed

    Jiang, Ling; Yang, Yun; Xu, Wei-Hong; Wang, Chong-Li; Chen, Rong; Xiong, Shi-Juan; Xie, Wen-Wen; Zhang, Jin-Zhong; Xiong, Zhi-Ting; Wang, Zheng-Yin; Xie, De-Ti

    2014-06-01

    Pot experiments were carried out to investigate the effects of ryegrass and arbuscular mycorrhiza on the plant growth, malondialdehyde (MDA), antioxidant enzyme activities of leaf and root, accumulation and chemical forms of cadmium (Cd) in tow varieties of tomato when exposed to Cd (20 mg x kg(-1)). The results showed that dry weights of fruit and plant, and contents of malondialdehyde (MDA) and antioxidant enzyme activities of leaf and root, and concentrations and accumulations of Cd significantly differed between two varieties of tomato. Dry weights of fruit, roots, stem, leaf and plant were increased by single or combined remediation of ryegrass and arbuscular mycorrhiza, while MDA contents and antioxidant enzyme activities of leaf and root reduced. The total extractable Cd, F(E), F(W), F(NaCl), F(HAc), F(HCl), and F(R) in fruit of two varieties of tomato reduced by 19.4% - 52.4%, 31.0% - 75.2%, 19.7% - 59.1%, 3.1% - 48.2%, 20.0% - 65.0%, 40.7% - 100.0% and 15.2% - 50.0%, respectively. Cadmium accumulations in tomato were in the order of leaf > stem > fruit > root. Cadmium concentrations in leaf, stem, root and fruit of both varieties decreased by single or combined remediation of ryegrass and arbuscular mycorrhiza, and Cd accumulations of stem and plant of two varieties also reduced. Cd accumulations in fruit of two varieties decreased by 42.9% and 43.7% in the combined remediation treatments, respectively. Tolerance and resistance of 'LUO BEI QI' on Cd was more than 'De Fu mm-8', and Cd concentrations and Cd accumulations in fruit and plant were in the order of 'LUO BEI QI' < 'De Fu mm-8' in the presence or absence of single or combined remediation of ryegrass and arbuscular mycorrhiza.

  10. Thyroid microsomal/thyroid peroxidase autoantibodies show discrete patterns of cross-reactivity to myeloperoxidase, lactoperoxidase and horseradish peroxidase.

    PubMed Central

    Banga, J P; Tomlinson, R W; Doble, N; Odell, E; McGregor, A M

    1989-01-01

    The recent cloning of the thyroid peroxidase (TPO) has shown that it is identical to the thyroid microsomal antigen (TMA), a potent antigen involved in autoimmune thyroid disease (ATD), which shares significant sequence homology with myeloperoxidase. The present study shows that autoantibodies (aAb) to the TMA/TPO antigen cross-react with human leucocyte myeloperoxidase, bovine lactoperoxidase and horseradish peroxidase. Cross-reactivity to myeloperoxidase was only apparent by ELISA using reduced and alkylated antigen preparations or by immunoblotting following denaturation with SDS. Sequential absorption of sera on SDS-denatured thyroid microsomes immobilized on Sepharose-4B followed by absorption on native microsomes removed all aAb specificities to TMA/TPO and the three peroxidase preparations, giving compelling evidence on the genuine cross-reactive nature of these aAbs. Sera from different patients contain different qualitative and quantitative specificities of aAb to the TMA/TPO antigen, confirming the polyclonal nature of this autoimmune response. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2546881

  11. Accumulate Repeat Accumulate Coded Modulation

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative coded modulation scheme called 'Accumulate Repeat Accumulate Coded Modulation' (ARA coded modulation). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes that are combined with high level modulation. Thus at the decoder belief propagation can be used for iterative decoding of ARA coded modulation on a graph, provided a demapper transforms the received in-phase and quadrature samples to reliability of the bits.

  12. The Induction of Tumor Necrosis Factor-alpha , Supeoxide Anion, Myeloperoxidase, and Superoxide Dismutase in the Peritoneal Lavage Cells of Mice after Prolonged Exposure to Dichloroacetate and Trichloroacetate

    PubMed Central

    Spildener, Jessica; Cearfoss, Jacquelyn

    2010-01-01

    The induction of phagocytic activation in response to prolonged treatment with different doses of dichloroacetate (DCA) and trichloroacetate (TCA) has been investigated in mice. Groups of B6C3F1 male mice were administered 7.7, 77, 154 and 410 mg of DCA or TCA/ kg/day , post orally, for 4- and 13-weeks. Peritoneal lavage cells (PLCs) were isolated and assayed for the different biomarkers of phagocytyic activation, including superoxide anion (SA), tumor necrosis factor-alpha (TNF-α), and myeloperoxidase (MPO). In addition, the role of superoxide dismutase (SOD) in the SA production was also assessed. DCA and TCA produced significant and dose-dependent increases in SA and TNF-α production and in MPO activity but the increases in response to the high doses of the compounds (> 77 mg/kg/day) in the 13-week treatment period were less significant than those produced in the 4-week treatment period. Also, dose-dependent increases in SOD activity were observed in both periods of treatments. In general, the results demonstrate significant induction of the biomarkers of phagocytic activation by doses of DCA and TCA that were previously shown to be non carcinogenic, with significantly greater increases observed at the earlier period of exposure, as compared with later period. These findings may argue against the contribution of those mechanisms to the hepatotoxicity/hepatocarcinogenicity of the compounds and suggest them to be early adaptive/ protective mechanisms against their long term effects. PMID:20391627

  13. Constant-load exercise decreases the serum concentration of myeloperoxidase in healthy smokers and smokers with COPD

    PubMed Central

    Holz, Olaf; Roepcke, Stefan; Watz, Henrik; Tegtbur, Uwe; Lahu, Gezim; Hohlfeld, Jens M

    2015-01-01

    There is an ongoing demand for easily accessible biomarkers related to pathophysiological processes in chronic obstructive pulmonary disease (COPD). Short-term intense exercise is known to increase the peripheral blood levels of cytokines. Therefore, we tested the potential and the repeatability of an exercise challenge to amplify seven serum biomarkers (interleukin 6 [IL6], C-reactive protein [CRP], myeloperoxidase [MPO], leukotriene B4, soluble intercellular adhesion molecule 1, soluble vascular cell adhesion molecule 1, and von Willebrand factor [VWF]) in smokers with and without COPD. Twenty-three smokers with moderate COPD (GOLD 2) and 23 sex- and age-matched healthy smokers underwent up to 30-minute submaximal, constant-load exercise (75% of maximum work load) on two occasions separated by 4 weeks (second challenge n=19/20). Serum samples were obtained before, 5 minutes after the start, at the end of exercise (maximum 30 minutes or until exhaustion), and after additional 20 minutes of rest. The median (interquartile range) exercise time until exhaustion in the two challenges was 10.0 (4.0) minutes and 10.0 (8.0) minutes in smokers with COPD and 22.0 (16.0) minutes and 26.5 (14.5) minutes in healthy smokers. The exercise challenge significantly increased the serum concentrations of IL6 and VWF, but decreased the concentrations of MPO. Healthy smokers showed a significantly greater increase (at the end of exercise compared to before exercise) in IL6 (P=0.01) and a larger decline (P=0.03) in MPO. The overall profile of the serum markers during the exercise challenge was shown to be repeatable in the second challenge. In summary, intense load exercise is capable of changing the concentration of inflammatory and endothelial function markers. Especially, the decline in the level of MPO, a marker closely related to cardiovascular risk, appears to be of clinical interest, as the exercise-induced decline might be related to the beneficial effects of physical activity

  14. Supplementation with Sodium Selenite and Selenium-Enriched Microalgae Biomass Show Varying Effects on Blood Enzymes Activities, Antioxidant Response, and Accumulation in Common Barbel (Barbus barbus)

    PubMed Central

    Kouba, Antonín; Velíšek, Josef; Stará, Alžběta; Masojídek, Jiří; Kozák, Pavel

    2014-01-01

    Yearling common barbel (Barbus barbus L.) were fed four purified casein-based diets for 6 weeks in outdoor cages. Besides control diet, these were supplemented with 0.3 mg kg−1 dw selenium (Se) from sodium selenite, or 0.3 and 1.0 mg kg−1 from Se-enriched microalgae biomass (Chlorella), a previously untested Se source for fish. Fish mortality, growth, Se accumulation in muscle and liver, and activity of selected enzymes in blood plasma, muscle, liver, and intestine were evaluated. There was no mortality, and no differences in fish growth, among groups. Se concentrations in muscle and liver, activity of alanine aminotransferase and creatine kinase in blood plasma, glutathione reductase (GR) in muscle, and GR and catalase in muscle and liver suggested that selenium from Se-enriched Chlorella is more readily accumulated and biologically active while being less toxic than sodium selenite. PMID:24772422

  15. [Myeloperoxidase, lactoferrin and elastase in bronchoalveolar lavage and plasma in pneumonia].

    PubMed

    Braun, J; Dalhoff, K; Lipp, R; Eckmann, C; Marre, R; Wood, W G; Wiessmann, K J

    1992-04-01

    Neutrophilic granulocytes in the lower respiratory tract are of decisive importance for the elimination of pathogenic germs in bacterial pneumonia. On the other hand, the liberation of phagocyte products (e.g. elastase) can result in tissue damage in the parenchyma of the lungs. For this reason, we determined in patients suffering from acute pneumonia (n = 21), in patients with acute pneumonia associated with immunosuppression (n = 12), in patients who had overcome their pneumonia (n = 9) and in controls (n = 17) in bronchoalveolar lavage (BALF) and in plasma, the concentration of the locally produced granulocyte products myeloperoxidase (MPO), lactoferrin (LF) and elastase-alpha 1 proteinase complex (ELA) as well as of the alpha 1 proteinase inhibitor (alpha 1 Pi) and alpha 2 proteinase inhibitor (alpha 2 Pi) via chemoluminescence immunoassay, and compared the same with the differential cell count in the BALF. The protein concentrations were referred to the albumin concentration (Alb) for standardisation. This concentration did not differ significantly between the various patients and control groups. The BALF concentration of ELA in the group with pneumonia (median: 86.3 micrograms/l or 8.5 micrograms/mg Alb) was about eight times higher than in the group of patients suffering from pneumonia with immunosuppression (median: 16 micrograms/l or 1.0 micrograms/l Alb, p less than 0.001) or in whom the pneumonia was no longer present (17.6 micrograms/l or 0.5 micrograms/mg), and approximately 40 times higher than in the control group (3 micrograms/l or 0.2 micrograms/mg, respectively). Similar results were obtained for LF (61 micrograms/mg Alb vs. 11.3; 16.8 and 5.9 micrograms/mg; p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1315965

  16. [Myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA) -associated glomerulonephritis with acute pancreatitis: a case report].

    PubMed

    Iida, Takeshi; Amari, Yoshifumi; Yurugi, Takatomi; Nakajima, Fumitaka

    2015-01-01

    We report here a case of a 64-year-old woman with myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA) -associated glomerulonephritis who developed acute pancreatitis. The patient was admitted to our hospital because of abnormal urinalysis findings, edema, and progressive renal failure. Laboratory studies showed a high white blood cell count (11,570/μL), anemia (hemoglobin 7.8 g/dL), and elevated serum creatinine (2.36 mg/dL) and C-reactive protein (12.20 mg/dL) levels. Furthermore, the MPO-ANCA titer was very high (1,625 U/mL, normal range < 10 U/mL). Histopathological findings of the renal biopsy were consistent with microscopic polyangiitis. Accordingly, we diagnosed MPO-ANCA-associated glomerulonephritis. On the day after the renal biopsy, the patient complained of low back pain. Computed tomography (CT) revealed postbiopsy hemorrhage. Thereafter, the patient's symptoms and laboratory studies gradually worsened. A repeat CT performed a few days later revealed no changes in the perirenal hematoma; however, an enlarged pancreas head was incidentally observed. There was no obvious cause of acute pancreatitis, and MPO-ANCA-associated vasculitis, although rare, was suspected as the cause. We initiated prednisolone pulse therapy for vasculitis along with the administration of nafamostat mesilate and ulinastatin for acute pancreatitis. Subsequently, the levels of pancreatic enzymes gradually increased, but several days later, abdominal magnetic resonance imaging showed improvement in the pancreas head. The pancreatitis gradually resolved over time. Acute pancreatitis occurring concurrently with MPO-ANCA-associated glomerulonephritis is extremely rare. To our knowledge, only a few such cases have been reported and have suggested that steroid therapy may play a role in triggering pancreatic involvement. In our case, however, an enlarged pancreas head was observed before steroid therapy was initiated. Therefore, we consider our case to be very rare. PMID

  17. Decreased cytochrome-c oxidase activity and lack of age-related accumulation of mitochondrial DNA deletions in the brains of schizophrenics

    SciTech Connect

    Cavelier, L.; Jazin, E.E.; Eriksson, I.

    1995-09-01

    Defects in mitochondrial energy production have been implicated in several neurodegenerative disorders, such as Parkinson disease and amyotrophic lateral sclerosis. To study the contribution of mitochondrial defects to Alzheimer disease and schizophrenia, cytochrome-c oxidase (COX) activity and levels of the mtDNA{sup 4977} deletion in postmortem brain tissue specimens of patients were compared with those of asymptomatic age-matched controls. No difference in COX activity was observed between Alzheimer patients and controls in any of five brain regions investigated. In contrast, schizophrenic patients had a 63% reduction of the COX activity in the nucleus caudatus (P<0.0001) and a 43% reduction in the cortex gyrus frontalis (P<0.05) as compared to controls. The average levels of the mtDNA{sup 4977} deletion did not differ significantly between Alzheimer patients and controls, and the deletion followed similar modes of accumulation with age in the two groups. In contrast, no age-related accumulation of mtDNA deletions was found in schizophrenic patients. The reduction in COX activity in schizophrenic patients did not correlate with changes in the total amount of mtDNA or levels of the mtDNA{sup 4977} deletion. The lack of age-related accumulation of the mtDNA{sup 4977} deletion and reduction in COX activity suggest that a mitochondrial dysfunction may be involved in the pathogenesis of schizophrenia. 41 refs., 3 figs., 1 tab.

  18. Enhanced Nrf2 Activity Worsens Insulin Resistance, Impairs Lipid Accumulation in Adipose Tissue, and Increases Hepatic Steatosis in Leptin-Deficient Mice

    PubMed Central

    Xu, Jialin; Kulkarni, Supriya R.; Donepudi, Ajay C.; More, Vijay R.; Slitt, Angela L.

    2012-01-01

    The study herein determined the role of nuclear factor erythoid 2–related factor 2 (Nrf2) in the pathogenesis of hepatic steatosis, insulin resistance, obesity, and type 2 diabetes. Lepob/ob-Keap1-knockdown (KD) mice, which have increased Nrf2 activity, were generated. Markers of obesity and type 2 diabetes were measured in C57Bl/6J, Keap1-KD, Lepob/ob, and Lepob/ob-Keap1-KD mice. Lepob/ob-Keap1-KD mice exhibited less lipid accumulation, smaller adipocytes, decreased food intake, and reduced lipogenic gene expression. Enhanced Nrf2 activity impaired insulin signaling, prolonged hyperglycemia in response to glucose challenge, and induced insulin resistance in Lepob/ob background. Nrf2 augmented hepatic steatosis and increased lipid deposition in liver. Next, C57Bl/6J and Keap1-KD mice were fed a high-fat diet (HFD) to determine whether Keap1 and Nrf2 impact HFD-induced obesity. HFD-induced obesity and lipid accumulation in white adipose tissue was decreased in Keap1-KD mice. Nrf2 activation via Keap1-KD or sulforaphane suppressed hormone-induced differentiation and decreased peroxisome proliferator–activated receptor-γ, CCAAT/enhancer–binding protein α, and fatty acid–binding protein 4 expression in mouse embryonic fibroblasts. Constitutive Nrf2 activation inhibited lipid accumulation in white adipose tissue, suppressed adipogenesis, induced insulin resistance and glucose intolerance, and increased hepatic steatosis in Lepob/ob mice. PMID:22936178

  19. Distributions and activities of ammonia oxidizing bacteria and polyphosphate accumulating organisms in a pumped-flow biofilm reactor.

    PubMed

    Wu, Guangxue; Nielsen, Michael; Sorensen, Ketil; Zhan, Xinmin; Rodgers, Michael

    2009-10-01

    The spatial distributions and activities of ammonia oxidizing bacteria (AOB) and polyphosphate accumulating organisms (PAOs) were investigated for a novel laboratory-scale sequencing batch pumped-flow biofilm reactor (PFBR) system that was operated for carbon, nitrogen and phosphorus removal. The PFBR comprised of two 16.5l tanks (Reactors 1 and 2), each with a biofilm module of 2m(2) surface area. To facilitate the growth of AOB and PAOs in the reactor biofilms, the influent wastewater was held in Reactor 1 under stagnant un-aerated conditions for 6 h after feeding, and was then pumped over and back between Reactors 1 and 2 for 12 h, creating aerobic conditions in the two reactors during this period; as a consequence, the biofilm in Reactor 2 was in an aerobic environment for almost all the 18.2 h operating cycle. A combination of micro-sensor measurements, molecular techniques, batch experiments and reactor studies were carried out to analyse the performance of the PFBR system. After 100 days operation at a filtered chemical oxygen demand (COD(f)) loading rate of 3.46 g/m(2) per day, the removal efficiencies were 95% COD(f), 87% TN(f) and 74% TP(f). While the PFBR microbial community structure and function were found to be highly diversified with substantial AOB and PAO populations, about 70% of the phosphorus release potential and almost 100% of the nitrification potential were located in Reactors 1 and 2, respectively. Co-enrichment of AOB and PAOs was realized in the Reactor 2 biofilm, where molecular analyses revealed unexpected microbial distributions at micro-scale, with population peaks of AOB in a 100-250 microm deep sub-surface zone and of PAOs in the 0-150 microm surface zone. The micro-distribution of AOB coincided with the position of the nitrification peak identified during micro-sensor analyses. The study demonstrates that enrichment of PAOs can be realized in a constant or near constant aerobic biofilm environment. Furthermore, the findings suggest

  20. Transcriptional activation of a MYB gene controls the tissue-specific anthocyanin accumulation in a purple cauliflower mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavonoids such as anthocyanins possess significant health benefits to humans and play important physiological roles in plants. An interesting Purple gene mutation in cauliflower confers an abnormal pattern of anthocyanin accumulation, giving intense purple color in very young leaves, curds, and see...

  1. Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation and pathogenicity.

    PubMed

    Shnaiderman, Chen; Miyara, Itay; Kobiler, Ilana; Sherman, Amir; Prusky, Dov

    2013-03-01

    Ammonium secreted by the post-harvest pathogen Colletotrichum gloeosporioides during host colonization accumulates in the host environment due to enhanced fungal nitrogen metabolism. Two types of ammonium transporter-encoding genes, AMET and MEP, are expressed during pathogenicity. Gene disruption of AMET, a gene modulating ammonia secretion, showed twofold reduced ammonia secretion and 45% less colonization on avocado fruit, suggesting a contribution to pathogenicity. MEPB, a gene modulating ammonium transport, is expressed by C. gloeosporioides during pathogenicity and starvation conditions in culture. Gene disruption of MEPB, the most highly expressed gene of the MEP family, resulted in twofold overexpression of MEPA and MEPC but reduced colonization, suggesting MEPB expression's contribution to pathogenicity. Analysis of internal and external ammonia accumulation by ΔmepB strains in mycelia and germinated spores showed rapid uptake and accumulation, and reduced secretion of ammonia in the mutant versus wild-type (WT) strains. Ammonia uptake by the WT germinating spores but not by the ΔmepB strain with compromised ammonium transport activated cAMP and transcription of PKA subunits PKAR and PKA2. ΔmepB mutants showed 75% less appressorium formation and colonization than the WT, which was partially restored by 10 mM exogenous ammonia. Thus, whereas both AMET and MEPB genes modulate ammonia secretion, only MEPB contributes to ammonia accumulation by mycelia and germinating spores that activate the cAMP pathways, inducing the morphogenetic processes contributing to C. gloeosporioides pathogenicity. PMID:23387470

  2. Heat accumulator

    SciTech Connect

    Bracht, A.

