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ScienceDirect Publication: Free Radical Biology and Medicine
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ScienceDirect Publication: Free Radical Biology and Medicine
  • nNOS uncoupling by oxidized LDL: Implications in atherosclerosis
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Juliana Maria Navia-Pelaez, Gianne Paul Campos-Mota, Jessica Cristina Araujo de Souza, Edenil Costa Aguilar, Nikos Stergiopulos, Jacqueline Isaura Alvarez-Leite, Luciano Santos Aggum Capettini


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  • Respiratory syncytial virus induces NRF2 degradation through a promyelocytic leukemia protein ‐ ring finger protein 4 dependent pathway
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Narayana Komaravelli, Maria Ansar, Roberto P. Garofalo, Antonella Casola

    Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or specific treatments are available. RSV causes airway mucosa inflammation and cellular oxidative damage by triggering production of reactive oxygen species and by inhibiting at the same time expression of antioxidant enzymes, via degradation of the transcription factor NF-E2-related factor 2 (NRF2). RSV infection induces NRF2 deacetylation, ubiquitination, and degradation through a proteasome-dependent pathway. Although degradation via KEAP1 is the most common mechanism, silencing KEAP1 expression did not rescue NRF2 levels during RSV infection. We found that RSV-induced NRF2 degradation occurs in an SUMO-specific E3 ubiquitin ligase - RING finger protein 4 (RNF4)-dependent manner. NRF2 is progressively SUMOylated in RSV infection and either blocking SUMOylation or silencing RNF4 expression rescued both NRF2 nuclear levels and transcriptional activity. RNF4 associates with promyelocytic leukemia – nuclear bodies (PML-NBs). RSV infection induces the expression of PML and PML-NBs formation in an interferon (INF)-dependent manner and also induces NRF2 – PMN-NBs association. Inhibition of PML-NB formation by blocking IFN pathway or silencing PML expression resulted in a significant reduction of RSV-associated NRF2 degradation and increased antioxidant enzyme expression, identifying the RNF4-PML pathway as a key regulator of antioxidant defenses in the course of viral infection.

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  • Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Trenton L. Place, Frederick E. Domann, Adam J. Case

    Molecular oxygen is one of the most important variables in modern cell culture systems. Fluctuations in its concentration can affect cell growth, differentiation, signaling, and free radical production. In order to maintain culture viability, experimental validity, and reproducibility, it is imperative that oxygen levels be consistently maintained within physiological “normoxic” limits. Use of the term normoxia, however, is not consistent among scientists who experiment in cell culture. It is typically used to describe the atmospheric conditions of a standard incubator, not the true microenvironment to which the cells are exposed. This error may lead to the situation where cells grown in a standard “normoxic” oxygen concentration may actually be experiencing a wide range of conditions ranging from hyperoxia to near-anoxic conditions at the cellular level. This apparent paradox is created by oxygen's sluggish rate of diffusion through aqueous medium, and the generally underappreciated effects that cell density, media volume, and barometric pressure can have on pericellular oxygen concentration in a cell culture system. This review aims to provide an overview of this phenomenon we have termed “consumptive oxygen depletion” (COD), and includes a basic review of the physics, potential consequences, and alternative culture methods currently available to help circumvent this largely unrecognized problem.

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  • White light emitting diode suppresses proliferation and induces apoptosis in hippocampal neuron cells through mitochondrial cytochrome c oxydase-mediated IGF-1 and TNF-α pathways
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Yang Yang, Yibo Zong, Qinwei Sun, Yimin Jia, Ruqian Zhao

