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ScienceDirect Publication: Free Radical Biology and Medicine
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ScienceDirect Publication: Free Radical Biology and Medicine
  • Crosstalk between Rac1-mediated actin regulation and ROS production
    Publication date: 20 February 2018
    Source:Free Radical Biology and Medicine, Volume 116

    Author(s): Alejandro Acevedo, Christian González-Billault

    The small RhoGTPase Rac1 is implicated in a variety of events related to actin cytoskeleton rearrangement. Remarkably, another event that is completely different from those related to actin regulation has the same relevance; the Rac1-mediated production of reactive oxygen species (ROS) through NADPH oxidases (NOX). Each outcome involves different Rac1 downstream effectors; on one hand, events related to the actin cytoskeleton require Rac1 to bind to WAVEs proteins and PAKs that ultimately promote actin branching and turnover, on the other, NOX-derived ROS production demands active Rac1 to be bound to a cytosolic activator of NOX. How Rac1-mediated signaling ends up promoting actin-related events, NOX-derived ROS, or both is poorly understood. Rac1 regulators, including scaffold proteins, are known to exert tight control over its functions. Hence, evidence of Rac1 regulatory events leading to both actin remodeling and NOX-mediated ROS generation are discussed. Moreover, cellular functions linked to physiological and pathological conditions that exhibit crosstalk between Rac1 outcomes are analyzed, while plausible roles in neuronal functions (and dysfunctions) are highlighted. Together, discussed evidence shed light on cellular mechanisms which requires Rac1 to direct either actin- and/or ROS-related events, helping to understand crucial roles of Rac1 dual functionality.

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  • REDOX STRESS IN MARFAN SYNDROME: DISSECTING THE ROLE OF THE NADPH OXIDASE NOX4 IN AORTIC ANEURYSM
    Publication date: Available online 20 February 2018
    Source:Free Radical Biology and Medicine

    Author(s): Francesc Jiménez-Altayó, Thayna Meirelles, Eva Crosas-Molist, M. Alba Sorolla, Darya Gorbenko del Blanco, Judit López-Luque, Aleksandra Mas-Stachurska, Ana-Maria Siegert, Fabio Bonorino, Laura Barberà, Carolina García, Enric Condom, Marta Sitges, Fernando Rodríguez-Pascual, Francisco Laurindo, Katrin Schröder, Joaquim Ros, Isabel Fabregat, Gustavo Egea

    Marfan syndrome (MFS) is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix fibrillin-containing microfibrils and dysfunction of TGF-β signaling. Here we identify the molecular targets of redox stress in aortic aneurysms from MFS patients, and investigate the role of NOX4, whose expression is strongly induced by TGF-β, in aneurysm formation and progression in a murine model of MFS. Working models included aortae and cultured vascular smooth muscle cells (VSMC) from MFS patients, and a NOX4-deficient Marfan mouse model (Fbn1 C1039G/+ -Nox4 -/- ). Increased S-nitrosylation and reactive oxygen species levels were found in the tunica media of human aortic aneurysms and in cultured VSMC. Proteomic analysis identified nitrated and carbonylated proteins, which included smooth muscle α-actin (αSMA) and annexin A2. NOX4 immunostaining increased in the tunica media of human Marfan aorta and was transcriptionally overexpressed in VSMC. Fbn1 C1039G/+-Nox4 -/- mice aortas showed a reduction of fragmented elastic fibers, which was accompanied by an amelioration in the Marfan-associated enlargement of the aortic root. Increase in the contractile phenotype marker calponin in the tunica media of MFS mice aortas was abrogated in Fbn1 C1039G/+-Nox4 -/- mice. Endothelial dysfunction evaluated by myography in the Marfan ascending aorta was prevented by the absence of Nox4 or catalase-induced H2O2 decomposition. We conclude that redox stress occurs in MFS, whose targets are actin-based cytoskeleton members and regulators of extracellular matrix homeostasis. Likewise, NOX4 have an impact in the progression of the aortic dilation in MFS and in the structural organization of the aortic tunica media, the VSMC phenotypic modulation, and endothelial function.