    1981-09-29

    A heat accumulator comprises a thermally-insulated reservoir full of paraffin wax mixture or other flowable or meltable heat storage mass, heat-exchangers immersed in the mass, a heat-trap connected to one of the heat-exchangers, and a heat user connected to the other heat-exchanger. Pumps circulate fluids through the heat-trap and the heat-using means and the respective heat-exchangers, and a stirrer agitates and circulates the mass, and the pumps and the stirrer and electric motors driving these devices are all immersed in the mass.

  3. Cellular cholesterol accumulation modulates high fat high sucrose (HFHS) diet-induced ER stress and hepatic inflammasome activation in the development of non-alcoholic steatohepatitis.

    PubMed

    Bashiri, Amir; Nesan, Dinushan; Tavallaee, Ghazaleh; Sue-Chue-Lam, Ian; Chien, Kevin; Maguire, Graham F; Naples, Mark; Zhang, Jing; Magomedova, Lilia; Adeli, Khosrow; Cummins, Carolyn L; Ng, Dominic S

    2016-07-01

    Non-alcoholic steatohepatitis (NASH), is the form of non-alcoholic fatty liver disease posing risk to progress into serious long term complications. Human and pre-clinical models implicate cellular cholesterol dysregulation playing important role in its development. Mouse model studies suggest synergism between dietary cholesterol and fat in contributing to NASH but the mechanisms remain poorly understood. Our laboratory previously reported the primary importance of hepatic endoplasmic reticulum cholesterol (ER-Chol) in regulating hepatic ER stress by comparing the responses of wild type, Ldlr-/-xLcat+/+ and Ldlr-/-xLcat-/- mice, to a 2% high cholesterol diet (HCD). Here we further investigated the roles of ER-Chol and ER stress in HFHS diet-induced NASH using the same strains. With HFHS diet feeding, both WT and Ldlr-/-xLcat+/+ accumulate ER-Chol in association with ER stress and inflammasome activation but the Ldlr-/-xLcat-/- mice are protected. By contrast, all three strains accumulate cholesterol crystal, in correlation with ER-Chol, albeit less so in Ldlr-/-xLcat-/- mice. By comparison, HCD feeding per se (i) is sufficient to promote steatosis and activate inflammasomes, and (ii) results in dramatic accumulation of cholesterol crystal which is linked to inflammasome activation in Ldlr-/-xLcat-/- mice, independent of ER-Chol. Our data suggest that both dietary fat and cholesterol each independently promote steatosis, cholesterol crystal accumulation and inflammasome activation through distinct but complementary pathways. In vitro studies using palmitate-induced hepatic steatosis in HepG2 cells confirm the key roles by cellular cholesterol in the induction of steatosis and inflammasome activations. These novel findings provide opportunities for exploring a cellular cholesterol-focused strategy for treatment of NASH. PMID:27090939

  4. Temporal Resolution of Misfolded Prion Protein Transport, Accumulation, Glial Activation, and Neuronal Death in the Retinas of Mice Inoculated with Scrapie.

    PubMed

    West Greenlee, M Heather; Lind, Melissa; Kokemuller, Robyn; Mammadova, Najiba; Kondru, Naveen; Manne, Sireesha; Smith, Jodi; Kanthasamy, Anumantha; Greenlee, Justin

    2016-09-01

    Currently, there is a lack of pathological landmarks to describe the progression of prion disease in vivo. Our goal was to use an experimental model to determine the temporal relationship between the transport of misfolded prion protein (PrP(Sc)) from the brain to the retina, the accumulation of PrP(Sc) in the retina, the response of the surrounding retinal tissue, and loss of neurons. Retinal samples from mice inoculated with RML scrapie were collected at 30, 60, 90, 105, and 120 days post inoculation (dpi) or at the onset of clinical signs of disease (153 dpi). Retinal homogenates were tested for prion seeding activity. Antibody staining was used to assess accumulation of PrP(Sc) and the resulting response of retinal tissue. Loss of photoreceptors was used as a measure of neuronal death. PrP(Sc) seeding activity was first detected in all samples at 60 dpi. Accumulation of PrP(Sc) and coincident activation of retinal glia were first detected at 90 dpi. Activation of microglia was first detected at 105 dpi, but neuronal death was not detectable until 120 dpi. Our results demonstrate that by using the retina we can resolve the temporal separation between several key events in the pathogenesis of prion disease. PMID:27521336

  5. AMPK-HDAC5 pathway facilitates nuclear accumulation of HIF-1α and functional activation of HIF-1 by deacetylating Hsp70 in the cytosol

    PubMed Central

    Chen, Shuyang; Yin, Chengqian; Lao, Taotao; Liang, Dongming; He, Dan; Wang, Chenguang; Sang, Nianli

    2015-01-01

    Hypoxia-inducible factor 1 (HIF-1) transcriptionally promotes production of adenosine triphosphate (ATP) whereas AMPK senses and regulates cellular energy homeostasis. A histone deacetylase (HDAC) activity has been proven to be critical for HIF-1 activation but the underlying mechanism and its role in energy homesostasis remain unclear. Here, we demonstrate that HIF-1 activation depends on a cytosolic, enzymatically active HDAC5. HDAC5 knockdown impairs hypoxia-induced HIF-1α accumulation and HIF-1 transactivation, whereas HDAC5 overexpression enhances HIF-1α stabilization and nuclear translocation. Mechanistically, we show that Hsp70 is a cytosolic substrate of HDAC5; and hyperacetylation renders Hsp70 higher affinity for HIF-1α binding, which correlates with accelerated degradation and attenuated nuclear accumulation of HIF-1α. Physiologically, AMPK-triggered cytosolic shuttling of HDAC5 is critical; inhibition of either AMPK or HDAC5 impairs HIF-1α nuclear accumulation under hypoxia or low glucose conditions. Finally, we show specifically suppressing HDAC5 is sufficient to inhibit tumor cell proliferation under hypoxic conditions. Our data delineate a novel link between AMPK, the energy sensor, and HIF-1, the major driver of ATP production, indicating that specifically inhibiting HDAC5 may selectively suppress the survival and proliferation of hypoxic tumor cells. PMID:26061431

  6. Increased Variation in Adh Enzyme Activity in Drosophila Mutation-Accumulation Experiment Is Not Due to Transposable Elements at the Adh Structural Gene

    PubMed Central

    Aquadro, C. F.; Tachida, H.; Langley, C. H.; Harada, K.; Mukai, T.

    1990-01-01

    We present here a molecular analysis of the region surrounding the structural gene encoding alcohol dehydrogenase (Adh) in 47 lines of Drosophila melanogaster that have each accumulated mutations for 300 generations. While these lines show a significant increase in variation of alcohol dehydrogenase enzyme activity compared to control lines, we found no restriction map variation in a 13-kb region including the complete Adh structural gene and roughly 5 kb of both 5' and 3' sequences. Thus, the rapid accumulation of ADH activity variation after 28,200 allele generations does not appear to have been due to the mobilization of transposable elements into or out of the Adh structural gene region. PMID:1963870

  7. The activity of HYDROPEROXIDE LYASE 1 regulates accumulation of galactolipids containing 12-oxo-phytodienoic acid in Arabidopsis

    PubMed Central

    Nilsson, Anders K.; Fahlberg, Per; Johansson, Oskar N.; Hamberg, Mats; Andersson, Mats X.; Ellerström, Mats

    2016-01-01

    Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids. PMID:27422994

  8. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation.

    PubMed

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  9. Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation

    SciTech Connect

    Yu, Jung Hwan; Lee, Yoo Jeong; Kim, Hyo Jung; Choi, Hyeonjin; Choi, Yoonjeong; Seok, Jo Woon; Kim, Jae-woo

    2015-05-08

    Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans. - Highlights: • PPARγ promotes MGAT1 expression in human primary hepatocytes. • PPARγ directly regulates MGAT1 promoter activity. • Human MGAT1 promoter has at least two PPARγ-binding elements. • Inhibition of MGAT1 expression attenuates hepatic lipid accumulation in humans.

  10. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

    PubMed Central

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  11. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

    SciTech Connect

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H{sub 2}O{sub 2}, rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  12. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells.

    PubMed

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H(2)O(2), rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  13. Maintenance of luminal pH and protease activity in lysosomes/late endosomes by vacuolar ATPase in chlorpromazine-treated RAW264 cells accumulating phospholipids.

    PubMed

    Hamaguchi, Ryohei; Haginaka, Jun; Tanimoto, Toshiko; Kuroda, Yukihiro

    2014-02-01

    Cationic amphiphilic drugs (CADs) inhibit phospholipases competitively/uncompetitively. It has also been reported that CADs spontaneously accumulate in acidic organelles and increase their luminal pH, which may lead to deactivation of phospholipid-metabolising enzymes, causing cellular phospholipid accumulation. Recently, however, contradictory results have also been reported in that the luminal pH is not increased by CAD treatment. In this study, we examined whether the lysosomal/late endosomal acidic pH was maintained by vacuolar ATPase (v-ATPase) after treatment with chlorpromazine (CPZ) as a model CAD. The activity of lysosomal protease after CPZ treatment was also measured. Oregon Green-dextran-tetramethylrhodamine conjugate was employed to determine the luminal pH of the lysosomes/late endosomes in RAW264 cells. The luminal pH remained acidic after treatment with CPZ for 23 h, and the lysosomal protease activity was not decreased by 5-min CPZ treatment. Co-treatment with CPZ and bafilomycin A1 (v-ATPase inhibitor) raised the luminal pH. These results suggest that the lysosomal/late endosomal pH is not affected by a 23-h CPZ treatment. In addition, lysosomal enzymes presumably maintain their activity when CPZ accumulates. Our results imply that the pH homeostasis in lysosomes/late endosomes is strictly maintained even after a longer treatment with CADs.

  14. An activator of protein kinase C (phorbol dibutyrate) attenuates atrial-natriuretic-factor-stimulated cyclic GMP accumulation in smooth-muscle cells.

    PubMed Central

    Nambi, P; Whitman, M; Aiyar, N; Stassen, F; Crooke, S T

    1987-01-01

    Rat thoracic aortic smooth-muscle cells (A-10; A.T.C.C. CRL 1476) displays a high density of vasopressin and atrial-natriuretic-factor (ANF) receptors and a low density of beta-adrenergic receptors. ANF stimulates cGMP (cyclic GMP) accumulation in a time- and dose-dependent fashion. Pretreatment of these cells with phorbol dibutyrate (PDBu), a known activator of protein kinase C, attenuated ANF-stimulated cGMP accumulation without affecting basal cGMP concentrations. This effect was concentration-dependent and was observed as early as 2 min after treatment. 4 alpha-Phorbol 12, 13-didecanoate (alpha PDD), which does not activate protein kinase C, did not inhibit the cGMP accumulation. PDBu pretreatment did not affect the density and affinity of ANF receptors. These data suggest that PDBu, presumably via activation of protein kinase C, might stimulate phosphorylation of a key regulatory protein in the ANF/cGMP pathway. PMID:2822009

  15. Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α' subunit of soybean β-conglycinin.

    PubMed

    Cabanos, Cerrone; Kato, Naoki; Amari, Yoshiki; Fujiwara, Keigo; Ohno, Tomoki; Shimizu, Kousuke; Goto, Tsuyoshi; Shimada, Masaya; Kuroda, Masaharu; Masuda, Taro; Takaiwa, Fumio; Utsumi, Shigeru; Nagaoka, Satoshi; Maruyama, Nobuyuki

    2014-08-01

    Soybean 7S globulin, known as β-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of β-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of β-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant 'Koshihikari' (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α' subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α' subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α' subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the β-conglycinin α' subunit.

  16. CD28/B7 Deficiency Attenuates Systolic Overload-Induced Congestive Heart Failure, Myocardial and Pulmonary Inflammation, and Activated T Cell Accumulation in the Heart and Lungs.

    PubMed

    Wang, Huan; Kwak, Dongmin; Fassett, John; Hou, Lei; Xu, Xin; Burbach, Brandon J; Thenappan, Thenappan; Xu, Yawei; Ge, Jun-Bo; Shimizu, Yoji; Bache, Robert J; Chen, Yingjie

    2016-09-01

    The inflammatory response regulates congestive heart failure (CHF) development. T cell activation plays an important role in tissue inflammation. We postulate that CD28 or B7 deficiency inhibits T cell activation and attenuates CHF development by reducing systemic, cardiac, and pulmonary inflammation. We demonstrated that chronic pressure overload-induced end-stage CHF in mice is characterized by profound accumulation of activated effector T cells (CD3(+)CD44(high) cells) in the lungs and a mild but significant increase of these cells in the heart. In knockout mice lacking either CD28 or B7, there was a dramatic reduction in the accumulation of activated effector T cells in both hearts and lungs of mice under control conditions and after transverse aortic constriction. CD28 or B7 knockout significantly attenuated transverse aortic constriction-induced CHF development, as indicated by less increase of heart and lung weight and less reduction of left ventricle contractility. CD28 or B7 knockout also significantly reduced transverse aortic constriction-induced CD45(+) leukocyte, T cell, and macrophage infiltration in hearts and lungs, lowered proinflammatory cytokine expression (such as tumor necrosis factor-α and interleukin-1β) in lungs. Furthermore, CD28/B7 blockade by CTLA4-Ig treatment (250 μg/mouse every 3 days) attenuated transverse aortic constriction-induced T cell activation, left ventricle hypertrophy, and left ventricle dysfunction. Our data indicate that CD28/B7 deficiency inhibits activated effector T cell accumulation, reduces myocardial and pulmonary inflammation, and attenuates the development of CHF. Our findings suggest that strategies targeting T cell activation may be useful in treating CHF. PMID:27432861

  17. CD28/B7 Deficiency Attenuates Systolic Overload-Induced Congestive Heart Failure, Myocardial and Pulmonary Inflammation, and Activated T Cell Accumulation in the Heart and Lungs.

    PubMed

    Wang, Huan; Kwak, Dongmin; Fassett, John; Hou, Lei; Xu, Xin; Burbach, Brandon J; Thenappan, Thenappan; Xu, Yawei; Ge, Jun-Bo; Shimizu, Yoji; Bache, Robert J; Chen, Yingjie

    2016-09-01

    The inflammatory response regulates congestive heart failure (CHF) development. T cell activation plays an important role in tissue inflammation. We postulate that CD28 or B7 deficiency inhibits T cell activation and attenuates CHF development by reducing systemic, cardiac, and pulmonary inflammation. We demonstrated that chronic pressure overload-induced end-stage CHF in mice is characterized by profound accumulation of activated effector T cells (CD3(+)CD44(high) cells) in the lungs and a mild but significant increase of these cells in the heart. In knockout mice lacking either CD28 or B7, there was a dramatic reduction in the accumulation of activated effector T cells in both hearts and lungs of mice under control conditions and after transverse aortic constriction. CD28 or B7 knockout significantly attenuated transverse aortic constriction-induced CHF development, as indicated by less increase of heart and lung weight and less reduction of left ventricle contractility. CD28 or B7 knockout also significantly reduced transverse aortic constriction-induced CD45(+) leukocyte, T cell, and macrophage infiltration in hearts and lungs, lowered proinflammatory cytokine expression (such as tumor necrosis factor-α and interleukin-1β) in lungs. Furthermore, CD28/B7 blockade by CTLA4-Ig treatment (250 μg/mouse every 3 days) attenuated transverse aortic constriction-induced T cell activation, left ventricle hypertrophy, and left ventricle dysfunction. Our data indicate that CD28/B7 deficiency inhibits activated effector T cell accumulation, reduces myocardial and pulmonary inflammation, and attenuates the development of CHF. Our findings suggest that strategies targeting T cell activation may be useful in treating CHF.

  18. Acceleration of large active earthflows triggered by massive snow accumulation events: evidences from monitoring the Corvara landslide in early 2014 (Dolomites, Italy)

    NASA Astrophysics Data System (ADS)

    Corsini, Alessandro; Mulas, Marco; Marcato, Gianluca; Chinellato, Giulia; Mair, Volkmar

    2015-04-01

    In the Dolomites of Italy, snowfall during winter 2013/2014 was exceptionally abundant. Major snowfall events occurred from late December 2013 to mid-March 2014. Snow accumulation in Badia Valley peaked in early February: from 2 to 4 meters with a positive gradient respect to altimetry and accordingly to wind accumulation zones. Below 2000 m asl, due to the mild temperatures recorded before the onset of snowfall, the relatively dry snow cover was mostly deposited on top of unfrozen soils. The Corvara landslide is a large active earthflow located close to Corvara in Badia, at an elevation from 2000 to 1600 m. It's displacement rate before, during and after the exceptional snowfall period was monitored at high temporal frequency. Surface displacement was measured bi-weekly by differential GPS in several benchmarks in the source, track and accumulation zone. Deep displacement was monitored semi-continuously by two in-place inclinometers at 48 m depth in the accumulation zone, across the main deep-seated sliding surface. Results show an acceleration of movements, both at surface and at depth, soon after the massive snow accumulation event of 31st January to 2nd February 2014, which suddenly increased snow thickness from 1 to more than 2 metres. Short time lags between the onset of the acceleration of movements in the source, the track and the accumulation zones were also recorded. The landslide then maintained a relatively constant velocity during the high snow cover period extended to earlyApril and underwent a progressive deceleration during the snowmelt period that lasted until mid-June. The fact that the acceleration of the Corvara earthflow was triggered by a massive and rapid snow accumulation event, provides a quite different perspective from the generally adopted one that considers the destabilizing effect of snow only in relation to the increase of groundwater level during rapid snowmelt. A full explanation of the processes associated to the dynamics observed

  19. Accumulation of biologically active furanocoumarins in agitated cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams.

    PubMed

    Ekiert, H; Czygan, F-Ch

    2005-08-01

    This study was designed to investigate the dynamics of accumulation of linear furanocoumarins (psoralen, bergapten, xanthotoxin, isopimpinellin, imperatorin) and their biogenetic precursor, umbelliferone, in agitated cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams during 6-week growth cycles. The metabolites under study were almost exclusively accumulated in the cultured biomass. The total content of all metabolites increased 4.8- and 2.0-fold, in R. graveolens and R. graveolens ssp. divaricata cultures, respectively. Xanthotoxin and bergapten, which are the most important therapeutic compounds, were the dominating metabolites in cultures of both plants. The maximum content of xanthotoxin (25.0 mg/100 g dry wt.) and bergapten (18.4 mg/100 g dry wt) and the maximum content of all metabolites (64.0 mg/100 g dry wt) in R. graveolens ssp. divaricata callus obtained on the 35th culture day were relatively low. However, maximum contents of xanthotoxin (136.8 mg/100 g dry wt), bergapten (210.4 mg/100 g dry wt.) and isopimpinellin (96.7 mg/100 g dry wt), and total content of all metabolites in R. graveolens shoots (520.8 mg/100 g dry wt) obtained on the 42nd culture day are interesting from a practical point of view.

  20. Increasing sediment accumulation rates in La Fonera (Palamós) submarine canyon axis and their relationship with bottom trawling activities

    NASA Astrophysics Data System (ADS)

    Puig, P.; Martín, J.; Masqué, P.; Palanques, A.

    2015-10-01

    Previous studies conducted in La Fonera (Palamós) submarine canyon (NW Mediterranean) found that trawling activities along the canyon flanks cause resuspension and transport of sediments toward the canyon axis. 210Pb chronology supported by 137Cs dating applied to a sediment core collected at 1750 m in 2002 suggested a doubling of the sediment accumulation rate since the 1970s, coincident with the rapid industrialization of the local trawling fleet. The same canyon area has been revisited a decade later, and new data are consistent with a sedimentary regime shift during the 1970s and also suggest that the accumulation rate during the last decade could be greater than expected, approaching ~2.4 cm yr-1 (compared to ~0.25 cm yr-1 pre-1970s). These results support the hypothesis that commercial bottom trawling can substantially affect sediment dynamics and budgets on continental margins, eventually initiating the formation of anthropogenic depocenters in submarine canyon environments.

