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Sample records for intracellular redox state

  1. Carbon-Dot-Based Nanosensors for the Detection of Intracellular Redox State.

    PubMed

    Liu, Ye; Tian, Ye; Tian, Yefei; Wang, Yajun; Yang, Wuli

    2015-11-25

    Carbon-dot-based nanosensors are prepared through sequentially assembling a polymer/carbon dot multilayer shell on mesoporous silica nanoparticles with different crosslinking densities of disulfide bonds; they can be utilized to evaluate the gluthathione (GSH) concentration. In vitro cell assays demonstrate the feasibility of using such nanosensors in evaluating the intracellular redox state of different cells. PMID:26450796

  2. Data on intracellular localization of RPSA upon alteration of its redox state

    PubMed Central

    Vilas-Boas, Filipe; Bagulho, Ana; Jerónimo, Ana; Tenente, Rita; Real, Carla

    2015-01-01

    Ribosomal Protein SA (RPSA), a component of the 40S ribosomal subunit, was identified as a H2O2 target in HeLa cells [1]. In order to analyze the intracellular localization of RPSA in different redox states we overexpressed wild-type RPSA (RPSAwt) or RPSA containing two cysteine to serine residue substitutions at positions 148 and 163 (RPSAmut) in HeLa cells. The transfected cells were exposed to H2O2 or N-acetylcysteine (NAC) and RPSA subcellular localization was assessed by immunofluorescence in permeabilized cells. In addition, co-immunofluorescence for RPSA and Ribosomal Protein S6 (RPS6) was performed in cells overexpressing RPSAwt or RPSAmut. Finally, the ribosomal expression of endogenous RPSA in the presence or absence of H2O2 was analyzed by Western blot. The data presented in this work is related to the research article entitled “Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA” [1]. PMID:26862576

  3. In vivo (31) P MRS assessment of intracellular NAD metabolites and NAD(+) /NADH redox state in human brain at 4 T.

    PubMed

    Lu, Ming; Zhu, Xiao-Hong; Chen, Wei

    2016-07-01

    NAD(+) and NADH play key roles in cellular respiration. Intracellular redox state defined by the NAD(+) /NADH ratio (RX) reflects the cellular metabolic and physiopathological status. By taking advantage of high/ultrahigh magnetic field strengths, we have recently established a novel in vivo (31) P MRS-based NAD assay for noninvasive and quantitative measurements of intracellular NAD concentrations and redox state in animal and human brains at 16.4 T, 9.4 T and 7 T. To explore its potential for clinical application, in this study we investigated the feasibility of assessing the NAD metabolism and redox state in human brain at a lower field of 4 T by incorporating the (1) H-decoupling technique with the in vivo (31) P NAD assay. The use of (1) H decoupling significantly narrowed the linewidths of NAD and α-ATP resonances, resulting in higher sensitivity and better spectral resolution as compared with the (1) H-coupled (31) P spectrum. These improvements made it possible to reliably quantify cerebral NAD concentrations and RX, consistent with previously reported results obtained from similar age human subjects at 7 T. In summary, this work demonstrates the capability and utility of the (1) H-decoupled (31) P MRS-based NAD assay at lower field strength; thus, it opens new opportunities for studying intracellular NAD metabolism and redox state in human brain at clinical settings. This conclusion is supported by the simulation results, indicating that similar performance and reliability as observed at 4T can be achieved at 3 T with the same signal-to-noise ratio. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27257783

  4. Redox-sensitive YFP sensors for monitoring dynamic compartment-specific glutathione redox state.

    PubMed

    Banach-Latapy, Agata; He, Tiantian; Dardalhon, Michèle; Vernis, Laurence; Chanet, Roland; Huang, Meng-Er

    2013-12-01

    Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox state and their dynamics in response to stress in human cells. RxYFP expressed in the cytosol, nucleus, or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady-state redox conditions among the cytosol, nucleus, and mitochondrial matrix. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by ultraviolet A radiation in a dose-dependent manner. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by 100 μM hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high-resolution readout of steady glutathione state and dynamics of redox changes. PMID:23891676

  5. Transient light-induced intracellular oxidation revealed by redox biosensor

    SciTech Connect

    Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  6. Global transcriptome analyses provide evidence that chloroplast redox state contributes to intracellular as well as long-distance signalling in response to stress and acclimation in Arabidopsis.

    PubMed

    Bode, Rainer; Ivanov, Alexander G; Hüner, Norman P A

    2016-06-01

    Global transcriptome analyses were used to assess the interactive effects of short-term stress versus long-term acclimation to high light (HL), low temperature (LT) and excitation pressure in Arabidopsis. Microarray analyses indicated that exposure to stress resulted in two times as many modulated transcripts in both, high-light-treated and low-temperature-treated plants, compared to plants that were fully acclimated to either one of these conditions. We showed that 10.9 % of all transcripts were regulated in the same way by both stress conditions, and hence, were categorized as excitation pressure regulated, rather than regulated by either high-light or low-temperature stress per se. This group of chloroplast redox-sensitive genes included various photosynthetic genes as well as genes known to be associated with cold acclimation (cbf3, cor15A, cor15B) and gibberellic acid (GA) metabolism and signalling (ga2ox1, gai). Chemical inhibition of the photosynthetic electron transport by either DCMU or DBMIB indicated that although the plastoquinone pool contributes significantly to redox regulation of the transcriptome (8.6 %), it appears that PSI represents the major source of redox signals (89 %), whereas PSII appears to contribute only 3.1 %. A comparison of the gene expression profiles between stress and acclimated plants indicated that 10 % of the genes induced by a short, 1-h stress were also associated with long-term acclimation to high excitation pressure. This included the APETALA2/ETHYLENE-RESPONSIVE-BINDING PROTEIN family, the MYB domain- and MYB-related transcription factor family as well as the GRAS transcription factor family important in GA signalling confirming that acclimation to stress is a time-nested phenomenon. We suggest that acclimation to photosynthetic redox imbalance extends beyond the chloroplast and the leaf cell to systemic ROS signalling. This is discussed in terms of the control of plant phenotype through regulation of the nuclear

  7. Monitoring Intracellular Redox Changes in Ozone-exposed airway epithelial cells

    EPA Science Inventory

    Background: The toxicity of many compounds involves oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically-encoded probes designed for monitoring intracellular redox s...

  8. Tumor intracellular redox status and drug resistance--serendipity or a causal relationship?

    PubMed

    Pervaiz, Shazib; Clement, Marie-Veronique

    2004-01-01

    Reducing tumor load by therapeutic induction of cell death in the transformed phenotype is the desirable goal of most chemotherapeutic regimens. Despite the tremendous strides made in our understanding of mechanisms that endow tumor cells with the ability to evade execution signals, development of chemo-resistance is still a major obstacle in the successful management of the disease. A host of factors have been implicated in the acquisition of the resistant phenotype, such as activation of drug efflux pumps, overexpression of proteins that inhibit cell death, absence of critical members of the death circuitry, and selective loss of cell cycle checkpoints. Consequently, it is now well established that the process of carcinogenesis is not only a result of an increase in cells' proliferative capacity, but a product of increased proliferation and defective or diminished cell death signaling. To that end, one of the critical determinants of cellular response to exogenous stimuli is the cellular redox status. Intracellular generation of reactive oxygen species (ROS) is tightly regulated by the intrinsic anti-oxidant defense systems. Despite the conventional dogma that ROS are harmful to the cell, experimental evidence over the last decade or so bear witness to the fact that ROS also play an important role as signaling molecules in diverse physiological processes. Indeed, low levels of intracellular ROS have been linked to cellular proliferation and cell cycle progression, which provides an explanation for the pro-oxidant state invariably associated with the transformed phenotype. Coupled to that are recent observations implicating pro-oxidant intracellular milieu in tumor cells' resistance to cell death signals delivered through the cell surface receptor or upon exposure to chemotherapeutic drugs. These studies provide convincing evidence to support a direct or indirect role for intracellular superoxide anion in creating an intracellular milieu non-permissive for cell

  9. Redox Regulation of Intracellular Zinc: Molecular Signaling in the Life and Death of Neurons

    PubMed Central

    Aizenman, Elias

    2011-01-01

    Abstract Zn2+ has emerged as a major regulator of neuronal physiology, as well as an important signaling agent in neural injury. The intracellular concentration of this metal is tightly regulated through the actions of Zn2+ transporters and the thiol-rich metal binding protein metallothionein, closely linking the redox status of the cell to cellular availability of Zn2+. Accordingly, oxidative and nitrosative stress during ischemic injury leads to an accumulation of neuronal free Zn2+ and the activation of several downstream cell death processes. While this Zn2+ rise is an established signaling event in neuronal cell death, recent evidence suggests that a transient, sublethal accumulation of free Zn2+ can also play a critical role in neuroprotective pathways activated during ischemic preconditioning. Thus, redox-sensitive proteins, like metallothioneins, may play a critical role in determining neuronal cell fate by regulating the localization and concentration of intracellular free Zn2+. Antioxid. Redox Signal. 15, 2249–2263. PMID:20849376

  10. Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal

    PubMed Central

    Putker, Marrit; O’Neill, John Stuart

    2016-01-01

    Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping. PMID:26810072

  11. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling1[OPEN

    PubMed Central

    2016-01-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. PMID:27208308

  12. Comparison of methods probing the intracellular redox milieu in Plasmodium falciparum.

    PubMed

    Mohring, Franziska; Jortzik, Esther; Becker, Katja

    2016-01-01

    Glutathione plays a crucial role in the redox regulation of the malaria parasite Plasmodium falciparum and is linked to drug resistance mechanisms, especially in resistance against the antimalarial drug chloroquine (CQ). The determination of the glutathione-dependent redox potential was recently established in living parasites using a cytosolically expressed biosensor comprising redox-sensitive green fluorescent protein coupled to human glutaredoxin 1 (hGrx1-roGFP2). In order to further elucidate redox changes induced by antimalarial drugs and to consolidate the application spectrum of the ratiometric biosensor we systematically compared it to other methods probing thiol and redox metabolism. Among these methods were cell disruptive and non-disruptive approaches including spectrophotometric assays with Ellman's reagent and naphthalene dicarboxyaldehyde as well as molecular probes such as ThiolTracker™ Violet and the dichlorofluorescein-based probe CM-H2DCFDA. To directly compare the methods, blood stages of the CQ-sensitive P. falciparum 3D7 strain were challenged with the oxidative agent diamide and the antimalarial drugs artemisinin and CQ for 1h, 4h, and 24h. For all conditions, dose-dependent changes in the different redox parameters could be monitored which are compared and discussed. We furthermore detected slight differences in thiol status of parasites transiently transfected with hGrx1-roGFP2 in comparison with control 3D7 cells. In conclusion, ThiolTracker™ Violet and, even more so, the hGrx1-roGFP2 probe reacted reliably and sensitively to drug induced changes in intracellular redox metabolism. These results were substantiated by classical cell disruptive methods. PMID:26593282

  13. Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Kasozi, Denis; Mohring, Franziska; Rahlfs, Stefan; Meyer, Andreas J.; Becker, Katja

    2013-01-01

    In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be −314.2±3.1 mV and −313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further

  14. Proteostasis and REDOX state in the heart

    PubMed Central

    Christians, Elisabeth S.