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  • Age-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression
    Publication date: April 2018
    Source:Free Radical Biology and Medicine, Volume 118

    Author(s): Yuxuan Zheng, Jeffrey D. Ritzenthaler, Tom J. Burke, Javier Otero, Jesse Roman, Walter H. Watson

    Aging is associated with progressive oxidation of the extracellular environment. The redox state of human plasma, defined by the concentrations of cysteine (Cys) and cystine (CySS), becomes more oxidized as we age. Recently, we showed that fibroblasts isolated from the lungs of young and old mice retain this differential phenotype; old cells produce and maintain a more oxidizing extracellular redox potential (Eh(Cys/CySS)) than young cells. Microarray analysis identified down-regulation of Slc7a11, the light subunit of the CySS/glutamate transporter, as a potential mediator of age-related oxidation in these cells. The purpose of the present study was to investigate the mechanistic link between Slc7a11 expression and extracellular Eh(Cys/CySS). Sulforaphane treatment or overexpression of Slc7a11 was used to increase Slc7a11 in lung fibroblasts from old mice, and sulfasalazine treatment or siRNA-mediated knock down was used to decrease Slc7a11 in young fibroblasts. Slc7a11 mRNA levels were measured by real-time PCR, Slc7a11 activity was determined by measuring the rate of glutamate release, Cys, CySS, glutathione (GSH) and its disulfide (GSSG) were measured by HPLC, and Eh(Cys/CySS) was calculated from the Nernst equation. The results showed that both Eh(Cys/CySS) and Eh(GSH/GSSG) were more oxidized in the conditioned media of old cells than in young cells. Up-regulation of Slc7a11 via overexpression or sulforaphane treatment restored extracellular Eh(Cys/CySS) in cultures of old cells, whereas down-regulation reproduced the oxidizing Eh(Cys/CySS) in young cells. Only sulforaphane treatment was able to increase total GSH and restore Eh(GSH/GSSG), whereas overexpression, knock down and sulfasalazine had no effect on these parameters. In addition, inhibition of GSH synthesis with buthionine sulfoximine had no effect on the ability of cells to restore their extracellular redox potential in response to an oxidative challenge. In conclusion, our study reveals Slc7a11 is the key regulator of age-dependent changes in extracellular Eh(Cys/CySS) in primary mouse lung fibroblasts, and its effects are not dependent on GSH synthesis.

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  • Superoxide dismutase protects ribonucleotide reductase from inactivation in yeast
    Publication date: 20 February 2018
    Source:Free Radical Biology and Medicine, Volume 116

    Author(s): Andrew B. Das, Izabela Sadowska-Bartosz, Andreas Königstorfer, Anthony J. Kettle, Christine C. Winterbourn

    Ribonucleotide reductase (RNR) catalyses the rate limiting step of DNA synthesis utilising a mechanism that requires a tyrosyl radical. We have previously shown that superoxide can quench protein tyrosyl radicals in vitro, either by oxidative addition, or reduction of the radical to tyrosine. Here, we observe that Saccharomyces cerevisiae strains lacking either copper-zincSOD (SOD1) or manganese SOD (SOD2) had decreased RNR activity compared to SOD-competent yeast. When superoxide production was increased by treatment with paraquat, RNR activity was further decreased, with yeast lacking SOD1 being the most sensitive. The growth of yeast lacking SOD1 was also the most sensitive to paraquat treatment. Using expressed recombinant RNR, superoxide addition was not detectable using mass-spectrometry. This suggests that oxidative addition is not the major route of inhibition in our system, but does not rule out reduction by superoxide as a possible mechanism. Our results demonstrate that protection of RNR from inactivation by superoxide is an important function of SOD, particularly cytoplasmic SOD1.