  1. Starch biosynthetic genes and enzymes are expressed and active in the absence of starch accumulation in sugar beet tap-root

    PubMed Central

    2014-01-01

    Background Starch is the predominant storage compound in underground plant tissues like roots and tubers. An exception is sugar beet tap-root (Beta vulgaris ssp altissima) which exclusively stores sucrose. The underlying mechanism behind this divergent storage accumulation in sugar beet is currently not fully known. From the general presence of starch in roots and tubers it could be speculated that the lack in sugar beet tap-roots would originate from deficiency in pathways leading to starch. Therefore with emphasis on starch accumulation, we studied tap-roots of sugar beet using parsnip (Pastinaca sativa) as a comparator. Results Metabolic and structural analyses of sugar beet tap-root confirmed sucrose as the exclusive storage component. No starch granules could be detected in tap-roots of sugar beet or the wild ancestor sea beet (Beta vulgaris ssp. maritima). Analyses of parsnip showed that the main storage component was starch but tap-root tissue was also found to contain significant levels of sugars. Surprisingly, activities of four main starch biosynthetic enzymes, phosphoglucomutase, ADP-glucose pyrophosphorylase, starch synthase and starch branching enzyme, were similar in sugar beet and parsnip tap-roots. Transcriptional analysis confirmed expression of corresponding genes. Additionally, expression of genes involved in starch accumulation such as for plastidial hexose transportation and starch tuning functions could be determined in tap-roots of both plant species. Conclusion Considering underground storage organs, sugar beet tap-root upholds a unique property in exclusively storing sucrose. Lack of starch also in the ancestor sea beet indicates an evolved trait of biological importance. Our findings in this study show that gene expression and enzymatic activity of main starch biosynthetic functions are present in sugar beet tap-root during storage accumulation. In view of this, the complete lack of starch in sugar beet tap-roots is enigmatic. PMID

  2. Waste activated sludge hydrolysis and short-chain fatty acids accumulation under mesophilic and thermophilic conditions: effect of pH.

    PubMed

    Zhang, Peng; Chen, Yinguang; Zhou, Qi

    2009-08-01

    The effect of pH (4.0-11.0) on waste activated sludge (WAS) hydrolysis and short-chain fatty acids (SCFAs) accumulation under mesophilic and thermophilic conditions were investigated. The WAS hydrolysis increased markedly in thermophilic fermentation compared to mesophilic fermentation at any pH investigated. The hydrolysis at alkaline pHs (8.0-11.0) was greater than that at acidic pHs, but both of the acidic and alkaline hydrolysis was higher than that pH uncontrolled under either mesophilic or thermophilic conditions. No matter in mesophilic or thermophilic fermentation, the accumulation of SCFAs at alkaline pHs was greater than at acidic or uncontrolled pHs. The optimum SCFAs accumulation was 0.298g COD/g volatile suspended solids (VSS) with mesophilic fermentation, and 0.368 with thermophilic fermentation, which was observed respectively at pH 9.0 and fermentation time 5 d and pH 8.0 and time 9 d. The maximum SCFAs productions reported in this study were much greater than that in the literature. The analysis of the SCFAs composition showed that acetic acid was the prevalent acid in the accumulated SCFAs at any pH investigated under both temperatures, followed by propionic acid and n-valeric acid. Nevertheless, during the entire mesophilic and thermophilic fermentation the activity of methanogens was inhibited severely at acid or alkaline pHs, and the highest methane concentration was obtained at pH 7.0 in most cases. The studies of carbon mass balance showed that during WAS fermentation the reduction of VSS decreased with the increase of pH, and the thermophilic VSS reduction was greater than the mesophilic one. Further investigation indicated that most of the reduced VSS was converted to soluble protein and carbohydrate and SCFAs in two fermentations systems, while little formed methane and carbon dioxide.

  3. Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals.

    PubMed

    Khan, Saifur R; Baghdasarian, Argishti; Nagar, Prarthna H; Fahlman, Richard; Jurasz, Paul; Michail, Karim; Aljuhani, Naif; Siraki, Arno G

    2015-09-01

    In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

  4. Heme-linked ionizations of myeloperoxidase detected by Raman difference spectroscopy. A comparison with plant and yeast peroxidases.

    PubMed Central

    Stump, R F; Deanin, G G; Oliver, J M; Shelnutt, J A

    1987-01-01

    The pH-dependence of the oxidation state marker line v4 of human leucocyte myeloperoxidase is determined in the absence of chloride using Raman difference spectroscopy (RDS). A transition in the frequency of v4 with pK of 4.2 +/- 0.3 is found. The pK compares favorably with that previously determined by spectrophotometric titration and kinetic studies. The shift in v4 across the transition is -1.3 cm-1. The shift in v4 and other Raman marker lines indicates enhanced pi charge in the chlorin ring below the transition. The low frequencies of the oxidation state marker lines indicate that a structural change occurs near the chromophore, which results in the formation of a more pi-charge donating protein environment for the chlorin ring at low pH. The Raman results are discussed in terms of a proposed catalytic control mechanism based on charge stabilization of the energy of ring charge-depleted ferryl intermediates of the reaction with peroxide. The myeloperoxidase findings are compared with similar RDS results for ferrous horseradish peroxidase and ferric cytochrome c peroxidase. PMID:3034344

  5. IL-21 Receptor Is Required for the Systemic Accumulation of Activated B and T Lymphocytes in MRL/MpJ-Faslpr/lpr/J Mice

    PubMed Central

    Rankin, Andrew L.; Guay, Heath; Herber, Deborah; Bertino, Sarah A.; Duzanski, Tatyana A.; Carrier, Yijun; Keegan, Sean; Senices, Mayra; Stedman, Nancy; Ryan, Mark; Bloom, Laird; Medley, Quintus; Collins, Mary; Nickerson-Nutter, Cheryl; Craft, Joe; Young, Deborah; Dunussi-Joannopoulos, Kyri

    2013-01-01

    MRL/MpJ-Faslpr/lpr/J (MRLlpr) mice develop lupus-like disease manifestations in an IL-21–dependent manner. IL-21 is a pleio-tropic cytokine that can influence the activation, differentiation, and expansion of B and T cell effector subsets. Notably, auto-reactive CD4+ T and B cells spontaneously accumulate in MRLlpr mice and mediate disease pathogenesis. We sought to identify the particular lymphocyte effector subsets regulated by IL-21 in the context of systemic autoimmunity and, thus, generated MRLlpr mice deficient in IL-21R (MRLlpr.IL-21R−/−). Lymphadenopathy and splenomegaly, which are characteristic traits of the MRLlpr model were significantly reduced in the absence of IL-21R, suggesting that immune activation was likewise decreased. Indeed, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R–deficient MRLlpr mice. Correspondingly, we observed a significant reduction in autoantibody titers. Activated CD4+ CD44+ CD62Llo T cells also failed to accumulate, and CD4+ Th cell differentiation was impaired, as evidenced by a significant reduction in CD4+ T cells that produced the pronephritogenic cytokine IFN-γ. T extrafollicular helper cells are a recently described subset of activated CD4+ T cells that function as the primary inducers of autoantibody production in MRLlpr mice. Importantly, we demonstrated that T extrafollicular helper cells are dependent on IL-21R for their generation. Together, our data highlighted the novel observation that IL-21 is a critical regulator of multiple pathogenic B and T cell effector subsets in MRLlpr mice. PMID:22231702

  6. Effect of operational strategies on activated sludge's acclimation to phenol, subsequent aerobic granulation, and accumulation of polyhydoxyalkanoates.

    PubMed

    Wosman, Afrida; Lu, Yuhao; Sun, Supu; Liu, Xiang; Wan, Chunli; Zhang, Yi; Lee, Duu-Jong; Tay, JooHwa

    2016-11-01

    Aerobic granules, a relative novel form of microbial aggregate, are capable of degrading many toxic organic pollutants. Appropriate strategy is needed to acclimate seed sludge to the toxic compounds for successful granulation. In this study, two distinct strategies, i.e. mixed or single carbon sources, were experimented to obtain phenol-acclimated sludge. Their effects on reactor performance, biomass characteristics, microbial population and the granulation process were analyzed. Sludge fed with phenol alone exhibited faster acclimation and earlier appearance of granules, but possibly lower microbial diversity and reactor stability. Using a mixture of acetate and phenol in the acclimation stage, on the other hand, led to a reactor with slower phenol degradation and granulation, but eventual formation of strong and stable aerobic granules. In addition, the content of intracellular polyhydoxyakanoates (PHA) was also monitored, and significant accumulation was observed during the pre-granulation stage, where PHA >50% of dry weight was observed in both reactors. PMID:27281169

  7. Differential regulation of grain sucrose accumulation and metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta) revealed through gene expression and enzyme activity analysis.

    PubMed

    Privat, Isabelle; Foucrier, Séverine; Prins, Anneke; Epalle, Thibaut; Eychenne, Magali; Kandalaft, Laurianne; Caillet, Victoria; Lin, Chenwei; Tanksley, Steve; Foyer, Christine; McCarthy, James

    2008-01-01

    * Coffea arabica (Arabica) and Coffea canephora (Robusta) are the two main cultivated species used for coffee bean production. Arabica genotypes generally produce a higher coffee quality than Robusta genotypes. Understanding the genetic basis for sucrose accumulation during coffee grain maturation is an important goal because sucrose is an important coffee flavor precursor. * Nine new Coffea genes encoding sucrose metabolism enzymes have been identified: sucrose phosphate synthase (CcSPS1, CcSPS2), sucrose phosphate phosphatase (CcSP1), cytoplasmic (CaInv3) and cell wall (CcInv4) invertases and four invertase inhibitors (CcInvI1, 2, 3, 4). * Activities and mRNA abundance of the sucrose metabolism enzymes were compared at different developmental stages in Arabica and Robusta grains, characterized by different sucrose contents in mature grain. * It is concluded that Robusta accumulates less sucrose than Arabica for two reasons: Robusta has higher sucrose synthase and acid invertase activities early in grain development - the expression of CcSS1 and CcInv2 appears to be crucial at this stage and Robusta has a lower SPS activity and low CcSPS1 expression at the final stages of grain development and hence has less capacity for sucrose re-synthesis. Regulation of vacuolar invertase CcInv2 activity by invertase inhibitors CcInvI2 and/or CcInvI3 during Arabica grain development is considered. PMID:18384509

  8. A Chimeric Cfh Transgene Leads to Increased Retinal Oxidative Stress, Inflammation, and Accumulation of Activated Subretinal Microglia in Mice

    PubMed Central

    Aredo, Bogale; Li, Tao; Chen, Xiao; Zhang, Kaiyan; Wang, Cynthia Xin-Zhao; Gou, Darlene; Zhao, Biren; He, Yuguang; Ufret-Vincenty, Rafael L.

    2015-01-01

    Purpose. Variants of complement factor H (Cfh) affecting short consensus repeats (SCRs) 6 to 8 increase the risk of age-related macular degeneration. Our aim was to explore the effect of expressing a Cfh variant on the in vivo susceptibility of the retina and RPE to oxidative stress and inflammation, using chimeric Cfh transgenic mice (chCfhTg). Methods. The chCfhTg and age-matched C57BL/6J (B6) mice were subjected to oxidative stress by either normal aging, or by exposure to a combination of oral hydroquinone (0.8% HQ) and increased light. Eyes were collected for immunohistochemistry of RPE–choroid flat mounts and of retinal sections, ELISA, electron microscopy, and RPE/microglia gene expression analysis. Results. Aging mice to 2 years led to an increased accumulation of basal laminar deposits, subretinal microglia/macrophages (MG/MΦ) staining for CD16 and for malondialdehyde (MDA), and MDA-modified proteins in the retina in chCfhTg compared to B6 mice. The chCfhTg mice maintained on HQ diet and increased light showed greater deposition of basal laminar deposits, more accumulation of fundus spots suggestive of MG/MΦ, and increased deposition of C3d in the sub-RPE space, compared to controls. In addition, chCfhTg mice demonstrated upregulation of NLRP3, IP-10, CD68, and TREM-2 in the RNA isolates from RPE/MG/MΦ. Conclusions. Expression of a Cfh transgene introducing a variant in SCRs 6 to 8 was sufficient to lead to increased retinal/RPE susceptibility to oxidative stress, a proinflammatory MG/MΦ phenotype, and a proinflammatory RPE/MG/MΦ gene expression profile in a transgenic mouse model. Our data suggest that altered interactions of Cfh with MDA-modified proteins may be relevant in explaining the effects of the Cfh variant. PMID:26030099

  9. Diversity of Arbuscular Mycorrhizal Fungi Associated with a Sb Accumulator Plant, Ramie (Boehmeria nivea), in an Active Sb Mining.

    PubMed

    Wei, Yuan; Chen, ZhiPeng; Wu, FengChang; Li, JiNing; ShangGuan, YuXian; Li, FaSheng; Zeng, Qing Ru; Hou, Hong

    2015-08-01

    Arbuscular mycorrhizal fungi (AMF) have great potential for assisting heavy metal hyperaccumulators in the remediation of contaminated soils. However, little information is available about the symbiosis of AMF associated with an antimony (Sb) accumulator plant under natural conditions. Therefore, the objective of this study was to investigate the colonization and molecular diversity of AMF associated with the Sb accumulator ramie (Boehmeria nivea) growing in Sb-contaminated soils. Four Sb mine spoils and one adjacent reference area were selected from Xikuangshan in southern China. PCR-DGGE was used to analyze the AMF community composition in ramie roots. Morphological identification was also used to analyze the species in the rhizosphere soil of ramie. Results obtained showed that mycorrhizal symbiosis was established successfully even in the most heavily polluted sites. From the unpolluted site Ref to the highest polluted site T4, the spore numbers and AMF diversity increased at first and then decreased. Colonization increased consistently with the increasing Sb concentrations in the soil. A total of 14 species were identified by morphological analysis. From the total number of species, 4 (29%) belonged to Glomus, 2 (14%) belonged to Acaulospora, 2 (14%) belonged to Funneliformis, 1 (7%) belonged to Claroideoglomus, 1 (7%) belonged to Gigaspora, 1 (7%) belonged to Paraglomus, 1 (7%) belonging to Rhizophagus, 1 (7%) belonging to Sclervocystis, and 1 (7%) belonged to Scutellospora. Some AMF sequences were present even in the most polluted site. Morphological identification and phylogenetic analysis both revealed that most species were affiliated withGlomus, suggesting that Glomus was the dominant genus in this AMF community. This study demonstrated that ramie associated with AMF may have great potential for remediation of Sb-contaminated soils.

  10. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

    PubMed

    Laura, Richard P; Dong, David; Reynolds, Wanda F; Maki, Richard A

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO. PMID:26890638

  11. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

    PubMed

    Laura, Richard P; Dong, David; Reynolds, Wanda F; Maki, Richard A

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.

  12. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation

    PubMed Central

    Laura, Richard P.; Dong, David; Reynolds, Wanda F.; Maki, Richard A.

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO. PMID:26890638

  13. Artesunate has its enhancement on antibacterial activity of β-lactams via increasing the antibiotic accumulation within methicillin-resistant Staphylococcus aureus (MRSA).

    PubMed

    Jiang, Weiwei; Li, Bin; Zheng, Xinchuan; Liu, Xin; Pan, Xichun; Qing, Rongxin; Cen, Yanyan; Zheng, Jiang; Zhou, Hong

    2013-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) has now emerged as a predominant and serious pathogen because of its resistance to a large group of antibiotics, leading to high morbidity and mortality. Therefore, to develop new agents against resistance is urgently required. Previously, artesunate (AS) was found to enhance the antibacterial effect of β-lactams against MRSA. In this study, AS was first found to increase the accumulation of antibiotics (daunorubicin and oxacillin) within MRSA by laser confocal microscopy and liquid chromatography-tandem MS method, suggesting the increased antibiotics accumulation might be related to the enhancement of AS on antibiotics. Furthermore, AS was found not to destroy the cell structure of MRSA by transmission electron microscope. AS was found to inhibit gene expressions of important efflux pumps such as NorA, NorB and NorC, but not MepA, SepA and MdeA. In conclusion, our results showed that AS was capable of enhancing the antibacterial activity of β-lactams via increasing antibiotic accumulations within MRSA through inhibiting gene expressions of efflux pumps such as NorA, NorB and NorC, but did not destroy the cell structure of MRSA. AS could be further investigated as a candidate drug for treatment of MRSA infection.

  14. The response of Hordeum spontaneum desert ecotype to drought and excessive light intensity is characterized by induction of O2 dependent photochemical activity and anthocyanin accumulation.

    PubMed

    Eppel, Amir; Keren, Nir; Salomon, Eitan; Volis, Sergei; Rachmilevitch, Shimon

    2013-03-01

    The goal of the current research was to study the role of anthocyanin accumulation, O(2)-related photochemical processes and non-photochemical quenching (NPQ) in the response of desert and Mediterranean plants to drought and excessive light. Plants of Hordeum spontaneum were collected from Mediterranean and desert environments and were subjected to terminal drought for 25 days and then measured for PSII yield at 2 and 21% O(2), NPQ, net carbon assimilation, stomatal conductance, leaf relative water content (LRWC), anthocyanin concentration and leaf absorbance. Under terminal drought, LRWC, carbon assimilation and stomatal conductance decreased similarly and significantly in both the Mediterranean and the desert ecotypes. Anthocyanin accumulated more in the desert ecotype than in the Mediterranean ecotype. NPQ increased more in the Mediterranean ecotype as compared with the desert ecotype. PSII yield decreased significantly in the Mediterranean ecotype under drought and was much lower than in the desert ecotype under drought. The relatively high PSII yield under drought in the desert ecotype was O(2) dependent. The response of the H. spontaneum ecotype from a desert environment to drought stress was characterized by anthocyanin accumulation and induction of O(2) dependent photochemical activity, while the response of the Mediterranean ecotype was based on a higher induction of NPQ.

  15. Subcellular cadmium distribution and antioxidant enzymatic activities in the leaves of two castor (Ricinus communis L.) cultivars exhibit differences in Cd accumulation.

    PubMed

    Zhang, Hanzhi; Guo, Qingjun; Yang, Junxing; Shen, Jianxiu; Chen, Tongbin; Zhu, Guangxu; Chen, Hui; Shao, Chunyan

    2015-10-01

    The aims of this study were: (1) the study of cadmium (Cd) accumulation and toxicity in different castor cultivars (Ricinus communis L.); (2) to investigate changes in antioxidant enzymatic activities and the subcellular distribution of Cd in young and old leaves from two different castor cultivars, after exposure to two different Cd concentrations, and explore the underlying mechanism of Cd detoxification focusing on antioxidant enzymes and subcellular compartmentalization. The Cd concentration, toxicity, and subcellular distribution, as well as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities were measured in Zibo-3 and Zibo-9 cultivars after exposure to two different concentrations of Cd (2mg/L and 5mg/L) for 10 days. This research revealed Cd accumulation characteristics in castor are root>stem>young leaf>old leaf. Castor tolerance was Cd dose exposure and the cultivars themselves dependent. Investigation of subcellular Cd partitioning showed that Cd accumulated mainly in the heat stable protein (HSP) and cellular debris fractions, followed by the Cd rich granule (MRG), heat denatured protein (HDP), and organelle fractions. With increasing Cd concentration in nutrient solution, the decreased detoxified fractions (BDM) and the increased Cd-sensitive fractions (MSF) in young leaves may indicate the increased Cd toxicity in castor cultivars. The BDM-Cd fractions or MSF-Cd in old leaves may be linked with Cd tolerance of different cultivars of castor. The antioxidant enzymes that govern Cd detoxification were not found to be active in leaves. Taken together, these results indicate Cd tolerance and toxicity in castor can be explained by subcellular partitioning.

  16. Quercetin and Allopurinol Ameliorate Kidney Injury in STZ-Treated Rats with Regulation of Renal NLRP3 Inflammasome Activation and Lipid Accumulation

    PubMed Central

    Zhang, Qing-Yu; Wang, Fu-Meng; Kong, Ling-Dong

    2012-01-01

    Hyperuricemia, hyperlipidemia and inflammation are associated with diabetic nephropathy. The NLRP3 inflammasome-mediated inflammation is recently recognized in the development of kidney injury. Urate and lipid are considered as danger signals in the NLRP3 inflammasome activation. Although dietary flavonoid quercetin and allopurinol alleviate hyperuricemia, dyslipidmia and inflammation, their nephroprotective effects are currently unknown. In this study, we used streptozotocin (STZ)-induced diabetic nephropathy model with hyperuricemia and dyslipidemia in rats, and found over-expression of renal inflammasome components NLRP3, apoptosis-associated speck-like protein and Caspase-1, resulting in elevation of IL-1β and IL-18, with subsequently deteriorated renal injury. These findings demonstrated the possible association between renal NLRP3 inflammasome activation and lipid accumulation to superimpose causes of nephrotoxicity in STZ-treated rats. The treatment of quercetin and allopurinol regulated renal urate transport-related proteins to reduce hyperuricemia, and lipid metabolism-related genes to alleviate kidney lipid accumulation in STZ-treated rats. Furthermore, quercetin and allopurinol were found to suppress renal NLRP3 inflammasome activation, at least partly, via their anti-hyperuricemic and anti-dyslipidemic effects, resulting in the amelioration of STZ-induced the superimposed nephrotoxicity in rats. These results may provide a basis for the prevention of diabetes-associated nephrotoxicity with urate-lowering agents such as quercetin and allopurinol. PMID:22701621

  17. Ethoxyresorufin-O-deethylase enzyme activities and accumulation of secondary/tertiary lysosomes in rabbitfish Siganus oramin as biomarkers for xenobiotic exposures.