    2012-01-01

    Force-generating contractile cells of the myocardium must achieve and maintain their primary function as an efficient mechanical pump over the life span of the organism. Because only half of the cardiomyocytes can be replaced during the entire human life span, the maintenance strategy elicited by cardiac cells relies on uninterrupted renewal of their components, including proteins whose specialized functions constitute this complex and sophisticated contractile apparatus. Thus cardiac proteins are continuously synthesized and degraded to ensure proteome homeostasis, also termed “proteostasis.” Once synthesized, proteins undergo additional folding, posttranslational modifications, and trafficking and/or become involved in protein-protein or protein-DNA interactions to exert their functions. This includes key transient interactions of cardiac proteins with molecular chaperones, which assist with quality control at multiple levels to prevent misfolding or to facilitate degradation. Importantly, cardiac proteome maintenance depends on the cellular environment and, in particular, the reduction-oxidation (REDOX) state, which is significantly different among cardiac organelles (e.g., mitochondria and endoplasmic reticulum). Taking into account the high metabolic activity for oxygen consumption and ATP production by mitochondria, it is a challenge for cardiac cells to maintain the REDOX state while preventing either excessive oxidative or reductive stress. A perturbed REDOX environment can affect protein handling and conformation (e.g., disulfide bonds), disrupt key structure-function relationships, and trigger a pathogenic cascade of protein aggregation, decreased cell survival, and increased organ dysfunction. This review covers current knowledge regarding the general domain of REDOX state and protein folding, specifically in cardiomyocytes under normal-healthy conditions and during disease states associated with morbidity and mortality in humans. PMID:22003057

  15. Redox state influence on human galectin-1 function.

    PubMed

    Yu, Xing; Scott, Stacy A; Pritchard, Rhys; Houston, Todd A; Ralph, Stephen J; Blanchard, Helen

    2015-09-01

    Intracellular and extracellular functions of human galectin-1 are influenced by its redox surroundings due to the presence of six cysteines within its amino acid sequence. Galectin-1 recognises intracellular-membrane-anchored Ras proteins that act as molecular switches regulating multiple signal transduction pathways. Human tumours frequently express Ras proteins that have become continuously activated due to point mutations, and this typically leads to deregulation of tumour cell growth, angiogenesis and invasion of metastatic cancer cells. Of significance is that galectin-1 preferably recognises H-Ras, one of the human Ras isoforms, and in particular galectin-1 recognition of the H-Ras farnesyl moiety is paramount to H-Ras membrane anchorage, a prerequisite step for H-Ras-mediated signal transduction regulating normal cell growth and malignant transformation. Herein the impact of the redox state on galectin-1's ability to interact with farnesyl analogues is explored. We demonstrate for the first time that reduced galectin-1 directly binds farnesyl and does so in a carbohydrate-independent manner. A K28T mutation abolishes farnesyl recognition by reduced dimeric galectin-1 whilst its carbohydrate-binding activity is retained, thus demonstrating the presence of an independent region on galectin-1 pertaining to growth inhibitory activity. Intriguingly, oxidised galectin-1 also recognises farnesyl, the biological implication of this novel finding is yet to be elucidated. Further, the redox effect on galectin-1 extracellular function was investigated and we discover that oxidised galectin-1 demonstrates a protective effect upon acute lymphoblastic leukaemia cells challenged by oxidative stress. PMID:26116885

  16. Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae

    PubMed Central

    Rawson, Frankie J.; Downard, Alison J.; Baronian, Keith H.

    2014-01-01

    Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators. PMID:24910017

  17. Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Rawson, Frankie J.; Downard, Alison J.; Baronian, Keith H.

    2014-06-01

    Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

  18. The Role of Copper Chaperone Atox1 in Coupling Redox Homeostasis to Intracellular Copper Distribution.

    PubMed

    Hatori, Yuta; Lutsenko, Svetlana

    2016-01-01

    Human antioxidant protein 1 (Atox1) is a small cytosolic protein with an essential role in copper homeostasis. Atox1 functions as a copper carrier facilitating copper transfer to the secretory pathway. This process is required for activation of copper dependent enzymes involved in neurotransmitter biosynthesis, iron efflux, neovascularization, wound healing, and regulation of blood pressure. Recently, new cellular roles for Atox1 have emerged. Changing levels of Atox1 were shown to modulate response to cancer therapies, contribute to inflammatory response, and protect cells against various oxidative stresses. It has also become apparent that the activity of Atox1 is tightly linked to the cellular redox status. In this review, we summarize biochemical information related to a dual role of Atox1 as a copper chaperone and an antioxidant. We discuss how these two activities could be linked and contribute to establishing the intracellular copper balance and functional identity of cells during differentiation. PMID:27472369

  19. [Monitoring the Redox States of Thioredoxin in Protein-Protein Interaction Using Intrinsic Fluorescence Probe].

    PubMed

    Wang, Pan; Guo, Ai-yu; Chang, Guan-xiao; Ran, Xia; Zhang, Yu; Guo, Li-jun

    2015-10-01

    The cellular redox states directly affect cell proliferation, differentiation and apoptosis, and the redox states changes is particularly important to the regulation of cell survival or death. Thioredoxin is a kind of oxidation regulatory protein which is widely exists in organisms, and the change of redox states is also an important process in redox regulation. In this work, we have used the site-directed mutagenesis of protein, SDS-polyacrylamide gel electrophoresis fluorescence spectroscopy and circular dichroism etc., to investigate redox states changes between TRX (E. coli) and glutathione peroxidase(GPX3) during their interaction. By observing the fluorescence spectra of TRX and its mutants, we have studied the protein interactions as well as the redox states switching between oxidation state TRX and the reduced state GPX3. The results demonstrate the presence of interactions and electron exchanges occurring between reduced GPX3 and oxidized TRX, which is of significance for revealing the physical and chemical mechanism of TRX in intracellular signal transduction. PMID:26904821

  20. Anti-cancer effects of cerium oxide nanoparticles and its intracellular redox activity.

    PubMed

    Pešić, Milica; Podolski-Renić, Ana; Stojković, Sonja; Matović, Branko; Zmejkoski, Danica; Kojić, Vesna; Bogdanović, Gordana; Pavićević, Aleksandra; Mojović, Miloš; Savić, Aleksandar; Milenković, Ivana; Kalauzi, Aleksandar; Radotić, Ksenija

    2015-05-01

    Data on medical applications of cerium oxide nanoparticles CeO2 (CONP) are promising, yet information regarding their action in cells is incomplete and there are conflicting reports about in vitro toxicity. Herein, we have studied cytotoxic effect of CONP in several cancer and normal cell lines and their potential to change intracellular redox status. The IC50 was achieved only in two of eight tested cell lines, melanoma 518A2 and colorectal adenocarcinoma HT-29. Self-propagating room temperature method was applied to produce CONP with an average crystalline size of 4 nm. The results confirmed presence of Ce(3+) and O(2-) vacancies. The induction of cell death by CONP and the production of reactive oxygen species (ROS) were analyzed by flow-cytometry. Free radicals related antioxidant capacity of the cells was studied by the reduction of stable free radical TEMPONE using electron spin resonance spectroscopy. CONP showed low or moderate cytotoxicity in cancer cell lines: adenocarcinoma DLD1 and multi-drug resistant DLD1-TxR, non-small cell lung carcinoma NCI-H460 and multi-drug resistant NCI-H460/R, while normal cell lines (keratinocytes HaCaT, lung fetal fibroblasts MRC-5) were insensitive. The most sensitive were 518A2 melanoma and HT-29 colorectal adenocarcinoma cell lines, with the IC50 values being between 100 and 200 μM. Decreased rate of TEMPONE reduction and increased production of certain ROS species (peroxynitrite and hydrogen peroxide anion) indicates that free radical metabolism, thus redox status was changed, and antioxidant capacity damaged in the CONP treated 518A2 and HT-29 cells. In conclusion, changes in intracellular redox status induced by CONP are partly attributed to the prooxidant activity of the nanoparticles. Further, ROS induced cell damages might eventually lead to the cell death. However, low inhibitory potential of CONP in the other human cell lines tested indicates that CONP may be safe for human usage in industry and medicine. PMID

  1. Redox and pH-responsive degradable micelles for dually activated intracellular anticancer drug release.

    PubMed

    Chen, Wei; Zhong, Ping; Meng, Fenghua; Cheng, Ru; Deng, Chao; Feijen, Jan; Zhong, Zhiyuan

    2013-08-10

    Redox and pH dual-responsive biodegradable micelles were developed based on poly(ethylene glycol)-SS-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate) (PEG-SS-PTMBPEC) copolymer and investigated for intracellular doxorubicin (DOX) release. PEG-SS-PTMBPEC copolymer with an Mn of 5.0-4.1kg/mol formed micellar particles with an average diameter of 140nm and a low polydispersity of 0.12. DOX was loaded into PEG-SS-PTMBPEC micelles with a decent drug loading content of 11.3wt.%. The in vitro release studies showed that under physiological conditions only ca. 24.5% DOX was released from DOX-loaded micelles in 21h. The release of DOX was significantly accelerated at pH5.0 or in the presence of 10mM glutathione (GSH) at pH7.4, in which 62.8% and 74.3% of DOX was released, respectively, in 21h. The drug release was further boosted under 10mM GSH and pH 5.0 conditions, with 94.2% of DOX released in 10h. Notably, DOX release was also facilitated by 2 or 4h incubation at pH 5.0 and then at pH 7.4 with 10mM GSH, which mimics the intracellular pathways of endocytosed micellar drugs. Confocal microscopy observation indicated that DOX was delivered and released into the nuclei of HeLa cells following 8h incubation with DOX-loaded PEG-SS-PTMBPEC micelles, while DOX was mainly located in the cytoplasm for reduction-insensitive PEG-PTMBPEC controls. MTT assays revealed that DOX-loaded PEG-SS-PTMBPEC micelles had higher anti-tumor activity than reduction-insensitive controls, with low IC50 of 0.75 and 0.60μg/mL for HeLa and RAW 264.7 cells, respectively, following 48h incubation. PEG-SS-PTMBPEC micelles displayed low cytotoxicity up to a concentration of 1.0mg/mL. These redox and pH dual-bioresponsive degradable micelles have appeared as a promising platform for targeted intracellular anticancer drug release. PMID:23306022

  2. Intracellular redox-activated anticancer drug delivery by functionalized hollow mesoporous silica nanoreservoirs with tumor specificity.

