Induction of synthesis of components of the hydrogen peroxide-generating oxidase during activation of the human monocytic cell line U937 by interferon-gamma.
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ABSTRACT:
SUBMITTER: Andrew PW
PROVIDER: S-EPMC1148532 | biostudies-other | 1987 Nov
REPOSITORIES: biostudies-other
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Project description:Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.
| S-EPMC84685 | biostudies-literature
Project description:Oxalate oxidase is a manganese containing enzyme that catalyzes the oxidation of oxalate to carbon dioxide in a reaction that is coupled with the reduction of oxygen to hydrogen peroxide. Oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx) is the first fungal and bicupin enzyme identified that catalyzes this reaction. Potential applications of oxalate oxidase for use in pancreatic cancer treatment, to prevent scaling in paper pulping, and in biofuel cells have highlighted the need to understand the extent of the hydrogen peroxide inhibition of the CsOxOx catalyzed oxidation of oxalate. We apply a membrane inlet mass spectrometry (MIMS) assay to directly measure initial rates of carbon dioxide formation and oxygen consumption in the presence and absence of hydrogen peroxide. This work demonstrates that hydrogen peroxide is both a reversible noncompetitive inhibitor of the CsOxOx catalyzed oxidation of oxalate and an irreversible inactivator. The build-up of the turnover-generated hydrogen peroxide product leads to the inactivation of the enzyme. The introduction of catalase to reaction mixtures protects the enzyme from inactivation allowing reactions to proceed to completion. Circular dichroism spectra indicate that no changes in global protein structure take place in the presence of hydrogen peroxide. Additionally, we show that the CsOxOx catalyzed reaction with the three carbon substrate mesoxalate consumes oxygen which is in contrast to previous proposals that it catalyzed a non-oxidative decarboxylation with this substrate.
| S-EPMC5423638 | biostudies-literature
Project description:Nox4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for Nox4 require upstream activators, either calcium or organizer/activator subunits (p47(phox), NOXO1/p67(phox), and NOXA1). Nox4 may also be unusual as it reportedly releases hydrogen peroxide (H?O?) in contrast to Nox1-Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection. Our studies were undertaken (1) to identify the Nox4 ROS product using a membrane-free, partially purified preparation of Nox4 and (2) to test the hypothesis that Nox4 activity is acutely regulated not by activator proteins or calcium, but by cellular pO?, allowing it to function as an O? sensor, the output of which is signaling H?O?. We find that approximately 90% of the electron flux through isolated Nox4 produces H?O? and 10% forms superoxide. The kinetic mechanism of H?O? formation is consistent with a mechanism involving binding of one oxygen molecule, which is then sequentially reduced by the heme in two one-electron reduction steps first to form a bound superoxide intermediate and then H?O?; kinetics are not consistent with a previously proposed internal superoxide dismutation mechanism involving two oxygen binding/reduction steps for each H?O? formed. Critically, Nox4 has an unusually high Km for oxygen (?18%), similar to the values of known oxygen-sensing enzymes, compared with a Km of 2-3% for Nox2, the phagocyte NADPH oxidase. This allows Nox4 to generate H?O? as a function of oxygen concentration throughout a physiological range of pO2 values and to respond rapidly to changes in pO?.
| S-EPMC4131900 | biostudies-other
Project description:The antibiofilm activity of a hydrogen peroxide-generating electrochemical scaffold (e-scaffold) was determined against mono- and trispecies biofilms of methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, and Candida albicans Significant time-dependent decreases were found in the overall CFU of biofilms of all three monospecies and the trispecies forms. Confocal laser scanning microscopy showed dramatic reductions in fluorescence intensities of biofilm matrix protein and polysaccharide components of e-scaffold-treated biofilms. The described e-scaffold has potential as a novel antibiotic-free strategy for treating wound biofilms.
| S-EPMC7179276 | biostudies-literature
Project description:Tumor-associated macrophages are immune cells with diverse functions in tumor development. Among other functions, they downregulate immune-mediated tumor rejection by depriving lymphocytes of nutrients. The essential amino acid tryptophan is metabolized by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase (TDO). Indoleamine 2,3-dioxygenase 1 is expressed in a large number of human tumors, and inhibitors are in development to improve immunotherapy. Tryptophan 2,3-dioxygenase was also found in human tumors and preclinical working models confirmed its immunosuppressive power. We explored a potential expression of TDO by macrophages. This enzyme could be induced in two human cell lines, THP-1 and U937, by incubation with phorbol myristate acetate, lipopolysaccharide, and interferon gamma. Phorbol-myristate-acetate-mediated induction was inhibited by rottlerin, a protein kinase C inhibitor. In contrast to these monocytic cell lines, other cell lines or fresh human monocytes isolated from peripheral blood mononuclear cells and differentiated into proinflammatory or anti-inflammatory macrophages could not be induced to express TDO. Our results suggest that TDO might play an immunosuppressive role in human monocytic leukemias but not in untransformed macrophages.