    PubMed

    Fang, James K H; Au, Doris W T; Chan, Alice K Y; So, Leo K Y; Liu, Wenhua; Mok, Helen O L; Yu, Wai-Yin; Wu, Rudolf S S; Shin, Paul K S

    2010-09-15

    The sensitivities of using hepatic and intestinal ethoxyresorufin-O-deethylase (EROD) activities and hepatic accumulation of secondary/tertiary (2 degrees/3 degrees) lysosomes to detect xenobiotic exposures were assessed in the rabbitfish Siganus oramin in a metropolitan harbour, subtropical Hong Kong, over a complete seasonal cycle of one year. Additional information on the body-burden pollutants and physiological indices in S. oramin, and seasonal variables in seawater quality, were extracted from published data and re-analyzed. Under the influences of pollutant cocktail and seasonal factors, neither the hepatic nor intestinal EROD activity was indicative of total polycyclic aromatic hydrocarbons (Sigma PAH), total polychlorinated biphenyls, condition factor and hepatosomatic index (HSI) in S. oramin. However, the relative ratio of hepatic to intestinal EROD activities provided an indication to differentiate the xenobiotic intake route in the fish through diffusion via gills/skin or consumption of contaminated food. In addition, the elevated hepatic accumulation of 2 degrees/3 degrees lysosomes was closely associated with the dominant temporal trends of zinc and Sigma PAH, as well as reduced HSI, in S. oramin. Being minimally influenced by any investigated seasonal factors, the hepatic 2 degrees/3 degrees lysosomes in S. oramin was recommended as an effective biomarker of xenobiotic exposures and toxic effects for use in coastal pollution monitoring programmes.

  18. Mild Electrical Stimulation Increases Stress Resistance and Suppresses Fat Accumulation via Activation of LKB1-AMPK Signaling Pathway in C. elegans

    PubMed Central

    Matsuyama, Shingo; Moriuchi, Masataka; Suico, Mary Ann; Yano, Shuichiro; Morino-Koga, Saori; Shuto, Tsuyoshi; Yamanaka, Kunitoshi; Kondo, Tatsuya; Araki, Eiichi; Kai, Hirofumi

    2014-01-01

    Electrical current at physiological strength has been applied as a therapeutic approach for various diseases. Several of our works showed that mild electrical stimulation (MES) at 0.1-ms pulse width has positive impact on organisms. But despite the growing evidence of the beneficial effects of MES, its effects on individual animals and the molecular underpinnings are poorly understood and rarely studied. Here, we examined the effects of MES on individual animal and its mechanisms by mainly using Caenorhabditis elegans, a powerful genetic model organism. Interestingly, MES increased stress resistance and suppressed excess fat accumulation in wild-type N2 worms but not in AMPK/AAK-2 and LKB1/PAR-4 mutant worms. MES promoted the nuclear localization of transcription factors DAF-16 and SKN-1 and consequently increased the expression of anti-stress genes, whereas MES inhibited the nuclear localization of SBP-1 and suppressed the expression of lipogenic genes. Moreover, we found that MES induced the activation of LKB1/PAR4-AMPK/AAK2 pathway in C. elegans and in several mammalian cell lines. The mitochondrial membrane potential and cellular ATP level were slightly and transiently decreased by MES leading to the activation of LKB1-AMPK signaling pathway. Together, we firstly and genetically demonstrated that MES exerts beneficial effects such as stress resistance and suppression of excess fat accumulation, via activation of LKB1-AMPK signaling pathway. PMID:25490091

  19. Effect of water withdrawal on formation of free radical, proline accumulation and activities of antioxidant enzymes in ZAT12-transformed transgenic tomato plants.

    PubMed

    Chandra Rai, Avinash; Singh, Major; Shah, Kavita

    2012-12-01

    Water stress often leads to the accumulation of reactive oxygen species (ROS) and their excessive production alters the activities of enzymes involved in their removal. ZAT12 is a member of stress-responsive C(2)H(2) type Zinc Finger Protein (ZFP) reported to control the expression of several stress-activated genes in plants through ROS signaling. The ZAT12-transformed tomato lines (cv. H-86 variety Kashi Vishesh) when subjected to water withdrawal for 7, 14 and 21 days revealed significant and consistent changes in activities of enzymes SOD, CAT, APX, GR and POD paralleled with an increased proline levels. Unlike that in wild-type tomato, the leaf superoxide anion and hydrogen peroxide concentrations in the transformed tomato plants did not alter much, suggesting a well regulated formation of free radicals suppressing oxidative stress in the latter. Results suggest BcZAT12-transformed tomato lines ZT1, ZT2 and ZT6 to be better adapted to drought stress tolerance by accumulation of osmolyte proline and increased antioxidant response triggered by the ZAT12 gene. Therefore, the ZAT12-transformed tomato cv. H-86 lines will prove useful for higher yield of tomato crop in regions affected with severe drought stress.

  20. Active accumulation of very diluted biomolecules by nano-dispensing for easy detection below the femtomolar range

    NASA Astrophysics Data System (ADS)

    Grilli, S.; Miccio, L.; Gennari, O.; Coppola, S.; Vespini, V.; Battista, L.; Orlando, P.; Ferraro, P.

    2014-11-01

    Highly sensitive detection of biomolecules is of paramount interest in many fields including biomedicine, safety and eco-pollution. Conventional analyses use well-established techniques with detection limits ~1 pM. Here we propose a pyro-concentrator able to accumulate biomolecules directly onto a conventional binding surface. The operation principle is relatively simple but very effective. Tiny droplets are drawn pyro-electro-dynamically and released onto a specific site, thus increasing the sensitivity. The reliability of the technique is demonstrated in case of labelled oligonucleotides diluted serially. The results show the possibility to detect very diluted oligonucleotides, down to a few hundreds of attomoles. Excellent results are shown also in case of a sample of clinical interest, the gliadin, where a 60-fold improved detection limit is reached, compared with standard ELISA. This method could open the way to a mass-based technology for sensing molecules at very low concentrations, in environmental as well as in diagnostics applications.

  1. Chemical PARP Inhibition Enhances Growth of Arabidopsis and Reduces Anthocyanin Accumulation and the Activation of Stress Protective Mechanisms

    PubMed Central

    Schulz, Philipp; Neukermans, Jenny; Van Der Kelen, Katrien; Mühlenbock, Per; Van Breusegem, Frank; Noctor, Graham; Teige, Markus; Metzlaff, Michael; Hannah, Matthew A.

    2012-01-01

    Poly-ADP-ribose polymerase (PARP) post-translationally modifies proteins through the addition of ADP-ribose polymers, yet its role in modulating plant development and stress responses is only poorly understood. The experiments presented here address some of the gaps in our understanding of its role in stress tolerance and thereby provide new insights into tolerance mechanisms and growth. Using a combination of chemical and genetic approaches, this study characterized phenotypes associated with PARP inhibition at the physiological level. Molecular analyses including gene expression analysis, measurement of primary metabolites and redox metabolites were used to understand the underlying processes. The analysis revealed that PARP inhibition represses anthocyanin and ascorbate accumulation under stress conditions. The reduction in defense is correlated with enhanced biomass production. Even in unstressed conditions protective genes and molecules are repressed by PARP inhibition. The reduced anthocyanin production was shown to be based on the repression of transcription of key regulatory and biosynthesis genes. PARP is a key factor for understanding growth and stress responses of plants. PARP inhibition allows plants to reduce protection such as anthocyanin, ascorbate or Non-Photochemical-Quenching whilst maintaining high energy levels likely enabling the observed enhancement of biomass production under stress, opening interesting perspectives for increasing crop productivity. PMID:22662141

  2. far4, far5, and far6 define three genes required for efficient activation of MAPKs Fus3 and Kss1 and accumulation of glycogen.

    PubMed

    Cherkasova, V; Elion, E A

    2001-08-01

    In Saccharomyces cerevisiae, mating pheromones induce G1 arrest through the activation of two MAP kinases, Fus3 and Kss1. Here we report the isolation of three mutants, far4, far5, and far6, that have the novel phenotype of regulating both the activity of Fus3 and Kss1 and the accumulation of glycogen. A far4 mutation constitutively activates Fus3 and Kss1, reduces glycogen, and blocks G1 arrest in the presence of alpha factor. In contrast, far5 and far6 mutations increase glycogen and reduce activation of Fus3 and Kss1 by pheromone. far4, far5, and far6 are recessive and not allelic to FAR1, FAR3, or 14 genes known to regulate the pheromone response. Non-allelic noncomplementation occurs between far6 and both far4 and far5, suggesting that FAR6 functionally interacts with FAR4 and FAR5. Additional observations suggest that FAR4 has functional overlap with FAR3, which we also find to regulate glycogen accumulation. Our results suggest that the activation of the mating MAPK cascade and subsequent G1 arrest is influenced by a signal transduction pathway that regulates glycogen. In support of this possibility, we find that Fus3 is activated to a greater extent in a "wimp" strain with defective protein kinase A. Finally, BIM1 and BIK1 have been identified as CEN suppressors of far5, suggesting that the microtubule apparatus may regulate the ability of the pheromone response pathway to promote G1 arrest. PMID:11570512

  3. [Influence of low dozes of ionizing radiation on accumulation of melanin pigments and catalase and superoxidedismutase activities in Cladosporium cladosporioides].

    PubMed

    Tuhaĭ, T I

    2007-01-01

    Influence of low dozes of ionizing radiation on melanin pigments synthesis and activity of antioxidant enzymes catalase and superoxidedismutase of two strains of Cladosporium cladosporioides 4, (isolated from radioactive soil) and 396 (control) were investigated. It was shown, that in C. cladosporioides 4 under the exposure of ionizing radiation an increase of melanin synthesis in a stationary growth phase and increase of superoxidedismutase activity in a logarithmic phase were observed; in the control strain C. cladosporioides 396 activation of melanin synthesis and superoxide dismutase activity in both growth phases was revealed. It was established that in C. cladosporioides 4 the endocellular catalase activity in a logarithmic phase is 3.2 times higher, than in control strain. Under the action of ionizing radiation a 2-fold increase of this enzyme activity unlike the control strain in which the activity inhibition was revealed. The obtained results testify to the complex response of antioxidant systems and melanin to the action of low dozes of radiation which depends on the growth phase and presence of radioadaptation properties in the investigated fungi.

  4. Inhibition of platelet activation prevents the P-selectin and integrin-dependent accumulation of cancer cell microparticles and reduces tumor growth and metastasis in vivo.

    PubMed

    Mezouar, Soraya; Darbousset, Roxane; Dignat-George, Françoise; Panicot-Dubois, Laurence; Dubois, Christophe

    2015-01-15

    Venous thromboembolism constitutes one of the main causes of death during the progression of a cancer. We previously demonstrated that tissue factor (TF)-bearing cancer cell-derived microparticles accumulate at the site of injury in mice developing a pancreatic cancer. The presence of these microparticles at the site of thrombosis correlates with the size of the platelet-rich thrombus. The objective of this study was to determine the involvement of TF expressed by cancer cell-derived microparticles on thrombosis associated with cancer. We observed that pancreatic cancer cell derived microparticles expressed TF, its inhibitor tissue factor pathway inhibitor (TFPI) as well as the integrins αvβ1 and αvβ3. In mice bearing a tumor under-expressing TF, a significant decrease in circulating TF activity associated with an increase bleeding time and a 100-fold diminished fibrin generation and platelet accumulation at the site of injury were observed. This was mainly due to the interaction of circulating cancer cell-derived microparticles expressing TFPI with activated platelets and fibrinogen. In an ectopic model of cancer, treatment of mice with Clopidogrel, an anti-platelet drug, decreased the size of the tumors and restored hemostasis by preventing the accumulation of cancer cell-derived microparticles at the site of thrombosis. In a syngeneic orthotopic model of pancreatic cancer Clopidogrel also significantly inhibited the development of metastases. Together, these results indicate that an anti-platelet strategy may efficiently treat thrombosis associated with cancer and reduce the progression of pancreatic cancer in mice.

  5. Accumulation and activation of epidermal γδ T cells in a mouse model of chronic dermatitis is not required for the inflammatory phenotype.

    PubMed

    Sulcova, Jitka; Maddaluno, Luigi; Meyer, Michael; Werner, Sabine

    2015-09-01

    Chronic skin inflammation resulting from a defective epidermal barrier is a hallmark of atopic dermatitis (AD). We previously demonstrated that mice lacking FGF receptors 1 and 2 in keratinocytes (K5-R1/R2 mice) develop an AD-like chronic dermatitis as a result of an impaired epidermal barrier. Here, we show that γδ T cells, which rapidly respond to various insults, accumulate in the epidermis of K5-R1/R2 mice before the development of histological abnormalities. Their number and activation further increase as the phenotype progresses, most likely as a consequence of increased expression of Il-2 and Il-7 and the stress-induced proteins Rae-1, H60c, Mult1, PlexinB2, and Skint1. To determine the role of γδ T cells in the skin phenotype, we generated quadruple mutant K5-R1/-R2 mice lacking γδ T cells. Surprisingly, loss of γδ T cells did not or only marginally affect keratinocyte proliferation, epidermal thickness, epidermal barrier function, and accumulation and activation of different immune cells in the skin of K5-R1/R2 mice, possibly due to partial compensation by αβ T cells. These results demonstrate that γδ T cells do not contribute to the development or maintenance of chronic inflammation in response to a defect in the epidermal barrier.

  6. Data on amyloid precursor protein accumulation, spontaneous physical activity, and motor learning after traumatic brain injury in the triple-transgenic mouse model of Alzheimer׳s disease.

    PubMed

    Kishimoto, Yasushi; Shishido, Hajime; Sawanishi, Mayumi; Toyota, Yasunori; Ueno, Masaki; Kubota, Takashi; Kirino, Yutaka; Tamiya, Takashi; Kawai, Nobuyuki

    2016-12-01

    This data article contains supporting information regarding the research article entitled "Traumatic brain injury accelerates amyloid-β deposition and impairs spatial learning in the triple-transgenic mouse model of Alzheimer׳s disease" (H. Shishido, Y. Kishimoto, N. Kawai, Y. Toyota, M. Ueno, T. Kubota, Y. Kirino, T. Tamiya, 2016) [1]. Triple-transgenic (3×Tg)-Alzheimer׳s disease (AD) model mice exhibited significantly poorer spatial learning than sham-treated 3×Tg-AD mice 28 days after traumatic brain injury (TBI). Correspondingly, amyloid-β (Aβ) deposition within the hippocampus was significantly greater in 3×Tg-AD mice 28 days after TBI. However, data regarding the short-term and long-term influences of TBI on amyloid precursor protein (APP) accumulation in AD model mice remain limited. Furthermore, there is little data showing whether physical activity and motor learning are affected by TBI in AD model mice. Here, we provide immunocytochemistry data confirming that TBI induces significant increases in APP accumulation in 3×Tg-AD mice at both 7 days and 28 days after TBI. Furthermore, 3×Tg-AD model mice exhibit a reduced ability to acquire conditioned responses (CRs) during delay eyeblink conditioning compared to sham-treated 3×Tg-AD model mice 28 days after TBI. However, physical activity and motor performance are not significantly changed in TBI-treated 3×Tg-AD model mice. PMID:27656663

  7. Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcriptional activator modulating reactive oxygen species accumulation and cell death.

    PubMed

    Niu, Fangfang; Wang, Chen; Yan, Jingli; Guo, Xiaohua; Wu, Feifei; Yang, Bo; Deyholos, Michael K; Jiang, Yuan-Qing

    2016-09-01

    NAC transcription factors (TFs) are plant-specific and play important roles in development, responses to biotic and abiotic cues and hormone signaling. So far, only a few NAC genes have been reported to regulate cell death. In this study, we identified and characterized a NAC55 gene isolated from oilseed rape (Brassica napus L.). BnaNAC55 responds to multiple stresses, including cold, heat, abscisic acid (ABA), jasmonic acid (JA) and a necrotrophic fungal pathogen Sclerotinia sclerotiorum. BnaNAC55 has transactivation activity and is located in the nucleus. BnaNAC55 is able to form homodimers in planta. Unlike ANAC055, full-length BnaNAC55, but not either the N-terminal NAC domain or C-terminal regulatory domain, induces ROS accumulation and hypersensitive response (HR)-like cell death when expressed both in oilseed rape protoplasts and Nicotiana benthamiana. Furthermore, BnaNAC55 expression causes obvious nuclear DNA fragmentation. Moreover, quantitative reverse transcription PCR (qRT-PCR) analysis identified that the expression levels of multiple genes regulating ROS production and scavenging, defense response as well as senescence are significantly induced. Using a dual luciferase reporter assay, we further confirm that BnaNAC55 could activate the expression of a few ROS and defense-related gene expression. Taken together, our work has identified a novel NAC TF from oilseed rape that modulates ROS accumulation and cell death. PMID:27312204

  8. Data on amyloid precursor protein accumulation, spontaneous physical activity, and motor learning after traumatic brain injury in the triple-transgenic mouse model of Alzheimer׳s disease.

    PubMed

    Kishimoto, Yasushi; Shishido, Hajime; Sawanishi, Mayumi; Toyota, Yasunori; Ueno, Masaki; Kubota, Takashi; Kirino, Yutaka; Tamiya, Takashi; Kawai, Nobuyuki

    2016-12-01

    This data article contains supporting information regarding the research article entitled "Traumatic brain injury accelerates amyloid-β deposition and impairs spatial learning in the triple-transgenic mouse model of Alzheimer׳s disease" (H. Shishido, Y. Kishimoto, N. Kawai, Y. Toyota, M. Ueno, T. Kubota, Y. Kirino, T. Tamiya, 2016) [1]. Triple-transgenic (3×Tg)-Alzheimer׳s disease (AD) model mice exhibited significantly poorer spatial learning than sham-treated 3×Tg-AD mice 28 days after traumatic brain injury (TBI). Correspondingly, amyloid-β (Aβ) deposition within the hippocampus was significantly greater in 3×Tg-AD mice 28 days after TBI. However, data regarding the short-term and long-term influences of TBI on amyloid precursor protein (APP) accumulation in AD model mice remain limited. Furthermore, there is little data showing whether physical activity and motor learning are affected by TBI in AD model mice. Here, we provide immunocytochemistry data confirming that TBI induces significant increases in APP accumulation in 3×Tg-AD mice at both 7 days and 28 days after TBI. Furthermore, 3×Tg-AD model mice exhibit a reduced ability to acquire conditioned responses (CRs) during delay eyeblink conditioning compared to sham-treated 3×Tg-AD model mice 28 days after TBI. However, physical activity and motor performance are not significantly changed in TBI-treated 3×Tg-AD model mice.

  9. Antioxidant activities of Se-SPI produced from soybean as accumulation and biotransformation reactor of natural selenium.

    PubMed

    Hu, Juwu; Zhao, Qiang; Cheng, Xiang; Selomulya, Cordelia; Bai, Chunqing; Zhu, Xuemei; Li, Xionghui; Xiong, Hua

    2014-03-01

    A study to compare the uptake, translocation, and distribution of selenium (Se) in soybean planted in natural seleniferous soil in Fengcheng city of China was conducted to clarify the relationship between the Se content levels of soybean proteins and their radical scavenging activity. The data showed that the total Se content in different parts of soybean plants varied with the growth periods. The selenoprotein (Se-SPI) content increased remarkably with the increase of Se content in seleniferous soils. The Se-SPI content obtained from the region with the highest Se level was almost 18 times higher than that of the control group, while antioxidant activities were about 4-fold compared to the control, suggesting that Se played a positive role in enhancing the antioxidant activity of Se-SPI. The increase in the Se level also led to changes in amino acids composition, but with nearly no effects on the subunit composition of soybean Se-SPI.