    PubMed

    Luo, Zhong; Hu, Yan; Cai, Kaiyong; Ding, Xingwei; Zhang, Quan; Li, Menghuan; Ma, Xing; Zhang, Beilu; Zeng, Yongfei; Li, Peizhou; Li, Jinghua; Liu, Junjie; Zhao, Yanli

    2014-09-01

    In this study, a type of intracellular redox-triggered hollow mesoporous silica nanoreservoirs (HMSNs) with tumor specificity was developed in order to deliver anticancer drug (i.e., doxorubicin (DOX)) to the target tumor cells with high therapeutic efficiency and reduced side effects. Firstly, adamantanamine was grafted onto the orifices of HMSNs using a redox-cleavable disulfide bond as an intermediate linker. Subsequently, a synthetic functional molecule, lactobionic acid-grafted-β-cyclodextrin (β-CD-LA), was immobilized on the surface of HMSNs through specific complexation with the adamantyl group, where β-CD served as an end-capper to keep the loaded drug within HMSNs. β-CD-LA on HMSNs could also act as a targeting agent towards tumor cells (i.e., HepG2 cells), since the lactose group in β-CD-LA is a specific ligand binding with the asialoglycoprotein receptor (ASGP-R) on HepG2 cells. In vitro studies demonstrated that DOX-loaded nanoreservoirs could be selectively endocytosed by HepG2 cells, releasing therapeutic DOX into cytoplasm and efficiently inducing the apoptosis and cell death. In vivo investigations further confirmed that DOX-loaded nanoreservoirs could permeate into the tumor sites and actively interact with tumor cells, which inhibited the tumor growth with the minimized side effect. On the whole, this drug delivery system exhibits a great potential as an efficient carrier for targeted tumor therapy in vitro and in vivo. PMID:24930850

  3. Thiol/disulfide redox states in signaling and sensing

    PubMed Central

    Go, Young-Mi; Jones, Dean P.

    2015-01-01

    Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady-states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling, and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities. PMID:23356510

  4. Glutamate-induced metabolic changes influence the cytoplasmic redox state of hippocampal neurons.

    PubMed

    Porras, Omar H; Stutzin, Andrés

    2011-07-22

    Brain cell metabolism is intimately associated with intracellular oxidation-reduction (redox) balance. Glutamatergic transmission is accompanied with changes in substrate preference in neurons. Therefore, we studied cytoplasmatic redox changes in hippocampal neurons in culture exposed to glutamate. Neurons were transfected with HyPer, a genetically encoded redox biosensor for hydrogen peroxide which allows real-time imaging of the redox state. The rate of fluorescence decay, corresponding to the reduction of the biosensor was found to be augmented by low doses of glutamate (10 μM) as well as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca(2+) abolished the glutamate-induced effect observed on HyPer fluorescence. Additional experiments indicated that mitochondrial function and hence energetic substrate availability commands the redox state of neurons and is required for the glutamate effect observed on the biosensor signal. Furthermore, our results implicated astrocytic metabolism in the changes of neuronal redox state observed with glutamate. PMID:21708127

  5. The Extracellular Redox State Modulates Mitochondrial Function, Gluconeogenesis, and Glycogen Synthesis in Murine Hepatocytes

    PubMed Central

    Nocito, Laura; Kleckner, Amber S.; Yoo, Elsia J.; Jones IV, Albert R.; Liesa, Marc; Corkey, Barbara E.

    2015-01-01

    Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases. PMID:25816337

  6. Redox-responsive micelles self-assembled from dynamic covalent block copolymers for intracellular drug delivery.

    PubMed

    Yang, Qinglai; Tan, Lianjiang; He, Changyu; Liu, Bingya; Xu, Yuhong; Zhu, Zhenggang; Shao, Zhifeng; Gong, Bing; Shen, Yu-Mei

    2015-04-01

    Redox-responsive micelles self-assembled from dynamic covalent block copolymers with double disulfide linkage in the backbone have been developed successfully. The amphiphilic block copolymers PEG-PLA associated with complementary H-bonding sequences can self-assemble into spherical micelles in aqueous media with sizes from 34 nm to 107 nm with different molar mass of PEG and PLA. Moreover, in vitro drug release analyses indicate that reductive environment can result in triggered drug release profiles. The glutathione (GSH) mediated intracellular drug delivery was investigated against HeLa human cervical carcinoma cell line. Flow cytometry and fluorescence microscopy measurements demonstrated that the micelles exhibited faster drug release in glutathione monoester (GSH-OEt) pretreated HeLa cells than that in the nonpretreated cells. Cytotoxicity assay of DOX-loaded micelles indicated the higher cellular proliferation inhibition against 10 mM of GSH-OEt pretreated HeLa cells than that of the nonpretreated ones. These reduction-responsive, biodegradable and biocompatibility micelles could provide a favorable platform to construct excellent drug delivery systems for cancer therapy. PMID:25662913

  7. Involvement of Redox State in the Aging of Drosophila melanogaster

    PubMed Central

    Radyuk, Svetlana N.; Sohal, Rajindar S.

    2013-01-01

    Abstract Significance: The main objective of this review was to provide an exposition of investigations, conducted in Drosophila melanogaster, on the role of reactive oxygen species and redox state in the aging process. While early transgenic studies did not clearly support the validity of the oxidative stress hypothesis of aging, predicated on the accumulation of structural damage, they spawned a broader search for redox-related effects that might impact the aging process. Recent Advances: Initial evidence implicating the thiol redox state as a possible causative factor in aging has been obtained in Drosophila. Overexpression of genes, such as GCL, G6PD, Prx2, and Prx5, which are involved in the maintenance of thiol redox homeostasis, has strong positive effects on longevity. Further, the depletion of peroxiredoxin activity in the mitochondria through the double knockdown of Prx5 and Prx3 not only results in a redox crisis but also elicits a rapid aging phenotype. Critical Issues: Herein, we summarize the present status of knowledge about the main components of the machinery controlling thiol redox homeostasis and describe how age-related redox fluctuations might impact aging more acutely through disruption of the redox-sensitive signaling mechanisms rather than via the simple accumulation of structural damage. Future Directions: Based on these initial insights into the plausible impact of redox fluctuations on redox signaling, future studies should focus on the pathways that have been explicitly implicated in aging, such as insulin signaling, TOR, and JNK/FOXO, with particular attention to elements that are redox sensitive. Antioxid. Redox Signal. 19, 788–803. PMID:23458359

  8. Redox State of the Neoarchean Earth Environment

    NASA Technical Reports Server (NTRS)

    Zerkle, Aubrey L.; Claire, Mark W.; Domagal-Goldman, Shawn; Farquhar, James; Poulton, Simon W.

    2011-01-01

    A Titan-like organic haze has been hypothesized for Earth's atmosphere prior to widespread surface oxygenation approx.2.45 billion years ago (Ga). We present a high-resolution record of quadruple sulfur isotopes, carbon isotopes, and Fe speciation from the approx.2.65-2.5 Ga Ghaap Group, South Africa, which suggest a linkage between organic haze and the biogeochemical cycling of carbon, sulfur, oxygen, and iron on the Archean Earth. These sediments provide evidence for oxygen production in microbial mats and localized oxygenation of surface waters. However, this oxygen production occurred under a reduced atmosphere which existed in multiple distinct redox states that correlate to changes in carbon and sulfur isotopes. The data are corroborated by photochemical model results that suggest bi-stable transitions between organic haze and haze-free atmospheric conditions in the Archean. These geochemical correlations also extend to other datasets, indicating that variations in the character of anomalous sulfur fractionation could provide insight into the role of carbon-bearing species in the reducing Archean atmosphere.

  9. Regulation of MMP-1 expression in response to hypoxia is dependent on the intracellular redox status of metastatic bladder cancer cells.

    PubMed

    Shin, Dong Hui; Dier, Usawadee; Melendez, Juan Andres; Hempel, Nadine

    2015-12-01

    High steady-state reactive oxygen species (ROS) production has been implicated with metastatic disease progression. We provide new evidence that this increased intracellular ROS milieu uniquely predisposes metastatic tumor cells to hypoxia-mediated regulation of the matrix metalloproteinase MMP-1. Using a cell culture metastatic progression model we previously reported that steady-state intracellular H2O2 levels are elevated in highly metastatic 253J-BV bladder cancer cells compared to their non-metastatic 253J parental cells. 253J-BV cells display higher basal MMP-1 expression, which is further enhanced under hypoxic conditions (1% O2). This hypoxia-mediated MMP-1 increase was not observed in the non-metastatic 253J cells. Hypoxia-induced MMP-1 increases are accompanied by the stabilization of hypoxia-inducible transcription factors (HIFs)-1α and HIF-2α, and a rise in intracellular ROS in metastatic 253J-BV cells. RNA interference studies show that hypoxia-mediated MMP-1 expression is primarily dependent on the presence of HIF-2α. Further, hypoxia promotes migration and spheroid outgrowth of only the metastatic 253J-BV cells and not the parental 253J cells. The observed HIF stabilization, MMP-1 expression and migration under hypoxia are dependent on increases in intracellular ROS, as these effects are attenuated by treatment with the antioxidant N-acetyl-L-cysteine. These data show that ROS play an important role in hypoxia-mediated MMP-1 expression and that an elevated intracellular redox environment, as observed in metastasis, predisposes tumor cells to an enhanced hypoxic response. It further supports the notion that metastatic tumor cells are uniquely able to utilize intracellular increases in ROS to drive pro-metastatic signaling events and highlights the important interplay between ROS and hypoxia in malignancy. PMID:26343184