    Light emitting diode (LED) light has been tested to treat traumatic brain injury, neural degenerative diseases and psychiatric disorders. Previous studies indicate that blue LED light affects cell proliferation and apoptosis in photosensitive cells and cancer cells. In this study, we demonstrate that white LED light exposure impaired proliferation and induced apoptosis in HeLa and HT-22 hippocampal neural cells, but not C2C12 cells. Furthermore, the mechanisms underlying the effect of white LED light exposure on HT-22 cells were elucidated. In HeLa and HT-22 cells, white LED light activated mitochondrial cytochrome c oxidase (Cco), in association with enhanced ATP synthase activity and elevated intracellular ATP concentration. Also, reactive oxygen species (ROS) and nitric oxide (NO) production were increased, accompanied by higher calcium concentration and lower mitochondrial membrane potential. HT-22 cells exposed to white LED light for 24h showed reduced viability, with higher apoptotic rate and a cell cycle arrest at G0/G1 phase. Concurrently, the mRNA expression and the concentration of IGF-1 were decreased, while that of TNF-α were increased, in light-exposed cells, which was supported by the luciferase activity of both gene promoters. The down-stream mitogen-activated protein kinase (MAPK), AKT/mTOR pathways were inhibited, in association with an activation of apoptotic caspase 3. N-Acetylcysteine, a ROS scavenger, protected the cells from LED light-induced cellular damage, with rescued cell viability and restored mRNA expression of IGF-1 and TNF-α. Our data demonstrate that white LED light suppresses proliferation and induces apoptosis in hippocampal neuron cells through mitochondrial Cco/ROS-mediated IGF-1 and TNF-α pathways.

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  • Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Sylvain Beaumel, Antoine Picciocchi, Franck Debeurme, Corinne Vivès, Anne-Marie Hesse, Myriam Ferro, Didier Grunwald, Heather Stieglitz, Pahk Thepchatri, Susan M.E. Smith, Franck Fieschi, Marie José Stasia

    NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47 phox and p67 phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b 558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.

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  • Differential carbonylation of proteins in end-stage human fatty and nonfatty NASH
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Colin T. Shearn, Laura M. Saba, James R. Roede, David J. Orlicky, Alisabeth H. Shearn, Dennis R. Petersen

    Objective In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease is oxidative stress leading to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine if significant differences were evident when evaluating carbonylation in human end-stage fatty nonalcoholic steatohepatitis (fNASH) compared to end-stage nonfatty NASH (nfNASH). Methods Using hepatic tissue obtained from healthy humans and patients diagnosed with end stage nfNASH or fNASH, overall carbonylation was assessed by immunohistochemistry (IHC) and LC-MS/MS followed by bioinformatics. Results Picrosirius red staining revealed extensive fibrosis in both fNASH and nfNASH which corresponded with increased reactive aldehyde staining. Although significantly elevated when compared to normal hepatic tissue, no significant differences in overall carbonylation and fibrosis were evident when comparing fNASH with nfNASH. Examining proteins that are critical for anti-oxidant defense revealed elevated expression of thioredoxin, thioredoxin interacting protein, glutathione S-transferase p1 and mitochondrial superoxide dismutase in human NASH. As important, using immunohistochemistry, significant colocalization of the aforementioned proteins occurred in cytokeratin 7 positive cells indicating that they are part of the ductular reaction. Expression of catalase and Hsp70 decreased in both groups when compared to normal human liver. Mass spectrometric analysis revealed a total of 778 carbonylated proteins. Of these, 194 were common to all groups, 124 unique to tissue prepared from healthy individuals, 357 proteins exclusive to NASH, 124 proteins distinct to samples from patients with fNASH and 178 unique to nfNASH. Using functional enrichment analysis of hepatic carbonylated proteins revealed a propensity for increased carbonylation of proteins regulating cholesterol and Huntington's disease related pathways occurred in nfNASH. Examining fNASH, increased carbonylation was evident in proteins regulating Rho cytoskeletal pathways, nicotinic acetylcholine receptor signaling and chemokine/cytokine inflammatory pathways. Using LC-MS/MS analysis and trypsin digests, sites of carbonylation were identified on peptides isolated from vimentin, endoplasmin and serum albumin in nfNASH and fNASH respectively. Conclusions These results indicate that cellular factors regulating mechanisms of protein carbonylation may be different depending on pathological diagnosis of NASH. Furthermore these studies are the first to use LC-MS/MS analysis of carbonylated proteins in human NAFLD and explore possible mechanistic links with end stage cirrhosis due to fatty liver disease and the generation of reactive aldehydes.