  10. Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

    PubMed

    Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

    2016-04-15

    The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.

  11. Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

    PubMed

    Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

    2016-04-15

    The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity. PMID:26867495

  12. Comparison of the inhibition of myeloperoxidase-catalyzed hypochlorite formation in vitro and in whole blood by different plant extracts contained in a phytopharmacon treating functional dyspepsia.

    PubMed

    Schempp, Harald; Hippeli, Susanne; Weiser, Dieter; Kelber, Olaf; Elstner, Erich F

    2004-01-01

    Ethanolic extracts from nine medicinal plants are combined in Iberogast (IG). This phytomedicine is successfully used in the treatment of gastrointestinal disorders. Functional gastrointestinal diseases such as non-ulcerous dyspepsia (NUD) are in many cases initiated by, or correlated to, inflammatory processes, where reactive oxygen species (ROS) play a crucial role. In this respect one prominent source of ROS are myeloperoxidase (MPO)-driven oxidation and chlorination reactions, assumed to be mainly responsible for tissue damage. In this study the contribution of the nine extracts to the overall performance of IG was compared with emphasis on MPO produced ROS. Concerning the influence on MPO-dependent chlorination reactions, it turned out that of the nine IG-components Iberis amara extract (IAE) exerted the highest activity. Furthermore, this can impressively be reproduced in an ex vivo experiment with whole blood, where neutrophilic leukocytes are activated by zymosan. Moreover, along with the extract of chamomile flowers, IAE counteracts the pro-oxidative properties of caraway, peppermint and celandine. As a consequence. IG was also efficiently inhibiting MPO-catalysed chlorinations. As shown by the addition of catalase, the pro-oxidative effects of caraway, peppermint and celandine are due to their content of hydrogen peroxide. The latter is probably an autoxidation product of certain monoterpenes in the essential oil part of these extracts. If one of the component extracts of IG is omitted, the antioxidant acitivity is reduced. Thus we conclude that all the single extracts combined in IG are of importance for the therapeutical effect, working in concert.

  13. Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.

    PubMed

    Zhang, Xiaobo; Liu, Chenghui; Wang, Honghong; Wang, Hui; Li, Zhengping

    2015-12-01

    A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level. PMID:26482714

  14. Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.

    PubMed

    Zhang, Xiaobo; Liu, Chenghui; Wang, Honghong; Wang, Hui; Li, Zhengping

    2015-12-01

    A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level.

  15. Accumulation of recalcitrant xylan in mushroom-compost is due to a lack of xylan substituent removing enzyme activities of Agaricus bisporus.

    PubMed

    Jurak, Edita; Patyshakuliyeva, Aleksandrina; Kapsokalyvas, Dimitris; Xing, Lia; van Zandvoort, Marc A M J; de Vries, Ronald P; Gruppen, Harry; Kabel, Mirjam A

    2015-11-01

    The ability of Agaricus bisporus to degrade xylan in wheat straw based compost during mushroom formation is unclear. In this paper, xylan was extracted from the compost with water, 1M and 4M alkali. Over the phases analyzed, the remaining xylan was increasingly substituted with (4-O-methyl-)glucuronic acid and arabinosyl residues, both one and two arabinosyl residues per xylosyl residue remained. In the 1M and 4M KOH soluble solids of spent compost, 33 and 49 out of 100 xylosyl residues, respectively, were substituted. The accumulation of glucuronic acid substituents matched with the analysis that the two A. bisporus genes encoding for α-glucuronidase activity (both GH115) were not expressed in the A. bisporus mycelium in the compost during fruiting. Also, in a maximum likelihood tree it was shown that it is not likely that A. bisporus possesses genes encoding for the activity to remove arabinose from xylosyl residues having two arabinosyl residues.

  16. A probable crosstalk between Ca⁺², reactive oxygen species accumulation and scavenging mechanisms and modulation of protein kinase C activity during seed development in sunflower.

    PubMed

    Thakur, Anita; Bhatla, Satish C

    2014-01-01

    Seed development in sunflower involves a gradual dehydration and accumulation of oil bodies in the cells of developing cotyledons during transition from 30 to 40 DAA stage. Reactive oxygen species (ROS) content decreased with seed maturation. NO content and NO contributed by putative nitric oxide synthase, however, did not change markedly. Superoxide dismutase (SOD) activity exhibited a peak at 30 DAA stage, indicating its scavenging role at the mid-stage of seed development. H₂O₂ produced as a result of SOD action is subsequently scavenged primarily by elevation of GR activity. Significant temporal differences were evident in GR and POD activity during seed development. Protein kinase C (PKC) activity also showed modulation during early stages of embryo and seed development. Use of PKC-specific fluorescent probe, Fim-1, and PKC inhibitors (staurosporine and bisindoylmaleamide) provided evidence for increase in PKC activity at 40 DAA stage with an increase in protein concentration (50 to 200 µg). Endogenous calcium content also increased with seed maturation. Tissue homogenates from 40 DAA stage showed enhanced fluorescence due to Fim-1-PKC binding in presence of calcium ions and its lowering due to calcium chelating agent (BAPTA). Western blot analysis revealed an increase in the intensity of 2 bands representing PKC with the advancement of seed maturation and their further upregulation by calcium. Present findings, thus, provide new information on the biochemical regulation of seed development in sunflower, with evidence for a possible correlation between calcium, ROS, their scavenging enzymes and "conventional" PKC activity.

  17. RKIP phosphorylation-dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3.

    PubMed

    Hahm, Jong Ryeal; Ahmed, Mahmoud; Kim, Deok Ryong

    2016-09-01

    3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-l-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-l-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity. PMID:27470585

  18. RKIP phosphorylation-dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3.

    PubMed

    Hahm, Jong Ryeal; Ahmed, Mahmoud; Kim, Deok Ryong

    2016-09-01

    3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-l-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-l-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity.

  19. Synthesis of a peroxidase activity by the cells of hairy cell leukemia: a study by ultrastructural cytochemistry.

    PubMed

    Reyes, F; Gourdin, M F; Farcet, J P; Dreyfus, B; Breton-Gorius, J

    1978-09-01

    The nature of cells present in the blood, marrow, and spleen of patients with hairy cell leukemia is largely debated. These cells have been tentatively categorized on the basis of either monocytic or lymphocytic markers, and the accumulating data points to the fact that they share some characteristics of both cell types. Although hairy cells are known to lack myeloperoxidase-positive granules, present in normal human monocytes, we investigated the possible presence of other peroxidase activities differing from the granule-bound myeloperoxidase. The study was carried out with several methods based on the incubation of fixed and unfixed cells in the presence of diaminobenzidine and hydrogen peroxide. A peroxidase activity was found in hairy cells, located always in the endoplasmic reticulum but not in the Golgi apparatus or in any granule. By its cytochemical characteristics it appears to be closely related to that of tissue macrophages, activated blood monocytes, and other nonlymphocytic hematopoietic cells. This peroxidase is not found in lymphocytes with B or T phenotypes. PMID:678670

  20. Molecular regulation of sinapate ester metabolism in Brassica napus: expression of genes, properties of the encoded proteins and correlation of enzyme activities with metabolite accumulation.

    PubMed

    Milkowski, Carsten; Baumert, Alfred; Schmidt, Diana; Nehlin, Lilian; Strack, Dieter

    2004-04-01

    Members of the Brassicaceae family accumulate specific sinapate esters, i.e. sinapoylcholine (sinapine), which is considered as a major antinutritive compound in seeds of important crop plants like Brassica napus, and sinapoylmalate, which is implicated in UV-B tolerance in leaves. We have studied the molecular regulation of the sinapate ester metabolism in B. napus, and we describe expression of genes, some properties of the encoded proteins and profiles of the metabolites and enzyme activities. The cloned cDNAs encoding the key enzymes of sinapine biosynthesis, UDP-glucose (UDP-Glc):B. napus sinapate glucosyltransferase (BnSGT1) and sinapoylglucose:B. napus choline sinapoyltransferase (BnSCT), were functionally expressed. BnSGT1 belongs to a subgroup of plant GTs catalysing the formation of 1-O-hydroxycinnamoyl-beta-d-glucoses. BnSCT is another member of serine carboxypeptidase-like (SCPL) family of acyltransferases. The B. napus genome contains at least two SGT and SCT genes, each derived from its progenitors B. oleracea and B. rapa. BnSGT1 and BnSCT activities are subjected to pronounced transcriptional regulation. BnSGT1 transcript level increases throughout early stages of seed development until the early cotyledonary stage, and stays constant in later stages. The highest level of BnSGT1 transcripts is reached in 2-day-old seedlings followed by a dramatic decrease. In contrast, expression of BnSCT is restricted to developing seeds. Regulation of gene expression at the transcript level seems to be responsible for changes of BnSGT1 and BnSCT activities during seed and seedling development of B. napus. Together with sinapine esterase (SCE) and sinapoylglucose:malate sinapoyltransferase (SMT), activities of BnSGT1 and BnSCT show a close correlation with the accumulation kinetics of the corresponding metabolites.

  1. Conformational and thermal stability of mature dimeric human myeloperoxidase and a recombinant monomeric form from CHO cells.

    PubMed

    Banerjee, Srijib; Stampler, Johanna; Furtmüller, Paul G; Obinger, Christian

    2011-02-01

    Myeloperoxidase (MPO) is a lysosomal heme enzyme present in the azurophilic granules of human neutrophils and monocytes. It is a critical element of the human innate immune system by exerting antimicrobial effects. It is a disulfide bridged dimer with each monomer containing a light and a heavy polypeptide and its biosynthesis and intracellular transport includes several posttranslational processing steps. By contrast, MPO recombinantly produced in Chinese hamster ovary cell lines is monomeric, partially unprocessed and contains a N-terminal propeptide (proMPO). It mirrors a second form of MPO constitutively secreted from normal bone marrow myeloid precursors. In order to clarify the impact of posttranslational modifications on the structural integrity and enzymology of these two forms of human myeloperoxidase, we have undertaken an investigation on the conformational and thermal stability of leukocyte MPO and recombinant proMPO by using complementary biophysical techniques including UV-Vis, circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Mature leucocyte MPO exhibits a peculiar high chemical and thermal stability under oxidizing conditions but is significantly destabilized by addition of dithiothreitol. Unfolding of secondary and tertiary structure occurs concomitantly with denaturation of the heme cavity, reflecting the role of three MPO-typical heme to protein linkages and of six intra-chain disulfides for structural integrity by bridging N- and C-terminal regions of the protein. Recombinant monomeric proMPO follows a similar unfolding pattern but has a lower conformational and thermal stability. Spectroscopic and thermodynamic data of unfolding are discussed with respect to the known three-dimensional structure of leukocyte MPO as well as to known physiological roles. PMID:20933108

  2. The involvement of polyphenols and peroxidase activities in heavy-metal accumulation by epidermal glands of the waterlily (Nymphaeaceae).

    PubMed

    Lavid, N; Schwartz, A; Yarden, O; Tel-Or, E

    2001-02-01

    Co-localization of polyphenols and peroxidase activity was demonstrated in epidermal glands of the waterlily (Nymphaea) by histochemistry. Total phenols, tannins and peroxidase activity were determined quantitatively in plant extracts. Polyphenols were partially identified and were found to consist mainly of hydrolyzable tannins, gallic and tannic acid derivatives. Nymphaea polyphenols were shown to chelate Cr, Hg, and Pb in vitro, and Cd-binding by polymerized polyphenols was demonstrated in leaves exposed to Cd in vivo. Both polyphenols and peroxidases were found at very high constitutive levels, which were not induced or altered by external conditions, such as light and heavy-metal stress. It is suggested that the polymerization of polyphenols by peroxidases, enhanced after heavy-metal uptake and detoxification, is responsible for the binding of heavy metals in Nymphaea epidermal glands.

  3. Effect of lipopolysaccharide on protein accumulation by murine peritoneal macrophages: the correlation to activation for macrophage tumoricidal function

    SciTech Connect

    Tannenbaum, C.S.

    1987-01-01

    The protein synthetic patterns of tumoricidal murine peritoneal macrophage populations have been compared to those of non-tumoricidal populations utilizing two dimensional polyacrylamide gel electrophoresis (2D PAGE) of (/sup 35/S)-methionine-labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory and activated macrophages had numerous common features which distinguished them from the other normal non-macrophage cell types examined, unique proteins also distinguished each macrophage population from the others. Peritoneal macrophages elicited by treatment with heat killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16h or 72h functional assays, and shared a common protein synthetic profile which differentiated them from the synthetic patterns characteristic of the non-tumoricidal resident and inflammatory macrophages.

  4. Sodium chloride enhances cadmium tolerance through reducing cadmium accumulation and increasing anti-oxidative enzyme activity in tobacco.

    PubMed

    Zhang, Bing-Lin; Shang, Sheng-Hua; Zhang, Hai-Tao; Jabeen, Zahra; Zhang, Guo-Ping

    2013-06-01

    The effect of sodium chloride (NaCl) on cadmium (Cd) uptake, translocation, and oxidative stress was investigated using 2 tobacco cultivars differing in Cd tolerance. The growth inhibition of the tobacco plants exposed to Cd toxicity was in part alleviated by moderate addition of NaCl in the culture solution. Cadmium concentration of shoots and roots in the 2 cultivars increased with increasing Cd levels in the solution and decreased with the addition of NaCl. The addition of NaCl could alleviate the oxidative stress caused by Cd toxicity, as reflected by reduced production of malondialdehyde and recovered or enhanced activities of antioxidative enzymes catalase and glutathione peroxidase. The results also showed that the enhancement of antioxidative enzyme activity by NaCl for the tobacco plants exposed to Cd stress is related to induced Ca signaling.

  5. Study of the accumulation of air pollution by the biological indicators, using 14 MeV neutron activation

    NASA Astrophysics Data System (ADS)

    Senhou, A.; Khoukhi, T. El; Chouak, A.; Cherkaoui, R. El Moursili; Yahiaoui, A. El; Lferde, M.

    2001-06-01

    14 MeV neutron activation analysis was used to determine air polluting elements in samples of mosses, lichens and tree barks, collected from different regions in Morocco. The analysis of spectra shows clearly that the elements Mg, Al, Si, Cl, J, Ca, Ti and Fe can easily be determined by 14 NAA with good precision, while results for Zn, Rb, Sr, Ba and La are less precise. Curves showing correlation between Al and Mg concentrations are given for different sites.

  6. Characterization of Phosphofructokinase Activity in Mycobacterium tuberculosis Reveals That a Functional Glycolytic Carbon Flow Is Necessary to Limit the Accumulation of Toxic Metabolic Intermediates under Hypoxia

    PubMed Central

    Phong, Wai Yee; Lin, Wenwei; Rao, Srinivasa P. S.; Dick, Thomas; Alonso, Sylvie; Pethe, Kevin

    2013-01-01

    Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK) activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7–10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when the glycolytic

  7. Activation of mTOR signaling leads to orthopedic surgery-induced cognitive decline in mice through β-amyloid accumulation and tau phosphorylation.

    PubMed

    Shen, Wenzhen; Lu, Keliang; Wang, Jiawan; Wu, Anshi; Yue, Yun

    2016-10-01

    Postoperative cognitive dysfunction (POCD) is a serious complication following surgery, however, the mechanism of POCD remains to be elucidated. Previous evidence has revealed that POCD may be associated with the pathogenesis of neurodegenerative processes. The mammalian target of rapamycin (mTOR) signaling pathway has been reported to be crucial in the pathophysiology of neurodegenerative diseases. However, the implications of mTOR in POCD remains to be fully elucidated. In the present study, western blotting and enzyme‑linked immunosorbent assay were used to determine the expression of mTOR and any associated downstream targets; contextual fear conditioning was used to estimate the learning and memory ability of mice. Using an animal model of orthopedic surgery, it was found that surgical injury impaired hippocampal‑dependent memory and enhanced the levels of phosphorylated mTOR at Serine‑2448, phosphorylated 70‑kDa ribosomal protein S6 kinase (p70S6K) at Threonine‑389 with accumulation of β‑amyloid (Aβ) and hyperphosphorylated tau at Serine-396, compared with the control group. Pretreatment with rapamycin, an mTOR inhibitor, restored the abnormal mTOR/p70S6K signaling induced by surgery, attenuated the accumulation of Aβ and reduced the phosphorylation of tau protein. Rapamycin also reversed the surgery‑induced cognitive dysfunction. The results of the present study suggested that the surgical stimulus activated mTOR/p70S6K signaling excessively, and that the inhibition of mTOR signaling with rapamycin may prevent postoperative cognitive deficits, partly through attenuating the accumulation of Aβ and hyperphosphorylation of tau protein. PMID:27599409

  8. Impact of high dietary zinc on zinc accumulation, enzyme activity and proteomic profiles in the pancreas of piglets.

    PubMed

    Pieper, R; Martin, L; Schunter, N; Villodre Tudela, C; Weise, C; Klopfleisch, R; Zentek, J; Einspanier, R; Bondzio, A

    2015-04-01

    The exocrine pancreas plays an important role in zinc homeostasis. Feeding very high (2000-3000mgzinc/kg diet) levels of zinc oxide to piglets for short periods is a common practice in the swine industry to improve performance and prevent diseases. The impact on pancreatic function and possible side effects during long-term feeding of high dietary zinc levels are still poorly understood. A total of 54 weaned piglets were either fed with low (57mg/kg, LZn), normal (164mg/kg, NZn) or high (2425mg/kg, HZn) zinc concentration in the diets. After 4 weeks of feeding, ten piglets per treatment were euthanized and pancreas samples were taken. Tissue zinc concentration and metallothionein abundance was greater with HZn compared with NZn and LZn (P<0.05). Similarly, activity of α-amylase, lipase, trypsin and chymotrypsin was higher with HZn as compared with NZn and LZn diets (P<0.05), whereas elastase activity was unchanged. Total trolox equivalent antioxidative capacity of pancreas tissue was higher with HZn diets compared with the other treatments (P<0.05). Pancreatic protein profiles of NZn and HZn fed piglets were obtained by 2D-DIGE technique and revealed 15 differentially expressed proteins out of 2100 detected spots (P<0.05). The differentially expressed proteins aldose reductase, eukaryotic elongation factor II and peroxiredoxin III were confirmed by immunoblotting. Identified proteins include zinc finger-containing transcription factors and proteins mainly associated with oxidative stress response and signal transduction in HZn compared with NZn pigs. Histologic examination however showed no morphologic changes. The results suggest that long-term supply of very high dietary zinc increases zinc and metallothionein concentration, and digestive enzyme activity, but also triggers oxidative stress reactions in the pancreas of young pigs. The data provide new insights into pancreatic function under outbalanced zinc homeostasis.

  9. Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species.

    PubMed

    Cingoz, Gunce Sahin; Verma, Sandeep Kumar; Gurel, Ekrem

    2014-09-01

    The effect of hydrogen peroxide (H2O2) on callus cultures of four Digitalis species (Digitalis lamarckii, Digitalis trojana, Digitalis davisiana and Digitalis cariensis) increased catalase (CAT), superoxide dismutase (SOD), total phenolic, proline activity and cardiotonic glycoside production. Callus derived from hypocotyl explants was cultured on Murashige and Skoog medium supplemented with 0.25 mg L(-1) indole-3-acetic acid (IAA) and 0.5 mg L(-1) thidiazuron (TDZ). After a month of culture, callus was transferred to MS medium containing 10 mM H2O2 and then incubated for 6 h. The amount of five cardenolides (Lanatoside C, Digitoxin, Digoxigenin, Gitoxigenin and Digoxin) as well as CAT, SOD, total phenolic, proline activity from Digitalis species were compared. No digoxin was detected in all treatments and control groups. The total cardenolides estimated were in the order of D. lamarckii (586.65  μg g(-1) dw), D. davisiana (506.79 μg g(-1) dw), D. cariensis (376.60 μg g(-1) dw) and D. trojana (282.39 μg g(-1) dw). It was clear that H2O2 pre-treatment resulted in an increase in enzymatic and nonenzymatic antioxidants. However, a significant negative relationship between cardenolides production and overall activities of CAT, SOD, total phenolic and proline was evident. The described protocol here will be useful for the development of new strategies for a large-scale production of cardenolides.