  10. Zn2+-dependent Redox Switch in the Intracellular T1-T1 Interface of a Kv Channel*†

    PubMed Central

    Wang, Guangyu; Strang, Candace; Pfaffinger, Paul J.; Covarrubias, Manuel

    2008-01-01

    The thiol-based redox regulation of proteins plays a central role in cellular signaling. Here, we investigated the redox regulation at the Zn2+ binding site (HX5CX20CC) in the intracellular T1-T1 inter-subunit interface of a Kv4 channel. This site undergoes conformational changes coupled to voltage-dependent gating, which may be sensitive to oxidative stress. The main results show that internally applied nitric oxide (NO) inhibits channel activity profoundly. This inhibition is reversed by reduced glutathione and suppressed by intracellular Zn2+, and at least two Zn2+ site cysteines are required to observe the NO-induced inhibition (Cys-110 from one subunit and Cys-132 from the neighboring subunit). Biochemical evidence suggests strongly that NO induces a disulfide bridge between Cys-110 and Cys-132 in intact cells. Finally, further mutational studies suggest that intra-subunit Zn2+ coordination involving His-104, Cys-131, and Cys-132 protects against the formation of the inhibitory disulfide bond. We propose that the interfacial T1 Zn2+ site of Kv4 channels acts as a Zn2+-dependent redox switch that may regulate the activity of neuronal and cardiac A-type K+ currents under physiological and pathological conditions. PMID:17331952

  11. Different redox states of metallothionein/thionein in biological tissue

    PubMed Central

    Krężel, Artur; Maret, Wolfgang

    2006-01-01

    Mammalian metallothioneins are redox-active metalloproteins. In the case of zinc metallothioneins, the redox activity resides in the cysteine sulfur ligands of zinc. Oxidation releases zinc, whereas reduction re-generates zinc-binding capacity. Attempts to demonstrate the presence of the apoprotein (thionein) and the oxidized protein (thionin) in tissues posed tremendous analytical challenges. One emerging strategy is differential chemical modification of cysteine residues in the protein. Chemical modification distinguishes three states of the cysteine ligands (reduced, oxidized and metal-bound) based on (i) quenched reactivity of the thiolates when bound to metal ions and restoration of thiol reactivity in the presence of metal-ion-chelating agents, and (ii) modification of free thiols with alkylating agents and subsequent reduction of disulfides to yield reactive thiols. Under normal physiological conditions, metallothionein exists in three states in rat liver and in cell lines. Ras-mediated oncogenic transformation of normal HOSE (human ovarian surface epithelial) cells induces oxidative stress and increases the amount of thionin and the availability of cellular zinc. These experiments support the notion that metallothionein is a dynamic protein in terms of its redox state and metal content and functions at a juncture of redox and zinc metabolism. Thus redox control of zinc availability from this protein establishes multiple methods of zinc-dependent cellular regulation, while the presence of both oxidized and reduced states of the apoprotein suggest that they serve as a redox couple, the generation of which is controlled by metal ion release from metallothionein. PMID:17134375

  12. Smart pH/Redox Dual-Responsive Nanogels for On-Demand Intracellular Anticancer Drug Release.

    PubMed

    Yang, Hao; Wang, Qin; Huang, Shan; Xiao, Ai; Li, Fuying; Gan, Lu; Yang, Xiangliang

    2016-03-30

    Efficient accumulation and intracellular drug release in cancer cells remain a crucial challenge in developing ideal anticancer drug delivery systems. Here, poly(N-isopropylacrylamide)-ss-acrylic acid (P(NIPAM-ss-AA)) nanogels based on NIPAM and AA cross-linked by N,N'-bis(acryloyl)cystamine (BAC) were constructed by precipitation polymerization. The nanogels exhibited pH/redox dual responsive doxorubicin (DOX) release behavior in vitro and in tumor cells, in which DOX release from nanogels was accelerated in lysosomal pH (pH 4.5) and cytosolic reduction (10 mM GSH) conditions. Moreover, intracellular tracking of DOX-loaded nanogels confirmed that after the nanogels and the loaded DOX entered the cells simultaneously mainly via lipid raft/caveolae-mediated endocytosis, DOX-loaded nanogels were transported to lysosomes and then the loaded DOX was released to nucleus triggered by lysosomal pH and cytoplasmic high GSH. MTT analysis showed that DOX-loaded nanogels could efficiently inhibit the proliferation of HepG2 cells. In vivo animal studies demonstrated that DOX-loaded nanogels were accumulated and penetrated in tumor tissues more efficiently than free DOX. Meanwhile, DOX-loaded nanogels exhibited stronger tumor inhibition activity and fewer side effects. This study indicated that pH/redox dual-responsive nanogels might present a prospective platform for intracellular drug controlled release in cancer therapy. PMID:26960600

  13. Changes in the redox state in the retina and brain during the onset of diabetes in rats.

    PubMed

    Salceda, R; Vilchis, C; Coffe, V; Hernández-Muñoz, R

    1998-06-01

    Diabetic retinopathy is thought to result from chronic changes in the metabolic pathways of the retina. Hyperglycemia leads to increased intracellular glucose concentrations, alterations in glucose degradation and an increase in lactate/pyruvate ratio. We measured lactate content in retina and other ocular and non-ocular tissues from normal and diabetic rats in the early stages of streptozotocin-induced diabetes. The intracellular redox state was calculated from the cytoplasmic [lactate]/[pyruvate] ratio. Elevated lactate concentration were found in retina and cerebral cortex from diabetic rats. These concentrations led to a significant and progressive decrease in the NAD+/NADH ratio, suggesting that altered glucose metabolism is an initial step of retinopathy. It is thus possible that tissues such as cerebral cortex have mechanisms that prevent the damaging effect of lactate produced by hyperglycemia and/or alterations of the intracellular redox state. PMID:9580389

  14. Engineered redox-responsive PEG detachment mechanism in PEGylated nano-graphene oxide for intracellular drug delivery.

    PubMed

    Wen, Huiyun; Dong, Chunyan; Dong, Haiqing; Shen, Aijun; Xia, Wenjuan; Cai, Xiaojun; Song, Yanyan; Li, Xuequan; Li, Yongyong; Shi, Donglu

    2012-03-12

    In biomedical applications, polyethylene glycol (PEG) functionalization has been a major approach to modify nanocarriers such as nano-graphene oxide for particular biological requirements. However, incorporation of a PEG shell poses a significant diffusion barrier that adversely affects the release of the loaded drugs. This study addresses this critical issue by employing a redox-responsive PEG detachment mechanism. A PEGylated nano-graphene oxide (NGO-SS-mPEG) with redox-responsive detachable PEG shell is developed that can rapidly release an encapsulated payload at tumor-relevant glutathione (GSH) levels. The PEG shell grafted onto NGO sheets gives the nanocomposite high physiological solubility and stability in circulation. It can selectively detach from NGO upon intracellular GSH stimulation. The surface-engineered structures are shown to accelerate the release of doxorubicin hydrochloride (DXR) from NGO-SS-mPEG 1.55 times faster than in the absence of GSH. Confocal microscopy shows clear evidence of NGO-SS-mPEG endocytosis in HeLa cells, mainly accumulated in cytoplasm. Furthermore, upon internalization of DXR-loaded NGO with a disulfide-linked PEG shell into HeLa cells, DXR is effectively released in the presence of an elevated GSH reducing environment, as observed in confocal microscopy and flow cytometric experiments. Importantly, inhibition of cell proliferation is directly correlated with increased intracellular GSH concentrations due to rapid DXR release. PMID:22228696

  15. A Low Glutathione Redox State Couples with a Decreased Ascorbate Redox Ratio to Accelerate Flowering in Oncidium Orchid.

    PubMed

    Chin, Dan-Chu; Hsieh, Chia-Chi; Lin, Hsin-Yi; Yeh, Kai-Wun

    2016-02-01

    Glutathione (GSH) plays multiple roles in plants, including stress defense and regulation of growth/development. Previous studies have demonstrated that the ascorbate (AsA) redox state is involved in flowering initiation in Oncidium orchid. In this study, we discovered that a significantly decreased GSH content and GSH redox ratio are correlated with a decline in the AsA redox state during flowering initiation and high ambient temperature-induced flowering. At the same time, the expression level and enzymatic activity of GSH redox-regulated genes, glutathione reductase (GR1), and the GSH biosynthesis genes γ-glutamylcysteine synthetase (GSH1) and glutathione synthase (GSH2), are down-regulated. Elevating dehydroascorbate (DHA) content in Oncidium by artificial addition of DHA resulted in a decreased AsA and GSH redox ratio, and enhanced dehydroascorbate reductase (DHAR) activity. This demonstrated that the lower GSH redox state could be influenced by the lower AsA redox ratio. Moreover, exogenous application of buthionine sulfoximine (BSO), to inhibit GSH biosynthesis, and glutathione disulfide (GSSG), to decrease the GSH redox ratio, also caused early flowering. However, spraying plants with GSH increased the GSH redox ratio and delayed flowering. Furthermore, transgenic Arabidopsis overexpressing Oncidium GSH1, GSH2 and GR1 displayed a high GSH redox ratio as well as delayed flowering under high ambient temperature treatment, while pad2, cad2 and gr1 mutants exhibited early flowering and a low GSH redox ratio. In conclusion, our results provide evidence that the decreased GSH redox state is linked to the decline in the AsA redox ratio and mediated by down-regulated expression of GSH metabolism-related genes to affect flowering time in Oncidium orchid. PMID:26738548

  16. Direct Determination of the Intracellular Oxidation State of Plutonium

    PubMed Central

    Gorman-Lewis, Drew; Aryal, Baikuntha P.; Paunesku, Tatjana; Vogt, Stefan; Lai, Barry; Woloschak, Gayle E.; Jensen, Mark P.

    2013-01-01

    Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1 μm2 areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 hours in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits was always consistent with tetravalent Pu even though the intracellular milieu is generally reducing. PMID:21755934

  17. Direct determination of the intracellular oxidation state of plutonium.

    PubMed

    Gorman-Lewis, Drew; Aryal, Baikuntha P; Paunesku, Tatjana; Vogt, Stefan; Lai, Barry; Woloschak, Gayle E; Jensen, Mark P

    2011-08-15

    Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1-μm(2) areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 h in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits were always consistent with tetravalent Pu even though the intracellular milieu is generally reducing. PMID:21755934

  18. Kinetics and intracellular location of intramolecular disulfide bond formation mediated by the cytoplasmic redox system encoded by vaccinia virus

    SciTech Connect

    Bisht, Himani; Brown, Erica; Moss, Bernard

    2010-03-15

    Poxviruses encode a redox system for intramolecular disulfide bond formation in cytoplasmic domains of viral proteins. Our objectives were to determine the kinetics and intracellular location of disulfide bond formation. The vaccinia virus L1 myristoylated membrane protein, used as an example, has three intramolecular disulfide bonds. Reduced and disulfide-bonded forms of L1 were distinguished by electrophoretic mobility and reactivity with monoclonal and polyclonal antibodies. Because disulfide bonds formed during 5 min pulse labeling with radioactive amino acids, a protocol was devised in which dithiothreitol was present at this step. Disulfide bond formation was detected by 2 min after removal of reducing agent and was nearly complete in 10 min. When the penultimate glycine residue was mutated to prevent myristoylation, L1 was mistargeted to the endoplasmic reticulum and disulfide bond formation failed to occur. These data suggested that viral membrane association was required for oxidation of L1, providing specificity for the process.