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  • Insulin-dependent metabolic and inotropic responses in the heart are modulated by hydrogen peroxide from NADPH-oxidase isoforms NOX2 and NOX4
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Benjamin Steinhorn, Juliano L. Sartoretto, Andrea Sorrentino, Natalia Romero, Hermann Kalwa, E. Dale Abel, Thomas Michel

    Rationale Hydrogen peroxide (H2O2) is a stable reactive oxygen species (ROS) that has long been implicated in insulin signal transduction in adipocytes. However, H2O2's role in mediating insulin's effects on the heart are unknown. Objective We investigated the role of H2O2 in activating insulin-dependent changes in cardiac myocyte metabolic and inotropic pathways. The sources of insulin-dependent H2O2 generation were also studied. Methods and results In addition to the canonical role of insulin in modulating cardiac metabolic pathways, we found that insulin also inhibited beta adrenergic-induced increases in cardiac contractility. Catalase and NADPH oxidase (NOX) inhibitors blunted activation of insulin-responsive kinases Akt and mTOR and attenuated beta adrenergic receptor-mediated responses. These insulin responses were lost in a mouse model of type 2 diabetes, suggesting a role for these H2O2-dependent pathways in the diabetic heart. The H2O2-sensitive fluorescent biosensor HyPer revealed rapid increases in cytosolic and caveolar H2O2 concentrations in response to insulin treatment, which were blocked by NOX inhibitors and attenuated in NOX2 KO and NOX4 KO mice. In NOX2 KO cardiac myocytes, insulin-mediated phosphorylation of Akt and mTOR was blocked, while these responses were unaffected in cardiac myocytes from NOX4 KO mice. In contrast, insulin's effects on contractility were lost in cardiac myocytes from NOX4 KO animals but were retained in NOX2 KO mice. Conclusions These studies identify a proximal point of bifurcation in cardiac insulin signaling through the simultaneous activation of both NOX2 and NOX4. Each NOX isoform generates H2O2 in cardiac myocytes with distinct time courses, with H2O2 derived from NOX2 augmenting Akt-dependent metabolic effects of insulin, while H2O2 from NOX4 blocks beta adrenergic increases in inotropy. These findings suggest that insulin resistance in the diabetic heart may lead to potentially deleterious potentiation of beta adrenergic responses.

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  • Dimerization and oxidation of tryptophan in UV-A photolysis sensitized by kynurenic acid
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Ekaterina D. Sormacheva, Peter S. Sherin, Yuri P. Tsentalovich

    Photoinduced generation of radicals in the eye lens may play an important role in the modification of proteins leading to their coloration, aggregation, and insolubilization. The radicals can be formed via the reactions of photoexcited endogenous chromophores of the human lens with lens proteins, in particular with tryptophan residues. In the present work we studied the reactions induced by UV-A (315–400nm) light between kynurenic acid (KNA), an effective photosensitizer present in the human lens, and N-acetyl-L-tryptophan (NTrpH) under aerobic and anaerobic conditions. Our results show that the reaction mechanism strongly depends on the presence of oxygen in solution. Under aerobic conditions, the generation of singlet oxygen is the major channel of the effective NTrpH oxidation. In argon-bubbled solutions, the quenching of triplet KNA by NTrpH results in the formation of KNA and NTrp radicals. Under laser pulse irradiation, when the radical concentration is high, the main pathway of the radical decay is the back electron transfer with the restoration of initial reagents. Other reactions include (i) the radical combination yielding NTrp dimers and (ii) the oxygen atom transfer from KNA to NTrp with the formation of oxidized NTrp species and deoxygenated KNA products. In continuous-wave photolysis, even trace amounts of molecular oxygen are sufficient to oxidize the majority of KNA radicals with the rate constant of (2.0 ± 0.2) × 109 M−1 s−1, leading to the restoration of KNA and the formation of superoxide radical O2 . The latter reacts with NTrp via either the radical combination to form oxidized NTrp (minor pathway), or the electron transfer to restore NTrpH in the ground state (major pathway). As the result, the quantum yields of the starting compound decomposition under continuous-wave anaerobic photolysis are rather low: 1.6% for NTrpH and 0.02% for KNA. The photolysis of KNA with alpha-crystallin yields the same deoxygenated KNA products as the photolysis of KNA with NTrpH, indicating the similarity of the photolysis mechanisms. Thus, inside the eye lens KNA can sensitize both protein photooxidation and protein covalent cross-linking with the minor self-degradation. This may play an important role in the lens protein modifications during the normal aging and cataract development.