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  • ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection
    Publication date: March 2018
    Source:Free Radical Biology and Medicine, Volume 117

    Author(s): Susana Cadenas

    Ischemia-reperfusion (IR) injury is central to the pathology of major cardiovascular diseases, such as stroke and myocardial infarction. IR injury is mediated by several factors including the elevated production of reactive oxygen species (ROS), which occurs particularly at reperfusion. The mitochondrial respiratory chain and NADPH oxidases of the NOX family are major sources of ROS in cardiomyocytes. The first part of this review discusses recent findings and controversies on the mechanisms of superoxide production by the mitochondrial electron transport chain during IR injury, as well as the contribution of the NOX isoforms expressed in cardiomyocytes, NOX1, NOX2 and NOX4, to this damage. It then focuses on the effects of ROS on the opening of the mitochondrial permeability transition pore (mPTP), an inner membrane non-selective pore that causes irreversible damage to the heart. The second part analyzes the redox mechanisms of cardiomyocyte mitochondrial protection; specifically, the activation of the hypoxia-inducible factor (HIF) pathway and the antioxidant transcription factor Nrf2, which are both regulated by the cellular redox state. Redox mechanisms involved in ischemic preconditioning, one of the most effective ways of protecting the heart against IR injury, are also reviewed. Interestingly, several of these protective pathways converge on the inhibition of mPTP opening during reperfusion. Finally, the clinical and translational implications of these cardioprotective mechanisms are discussed.

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  • Analysis of Eicosanoid Oxidation Products in Alzheimer Brain by LC-MS with Uniformly 13C-Labeled Internal Standards
    Publication date: Available online 21 February 2018
    Source:Free Radical Biology and Medicine

    Author(s): Ran Furman, Jin V. Lee, Paul H. Axelsen

    The quantitative analysis of polyunsaturated fatty acyl (PUFA) chain oxidation products in tissue samples by mass spectrometry is hindered by the lack of durable internal standards for the large number of possible products. To address this problem in a study of oxidative PUFA degradation in Alzheimer's disease (AD) brain, uniformly 13C-labeled arachidonic acid (ARA) was produced biosynthetically, and allowed to oxidize under controlled conditions into a mixture of U-13C-labeled ARA oxidation products. The components of this mixture were characterized with respect to their partitioning behavior during lipid extraction, their durability during saponification, trends in mouse brain tissue concentrations during post mortem intervals, and their overall suitability as internal standards for multiple-reaction monitoring tandem mass spectrometry. This mixture has now been used as a set of internal standards to determine the relative abundance of ARA and 54 non-stereospecific oxidation products in milligram samples of brain tissue. Many of these oxidation products were recovered from both healthy mouse and healthy human brain, although some of them were unique to each source, and some have not heretofore been described. The list of oxidation products detected in AD brain tissue was the same as in healthy human brain, although simple hydroxy-eicosanoids were significantly increased in AD brain. while more complex oxidation products were not. These results are consistent with an increased level of chemically-mediated oxidative ARA degradation in Alzheimer's disease. However, they also point to the existence of processes that selectively produce or eliminate specific oxidation products, and those processes may account for some of the inconsistencies in previously reported results.

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  • The potential therapeutic effects of ergothioneine in pre-eclampsia
    Publication date: March 2018
    Source:Free Radical Biology and Medicine, Volume 117

    Author(s): Robert N. Kerley, Cathal McCarthy, Douglas B. Kell, Louise C. Kenny

    Ergothioneine (ERG), is a water-soluble amino acid that is derived entirely from dietary sources. It has received much attention as a therapeutic agent due to its anti-oxidant properties, and there are claims of preferential accumulation within high oxidative stress organs. Pre-eclampsia, a condition accompanied by increased oxidative stress, is one of the leading causes of maternal morbidity and mortality. Despite intense research efforts, its aetiologies remain somewhat unclear and there are still no effective treatment options. Clinical trials of the anti-oxidants vitamin C and vitamin E have proven largely ineffective with little improvement in clinical outcome or even a negative response. This could be explained in part by their inability to permeate the plasma and mitochondrial membranes and scavenge mitochondria-derived superoxide species, and for the former by the fact that it is actually a pro-oxidant in the presence of unliganded iron. ERG accumulates within tissues through the action of a specific organic cation transporter, SLC22A4 (previously referred to as OCTN1), which is possibly also expressed in mammalian mitochondria. Mitochondrial dysfunction has been implicated in a variety of vascular diseases including pre-eclampsia. This review discusses the use of ERG as a possibly mitochondrial-targeted anti-oxidant, focusing on its physical properties, potential mechanisms of action, safety profile and administration in relation to pregnancies complicated by pre-eclampsia.