  19. An intracellular redox sensor for reactive oxygen species at the M3-M4 linker of GABAAρ1 receptors

    PubMed Central

    Beltrán González, Andrea N; Gasulla, Javier; Calvo, Daniel J

    2014-01-01

    Background and Purpose Reactive oxygen species (ROS) are normally involved in cell oxidative stress but also play a role as cellular messengers in redox signalling; for example, modulating the activity of neurotransmitter receptors and ion channels. However, the direct actions of ROS on GABAA receptors were not previously demonstrated. In the present work, we studied the effects of ROS on GABAAρ1 receptor function. Experimental Approach GABAAρ1 receptors were expressed in oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of ROS. Chemical protection of cysteines by selective sulfhydryl reagents and site-directed mutagenesis studies were used to identify protein residues involved in ROS actions. Key Results GABAAρ1 receptor-mediated responses were significantly enhanced in a concentration-dependent and reversible manner by H2O2. Potentiating effects were attenuated by a free radical scavenger, lipoic acid or an inhibitor of the Fenton reaction, deferoxamine. Each ρ1 subunit contains only three cysteine residues, two extracellular at the Cys-loop (C177 and C191) and one intracellular (C364) at the M3-M4 linker. Mutant GABAAρ1 receptors in which C364 was exchanged by alanine were completely insensitive to modulation, implying that this site, rather than a cysteine in the Cys-loop, is essential for ROS modulation. Conclusion and Implications Our results show that the function of GABAAρ1 receptors is enhanced by ROS and that the intracellular C364 is the sensor for ROS actions. PMID:24428763

  20. Zinc and calcium modulate mitochondrial redox state and morphofunctional integrity.

    PubMed

    Sharaf, Mahmoud S; van den Heuvel, Michael R; Stevens, Don; Kamunde, Collins

    2015-07-01

    Zinc and calcium have highly interwoven functions that are essential for cellular homeostasis. Here we first present a novel real-time flow cytometric technique to measure mitochondrial redox state and show it is modulated by zinc and calcium, individually and combined. We then assess the interactions of zinc and calcium on mitochondrial H2O2 production, membrane potential (ΔΨm), morphological status, oxidative phosphorylation (OXPHOS), complex I activity, and structural integrity. Whereas zinc at low doses and both cations at high doses individually and combined promoted H2O2 production, the two cations individually did not alter mitochondrial redox state. However, when combined at low and high doses the two cations synergistically suppressed and promoted, respectively, mitochondrial shift to a more oxidized state. Surprisingly, the antioxidants vitamin E and N-acetylcysteine showed pro-oxidant activity at low doses, whereas at high antioxidant doses NAC inhibited OXPHOS and dyscoupled mitochondria. Individually, zinc was more potent than calcium in inhibiting OXPHOS, whereas calcium more potently dissipated the ΔΨm and altered mitochondrial volume and ultrastructure. The two cations synergistically inhibited OXPHOS but antagonistically dissipated ΔΨm and altered mitochondrial volume and morphology. Overall, our study highlights the importance of zinc and calcium in mitochondrial redox regulation and functional integrity. Importantly, we uncovered previously unrecognized bidirectional interactions of zinc and calcium that reveal distinctive foci for modulating mitochondrial function in normal and disease states because they are potentially protective or damaging depending on conditions. PMID:25841782

  1. Redox state of earth's upper mantle from kimberlitic ilmenites

    NASA Technical Reports Server (NTRS)

    Haggerty, S. E.; Tompkins, L. A.

    1983-01-01

    Temperatures and oxygen fugacities are reported on discrete ilmenite nodules in kimberlites from West Africa which demonstrate that the source region in the upper mantle is moderately oxidized, consistent with other nodule suites in kimberlites from southern Africa and the United States. A model is presented for a variety of tectonic settings, proposing that the upper mantle is profiled in redox potential, oxidized in the fertile asthenosphere but reduced in the depleted lithosphere.

  2. Dietary restriction in cerebral bioenergetics and redox state.

    PubMed

    Amigo, Ignacio; Kowaltowski, Alicia J

    2014-01-01

    The brain has a central role in the regulation of energy stability of the organism. It is the organ with the highest energetic demands, the most susceptible to energy deficits, and is responsible for coordinating behavioral and physiological responses related to food foraging and intake. Dietary interventions have been shown to be a very effective means to extend lifespan and delay the appearance of age-related pathological conditions, notably those associated with brain functional decline. The present review focuses on the effects of these interventions on brain metabolism and cerebral redox state, and summarizes the current literature dealing with dietary interventions on brain pathology. PMID:24563846

  3. Establishment of a system for monitoring endoplasmic reticulum redox state in mammalian cells

    PubMed Central

    Kanekura, Kohsuke; Ishigaki, Shinsuke; Merksamer, Philip I.; Papa, Feroz R.; Urano, Fumihiko

    2014-01-01

    The endoplasmic reticulum (ER) performs a critical role in the oxidative folding of nascent proteins such that perturbations to ER homeostasis may lead to protein misfolding and subsequent pathological processes. Among the mechanisms for maintaining ER homeostasis is a redox regulation, which is a critical determinant of the fate of ER stressed cells. Here we report the establishment of a system for monitoring ER redox state in mammalian cells. The new ER redox sensing system was developed based on the previously described monitoring system in yeast. Our system could successfully monitor the dynamic ER redox state in mammalian cells. Using this system, we find that manipulation of ER oxidases changes ER redox state. The mammalian ER redox sensing system could be used to study the mechanisms of ER redox regulation and provide a foundation for an approach to develop novel therapeutic modalities for human diseases related to dysregulated ER homeostasis including diabetes, neurodegeneration and Wolfram syndrome. PMID:24042438

  4. Chasing stress signals - Exposure to extracellular stimuli differentially affects the redox state of cell compartments in the wild type and signaling mutants of Botrytis cinerea.

    PubMed

    Marschall, Robert; Schumacher, Julia; Siegmund, Ulrike; Tudzynski, Paul

    2016-05-01

    Reactive oxygen species (ROS) are important molecules influencing intracellular developmental processes as well as plant pathogen interactions. They are produced at the infection site and affect the intracellular redox homeostasis. However, knowledge of ROS signaling pathways, their connection to other signaling cascades, and tools for the visualization of intra- and extracellular ROS levels and their impact on the redox state are scarce. By using the genetically encoded biosensor roGFP2 we studied for the first time the differences between the redox states of the cytosol, the intermembrane space of mitochondria and the ER in the filamentous fungus Botrytis cinerea. We showed that the ratio of oxidized to reduced glutathione inside of the cellular compartments differ and that the addition of hydrogen peroxide (H2O2), calcium chloride (CaCl2) and the fluorescent dye calcofluor white (CFW) have a direct impact on the cellular redox states. Dependent on the type of stress agents applied, the redox states were affected in the different cellular compartments in a temporally shifted manner. By integrating the biosensor in deletion mutants of bcnoxA, bcnoxB, bctrx1 and bcltf1 we further elucidated the putative roles of the different proteins in distinct stress-response pathways. We showed that the redox states of ΔbcnoxA and ΔbcnoxB display a wild-type pattern upon exposure to H2O2, but appear to be strongly affected by CaCl2 and CFW. Moreover, we demonstrated the involvement of the light-responsive transcription factor BcLtf1 in the maintenance of the redox state in the intermembrane space of the mitochondria. Finally, we report that CaCl2 as well as cell wall stress-inducing agents stimulate ROS production and that ΔbcnoxB produces significantly less ROS than the wild type and ΔbcnoxA. PMID:26988904

  5. Dual stimulus of hyperthermia and intracellular redox environment triggered release of siRNA for tumor-specific therapy.

    PubMed

    Yang, Yanfang; Yang, Yang; Xie, Xiangyang; Xu, Xueqing; Xia, Xuejun; Wang, Hongliang; Li, Lin; Dong, Wujun; Ma, Panpan; Liu, Yuling

    2016-06-15

    Small interfering RNA (siRNA) offers a new and potential therapeutic strategy for tackling many diseases at the molecular level. Recently, cell-penetrating peptides (CPPs) conjugated with siRNA via disulfide-bonds (designated as siRNA-CPPs) were reported to form glutathione-sensitive carriers. However, non-cell specificity, CPPs degradation and the unwanted reduction of siRNA-CPPs before reaching the targeted tissue in vivo hampered the development of siRNA-CPPs. Herein, utilizing the dual stimulus of hyperthermia and the intracellular redox environment, we devised a thermosensitive liposome (TSL) containing an Asparagine-Glycine-Arginine (NGR) peptide and reducible siRNA-CPPs for tumor-specific siRNA transfection (siRNA-CPPs/NGR-TSL), in which siRNA-CPPs were "caged" in NGR-TSL to overcome their limitations in vivo. The functional nanocarrier possessed a small particle size of approximately 90nm, a high drug encapsulation efficiency of approximately 86% and good serum stability. Both free siRNA-CPPs and siRNA-CPPs/NGR-TSL (preheated) silenced c-myc in human fibrosarcoma (HT-1080) cells in vitro. However, in an HT-1080 xenograft murine model, siRNA-CPPs/NGR-TSL with hyperthermia displayed superior in vivo antitumor efficacy (about 3-fold) and gene silencing efficiency (about 2-fold) compared with free siRNA-CPPs under hyperthermia. This study demonstrates that the constructed vesicle in combination with hyperthermia could greatly improve the in vivo stability of siRNA-CPPs and synergistically enhance its cancer therapy efficiency. PMID:27106526

  6. Redox states of underground brine system along the southern coast of the Laizhou Bay

    NASA Astrophysics Data System (ADS)

    Jiang, Xueyan; Yu, Zhigang; Ning, Jinsong; Chen, Hongtao; Mi, Tiezhu

    2008-05-01

    Underground brine samples were collected along the southern coast of the Laizhou Bay, Shangdong, China in two field investigations in 2003. The brines are confined in the Quaternary sediment and underwent a series of geochemical changes. The redox states of these brines were assessed qualitatively based on the measurements of Eh and redox-sensitive species such as DO, NO NO{3/-}, Mn2+, Fe2+, SO{4/2-} in the brines. The redox condition of the underground brine is anoxic, and the redox reactions that controlled the redox potential of brines should be Fe(III) reduction and sulfate reduction.