| S-EPMC6933542 | biostudies-literature
Project description:Increasing rates of chronic wound infections caused by antibiotic-resistant bacteria are a crisis in healthcare settings. Biofilms formed by bacterial communities in these wounds create a complex environment, enabling bacteria to persist, even with antibiotic treatment. Wound infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are major causes of morbidity in clinical practice. There is a need for new therapeutic interventions not based on antibiotics. Hydrogen peroxide (H2O2) is a known antibacterial/antibiofilm agent, continuous delivery of which has been challenging. A conductive electrochemical scaffold (e-scaffold) is developed, which is composed of carbon fabric that electrochemically reduces dissolved oxygen into H2O2 when polarized at -0.6 VAg/AgCl, as a novel antibiofilm wound dressing material. In this study, the in vitro antibiofilm activity of the e-scaffold against MRSA is investigated. The developed e-scaffold efficiently eradicates MRSA biofilms, based on bacterial quantitation and ATP measurements. Moreover, imaging hinted at the possibility of cell-membrane damage as a mechanism of action. These results suggest that an H2O2-generating e-scaffold may be a novel platform for eliminating MRSA biofilms without using antibiotics and may be useful to treat chronic MRSA wound infections.
| S-EPMC6551415 | biostudies-literature
Project description:In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c) is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+ cyt c) in the absence and presence of H2O2. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with sulfite, Fe3+ cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3-) was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+ cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+ cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+ cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+ cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which occur to humans born with molybdenum cofactor and sulfite oxidase deficiencies.
| S-EPMC4689149 | biostudies-literature
Project description:AimsOur objective was to investigate whether pro-oxidant properties of ascorbic acid (AA) and tetrahydrobiopterin (BH(4)) modulate endothelium-dependent, electrotonically mediated arterial relaxation.Methods and resultsIn studies with rabbit iliac artery (RIA) rings, NO-independent, endothelium-derived hyperpolarizing factor (EDHF)-type relaxations evoked by the sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and the G protein-coupled agonist acetylcholine (ACh) were enhanced by AA (1 mM) and BH(4) (200 microM), which generated buffer concentrations of H(2)O(2) in the range of 40-80 microM. Exogenous H(2)O(2) potentiated cyclopiazonic acid (CPA)- and ACh-evoked relaxations with a threshold of 10-30 microM, and potentiation by AA and BH(4) was abolished by catalase, which destroyed H(2)O(2) generated by oxidation of these agents in the organ chamber. Adventitial application of H(2)O(2) also enhanced EDHF-type dilator responses evoked by CPA and ACh in RIA segments perfused intraluminally with H(2)O(2)-free buffer, albeit with reduced efficacy. In RIA rings, both control relaxations and their potentiation by H(2)O(2) were overcome by blockade of gap junctions by connexin-mimetic peptides (YDKSFPISHVR and SRPTEK) targeted to the first and second extracellular loops of the dominant vascular connexins expressed in the RIA. Superoxide dismutase attenuated the potentiation of EDHF-type relaxations by BH(4), but not AA, consistent with findings demonstrating a differential role for superoxide anions in the generation of H(2)O(2) by the two agents.ConclusionPro-oxidant effects of AA and BH(4) can enhance the EDHF phenomenon by generating H(2)O(2), which has previously been shown to amplify electrotonic hyperpolarization-mediated relaxation by facilitating Ca(2+) release from endothelial stores.
| S-EPMC2761203 | biostudies-literature
Project description:Redox status is a central determinant of cellular activities and redox imbalance is correlated with numerous diseases. NADPH oxidase activity results in formation of H2O2, that, in turn, sets cellular redox status, a key regulator of cellular homeostasis and responses to external stimuli. Hydrogen peroxide metabolism regulates cell redox status by driving changes in protein cysteine oxidation often via cycling of thioredoxin/peroxiredoxin and glutathione; however, regulation of enzymes controlling synthesis and utilization of H2O2 is not understood beyond broad outlines. The data presented here show that calcium-stimulated epithelial Duox H2O2 synthesis is transient, independent of intracellular calcium renormalization, H2O2 scavenging by antioxidant enzymes, or substrate depletion. The data support existence of a separate mechanism that restricts epithelial H2O2 synthesis to a burst and prevents harmful changes in redox tone following continuous stimulation. Elucidation of this H2O2 synthesis tempering mechanism is key to understanding cellular redox regulation and control of downstream effectors, and this observation provides a starting point for investigation of the mechanism that controls H2O2-mediated increases in redox tone.
| S-EPMC8010520 | biostudies-literature
Project description:Hydrogen peroxide (H2O2) functions as a signaling molecule in diverse cellular processes. While cells have evolved the capability to detect and manage changes in H2O2 levels, the mechanisms regulating key H2O2-producing enzymes to maintain optimal levels, especially in pancreatic beta cells with notably weak antioxidative defense, remain unclear. We found that the protein EI24 responds to changes in H2O2 concentration and regulates the production of H2O2 by controlling the translation of NOX4, an enzyme that is constitutively active, achieved by recruiting an RNA-binding protein, RTRAF, to the 3'-UTR of Nox4. Depleting EI24 results in RTRAF relocating into the nucleus, releasing the brake on NOX4 translation. The excessive production of H2O2 by liberated NOX4 further suppresses the translation of the key transcription factor MafA, ultimately preventing its binding to the Ins2 gene promoter and subsequent transcription of insulin. Treatment with a specific NOX4 inhibitor or the antioxidant NAC reversed these effects and alleviated the diabetic symptoms in beta-cell specific Ei24-KO mice. This study revealed a new mechanism through which cells regulate oxidative stress at the translational level, involving an ER-tethered RNA-binding protein that controls the expression of the key H2O2-producing enzyme NOX4.
| S-EPMC10963860 | biostudies-literature