  10. Enhancing Cell Nucleus Accumulation and DNA Cleavage Activity of Anti-Cancer Drug via Graphene Quantum Dots

    PubMed Central

    Wang, Chong; Wu, Congyu; Zhou, Xuejiao; Han, Ting; Xin, Xiaozhen; Wu, Jiaying; Zhang, Jingyan; Guo, Shouwu

    2013-01-01

    Graphene quantum dots (GQDs) maintain the intrinsic layered structural motif of graphene but with smaller lateral size and abundant periphery carboxylic groups, and are more compatible with biological system, thus are promising nanomaterials for therapeutic applications. Here we show that GQDs have a superb ability in drug delivery and anti-cancer activity boost without any pre-modification due to their unique structural properties. They could efficiently deliver doxorubicin (DOX) to the nucleus through DOX/GQD conjugates, because the conjugates assume different cellular and nuclear internalization pathways comparing to free DOX. Also, the conjugates could enhance DNA cleavage activity of DOX markedly. This enhancement combining with efficient nuclear delivery improved cytotoxicity of DOX dramatically. Furthermore, the DOX/GQD conjugates could also increase the nuclear uptake and cytotoxicity of DOX to drug-resistant cancer cells indicating that the conjugates may be capable to increase chemotherapy efficacy of anti-cancer drugs that are suboptimal due to the drug resistance. PMID:24092333

  11. Hydrolysis and volatile fatty acids accumulation of waste activated sludge enhanced by the combined use of nitrite and alkaline pH.

    PubMed

    Huang, Cheng; Liu, Congcong; Sun, Xiuyun; Sun, Yinglu; Li, Rui; Li, Jiansheng; Shen, Jinyou; Han, Weiqing; Liu, Xiaodong; Wang, Lianjun

    2015-12-01

    Volatile fatty acids (VFAs) production from anaerobic digestion of waste activated sludge (WAS) is often limited by the slow hydrolysis and/or poor substrate availability. Increased attention has been given to enhance the hydrolysis and acidification of WAS recently. This study presented an efficient and green strategy based on the combined use of nitrite pretreatment and alkaline pH to stimulate hydrolysis and VFA accumulation from WAS. Results showed that both proteins and polysaccharides increased in the presence of nitrite, indicating the enhancement of sludge solubilization and hydrolysis processes. Mechanism investigations showed that nitrite pretreatment could disintegrate the sludge particle and disperse extracellular polymeric substances (EPS). Then, anaerobic digestion tests demonstrated VFA production increased with nitrite treatment. The maximal VFA accumulation was achieved with 0.1 g N/L nitrite dosage and pH 10.0 at a sludge retention time (SRT) of 7 days, which was much higher VFA production in comparison with the blank, sole nitrite pretreatment, or sole pH 10. The potential analysis suggested that the combined nitrite pretreatment and alkaline pH is capable of enhancing WAS digestion with a great benefit for biological nutrient removal (BNR).

  12. Effect of Ethanol Accumulation on Porcine Interferon-α Production by Pichia pastoris and Activities of Key Enzymes in Carbon Metabolism.

    PubMed

    Ding, Jian; Gao, Minjie; Hou, Guoli; Liang, Kexue

    2015-08-01

    In production of porcine interferon α (pIFN-α) by Pichia pastoris, improper glycerol feeding strategy leads to ethanol accumulation in the last stage of growth phase. In the present study, taking two runs with low ethanol accumulation under 2 g/L as control group, effects of long-term (>4 h) and instantaneous high ethanol concentration (>10 g/L) on pIFN-α production, and activities of key enzymes in carbon metabolism were discussed. As a result, compared with control group, pIFN-α expression level was decreased about 4~12 folds under long-term high ethanol concentration, from the level above 3 g/L to the level under 1 g/L; pIFN-α expression level was decreased about 8 folds under instantaneous high ethanol concentration, reaching to the low level of 0.42 g/L. The low production of pIFN-α was caused by the severe inhibitory effect of ethanol on these enzymes.

  13. Increased peroxisome proliferator-activated receptor γ expression levels in visceral adipose tissue, and serum CCL2 and interleukin-6 levels during visceral adipose tissue accumulation.

    PubMed

    Yogarajah, Thaneswary; Bee, Yvonne-Tee Get; Noordin, Rahmah; Yin, Khoo Boon

    2015-01-01

    This study was conducted to determine the mRNA and protein expression levels of peroxisome proliferator-activated receptors (PPARs) in visceral adipose tissue, as well as serum adipokine levels, in Sprague Dawley rats. The rats were fed either a normal (control rats) or excessive (experimental rats) intake of food for 8 or 16 weeks, then sacrificed, at which time visceral and subcutaneous adipose tissues, as well as blood samples, were collected. The mRNA and protein expression levels of PPARs in the visceral adipose tissues were determined using reverse transcription-polymerase chain reaction and Western blotting, respectively. In addition, the levels of adipokines in the serum samples were determined using commercial ELISA kits. The results revealed that at 8 weeks, the mass of subcutaneous adipose tissue was higher than that of the visceral adipose tissue in the experimental rats, but the reverse occurred at 16 weeks. Furthermore, at 16 weeks the experimental rats exhibited an upregulation of PPARγ mRNA and protein expression levels in the visceral adipose tissues, and significant increases in the serum levels of CCL2 and interleukin (IL)-6 were observed, compared with those measured at 8 weeks. In conclusion, this study demonstrated that the PPARγ expression level was likely correlated with serum levels of CCL2 and IL-6, molecules that may facilitate visceral adipose tissue accumulation. In addition, the levels of the two adipokines in the serum may be useful as surrogate biomarkers for the expression levels of PPARγ in accumulated visceral adipose tissues.

  14. Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro.

    PubMed

    Leonardsen, L; Strömstedt, M; Jacobsen, D; Kristensen, K S; Baltsen, M; Andersen, C Y; Byskov, A G

    2000-01-01

    Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in

  15. Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha

    PubMed Central

    Silvestrini, Lucia; Rossi, Beatrice; Gallmetzer, Andreas; Mathieu, Martine; Scazzocchio, Claudio; Berardi, Enrico; Strauss, Joseph

    2015-01-01

    A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems. PMID:26335797

  16. The snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism.

    PubMed

    Chew, Shit F; Wong, Mei Y; Tam, Wai L; Ip, Yuen K

    2003-02-01

    The freshwater snakehead Channa asiatica is an obligatory air-breather that resides in slow-flowing streams and in crevices near riverbanks in Southern China. In its natural habitat, it may encounter bouts of aerial exposure during the dry seasons. In the laboratory, the ammonia excretion rate of C. asiatica exposed to terrestrial conditions in a 12 h:12 h dark:light regime was one quarter that of the submerged control. Consequently, the ammonia contents in the muscle, liver and plasma increased significantly, and C. asiatica was able to tolerate quite high levels of ammonia in its tissues. Urea was not the major product of ammonia detoxification in C. asiatica, which apparently did not possess a functioning ornithine urea cycle. Rather, alanine increased fourfold to 12.6 micromol g(-1) in the muscle after 48 h of aerial exposure. This is the highest level known in adult teleosts exposed to air or an ammonia-loading situation. The accumulated alanine could account for 70% of the deficit in ammonia excretion during this period, indicating that partial amino acid catabolism had occurred. This would allow the utilization of certain amino acids as energy sources and, at the same time, maintain the new steady state levels of ammonia in various tissues, preventing them from rising further. There was a reduction in the aminating activity of glutamate dehydrogenase from the muscle and liver of specimens exposed to terrestrial conditions. Such a phenomenon has not been reported before and could, presumably, facilitate the entry of alpha-ketoglutarate into the Krebs cycle instead of its amination to glutamate, as has been suggested elsewhere. However, in contrast to mudskippers, C. asiatica was apparently unable to reduce the rates of proteolysis and amino acid catabolism, because the reduction in nitrogenous excretion during 48 h of aerial exposure was completely balanced by nitrogenous accumulation in the body. Alanine accumulation also occurred in specimens exposed to

  17. Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha.

    PubMed

    Silvestrini, Lucia; Rossi, Beatrice; Gallmetzer, Andreas; Mathieu, Martine; Scazzocchio, Claudio; Berardi, Enrico; Strauss, Joseph

    2015-01-01

    A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.

  18. A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity

    PubMed Central

    Boesler, Carsten; Rigo, Norbert; Anokhina, Maria M.; Tauchert, Marcel J.; Agafonov, Dmitry E.; Kastner, Berthold; Urlaub, Henning; Ficner, Ralf; Will, Cindy L.; Lührmann, Reinhard

    2016-01-01

    The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5′ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5′ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation. PMID:27377154

  19. Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

    PubMed

    Itaya, Nair M; Asega, Amanda F; Carvalho, Maria Angela M; Figueiredo-Ribeiro, Rita de Cássia L

    2007-09-01

    Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.

  20. Photosynthesis and assimilate partitioning between carbohydrates and isoprenoid products in vegetatively active and dormant guayule: physiological and environmental constraints on rubber accumulation in a semiarid shrub.

    PubMed

    Salvucci, Michael E; Barta, Csengele; Byers, John A; Canarini, Alberto

    2010-12-01

    The stems and roots of the semiarid shrub guayule, Parthenium argentatum, contain a significant amount of natural rubber. Rubber accumulates in guayule when plants are vegetatively and reproductively dormant, complicating the relationship between growth/reproduction and product synthesis. To evaluate the factors regulating the partitioning of carbon to rubber, carbon assimilation and partitioning were measured in guayule plants that were grown under simulated summer- and winter-like conditions and under winter-like conditions with CO(2) enrichment. These conditions were used to induce vegetatively active and dormant states and to increase the source strength of vegetatively dormant plants, respectively. Rates of CO(2) assimilation, measured under growth temperatures and CO(2) , were similar for plants grown under summer- and winter-like conditions, but were higher with elevated CO(2) . After 5 months, plants grown under summer-like conditions had the greatest aboveground biomass, but the lowest levels of non-structural carbohydrates and rubber. In contrast, the amount of resin in the stems was similar under all growth conditions. Emission of biogenic volatile compounds was more than three-fold higher in plants grown under summer- compared with winter-like conditions. Taken together, the results show that guayule plants maintain a high rate of photosynthesis and accumulate non-structural carbohydrates and rubber in the vegetatively dormant state, but emit volatile compounds at a lower rate when compared with more vegetatively active plants. Enrichment with CO(2) in the vegetatively dormant state increased carbohydrate content but not the amount of rubber, suggesting that partitioning of assimilate to rubber is limited by sink strength in guayule.

  1. Soil organic carbon (SOC) accumulation in rice paddies under long-term agro-ecosystem experiments in southern China - VI. Changes in microbial community structure and respiratory activity

    NASA Astrophysics Data System (ADS)

    Liu, D.; Liu, X.; Liu, Y.; Li, L.; Pan, G.; Crowley, D.; Tippkötter, R.

    2011-02-01

    Biological stabilization within accumulated soil organic carbon (SOC) has not been well understood, while its role in physical and chemical protection as well as of chemical recalcitrance had been addressed in Chinese rice paddies. In this study, topsoil samples were collected and respiratory activity measured in situ following rice harvest under different fertilization treatments of three long-term experimental sites across southern China in 2009. The SOC contents, microbial biomass carbon (SMBC) and nitrogen (SMBN) were analysed using chemical digestion and microbial community structure assessment via clony dilute plate counting methods. While SOC contents were consistently higher under compound chemical fertilization (Comp-Fert) or combined organic and inorganic fertilization (Comb-Fert) compared to N fertilization only (N-Fert), there was significantly higher fungal-bacterial ratio under Comb-Fert than under N-Fert and Comp-Fert. When subtracting the background effect under no fertilization treatment (Non-Fert), the increase both in SMBC and SMBN under fertilization treatment was found very significantly correlated to the increase in SOC over controls across the sites. Also, the ratio of culturable fungal to bacterial population numbers (F/B ratio) was well correlated with soil organic carbon contents in all samples across the sites studied. SOC accumulation favoured a build-up the microbial community with increasing fungal dominance in the rice paddies under fertilization treatments. While soil respiration rates were high under Comb-Fert as a result of enhanced microbial community build-up, the specific soil respiratory activity based on microbial biomass carbon was found in a significantly negatively correlation with the SOC contents for overall samples. Thus, a fungal-dominated microbial community seemed to slow SOC turnover, thereby favouring SOC accumulation under Comp-Fert or under Comb-Fert in the rice paddies. Therefore, the biological stabilization

  2. BraSto, a Stowaway MITE from Brassica: recently active copies preferentially accumulate in the gene space.

    PubMed

    Sarilar, Véronique; Marmagne, Anne; Brabant, Philippe; Joets, Johann; Alix, Karine

    2011-09-01

    We characterized a Brassica miniature inverted repeat transposable element (MITE) from the Stowaway superfamily, designated BraSto (Bra ssica Sto waway). BraSto copy number was assessed using real-time quantitative PCR in the two diploid species B. rapa (genome A) and B. oleracea (genome C) and the corresponding allotetraploid species B. napus (genome AC). Phylogenetic relationships among a set of 131 BraSto copies were then analyzed. BraSto appears to have been only moderately amplified in the Brassica genome and was still active recently with marks of proliferation in both diploid Brassica species, which diverged 3.75 million years ago, but also in the allotetraploid species after reuniting of the two differentiated genomes. We characterized insertion sites for low-divergence BraSto copies among the gene space of the B. rapa genome using bioinformatics approaches. For BraSto copies localized nearby or within genes, we observed frequent associations of BraSto with putative promoters and regulatory regions of genes, but exclusion from coding regions. In addition, BraSto was significantly similar to several Brassica expressed sequence tags (ESTs), including stress-induced ESTs. We also demonstrated the enrichment of BraSto sequences in binding sites for transcription factors and other regulatory elements. Our results lead to the question of a role for BraSto in the regulation of gene expression: this putative role, if further confirmed experimentally, would help to obtain a new insight into the significance of MITEs in the functional plant genome.

  3. Accumulation of activated CD4+ lymphocytes in the lung of individuals infected with HIV accompanied by increased virus production in patients with secondary infections.

    PubMed Central

    Franchini, M; Walker, C; Henrard, D R; Suter-Gut, D; Braun, P; Villiger, B; Suter, M

    1995-01-01

    The lung is continuously exposed to infectious and non-infectious agents causing cell activation. Activated cells in the lung such as antigen-presenting cells which harbour HIV may favour this organ as a site for virus production. To test this hypothesis, cells from blood and bronchoalveolar lavage (BAL) of HIV-infected patients and healthy controls were obtained and the activation of the cells were analysed by measuring the expression of IL-2 receptor, HLA-DR and VLA-1. The HIV-infected individuals were subdivided into 'lung symptomatic' or 'lung asymptomatic' patients, depending on the presence or absence of secondary lung diseases besides HIV. All HIV-infected individuals demonstrated a decreased number of CD4+ lymphocytes in blood; however, normal numbers of these cells were found in BAL. The activation state of CD4+ and CD8+ T lymphocytes in blood and BAL was higher in lymphocytes from HIV-infected patients compared with controls. The activation state was highest in the lung symptomatic group. Lung symptomatic patients and lung asymptomatic patients with extrapulmonary infections had increased levels of free virus in plasma. Four out of four individuals without or with only low amounts of cell-free HIV in plasma belonged to the symptom-free subgroup. These results suggest that microorganisms other than HIV may promote viral replication via antigen-driven accumulation and activation of CD4+ cells in the lung or other organs, and thus may be responsible for the loss of helper T cells and the progression of the disease. PMID:7586671

  4. A probable crosstalk between Ca+2, reactive oxygen species accumulation and scavenging mechanisms and modulation of protein kinase C activity during seed development in sunflower

    PubMed Central

    Thakur, Anita; Bhatla, Satish C

    2014-01-01

    Seed development in sunflower involves a gradual dehydration and accumulation of oil bodies in the cells of developing cotyledons during transition from 30 to 40 DAA stage. Reactive oxygen species (ROS) content decreased with seed maturation. NO content and NO contributed by putative nitric oxide synthase, however, did not change markedly. Superoxide dismutase (SOD) activity exhibited a peak at 30 DAA stage, indicating its scavenging role at the mid-stage of seed development. H2O2 produced as a result of SOD action is subsequently scavenged primarily by elevation of GR activity. Significant temporal differences were evident in GR and POD activity during seed development. Protein kinase C (PKC) activity also showed modulation during early stages of embryo and seed development. Use of PKC-specific fluorescent probe, Fim-1, and PKC inhibitors (staurosporine and bisindoylmaleamide) provided evidence for increase in PKC activity at 40 DAA stage with an increase in protein concentration (50 to 200 µg). Endogenous calcium content also increased with seed maturation. Tissue homogenates from 40 DAA stage showed enhanced fluorescence due to Fim-1-PKC binding in presence of calcium ions and its lowering due to calcium chelating agent (BAPTA). Western blot analysis revealed an increase in the intensity of 2 bands representing PKC with the advancement of seed maturation and their further upregulation by calcium. Present findings, thus, provide new information on the biochemical regulation of seed development in sunflower, with evidence for a possible correlation between calcium, ROS, their scavenging enzymes and “conventional” PKC activity. PMID:24521818

  5. Quantification of fecal neutrophils by MPO determination (myeloperoxidase) in patients with invasive diarrhea. Cuantificación de neutrofilos fecales mediante la determinación de MPO (Mieloperoxidasa) en pacientes con diarrea invasiva.

    PubMed

    Bustos, D; Greco, G; Yapur, V; Facente, A; Di Carlo, M; Bustos, F; Dapia, L; Ascione, A; Negri, G

    2000-01-01

    Myeloperoxidase (MPO), a specific polymorphonuclear leukocyte enzyme, has been used previously to quantify the number of neutrophils in tissue. MPO activity was found to be linearly related to the number of neutrophil cells. In an attempt to use this method in leukocytes measuring in stool, fecal MPO was solubilized with hexadecyltrimethylammonium bromide and the MPO activity was measured by a dianisidine-H2O2 assay. Stools from 10 normal subjects and 39 patients with diarrhea produced by enteropathogenic bacteria were examined for leukocytes by MPO activity as well as microscopically using methylene blue stain, MPO activity was positive in 36 patients (92%) and leukocytes were present by microscopic observation in 30 (77%). Fecal leukocytes were not found in healthy controls and the MPO activity was undectable. Stool MPO activity had a range of from 1.6 to 2,830.0 x 10(3) UMPO per gram of feces (median 460.0). The number of neutrophils obtained through MPO activity had a range of 6.0 to 13,216.0/ mm3 (median 1,261.0). Fecal MPO activity is a simple biochemical assay for the detection and quantification of fecal leukocytes.

  6. Biochemical and ultrastructural effects of monensin on myeloperoxidase (MPO) processing in human leukemic HL-60 cells

    SciTech Connect

    Akin, D.T.; Kinkade J.M. Jr.; Parmley, R.T.

    1986-05-01

    Pulse-chase experiments using /sup 35/S-methionine, immunoprecipitation, and SDS-PAGE fluorography were used to study the effects of monensin (1 ..mu..M) on the post-translational processing and packaging of MPO into azurophil granules (AG). After 20 hr, maturation of MPO was inhibited by 80% and a large intermediate accumulated. Electron microscopy of treated cells revealed striking changes in morphology (formation of large vacuoles with small electron dense cores in the Golgi region) and staining patterns for complex glycoconjugates (PA-TCH-SP) and sulfate (HID), indicating qualitative changes in granule and Golgi characteristics. The distribution of DAB-reactive peroxidase was relatively unchanged. Subcellular fractionation using Percoll density gradient centrifugation showed labeled MPO remained in a lower density region and did not chase into higher density AG as seen with untreated cells. These data indicate that monensin inhibits both the maturation of MPO and the AG. Further studies are required to determine how closely these two processes are related.

  7. Influence of chloride on modification of unsaturated phosphatidylcholines by the myeloperoxidase/hydrogen peroxide/bromide system.