  7. Multiple redox states of multiheme cytochromes may enable bacterial response to changing redox environments

    NASA Astrophysics Data System (ADS)

    Arbour, T.; Wrighton, K. C.; Mullin, S. W.; Castelle, C.; Luef, B.; Gilbert, B.; Banfield, J. F.

    2013-12-01

    Multiheme c-type cytochromes (MHCs) are key components in electron-transport pathways that enable some microorganisms to transfer electron byproducts of metabolism to a variety of minerals. As a response to changes in mineral redox potential, microbial communities may shift their membership, or individual organisms may adjust protein expression. Alternatively, the ability to respond may be conferred by the innate characteristics of certain electron-transport-chain components. Here, we used potentiostat-controlled microbial fuel cells (MFCs) to measure the timescale of response to imposed changes in redox conditions, thus placing constraints on the importance of these different mechanisms. In the experiments, a solid electrode acts as an electron-accepting mineral whose redox potential can be precisely controlled. We inoculated duplicate MFCs with a sediment/groundwater mixture from an aquifer at Rifle, Colorado, supplied acetate as an electron donor, and obtained stable, mixed-species biofilms dominated by Geobacter and a novel Geobacter-related family. We poised the anode at potentials spanning the range of natural Fe(III)-reduction, then performed cyclic voltammetry (CV) to characterize the overall biofilm redox signature. The apparent biofilm midpoint potential shifted directly with anode set potential when the latter was changed within the range from about -250 to -50 mV vs. SHE. Following a jump in set potential by 200 mV, the CV-midpoint shift by ~100 mV over a timescale of ~30 minutes to a few hours, depending on the direction of the potential change. The extracellular electron transfer molecules, whose overall CV signature is very similar to those of purified MHCs, appear to span a broad redox range (~200 mV), supporting the hypothesis that MHCs confer substantial redox flexibility. This flexibility may be a principle reason for the abundance of MHCs expressed by microorganisms capable of extracellular electron transfer to minerals.

  8. Feasibility of assessing health state by detecting redox state of human body based on Chinese medicine constitution.

    PubMed

    Li, Ling-Ru; Wang, Qi; Wang, Ji; Wang, Qian-Fei; Yang, Ling-Ling; Zheng, Lu-Yu; Zhang, Yan

    2016-08-01

    This article discussed the feasibility of assessing health state by detecting redox state of human body. Firstly, the balance of redox state is the basis of homeostasis, and the balance ability of redox can reflflect health state of human body. Secondly, the redox state of human body is a sensitive index of multiple risk factors of health such as age, external environment and psychological factors. It participates in the occurrence and development of multiple diseases involving metabolic diseases and nervous system diseases, and can serve as a cut-in point for treatment of these diseases. Detecting the redox state of high risk people is signifificantly important for early detection and treatment of disease. The blood plasma and urine could be selected to detect, which is convenient. It is pointed that the indexes not only involve oxidation product and antioxidant enzyme but also redox couple. Chinese medicine constitution reflflects the state of body itself and the ability of adapting to external environment, which is consistent with the connotation of health. It is found that there are nine basic types of constitution in Chinese population, which provides a theoretical basis of health preservation, preventive treatment of disease and personalized treatment. With the combination of redox state detection and the Chinese medicine constitution theory, the heath state can be systemically assessed by conducting large-scale epidemiological survey with classifified detection on redox state of human body. PMID:26712210

  9. Exogenous antioxidants—Double-edged swords in cellular redox state

    PubMed Central

    Bohn, Torsten

    2010-01-01

    The balance between oxidation and antioxidation is believed to be critical in maintaining healthy biological systems. Under physiological conditions, the human antioxidative defense system including e.g., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH) and others, allows the elimination of excess reactive oxygen species (ROS) including, among others superoxide anions (O2.-), hydroxyl radicals (OH.), alkoxyl radicals (RO.) and peroxyradicals (ROO.). However, our endogenous antioxidant defense systems are incomplete without exogenous originating reducing compounds such as vitamin C, vitamin E, carotenoids and polyphenols, playing an essential role in many antioxidant mechanisms in living organisms. Therefore, there is continuous demand for exogenous antioxidants in order to prevent oxidative stress, representing a disequilibrium redox state in favor of oxidation. However, high doses of isolated compounds may be toxic, owing to prooxidative effects at high concentrations or their potential to react with beneficial concentrations of ROS normally present at physiological conditions that are required for optimal cellular functioning. This review aims to examine the double-edged effects of dietary originating antioxidants with a focus on the most abundant compounds, especially polyphenols, vitamin C, vitamin E and carotenoids. Different approaches to enrich our body with exogenous antioxidants such as via synthetic antioxidants, diets rich in fruits and vegetables and taking supplements will be reviewed and experimental and epidemiological evidences discussed, highlighting that antioxidants at physiological doses are generally safe, exhibiting interesting health beneficial effects. PMID:20972369

  10. An evaluation of the redox state in professional scuba divers.

    PubMed

    Radojevic-Popovic, Radmila; Zivkovic, Vladimir; Jeremic, Nevena; Sretenovic, Jasmina; Velicanin, Nevena; Bradic, Jovana; Jakovljevic, Vladimir

    2015-01-01

    Taking into consideration limited data regarding molecular interactions during and after diving, this investigation was intended to determine the oxidative status of divers before and after scuba diving by monitoring the oxidative status parameters. The prevalence study included a group of 32 male professional police scuba divers, 32 ± 5.1 years old. The examination took place twice: in a resting state before scuba diving and immediately after the dive (to 30 meters for 30 minutes). The oxidative status of the scuba divers was determined by measuring levels of the following oxidative stress markers: the index of lipid peroxidation (measured as TBARS), nitrites (NO2-), superoxide anion radical (O2*-), hydrogen peroxide (H2O2), superoxide dismutase (SOD) and catalase (CAT). Statistically significant increases in levels of NO2- and TBARS were observed after the dive, while there were no statistically relevant changes in levels of O2*-, H2O2, SOD and CAT. Our results have shown that a dive with these characteristics only slightly disturbs redox homeostasis, without serious intermolecular changes that can lead to prominent oxidative stress. PMID:26591980

  11. Redox state of plutonium in irradiated mixed oxide fuels

    NASA Astrophysics Data System (ADS)

    Degueldre, C.; Pin, S.; Poonoosamy, J.; Kulik, D. A.

    2014-03-01

    Nowadays, MOX fuels are used in about 20 nuclear power plants around the world. After irradiation, plutonium co-exists with uranium oxide. Due to the redox sensitive nature of UO2 other plutonium oxides than PuO2 potentially present in the fuel may interact with the matrix. The aim of this study is to determine which plutonium species are present in heterogeneous and homogeneous MOX. The results provided by X-ray Absorption Near Edge Spectroscopy (XANES) for non-irradiated as well as irradiated (center and periphery) homogeneous MOX fuel were published earlier and are completed by Extended X-ray Fine Structure (EXAFS) analysis in this work. The EXAFS signals have been extracted using the ATHENA code and the analyses were carried using EXCURE98 as performed earlier for an analogous element. EXAFS shows that plutonium redox state remains tetravalent in the solid solution and that the minor fraction of trivalent Pu must be below 10%. Independently, the study of homogeneous MOX was also approached by thermodynamics of solid solution of (U,Pu)O2. Such solid solutions were modeled using the Gibbs Energy Minimisation (GEM)-Selektor code (developed at LES, NES, PSI) supported by the literature data on such solid solutions. A comparative study was performed showing which plutonium oxides in their respective mole fractions are more likely to occur in (U,Pu)O2. In the modeling, these oxides were set as ideal and non-ideal solid solutions, as well as separate pure phases. Pu exists mainly as PuO2 in the case of separate phases, but can exist under its reduced forms, PuO1.61 and PuO1.5 in minor fraction i.e. ~15% in ideal solid solution (unlikely) and ~10% in non-ideal solid solution (likely) and at temperature around 1300 K. This combined thermodynamic and EXAFS studies confirm independently the results obtained so far by Pu XANES for the same MOX samples.

  12. Integrated Stress Response Modulates Cellular Redox State via Induction of Cystathionine γ-Lyase

    PubMed Central

    Dickhout, Jeffrey G.; Carlisle, Rachel E.; Jerome, Danielle E.; Mohammed-Ali, Zahraa; Jiang, Hua; Yang, Guangdong; Mani, Sarathi; Garg, Sanjay K.; Banerjee, Ruma; Kaufman, Randal J.; Maclean, Kenneth N.; Wang, Rui; Austin, Richard C.

    2012-01-01

    The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine β-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4−/−) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4−/− MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4−/− MEFs could be rescued from l-Hcy-induced apoptosis by β-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4−/− MEFs showed little or no CSE protein but did express cystathionine β-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4+/+ MEFs. Consistent with ATF4−/− MEFs, CSE−/− MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE+/+ MEFs. Liver and kidney GSH levels were also reduced in CSE−/− mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress. PMID:22215680

  13. Mantle redox evolution and the oxidation state of the Archean atmosphere

    NASA Technical Reports Server (NTRS)

    Kasting, J. F.; Eggler, D. H.; Raeburn, S. P.

    1993-01-01

    Current models predict that the early atmosphere consisted mostly of CO2, N2, and H2O, along with traces of H2 and CO. Such models are based on the assumption that the redox state of the upper mantle has not changed, so that volcanic gas composition has remained approximately constant with time. We argue here that this assumption is probably incorrect: the upper mantle was originally more reduced than today, although not as reduced as the metal arrest level, and has become progressively more oxidized as a consequence of the release of reduced volcanic gases and the subduction of hydrated, oxidized seafloor. Data on the redox state of sulfide and chromite inclusions in diamonds imply that the process of mantle oxidation was slow, so that reduced conditions could have prevailed for as much as half of the earth's history. To be sure, other oxybarometers of ancient rocks give different results, so the question of when the mantle redox state has changed remains unresolved. Mantle redox evolution is intimately linked to the oxidation state of the primitive atmosphere: A reduced Archean atmosphere would have had a high hydrogen escape rate and should correspond to a changing mantle redox state; an oxidized Archean atmosphere should be associated with a constant mantle redox state. The converses of these statements are also true. Finally, our theory of mantle redox evolution may explain why the Archean atmosphere remained oxygen-deficient until approximately 2.0 billion years ago (Ga) despite a probable early origin for photosynthesis.

  14. Intracellular Delivery: Redox-Triggered Release of Moxifloxacin from Mesoporous Silica Nanoparticles Functionalized with Disulfide Snap-Tops Enhances Efficacy Against Pneumonic Tularemia in Mice (Small 27/2016).