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  • Interaction between p22phox and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation
    Publication date: 20 February 2018
    Source:Free Radical Biology and Medicine, Volume 116

    Author(s): Melinda Zana, Zalán Péterfi, Hajnal A. Kovács, Zsuzsanna E. Tóth, Balázs Enyedi, Françoise Morel, Marie-Hélène Paclet, Ágnes Donkó, Stanislas Morand, Thomas L. Leto, Miklós Geiszt

    The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the TGF-β1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-β1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-β1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-β1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.

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  • Carbonic anhydrase II does not exhibit Nitrite reductase or Nitrous Anhydrase Activity
    Publication date: March 2018
    Source:Free Radical Biology and Medicine, Volume 117

    Author(s): Jacob T. Andring, Carrie L. Lomelino, Chingkuang Tu, David N. Silverman, Robert McKenna, Erik R. Swenson

    Carbonic anhydrase II (CA II) is a zinc metalloenzyme that catalyzes the reversible interconversion of water and CO2 to bicarbonate and a proton. CA II is abundant in most cells, and plays a role in numerous processes including gas exchange, epithelial ion transport, respiration, extra- and intracellular pH control, and vascular regulation. Beyond these CO2 and pH-linked roles, it has been postulated that CA II might also reduce nitrite (NO2 -) to nitric oxide (NO), as bicarbonate and NO2 - both exhibit sp 2 molecular geometry and NO also plays an important role in vasodilation and regulation of blood pressure. Indeed, previous studies by Aamand et al. have shown that bovine CA II (BCA II) possesses nitrite dehydration activity and paradoxically demonstrated that CA inhibitors (CAIs) such as dorzolamide and acetazolamide significantly increased NO production (Aamand et al., 2009; Nielsen and Fago, 2015) [1,2]. Hence, the goal of this work was to revisit these studies using the same experimental conditions as Aamand et al. measuring NO generation by two methods, and to examine the structure of CA II in complex with NO2 - in the presence and absence of dorzolamide. Our results contradict the previous findings and indicate that CA II does not exhibit nitrite reductase or dehydration activity, and that this is not enhanced in the presence of CA inhibitors. In addition, a structural examination of BCA II in complex with NO2 - and superimposed with dorzolamide demonstrates that CA inhibitor binding at the active site to the zinc moiety blocks potential NO2 - binding.

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  • An improved cell-permeable fluorogenic substrate as the basis for a highly sensitive test for NAD(P)H quinone oxidoreductase 1 (NQO1) in living cells
    Publication date: 20 February 2018
    Source:Free Radical Biology and Medicine, Volume 116

    Author(s): Simone Cuff, Ruth D. Lewis, Edwin Chinje, Mohammed Jaffar, Richard Knox, Ian Weeks

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme upregulated in response to oxidative stress and in some cancers. Its upregulation by compounds has been used as an indicator of their potential anti-cancer properties. In this study we have designed, produced and tested a fluorogenic coumarin conjugate which selectively releases highly fluorescent 4-methylumbelliferone (4-MU) in the presence of NQO1. It was found that measuring 4-MU release rapidly and specifically quantitated NQO1 levels in vitro and in live cells. Both the substrate and its products freely perfused through cell membranes and were non-toxic. The substrate was very specific with low background, and the assay itself could be done in less than 10minutes. This is the first assay to allow the quantitation of NQO1 in live cells which can then be retained for further experiments.

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