    PubMed

    Panasenko, Oleg M; Vakhrusheva, Tatyana; Tretyakov, Vadim; Spalteholz, Holger; Arnhold, Juergen

    2007-01-01

    The leukocyte enzyme myeloperoxidase (MPO) is capable of catalyzing the oxidation of chloride and bromide ions, at physiological concentrations of these substrates, by hydrogen peroxide, generating hypochlorous acid (HOCl) and hypobromous acid (HOBr), respectively. Our previous results showed that the hypohalous acids formed react with double bonds in phosphatidylcholines (PCs) to produce chloro- and bromohydrins. Lysophosphatidylcholine (lyso-PC) is additionally formed in PCs with two or more double bonds. This study was conducted to determine the effect physiological chloride concentration (140 mM) has on the formation of bromohydrins and lyso-PC from unsaturated PC upon treatment with the myeloperoxidase/hydrogen peroxide/bromide (MPO/H2O2/Br-) system using physiological bromide concentrations (20-100 microM). The composition of reaction products was analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). With monounsaturated PC, we demonstrated that the rate and extent of mono-bromohydrin formation were higher in the samples with 140 mM chloride compared to those with no added chloride. Moreover, mono-bromohydrin came to be the major product and no mono-chlorohydrin was observed already at 60 microM bromide. We attributed these effects to the involvement of HOBr arising from the reaction of MPO-derived HOCl with bromide rather than to the exchange of bromide with chlorine atoms of chlorohydrins or direct formation of HOBr by MPO. The presence of chloride shifted the pH optimum for mono-bromohydrin formation (pH 5.0) toward neutral values, and a significant yield of mono-bromohydrin was detected at physiological pH values (7.0-7.4). For polyunsaturated PC, chloride enhanced also lyso-PC production, the effect being pronounced at bromide concentrations below 40 microM. The results indicate that at physiological levels of chloride and bromide, chloride promotes MPO-mediated formation of bromohydrins and lyso

  8. A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule.

    PubMed

    Gisby, Martin F; Mellors, Philip; Madesis, Panagiotis; Ellin, Marianne; Laverty, Hugh; O'Kane, Sharon; Ferguson, Mark W J; Day, Anil

    2011-06-01

    Human transforming growth factor-β3 (TGFβ3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFβ3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFβ3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFβ3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFβ3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFβ3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFβ3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFβ3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFβ3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFβ3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFβ3. PMID:21535357

  9. Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro

    PubMed Central

    Podrez, Eugene A.; Schmitt, David; Hoff, Henry F.; Hazen, Stanley L.

    1999-01-01

    Oxidized LDL is implicated in atherosclerosis; however, the pathways that convert LDL into an atherogenic form in vivo are not established. Production of reactive nitrogen species may be one important pathway, since LDL recovered from human atherosclerotic aorta is enriched in nitrotyrosine. We now report that reactive nitrogen species generated by the MPO-H2O2-NO2– system of monocytes convert LDL into a form (NO2-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation, essential steps in lesion development. Incubation of LDL with isolated MPO, an H2O2-generating system, and nitrite (NO2–)— a major end-product of NO metabolism—resulted in nitration of apolipoprotein B 100 tyrosyl residues and initiation of LDL lipid peroxidation. The time course of LDL protein nitration and lipid peroxidation paralleled the acquisition of high-affinity, concentration-dependent, and saturable binding of NO2-LDL to human monocyte–derived macrophages and mouse peritoneal macrophages. LDL modification and conversion into a high-uptake form occurred in the absence of free metal ions, required NO2–, occurred at physiological levels of Cl–, and was inhibited by heme poisons, catalase, and BHT. Macrophage binding of NO2-LDL was specific and mediated by neither the LDL receptor nor the scavenger receptor class A type I. Exposure of macrophages to NO2-LDL promoted cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation. Collectively, these results identify MPO-generated reactive nitrogen species as a physiologically plausible pathway for converting LDL into an atherogenic form. PMID:10359564

  10. Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco-specific nitrosamine accumulation in cured leaves and cigarette smoke.

    PubMed

    Lu, Jianli; Zhang, Leichen; Lewis, Ramsey S; Bovet, Lucien; Goepfert, Simon; Jack, Anne M; Crutchfield, James D; Ji, Huihua; Dewey, Ralph E

    2016-07-01

    Burley tobaccos (Nicotiana tabacum) display a nitrogen-use-deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco-specific nitrosamines (TSNAs). Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen-assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field-grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well-documented animal carcinogens found in tobacco products. PMID:26800860

  11. Inborn Error of Cobalamin Metabolism Associated with the Intracellular Accumulation of Transcobalamin-Bound Cobalamin and Mutations in ZNF143, Which Codes for a Transcriptional Activator.

    PubMed

    Pupavac, Mihaela; Watkins, David; Petrella, Francis; Fahiminiya, Somayyeh; Janer, Alexandre; Cheung, Warren; Gingras, Anne-Claude; Pastinen, Tomi; Muenzer, Joseph; Majewski, Jacek; Shoubridge, Eric A; Rosenblatt, David S

    2016-09-01

    Vitamin B12 (cobalamin, Cbl) cofactors adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) are required for the activity of the enzymes methylmalonyl-CoA mutase (MCM) and methionine synthase (MS). Inborn errors of Cbl metabolism are rare Mendelian disorders associated with hematological and neurological manifestations, and elevations of methylmalonic acid and/or homocysteine in the blood and urine. We describe a patient whose fibroblasts had decreased functional activity of MCM and MS and decreased synthesis of AdoCbl and MeCbl (3.4% and 1.0% of cellular Cbl, respectively). The defect in cultured patient fibroblasts complemented those from all known complementation groups. Patient cells accumulated transcobalamin-bound-Cbl, a complex which usually dissociates in the lysosome to release free Cbl. Whole-exome sequencing identified putative disease-causing variants c.851T>G (p.L284*) and c.1019C>T (p.T340I) in transcription factor ZNF143. Proximity biotinylation analysis confirmed the interaction between ZNF143 and HCFC1, a protein that regulates expression of the Cbl trafficking enzyme MMACHC. qRT-PCR analysis revealed low MMACHC expression levels both in patient fibroblasts, and in control fibroblasts incubated with ZNF143 siRNA. PMID:27349184

  12. Increased neuronal PreP activity reduces Aβ accumulation, attenuates neuroinflammation and improves mitochondrial and synaptic function in Alzheimer disease's mouse model.

    PubMed

    Fang, Du; Wang, Yongfu; Zhang, Zhihua; Du, Heng; Yan, Shiqiang; Sun, Qinru; Zhong, Changjia; Wu, Long; Vangavaragu, Jhansi Rani; Yan, Shijun; Hu, Gang; Guo, Lan; Rabinowitz, Molly; Glaser, Elzbieta; Arancio, Ottavio; Sosunov, Alexander A; McKhann, Guy M; Chen, John Xi; Yan, Shirley ShiDu

    2015-09-15

    Accumulation of amyloid-β (Aβ) in synaptic mitochondria is associated with mitochondrial and synaptic injury. The underlying mechanisms and strategies to eliminate Aβ and rescue mitochondrial and synaptic defects remain elusive. Presequence protease (PreP), a mitochondrial peptidasome, is a novel mitochondrial Aβ degrading enzyme. Here, we demonstrate for the first time that increased expression of active human PreP in cortical neurons attenuates Alzheimer disease's (AD)-like mitochondrial amyloid pathology and synaptic mitochondrial dysfunction, and suppresses mitochondrial oxidative stress. Notably, PreP-overexpressed AD mice show significant reduction in the production of proinflammatory mediators. Accordingly, increased neuronal PreP expression improves learning and memory and synaptic function in vivo AD mice, and alleviates Aβ-mediated reduction of long-term potentiation (LTP). Our results provide in vivo evidence that PreP may play an important role in maintaining mitochondrial integrity and function by clearance and degradation of mitochondrial Aβ along with the improvement in synaptic and behavioral function in AD mouse model. Thus, enhancing PreP activity/expression may be a new therapeutic avenue for treatment of AD.

  13. Bidens pilosa and its active compound inhibit adipogenesis and lipid accumulation via down-modulation of the C/EBP and PPARγ pathways

    PubMed Central

    Liang, Yu-Chuan; Yang, Meng-Ting; Lin, Chuan-Ju; Chang, Cicero Lee-Tian; Yang, Wen-Chin

    2016-01-01

    Obesity and its complications are a major global health problem. In this study, we investigated the anti-obesity effect and mechanism of an edible plant, Bidens pilosa, and its active constituent. We first assessed the long-term effect of B. pilosa on body composition, body weight, blood parameters in ICR mice. We observed that it significantly decreased fat content and increased protein content in ICR mice. Next, we verified the anti-obesity effect of B. pilosa in ob/ob mice. It effectively and dose-dependently reduced fat content, adipocyte size and/or body weight in mice. Moreover, mechanistic studies showed that B. pilosa inhibited the expression of peroxisome proliferator activated receptor γ (PPARγ), CCAAT/enhancer binding proteins (C/EBPs) and Egr2 in adipose tissue. Finally, we examined the effect of 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHT) on adipogenesis in adipocytes. We found that B. pilosa significantly decreased the adipogenesis and lipid accumulation. This decrease was associated with the down-regulation of expression of Egr2, C/EBPs, PPARγ, adipocyte Protein 2 (aP2) and adiponectin. In summary, this work demonstrated that B. pilosa and GHT suppressed adipogenesis and lipid content in adipocytes and/or animals via the down-regulation of the Egr2, C/EBPs and PPARγ pathways, suggesting a novel application of B. pilosa and GHT against obesity. PMID:27063434

  14. Different-Sized Gold Nanoparticle Activator/Antigen Increases Dendritic Cells Accumulation in Liver-Draining Lymph Nodes and CD8+ T Cell Responses.

    PubMed

    Zhou, Qianqian; Zhang, Yulong; Du, Juan; Li, Yuan; Zhou, Yong; Fu, Qiuxia; Zhang, Jingang; Wang, Xiaohui; Zhan, Linsheng

    2016-02-23

    The lack of efficient antigen and activator delivery systems, as well as the restricted migration of dendritic cells (DCs) to secondary lymph organs, dramatically limits DC-based adoptive immunotherapy. We selected two spherical gold nanoparticle (AuNP)-based vehicles of optimal size for activator and antigen delivery. Their combination (termed the NanoAu-Cocktail) was associated with the dual targeting of CpG oligonucleotides (CpG-ODNs) and an OVA peptide (OVAp) to DC subcellular compartments, inducing enhanced antigen cross-presentation, upregulated expression of costimulatory molecules and elevated secretion of T helper1 cytokines. We demonstrated that the intravenously transfused NanoAu-Cocktail pulsed DCs showed dramatically improved in vivo homing ability to lymphoid tissues and were settled in T cell area. Especially, by tissue-distribution analysis, we found that more than 60% of lymphoid tissues-homing DCs accumulated in liver-draining lymph nodes (LLNs). The improved homing ability of NanoAu-Cocktail pulsed DCs was associated with the high expression of chemokine receptor 7 (CCR7) and rearrangement of the cytoskeletons. In addition, by antigen-specific tetramers detection, NanoAu-Cocktail pulsed DCs were proved able to elicit strong antigen-specific CD8+ T cell responses, which provided enhanced protection from viral invasions. This study highlights the importance of codelivering antigen/adjuvant using different sized gold nanoparticles to improve DC homing and therapy. PMID:26771692

  15. The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

    PubMed

    Ip, Y K; Loong, A M; Ching, B; Tham, G H Y; Wong, W P; Chew, S F

    2009-12-01

    This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.

  16. Perennial peanut (Arachis glabrata Benth.) leaves contain hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase activity and accumulate hydroxycinnamoyl-tartaric acid esters.

    PubMed

    Sullivan, Michael L

    2014-05-01

    Many plants accumulate hydroxycinnamoyl esters to protect against abiotic and biotic stresses. Caffeoyl esters in particular can be substrates for endogenous polyphenol oxidases (PPOs). Recently, we showed that perennial peanut (Arachis glabrata Benth.) leaves contain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were believed to be cis- and trans-p-coumaroyl tartaric acid and cis- and trans-feruloyl-tartaric acid, but lack of standards prevented definitive identifications. Here we characterize enzymatic activities in peanut leaves to understand how caftaric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid (specific activities of 11 ± 2.8, 8 ± 1.8, 4 ± 0.8 pkat mg(-1) crude protein, respectively). The HTT activity was used to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric acid in vitro. These products allowed definitive identification of the corresponding cis- and trans-hydroxycinnamoyl esters extracted from leaves. We tentatively identified sinapoyl-tartaric acid as another major phenolic compound in peanut leaves that likely participates in secondary reactions with PPO-generated quinones. These results suggest hydroxycinnamoyl-tartaric acid esters are made by an acyltransferase, possibly a BAHD family member, in perennial peanut. Identification of a gene encoding HTT and further characterization of the enzyme will aid in identifying determinants of donor and acceptor substrate specificity for this important class of biosynthetic enzymes. An HTT gene could also provide a means by genetic engineering for producing caffeoyl- and other hydroxycinnamoyl-tartaric acid esters in forage crops that lack them.

  17. [A case of myeloperoxidase-antineutrophil cytoplasmic antibody (ANCA)-positive crescentic glomerulonephritis induced by propylthiouracil (PTU)].

    PubMed

    Kato, H; Osajima, A; Tanaka, H; Serino, R; Kabashima, N; Tamura, M; Segawa, K; Anai, H; Takasugi, M; Nakajima, Y

    1997-07-01

    We have experienced a case of myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-related glomerulonephritis induced by propylthiouracil (PTU). A 45-year-old female had been treated with PTU for 4 years after the diagnosis of hyperthyroidism. She was referred to out hospital because of abrupt macroscopic hematuria and moderate proteinuria after several days of upper respiratory tract infection. On admission, her laboratory findings showed deterioration of renal function. Renal biopsy revealed crescentic glomerulonephritis without deposition of immune complexes. Her serology was found to be MPO-ANCA-positive and cytoplasmic-ANCA-negative. Based of these findings, we diagnosed idiopathic crescentic glomerulonephritis. Following the initiation of steroid pulse therapy, her urinary protein excretion and renal function gradually improved in parallel with a decrease in the MPO-ANCA titer. Although steroid therapy effectively responded to their renal function without the withdrawal of PTU, it seems that PTU may be closely associated with the development of (MPO-ANCA)-related glomerulonephritis in this case. Therefore, hyperthyroidism patients treated with PTU should be paced under vigilant observation by monitoring their urinalysis and serum creatinine level.

  18. Distinct regulation of the anterior and posterior myeloperoxidase expression by Etv2 and Gata1 during primitive Granulopoiesis in zebrafish.

    PubMed

    Glenn, Nicole O; Schumacher, Jennifer A; Kim, Hyon J; Zhao, Emma J; Skerniskyte, Jurate; Sumanas, Saulius

    2014-09-01

    Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.

  19. Detection of anti-lactoferrin antibodies and anti-myeloperoxidase antibodies in autoimmune hepatitis: a retrospective study.

    PubMed

    Tan, Liming; Zhang, Yuhong; Peng, Weihua; Chen, Juanjuan; Li, Hua; Ming, Feng

    2014-01-01

    Anti-lactoferrin antibodies (ALA) and anti-myeloperoxidase antibodies (AMPA) are specific serological markers for autoimmune hepatitis (AIH). The project aimed to detect ALA and AMPA and explore their clinical significances in AIH patients. 59 AIH patients, 217 non AIH patients, and 50 healthy controls were enrolled in this study. ALA and AMPA were detected by ELISA. Antineutropil cytoplasmic antibodies (ANCA) and anti-smooth muscle antibodies (ASMA) were examined by indirect immunofluorescence. Antimitochondrial antibody M2 subtype (AMA-M2), anti-liver kidney microsomal antibody Type 1 (LKM1), anti-liver cytosol antibody Type 1 (LC1), and anti-soluble liver antigen/liver-pancreas antibodies (SLA/LP) were tested by immunoblot. The positivity for ALA was 18.6% in AIH group, only one patient in non-AIH group was positive for ALA; the positivity for AMPA was 59.3% in AIH group, with significant differences (P < 0.01) compared with other groups. The specificities for ALA and AMPA were 99.63% and 97.75%; the sensitivities were 18.64% and 59.32%; and the accuracy rates were 84.97% and 90.80%, respectively. A certain correlation was observed between ALA and SLA/LP, AMPA and ANCA, ASMA in AIH group. ALA and AMPA were associated with AIH, and had high clinical diagnostic value. Co-detection with other relative autoantibodies could play an important role in differential diagnosis of AIH.

  20. Comparisons between myeloperoxidase, lactoferrin, calprotectin and lipocalin-2, as fecal biomarkers of intestinal inflammation in malnourished children

    PubMed Central

    Prata, Mara de Moura Gondim; Havt, A; Bolick, DT; Pinkerton, R; Lima, AAM; Guerrant, RL

    2016-01-01

    Fecal biomarkers have emerged as important tools to assess intestinal inflammation and enteropathy. The aim of this study was to investigate the correlations between the fecal markers, myeloperoxidase (MPO), lactoferrin (FL), calprotectin (FC) and lipocalin-2 (Lcn-2), and to compare differences by breastfeeding status as well as normalization by fecal protein or by fecal weight. Simultaneous, quantitative MPO, FL, FC and Lcn-2, levels were determined in frozen fecal specimens collected from 78 children (mean age 15.2 ± 5.3 months) in a case-control study of childhood malnutrition in Brazil. The biomarker concentrations were measured by enzymelinked immunosorbent assay. The correlations among all biomarkers were significant (P<0.01). There were stronger correlations of fecal MPO with fecal lactoferrin and calprotectin, with lower, but still highly significant correlations of all 3 inflammatory biomarkers with Lcn-2 likely because the latter may also reflect enterocyte damage as well as neutrophil presence. Furthermore, the biomarker results with protein normalized compared to simple fecal weight normalized values showed only a slightly better correlation suggesting that the added cost and time for protein normalization added little to carefully measured fecal weights as denominators. In conclusion, fecal MPO correlates tightly with fecal lactoferrin and calprotectin irrespective of breastfeeding status and provides a common, available biomarker for comparison of human and animal model studies.

  1. Major Basic Protein from Eosinophils and Myeloperoxidase from Neutrophils Are Required for Protective Immunity to Strongyloides stercoralis in Mice ▿

    PubMed Central

    O'Connell, Amy E.; Hess, Jessica A.; Santiago, Gilberto A.; Nolan, Thomas J.; Lok, James B.; Lee, James J.; Abraham, David

    2011-01-01

    Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity. PMID:21482685

  2. [Simultaneous presence of antibodies against the glomerular basement membrane and anti-myeloperoxidase antibodies in 2 patients with rapidly progressive glomerulonephritis].

    PubMed

    Díaz Rodríguez, C; Costero, O; Torre, A; De Alvaro, F; Gil, F; Picazo, M L; Martínez-Ara, J

    2002-01-01

    We report two patients with rapidly progressive glomerulonephritis without alveolar hemorrhage. Renal biopsy showed extracapillary glomerulonephritis with linear deposits of immunoglobulin G. Serologically anti-glomerular basement membrane antibodies (Ac AMBG) and ANCA anti-myeloperoxidase were present. All patients were treated with steroids, cyclophosphamide and plasma exchange. One patient needed dialysis, and other one died from a renal biopsy complication. We discuss the epidemiologic, pathogenic and prognostic aspects of this association.