    PubMed

    Lee, Bai-Yu; Li, Zilu; Clemens, Daniel L; Dillon, Barbara Jane; Hwang, Angela A; Zink, Jeffrey I; Horwitz, Marcus A

    2016-07-01

    The drug trapping and intracellular release mechanism of redox-responsive disulfide snap-top mesoporous silica nanoparticles (MSN-SS-MXF) is depicted by J. I. Zink, M. A. Horwitz and co-workers on page 3690. Mesoporous silica nanoparticles with antibiotic (cyan) trapped within their pores by disulfide snap-tops are avidly ingested by macrophages. The intracellular redox potential reduces the disulfide (yellow) in the stalk (green/blue), releases the caps (orange) and frees drug to kill Francisella tularensis (green). Artwork by Bastian Ruehle. PMID:27412305

  15. Noninvasive optical cytochrome c oxidase redox state measurements using diffuse optical spectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, Jangwoen; Kim, Jae G.; Mahon, Sari B.; Mukai, David; Yoon, David; Boss, Gerry R.; Patterson, Steven E.; Rockwood, Gary; Isom, Gary; Brenner, Matthew

    2014-05-01

    A major need exists for methods to assess organ oxidative metabolic states in vivo. By contrasting the responses to cyanide (CN) poisoning versus hemorrhage in animal models, we demonstrate that diffuse optical spectroscopy (DOS) can detect cytochrome c oxidase (CcO) redox states. Intermittent decreases in inspired O2 from 100% to 21% were applied before, during, and after CN poisoning, hemorrhage, and resuscitation in rabbits. Continuous DOS measurements of total hemoglobin, oxyhemoglobin, deoxyhemoglobin, and oxidized and reduced CcO from muscle were obtained. Rabbit hemorrhage was accomplished with stepwise removal of blood, followed by blood resuscitation. CN treated rabbits received 0.166 mg/min NaCN infusion. During hemorrhage, CcO redox state became reduced concurrently with decreases in oxyhemoglobin, resulting from reduced tissue oxygen delivery and hypoxia. In contrast, during CN infusion, CcO redox state decreased while oxyhemoglobin concentration increased due to CN binding and reduction of CcO with resultant inhibition of the electron transport chain. Spectral absorption similarities between hemoglobin and CcO make noninvasive spectroscopic distinction of CcO redox states difficult. By contrasting physiological perturbations of CN poisoning versus hemorrhage, we demonstrate that DOS measured CcO redox state changes are decoupled from hemoglobin concentration measurement changes.

  16. Noninvasive optical cytochrome c oxidase redox state measurements using diffuse optical spectroscopy

    PubMed Central

    Lee, Jangwoen; Kim, Jae G.; Mahon, Sari B.; Mukai, David; Yoon, David; Boss, Gerry R.; Patterson, Steven E.; Rockwood, Gary; Isom, Gary; Brenner, Matthew

    2014-01-01

    Abstract. A major need exists for methods to assess organ oxidative metabolic states in vivo. By contrasting the responses to cyanide (CN) poisoning versus hemorrhage in animal models, we demonstrate that diffuse optical spectroscopy (DOS) can detect cytochrome c oxidase (CcO) redox states. Intermittent decreases in inspired O2 from 100% to 21% were applied before, during, and after CN poisoning, hemorrhage, and resuscitation in rabbits. Continuous DOS measurements of total hemoglobin, oxyhemoglobin, deoxyhemoglobin, and oxidized and reduced CcO from muscle were obtained. Rabbit hemorrhage was accomplished with stepwise removal of blood, followed by blood resuscitation. CN treated rabbits received 0.166  mg/min NaCN infusion. During hemorrhage, CcO redox state became reduced concurrently with decreases in oxyhemoglobin, resulting from reduced tissue oxygen delivery and hypoxia. In contrast, during CN infusion, CcO redox state decreased while oxyhemoglobin concentration increased due to CN binding and reduction of CcO with resultant inhibition of the electron transport chain. Spectral absorption similarities between hemoglobin and CcO make noninvasive spectroscopic distinction of CcO redox states difficult. By contrasting physiological perturbations of CN poisoning versus hemorrhage, we demonstrate that DOS measured CcO redox state changes are decoupled from hemoglobin concentration measurement changes. PMID:24788369

  17. Quantum-dot/dopamine bioconjugates function as redox coupled assemblies for in vitro and intracellular pH sensing

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Stewart, Michael H.; Trammell, Scott A.; Susumu, Kimihiro; Delehanty, James B.; Mei, Bing C.; Melinger, Joseph S.; Blanco-Canosa, Juan B.; Dawson, Philip E.; Mattoussi, Hedi

    2010-08-01

    The use of semiconductor quantum dots (QDs) for bioimaging and sensing has progressively matured over the past decade. QDs are highly sensitive to charge-transfer processes, which can alter their optical properties. Here, we demonstrate that QD-dopamine-peptide bioconjugates can function as charge-transfer coupled pH sensors. Dopamine is normally characterized by two intrinsic redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O2 at basic pH. We show that the latter quinone can function as an electron acceptor quenching QD photoluminescence in a manner that depends directly on pH. We characterize the pH-dependent QD quenching using both electrochemistry and spectroscopy. QD-dopamine conjugates were also used as pH sensors that measured changes in cytoplasmic pH as cells underwent drug-induced alkalosis. A detailed mechanism describing the QD quenching processes that is consistent with dopamine's inherent redox chemistry is presented.

  18. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State.

    PubMed

    Folda, Alessandra; Citta, Anna; Scalcon, Valeria; Calì, Tito; Zonta, Francesco; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

    2016-01-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release. PMID:26975474

  19. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State

    NASA Astrophysics Data System (ADS)

    Folda, Alessandra; Citta, Anna; Scalcon, Valeria; Calì, Tito; Zonta, Francesco; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

    2016-03-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release.

  20. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State

    PubMed Central

    Folda, Alessandra; Citta, Anna; Scalcon, Valeria; Calì, Tito; Zonta, Francesco; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

    2016-01-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release. PMID:26975474

  1. New redox states observed in [FeFe] hydrogenases reveal redox coupling within the H-cluster.

    PubMed

    Adamska-Venkatesh, Agnieszka; Krawietz, Danuta; Siebel, Judith; Weber, Katharina; Happe, Thomas; Reijerse, Edward; Lubitz, Wolfgang

    2014-08-13

    Active [FeFe] hydrogenases can be obtained by expressing the unmaturated enzyme in Escherichia coli followed by incubation with a synthetic precursor of the binuclear [2Fe] subcluster, namely: [NEt4]2[Fe2(adt)(CO)4(CN)2] (adt = [S-CH2-NH-CH2-S](2-)). The binuclear subsite Fe2(adt)(CO)3(CN)2 is attached through a bridging cysteine side chain to a [4Fe-4S] subcluster already present in the unmaturated enzyme thus yielding the intact native "H-cluster". We present FTIR electrochemical studies of the [FeFe] hydrogenase from Chlamydomonas reinhardtii, CrHydA1, maturated with the precursor of the native cofactor [Fe2(adt)(CO)4(CN)2](2-) as well as a non-natural variant [Fe2(pdt)(CO)4(CN)2](2-) in which the bridging amine functionality is replaced by CH2. The obtained active enzyme CrHydA1(adt) shows the same redox states in the respective potential range as observed for the native system (E(ox/red) = -400 mV, E(red/sred) = -470 mV). For the Hox → Hred transition the reducing equivalent is stored on the binuclear part, ([4Fe-4S](2+)Fe(II)Fe(I) → [4Fe-4S](2+)Fe(I)Fe(I)), while the Hred → Hsred transition is characterized by a reduction of the [4Fe-4S] part of the H-cluster ([4Fe-4S](2+)Fe(I)Fe(I) → [4Fe-4S](+)Fe(I)Fe(I)). A similar transition is reported here for the CO inhibited state of the H-cluster: ([4Fe-4S](2+)Fe(I)Fe(II)CO → [4Fe-4S](+)Fe(I)Fe(II)CO). An FTIR electrochemical study of the inactive variant with the pdt ligand, CrHydA1(pdt), identified two redox states H(pdt)-ox and H(pdt)-"red". Both EPR and FTIR spectra of H(pdt)-ox are virtually identical to those of the H(adt)-ox and the native Hox state. The H(pdt)-"red" state is also characterized by a reduced [4Fe-4S] subcluster. In contrast to CrHydA1(adt), the H(pdt)-ox state of CrHydA1(pdt) is stable up to rather high potentials (+200 mV). This study demonstrates the distinct redox coupling between the two parts of the H-cluster and confirms that the [4Fe-4S]H subsite is also redox active and as

  2. Extrinsic periodic information interpolates between monostable and bistable states in intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Lin, Ling; Duan, Wei-Long

    2015-06-01

    Extrinsic periodic information including physiological cyclical and circadian replacement would affect inevitably a real cell, in this paper we investigate the effect of extrinsic periodic information on intracellular calcium dynamics by means of second-order algorithm for stochastic simulation colored noises. By simulating time evolutions and stationary probability distribution of intracellular Ca2+ concentrations, the results show: (i) intracellular calcium oscillation between cytosol and calcium store shows synchronous and anti-synchronous oscillation as intensity and frequency of extrinsic periodic information vary; (ii) extrinsic periodic information interpolates stability from bistable state → monostable state → bistable state → monostable state as frequency of extrinsic periodic information increases; (iii) extrinsic periodic information interpolates stability from monostable state → bistable state as intensity of extrinsic periodic information increases.