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  • Nox4 regulates the eNOS uncoupling process in aging endothelial cells
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Hwa-Young Lee, Hafiz Maher Ali Zeeshan, Hyung-Ryong Kim, Han-Jung Chae

    ROS and its associated signaling contribute to vascular aging-associated endothelial disturbance. Since the non-effective endothelial nitric oxide synthase (eNOS) coupling status is related to vascular aging-related phenotypes, eNOS coupled/uncoupled system signaling was studied in human umbilical vein endothelial cells (HUVEC). Nitric oxide (NO) and eNOS Ser1177 were significantly decreased, whereas O2 - (superoxide anion radical) increased with passage number. In aging cells, NADPH oxidase 4 (Nox4), one of the main superoxide generating enzymes, and its associated protein disulfide isomerase (PDI) chaperone were highly activated, and the resultant ER redox imbalance leads to disturbance of protein folding capability, namely endoplasmic reticulum (ER) stress, ultimately inducing dissociation between HSP90 and IRE-1α or PERK, decreasing HSP90 stability and dissociating the binding of eNOS from the HSP90 and leading to eNOS uncoupling. Through chemical and Nox4 siRNA approaches, Nox4 and its linked ER stress were shown to mainly contribute to eNOS uncoupling and its associated signaling, suggesting that Nox4 and its related ER stress signaling are key signals of the aging process in endothelial cells.

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  • Increased serotransferrin and ceruloplasmin turnover in diet-controlled patients with type 2 diabetes
    Publication date: December 2017
    Source:Free Radical Biology and Medicine, Volume 113

    Author(s): Makan Golizeh, Kwangwon Lee, Serguei Ilchenko, Abdullah Ösme, James Bena, Rovshan G. Sadygov, Sangeeta Kashyap, Takhar Kasumov

    Type 2 diabetes mellitus (T2DM) is associated with oxidative stress and perturbed iron metabolism. Serotransferrin (Trf) and ceruloplasmin (Cp) are two key proteins involved in iron metabolism and anti-oxidant defense. Non-enzymatic glycation and oxidative modification of plasma proteins are known to occur under hyperglycemia and oxidative stress. In this study, shotgun proteomics and 2H2O-based metabolic labeling were used to characterize post-translational modifications and assess the kinetics of Trf and Cp in T2DM patients and matched controls in vivo. Six early lysine (Amadori) and one advanced arginine glycation were detected in Trf. No glycation, but five asparagine deamidations, were found in Cp. T2DM patients had increased fractional catabolic rates of both Trf and Cp that correlated with HbA1c (p < 0.05). The glycated Trf population was subject to an even faster degradation compared to the total Trf pool, suggesting that hyperglycemia contributed to an increased Trf degradation in T2DM patients. Enhanced production of Trf and Cp kept their levels stable. The changes in Trf and Cp turnover were associated with increased systemic oxidative stress without any alteration in iron status in T2DM. These findings can help better understand the potential role of altered Trf and Cp metabolism in the pathogenesis of T2DM and other diseases.

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