  3. Selective V(1a) agonism attenuates vascular dysfunction and fluid accumulation in ovine severe sepsis.

    PubMed

    Rehberg, Sebastian; Yamamoto, Yusuke; Sousse, Linda; Bartha, Eva; Jonkam, Collette; Hasselbach, Anthony K; Traber, Lillian D; Cox, Robert A; Westphal, Martin; Enkhbaatar, Perenlei; Traber, Daniel L

    2012-11-15

    Vasopressin analogs are used as a supplement to norepinephrine in septic shock. The isolated effects of vasopressin agonists on sepsis-induced vascular dysfunction, however, remain controversial. Because V(2)-receptor stimulation induces vasodilation and procoagulant effects, a higher V(1a)- versus V(2)-receptor selectivity might be advantageous. We therefore hypothesized that a sole, titrated infusion of the selective V(1a)-agonist Phe(2)-Orn(8)-Vasotocin (POV) is more effective than the mixed V(1a)-/V(2)-agonist AVP for the treatment of vascular and cardiopulmonary dysfunction in methicillin resistant staphylococcus aureus pneumonia-induced, ovine sepsis. After the onset of hemodynamic instability, awake, chronically instrumented, mechanically ventilated, and fluid resuscitated sheep were randomly assigned to receive continuous infusions of either POV, AVP, or saline solution (control; each n = 6). AVP and POV were titrated to maintain mean arterial pressure above baseline - 10 mmHg. When compared with that of control animals, AVP and POV reduced neutrophil migration (myeloperoxidase activity, alveolar neutrophils) and plasma levels of nitric oxide, resulting in higher mean arterial pressures and a reduced vascular leakage (net fluid balance, chest and abdominal fluid, pulmonary bloodless wet-to-dry-weight ratio, alveolar and septal edema). Notably, POV stabilized hemodynamics at lower doses than AVP. In addition, POV, but not AVP, reduced myocardial and pulmonary tissue concentrations of 3-nitrotyrosine, VEGF, and angiopoietin-2, thereby leading to an abolishment of cumulative fluid accumulation (POV, 9 ± 15 ml/kg vs. AVP, 110 ± 13 ml/kg vs. control, 213 ± 16 ml/kg; P < 0.001 each) and an attenuated cardiopulmonary dysfunction (left ventricular stroke work index, PaO(2)-to-FiO(2) ratio) versus control animals. Highly selective V(1a)-agonism appears to be superior to unselective vasopressin analogs for the treatment of sepsis-induced vascular dysfunction.

  4. Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages

    PubMed Central

    Lanuti, Mirko; Talamonti, Emanuela; Maccarrone, Mauro; Chiurchiù, Valerio

    2015-01-01

    The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases. PMID:25970609

  5. Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages.

    PubMed

    Lanuti, Mirko; Talamonti, Emanuela; Maccarrone, Mauro; Chiurchiù, Valerio

    2015-01-01

    The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases. PMID:25970609

  6. Flavonol glycosides with lipid accumulation inhibitory activity and simultaneous quantitative analysis of 15 polyphenols and caffeine in the flower buds of Camellia sinensis from different regions by LCMS.

    PubMed

    Morikawa, Toshio; Ninomiya, Kiyofumi; Miyake, Sohachiro; Miki, Yoshinobu; Okamoto, Masaki; Yoshikawa, Masayuki; Muraoka, Osamu

    2013-09-01

    A simultaneous quantitative analytical method for 15 major polyphenols, e.g. five catechins (1-5) and 10 flavonols (6-15), as functional constituents in the extracts of "tea flowers", the flower buds of Camellia sinensis (Theaceae), has been developed. The content of caffeine (16), which showed similar chromatographic behaviour under the analytical conditions, was also determined. To approve the validity of the newly developed protocol, thirteen extracts of the plant's flower buds collected from different regions, i.e. China, Taiwan, Japan and India, were evaluated. The results indicated that the assay was reproducible and precise, and could be readily underutilised for the quality evaluation of tea flowers on the basis of polyphenols' contents. It was noteworthy that the contents of two major constituents, kaempferol 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (10) and kaempferol 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside (11), varied by region where the flower buds were produced. A new flavonol glycoside, chakaflavonoside B (17), which was isolated in the course of this analytical study, was found to show oleic acid-albumin-induced lipid accumulation inhibitory activity.

  7. Protein cryoprotective activity of a cytosolic small heat shock protein that accumulates constitutively in chestnut stems and is up-regulated by low and high temperatures.

    PubMed

    Lopez-Matas, Maria-Angeles; Nuñez, Paulina; Soto, Alvaro; Allona, Isabel; Casado, Rosa; Collada, Carmen; Guevara, Maria-Angeles; Aragoncillo, Cipriano; Gomez, Luis

    2004-04-01

    Heat shock, and other stresses that cause protein misfolding and aggregation, trigger the accumulation of heat shock proteins (HSPs) in virtually all organisms. Among the HSPs of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. We analyzed the occurrence of sHSPs in vegetative organs of Castanea sativa (sweet chestnut), a temperate woody species that exhibits remarkable freezing tolerance. A constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously described in cotyledons. Expression of the corresponding gene in stems was confirmed through cDNA cloning and reverse transcription-PCR. Stem protein and mRNA profiles indicated that CsHSP17.5 is significantly up-regulated in spring and fall, reaching maximal levels in late summer and, especially, in winter. In addition, cold exposure was found to quickly activate shsp gene expression in both stems and roots of chestnut seedlings kept in growth chambers. Our main finding is that purified CsHSP17.5 is very effective in protecting the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (on a molar basis, CsHSP17.5 is about 400 times more effective as cryoprotectant than hen egg-white lysozyme). Consistent with these observations, repeated freezing/thawing did not affect appreciably the chaperone activity of diluted CsHSP17.5 nor its ability to form dodecameric complexes in vitro. Taken together, these results substantiate the hypothesis that sHSPs can play relevant roles in the acquisition of freezing tolerance.

  8. Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor.

    PubMed

    Bülow, E; Nauseef, W M; Goedken, M; McCormick, S; Calafat, J; Gullberg, U; Olsson, I

    2002-02-01

    During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein alpha(1)-microglobulin (alpha(1)-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to alpha(1)-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of alpha(1)-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.

  9. Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: mechanical loading regulates osteopontin and myeloperoxidase.

    PubMed

    Miles, R R; Turner, C H; Santerre, R; Tu, Y; McClelland, P; Argot, J; DeHoff, B S; Mundy, C W; Rosteck, P R; Bidwell, J; Sluka, J P; Hock, J; Onyia, J E

    1998-03-01

    The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone

  10. Bilirubin scavenges chloramines and inhibits myeloperoxidase-induced protein/lipid oxidation in physiologically relevant hyperbilirubinemic serum.

    PubMed

    Boon, A C; Hawkins, C L; Coombes, J S; Wagner, K H; Bulmer, A C

    2015-09-01

    Hypochlorous acid (HOCl), an oxidant produced by myeloperoxidase (MPO), induces protein and lipid oxidation, which is implicated in the pathogenesis of atherosclerosis. Individuals with mildly elevated bilirubin concentrations (i.e., Gilbert syndrome; GS) are protected from atherosclerosis, cardiovascular disease, and related mortality. We aimed to investigate whether exogenous/endogenous unconjugated bilirubin (UCB), at physiological concentrations, can protect proteins/lipids from oxidation induced by reagent and enzymatically generated HOCl. Serum/plasma samples supplemented with exogenous UCB (≤250µM) were assessed for their susceptibility to HOCl and MPO/H2O2/Cl(-) oxidation, by measuring chloramine, protein carbonyl, and malondialdehyde (MDA) formation. Serum/plasma samples from hyperbilirubinemic Gunn rats and humans with GS were also exposed to MPO/H2O2/Cl(-) to: (1) validate in vitro data and (2) determine the relevance of endogenously elevated UCB in preventing protein and lipid oxidation. Exogenous UCB dose-dependently (P<0.05) inhibited HOCl and MPO/H2O2/Cl(-)-induced chloramine formation. Albumin-bound UCB efficiently and specifically (3.9-125µM; P<0.05) scavenged taurine, glycine, and N-α-acetyllysine chloramines. These results were translated into Gunn rat and GS serum/plasma, which showed significantly (P<0.01) reduced chloramine formation after MPO-induced oxidation. Protein carbonyl and MDA formation was also reduced after MPO oxidation in plasma supplemented with UCB (P<0.05; 25 and 50µM, respectively). Significant inhibition of protein and lipid oxidation was demonstrated within the physiological range of UCB, providing a hypothetical link to protection from atherosclerosis in hyperbilirubinemic individuals. These data demonstrate a novel and physiologically relevant mechanism whereby UCB could inhibit protein and lipid modification by quenching chloramines induced by MPO-induced HOCl. PMID:26057938

  11. Organic UV filters inhibit multixenobiotic resistance (MXR) activity in Tetrahymena thermophila: investigations by the Rhodamine 123 accumulation assay and molecular docking.

    PubMed

    Gao, Li; Yuan, Tao; Cheng, Peng; Zhou, Chuanqi; Ao, Junjie; Wang, Wenhua; Zhang, Haimou

    2016-09-01

    Multixenobiotic resistance (MXR) transporters, which belong to ATP-binding cassette (ABC) family proteins, are present in living organisms as a first line of defense system against xenobiotics and environmental contaminants. The effects of six organic UV filters (4-methyl -benzylidene camphor, 4-MBC; benzophenone-3, BP-3; butyl methoxydibenzoyl-methane, BM-DBM; ethylhexyl methoxy cinnamate, EHMC; octocrylene, OC and homosalate, HMS) on multixenobiotic resistance (MXR) in Tetrahymena thermophila were investigated in this study. It was found that 4-MBC, BP-3 and BM-DBM could significantly inhibit activity of the MXR system, causing concentration dependent accumulation of rhodamine 123; while EHMC, OC and HMS had weak MXR inhibition. The IC50 (50 % inhibition concentration) values of 4-MBC, BP-3 and BM-DBM were 23.54, 40.59 and 26.37 μM, respectively, with inhibitory potentials of 23.1, 13.4 and 20.6 % relative to verapamil (VER, a model inhibitor of P-glycoprotein). Our results firstly provide the evidence for UV filters inhibition effect on MXR in aquatic organisms. In addition, it was revealed by molecular docking analysis that the selected six UV filters can occupy the same binding site on T. thermophila P-gp as VER does; and form H-bonds with residues Ser 328 and/or Asn 281. This study raises the awareness of aquatic ecological risk from the organic UV filters exposure, as they would be involved in potentiating toxic effects by chemosensitizing. PMID:27315091

  12. Real-time monitoring of superoxide accumulation and antioxidant activity in a brain slice model using an electrochemical cytochrome c biosensor.

    PubMed

    Ganesana, Mallikarjunarao; Erlichman, Joseph S; Andreescu, Silvana

    2012-12-15

    The overproduction of reactive oxygen species and the resulting damage are central to the pathology of many diseases. The study of the temporal and spatial accumulation of reactive oxygen species has been limited because of the lack of specific probes and techniques capable of continuous measurement. We demonstrate the use of a miniaturized electrochemical cytochrome c (Cyt c) biosensor for real-time measurements and quantitative assessment of superoxide production and inactivation by natural and engineered antioxidants in acutely prepared brain slices from mice. Under control conditions, superoxide radicals produced from the hippocampal region of the brain in 400-μm-thick sections were well within the range of detection of the electrode. Exposure of the slices to ischemic conditions increased the superoxide production twofold and measurements from the slices were stable over a 3- to 4-h period. The stilbene derivative and anion channel inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilbene markedly reduced the extracellular superoxide signal under control conditions, suggesting that a transmembrane flux of superoxide into the extracellular space may occur as part of normal redox signaling. The specificity of the electrode for superoxide released by cells in the hippocampus was verified by the exogenous addition of superoxide dismutase (SOD), which decreased the superoxide signal in a dose-dependent manner. Similar results were seen with the addition of the SOD mimetic cerium oxide nanoparticles (nanoceria), in that the superoxide anion radical scavenging activity of nanoceria with an average diameter of 15 nm was equivalent to 527 U of SOD for each 1 μg/ml of nanoceria added. This study demonstrates the potential of electrochemical biosensors for studying real-time dynamics of reactive oxygen species in a biological model and the utility of these measurements in defining the relative contribution of superoxide to oxidative injury. PMID:23085519

  13. Real-time monitoring of superoxide accumulation and antioxidant activity in a brain slice model using an electrochemical cytochrome c biosensor

    PubMed Central

    Ganesana, Mallikarjunarao; Erlichman, Joseph S.; Andreescu, Silvana

    2012-01-01

    The overproduction of reactive oxygen species and resulting damage are central to the pathology of many diseases. The study of the temporal and spatial accumulation of reactive oxygen species has been limited due to the lack of specific probes and techniques capable of continuous measurement. We demonstrate the use of a miniaturized electrochemical cytochrome C (Cyt C) biosensor for real-time measurements and quantitative assessment of superoxide production and inactivation by natural and engineered antioxidants in acutely prepared brain slices from mice. During control conditions, superoxide radicals produced from the hippocampal region of the brain in 400 μm thick sections were well within the range of detection of the electrode. Exposure of the slices to ischemic conditions increased the superoxide production two fold and measurements from the slices were stable over a 3–4 hour period. The stilbene derivative and anion channel inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic stilbene (DIDS), markedly reduced the extracellular superoxide signal under control conditions suggesting that a transmembrane flux of superoxide into the extracellular space may occur as part of normal redox signaling. The specificity of the electrode for superoxide released by cells in the hippocampus was verified by the exogenous addition of superoxide dismutase (SOD) which decreased the superoxide signal in a dose-dependent manner. Similar results were seen with the addition of the SOD-mimetic, cerium oxide nanoparticles (nanoceria) where the superoxide anion radical scavenging activity of nanoceria with an average diameter of 15 nm was equivalent to 527 U of SOD for each 1 μg/ml of nanoceria added. This study demonstrates the potential of electrochemical biosensors for studying real-time dynamics of reactive oxygen species in a biological model and the utility of these measurements in defining the relative contribution of superoxide to oxidative injury. PMID:23085519

  14. Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

    PubMed

    Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

    2014-12-01

    The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga.

  15. Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

    PubMed

    Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

    2014-12-01

    The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga. PMID:25210079

  16. Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii

    PubMed Central

    Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

    2014-01-01

    The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga. PMID:25210079

  17. Nutrient-contaminant (Pu) plant accumulation model

    SciTech Connect

    Cowan, C.E.; Jenne, E.A.; Simpson, J.C.; Cataldo, D.A.

    1981-12-01

    A model was developed which simulates the movement and daily accumulation of nutrients and contaminants in crop plants resulting from known physiological processes in the plant. In the model, the daily contaminant accumulation is governed by daily increase in plant biomass derived from photosynthesis and by the specified thermodynamic activity of the bioavailable contaminant species in soil or hydroponic solutin. Total accumulation and resulting concentration in the plant's root, stem and branch, leaf, and reproductive compartments can be simulated any time during the growing season. Parameters were estimated from data on plutonium accumulation in soybeans and the model was calibrated against this same data set. The plutonium distribution in the plant was found to be most sensitive to parameters related to leaf accumulation. Contamination at different times during the growing season resulted in a large change in predicted leaf accumulation but very little change in predicted accumulation in other plant parts except when contamination occurred very late in the growing season.

  18. Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

    SciTech Connect

    Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.; Akita, Geoffrey Y.; Cheng, Seng H.; Gregory, Richard J.; Jiang Canwen

    2007-12-21

    In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.

  19. Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase Inhibitor for the Treatment of Cardiovascular Diseases.

    PubMed

    Ruggeri, Roger B; Buckbinder, Leonard; Bagley, Scott W; Carpino, Philip A; Conn, Edward L; Dowling, Matthew S; Fernando, Dilinie P; Jiao, Wenhua; Kung, Daniel W; Orr, Suvi T M; Qi, Yingmei; Rocke, Benjamin N; Smith, Aaron; Warmus, Joseph S; Zhang, Yan; Bowles, Daniel; Widlicka, Daniel W; Eng, Heather; Ryder, Tim; Sharma, Raman; Wolford, Angela; Okerberg, Carlin; Walters, Karen; Maurer, Tristan S; Zhang, Yanwei; Bonin, Paul D; Spath, Samantha N; Xing, Gang; Hepworth, David; Ahn, Kay; Kalgutkar, Amit S

    2015-11-12

    Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies. PMID:26509551

  20. Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death.

    PubMed

    Li, Liang; Ye, Chaofei; Zhao, Rui; Li, Xin; Liu, Wu-zhen; Wu, Feifei; Yan, Jingli; Jiang, Yuan-Qing; Yang, Bo

    2015-11-27

    MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death.

  1. Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death.

    PubMed

    Li, Liang; Ye, Chaofei; Zhao, Rui; Li, Xin; Liu, Wu-zhen; Wu, Feifei; Yan, Jingli; Jiang, Yuan-Qing; Yang, Bo

    2015-11-27

    MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death. PMID:26498521

  2. Genetic Variation in Myeloperoxidase Modifies the Association of Serum α-Tocopherol with Aggressive Prostate Cancer among Current Smokers12

    PubMed Central

    Cheng, Ting-Yuan David; Barnett, Matt J.; Kristal, Alan R.; Ambrosone, Christine B.; King, Irena B.; Thornquist, Mark D.; Goodman, Gary E.; Neuhouser, Marian L.

    2011-01-01

    We investigated associations of serum α- and γ-tocopherols and their effect modification by polymorphisms in oxidative stress regulatory enzymes in relation to prostate cancer risk. In a nested case-control study in the Carotene and Retinol Efficacy Trial, prerandomized serum α- and γ-tocopherol were assayed among 684 men with incident prostate cancer [375 nonaggressive and 284 aggressive cancer (stage III/IV or Gleason score ≥7)] and 1441 controls. Manganese superoxide dismutase Ala-16Val (rs4880), glutathione peroxidase 1 Pro200Leu (rs1050450), catalase −262 C > T (rs1001179), and myeloperoxidase (MPO) G–463A (rs2333227) were genotyped. A multivariate-adjusted inverse association of serum α-tocopherol with total prostate cancer risk was observed in current smokers (OR = 0.62, 95% CI = 0.40–0.96, 4th vs. 1st quartiles). High (≥median) compared to low serum concentrations of α- and γ-tocopherol were inversely associated with aggressive prostate cancer in current smokers (OR = 0.50, 95% CI = 0.32–0.78 and OR = 0.64, 95% CI = 0.43–0.95, respectively). The association was stronger among those with MPO G/A+A/A genotypes. Among current smokers with low serum α-tocopherol concentrations, MPO G/A+A/A, the genotypes downregulating oxidative stress, were associated with an increased risk for aggressive prostate cancer (OR = 2.06, 95% CI = 1.22–3.46). Conversely, current smokers with these genotypes who had high α-tocopherol concentrations had a reduced risk for aggressive prostate cancer (OR = 0.34, 95% CI = 0.15–0.80; P-interaction = 0.001). In conclusion, among current smokers, both high serum α- and γ-tocopherol concentrations were associated with reduced risks of aggressive prostate cancer. The α-tocopherol–associated risks are modified by polymorphism in MPO G–463A. PMID:21795425

  3. Fenofibrate improves endothelial function and plasma myeloperoxidase in patients with type 2 diabetes mellitus: an open-label interventional study

    PubMed Central

    2014-01-01

    Background Fenofibrate offers a number of benefits on the cardiovascular system and it is plausible that its anti-inflammatory, anti-oxidant and anti-fibrotic effects and enhancement of cardiac metabolic performances may account for its direct cardioprotective effects. In this study we aimed to investigate the effect of fenofibrate on endothelial function assesed by vascular studies and levels of soluble E-selectin (sE-selectin) as well as the effect on plasma myeloperoxidase (MPO) in patients with type 2 diabetes mellitus (T2DM) without previous use of lipid-lowering medication. Methods 27 patients (14 men and 13 women) with T2DM and good glycemic control (HbA1c: min 5.9%, max: 7.1%) treated with metformin monotherapy, without previous use of lipid-lowering medication were enrolled in this study. Vascular studies included measures of brachial artery diameter before and after release of a suprasystolic ischemia. FMD was calculated as the percent (%) change in arterial diameter following reactive hyperemia. Student’s paired t test and Wilcoxon Signed Ranks Test were used to compare values before and after fenofibrate therapy. Results Fenofibrate therapy significantly increased post ischemia mean brachial artery diameter at 60 s (from 4.7 [4.4; 5.0] mm to 4.9 [4.6; 5.2] mm, p = 0.01) and at 90 s (from 4.7 [4.4; 5.0] mm to 4.9 [4.6; 5.1], p = 0.02). FMD response to hyperaemia at 60 s increased with 4.5 ± 13.7% (median value pre- treatment: 22.2%, median value post- treatment 25.0%, z = −2.9, p = 0.004). After 8 weeks of fenofibrate therapy, plasma MPO levels decreased to 49.5 [30.3; 71.5] ng/ml (% change from baseline = 4.6%, z = −2.2, p = 0.03) and mean plasma sE-selectin levels decreased to 67.1 [54.4; 79.8] ng/ml, (% change from baseline = 2.6%, p = 0.03). Conclusion In patients with T2DM without previous treatment for dyslipidemia, short-term treatment with fenofibrate improved vascular endothelial function as demonstrated by increased post ischemia mean

  4. Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

    PubMed

    Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

    2016-08-01

    N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more