  3. Measuring the redox state of cellular peroxiredoxins by immunoblotting.

    PubMed

    Cox, Andrew G; Winterbourn, Christine C; Hampton, Mark B

    2010-01-01

    The peroxiredoxins (Prxs) are a family of thiol peroxidases that scavenge hydroperoxides and peroxynitrite. The abundance and reactivity of these proteins makes them primary targets for cellular H(2)O(2). The catalytic cycle of typical 2-Cys Prxs involves formation of an intermolecular disulfide bond between peroxidatic and resolving cysteines on opposing subunits. Rapid alterations in the ratio of reduced monomer and oxidized dimer have been detected in the cytoplasm and mitochondria of cultured cells exposed to various exogenous and endogenous sources of oxidative stress. Here we describe immunoblot methods to monitor the interconversion of individual 2-Cys Prxs in cultured cells. We also outline an adaptation of this method to measure the extent to which individual 2-Cys Prxs become hyper oxidized in treated cells. Together, these methods enable the redox status of cellular Prxs to be assessed and quantified in a rapid and robust manner. PMID:20609904

  4. Intermittent fasting results in tissue-specific changes in bioenergetics and redox state.

    PubMed

    Chausse, Bruno; Vieira-Lara, Marcel A; Sanchez, Angélica B; Medeiros, Marisa H G; Kowaltowski, Alicia J

    2015-01-01

    Intermittent fasting (IF) is a dietary intervention often used as an alternative to caloric restriction (CR) and characterized by 24 hour cycles alternating ad libitum feeding and fasting. Although the consequences of CR are well studied, the effects of IF on redox status are not. Here, we address the effects of IF on redox state markers in different tissues in order to uncover how changes in feeding frequency alter redox balance in rats. IF rats displayed lower body mass due to decreased energy conversion efficiency. Livers in IF rats presented increased mitochondrial respiratory capacity and enhanced levels of protein carbonyls. Surprisingly, IF animals also presented an increase in oxidative damage in the brain that was not related to changes in mitochondrial bioenergetics. Conversely, IF promoted a substantial protection against oxidative damage in the heart. No difference in mitochondrial bioenergetics or redox homeostasis was observed in skeletal muscles of IF animals. Overall, IF affects redox balance in a tissue-specific manner, leading to redox imbalance in the liver and brain and protection against oxidative damage in the heart. PMID:25749501

  5. Redox biology of tuberculosis pathogenesis.

    PubMed

    Trivedi, Abhishek; Singh, Nisha; Bhat, Shabir Ahmed; Gupta, Pawan; Kumar, Ashwani

    2012-01-01

    Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens. Mtb is persistently exposed to numerous oxidoreductive stresses during its pathogenic cycle of infection and transmission. The distinctive ability of Mtb, not only to survive the redox stress manifested by the host but also to use it for synchronizing the metabolic pathways and expression of virulence factors, is central to its success as a pathogen. This review describes the paradigmatic redox and hypoxia sensors employed by Mtb to continuously monitor variations in the intracellular redox state and the surrounding microenvironment. Two component proteins, namely, DosS and DosT, are employed by Mtb to sense changes in oxygen, nitric oxide, and carbon monoxide levels, while WhiB3 and anti-sigma factor RsrA are used to monitor changes in intracellular redox state. Using these and other unidentified redox sensors, Mtb orchestrates its metabolic pathways to survive in nutrient-deficient, acidic, oxidative, nitrosative, and hypoxic environments inside granulomas or infectious lesions. A number of these metabolic pathways are unique to mycobacteria and thus represent potential drug targets. In addition, Mtb employs versatile machinery of the mycothiol and thioredoxin systems to ensure a reductive intracellular environment for optimal functioning of its proteins even upon exposure to oxidative stress. Mtb also utilizes a battery of protective enzymes, such as superoxide dismutase (SOD), catalase (KatG), alkyl hydroperoxidase (AhpC), and peroxiredoxins, to neutralize the redox stress generated by the host immune system. This chapter reviews the current understanding of mechanisms employed by Mtb to sense and neutralize redox stress and their importance in TB pathogenesis and drug development. PMID:22633061

  6. Iron isotope constraints on the Archean and Paleoproterozoic ocean redox state.

    PubMed

    Rouxel, Olivier J; Bekker, Andrey; Edwards, Katrina J

    2005-02-18

    The response of the ocean redox state to the rise of atmospheric oxygen about 2.3 billion years ago (Ga) is a matter of controversy. Here we provide iron isotope evidence that the change in the ocean iron cycle occurred at the same time as the change in the atmospheric redox state. Variable and negative iron isotope values in pyrites older than about 2.3 Ga suggest that an iron-rich global ocean was strongly affected by the deposition of iron oxides. Between 2.3 and 1.8 Ga, positive iron isotope values of pyrite likely reflect an increase in the precipitation of iron sulfides relative to iron oxides in a redox stratified ocean. PMID:15718467

  7. Mapping the Redox State of CHOP-Treated Non-Hodgkin’s Lymphoma Xenografts in Mice

    PubMed Central

    Xu, He N.; Mir, Tahreem A.; Lee, Seung-Cheol; Feng, Min; Farhad, Namisa; Choe, Regine; Glickson, Jerry D.; Li, Lin Z.

    2015-01-01

    Drug treatment may alter the metabolism of cancer cells and may alter the mitochondrial redox state. Using the redox scanner that collects the fluorescence signals from both the oxidized flavoproteins (Fp) and the reduced form of nicotin-amide adenine dinucleotide (NADH) in snap-frozen tumor tissues, we investigated the effects of chemotherapy on mouse xenografts of a human diffuse large B-cell lymphoma cell line (DLCL2). The mice in the treatment group were treated with CHOP – cyclophosphamide (C) + hydroxydoxorubicin (H) + Oncovin (O) + prednisone (P) using the following regimen: CHO administration on day 1 followed by prednisone administration on day 1–5. On day 5 the mitochondrial redox state of the treated group was slightly more reduced than that of the control group (p = 0.049), and the Fp content of the treated group was significantly decreased (p = 0.033). PMID:23852501

  8. Fluorescence spectroscopy and cryoimaging of rat lung tissue mitochondrial redox state

    NASA Astrophysics Data System (ADS)

    Sepehr, R.; Audi, S.; Staniszewski, K.; Maleki, S.; Ranji, M.

    2011-07-01

    The objective of this study was to demonstrate the utility of optical cryoimaging and fluorometry to evaluate tissue redox state of the mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavin Adenine Dinucleotide) in intact rat lungs. The ratio (NADH/FAD), referred to as mitochondrial redox ratio (RR), is a measure of the lung tissue mitochondrial redox state. Isolated rat lungs were connected to a ventilation-perfused system. Surface NADH and FAD fluorescence signals were acquired before and after lung perfusion in the absence (control perfusate) or presence of potassium cyanide (KCN, complex IV inhibitor) to reduce the mitochondrial respiratory chain (state 5 respiration). Another group of lungs were perfused with control perfusate or KCN-containing perfusate as above, after which the lungs were deflated and frozen rapidly for subsequent 3D cryoimaging. Results demonstrate that lung treatment with KCN increased lung surface NADH signal by 22%, decreased FAD signal by 8%, and as result increased RR by 31% as compared to control perfusate (baseline) values. Cryoimaging results also show that KCN increased mean lung tissue NADH signal by 37%, decreased mean FAD signal by 4%, and increased mean RR by 47%. These results demonstrate the utility of these optical techniques to evaluate the effect of pulmonary oxidative stress on tissue mitochondrial redox state in intact lungs.

  9. Redox states of Desulfovibrio vulgaris DsrC, a key protein in dissimilatory sulfite reduction

    SciTech Connect

    Venceslau, Sofia S.; Cort, John R.; Baker, Erin S.; Chu, Rosalie K.; Robinson, Errol W.; Dahl, Christiane; Saraiva, Lígia M.; Pereira, Inês A.C.

    2013-11-29

    Highlights: •DsrC is known to interact with the dissimilatory sulfite reductase enzyme (DsrAB). •We show that, however, most cellular DsrC is not associated with DsrAB. •A gel-shift assay was developed that allows monitoring of the DsrC redox state. •The DsrC intramolecularly oxidized state could only be produced by arginine treatment. -- Abstract: Dissimilatory reduction of sulfite is carried out by the siroheme enzyme DsrAB, with the involvement of the protein DsrC, which has two conserved redox-active cysteines. DsrC was initially believed to be a third subunit of DsrAB. Here, we report a study of the distribution of DsrC in cell extracts to show that, in the model sulfate reducer Desulfovibrio vulgaris, the majority of DsrC is not associated with DsrAB and is thus free to interact with other proteins. In addition, we developed a cysteine-labelling gel-shift assay to monitor the DsrC redox state and behaviour, and procedures to produce the different redox forms. The oxidized state of DsrC with an intramolecular disulfide bond, which is proposed to be a key metabolic intermediate, could be successfully produced for the first time by treatment with arginine.

  10. Ascorbate distribution during hibernation is independent of ascorbate redox state.

    PubMed

    Ma, Yi Long; Rice, Margaret E; Chao, Mei Lan; Rivera, Patricia M; Zhao, Huiwen W; Ross, Austin P; Zhu, Xiongwei; Smith, Mark A; Drew, Kelly L

    2004-08-15

    Distribution of ascorbate into tissues is an essential process in ascorbate antioxidant defense. Hibernating animals are studied as a model of tolerance to ischemia-reperfusion because of their tolerance to fluctuations in blood flow associated with prolonged torpor and periodic arousal episodes. Throughout hibernation, plasma ascorbate concentration ([Asc](p)) repetitively increases during torpor, then falls during periodic arousal bouts. We previously proposed that high [Asc](p) provides a ready source of antioxidant protection for distribution to the central nervous system and peripheral tissues during arousal. Here we tested whether deliberate oxidation of plasma ascorbate by intravenous administration of ascorbate oxidase (AO), prior to arousal, compromised tissue levels of ascorbate or the other water-soluble antioxidants, glutathione (GSH) and urate. Although AO decreased [Asc](p) to below the level of detection during torpor and after arousal, ascorbate oxidation did not decrease post-arousal tissue levels of reduced ascorbate, glutathione, or urate in any tissue examined, except liver. The data imply that ascorbate is taken up equally well into brain and other tissues as either ascorbate or its oxidized product dehydroascorbate, with subsequent intracellular reduction of dehydroascorbate. Lack of effect of ascorbate oxidation on tissue levels of GSH or urate indicates that dehydroascorbate uptake and reduction do not compromise tissue concentrations of these other water-soluble antioxidants. Thus, we show equal availability of reduced and oxidized plasma ascorbate during metabolically demanding thermogenesis and reperfusion associated with arousal from hibernation. PMID:15256222

  11. Redox State of Cytochromes in Frozen Yeast Cells Probed by Resonance Raman Spectroscopy.

    PubMed

    Okotrub, Konstantin A; Surovtsev, Nikolay V

    2015-12-01

    Cryopreservation is a well-established technique used for the long-term storage of biological materials whose biological activity is effectively stopped under low temperatures (suspended animation). Since most biological methods do not work in a low-temperature frozen environment, the mechanism and details of the depression of cellular activity in the frozen state remain largely uncharacterized. In this work, we propose, to our knowledge, a new approach to study the downregulation of the redox activity of cytochromes b and c in freezing yeast cells in a contactless, label-free manner. Our approach is based on cytochrome photobleaching effects observed in the resonance Raman spectra of live cells. Photoinduced and native redox reactions that contributed to the photobleaching rate were studied over a wide temperature range (from -173 to +25 °C). We found that ice formation influences both the rate of cytochrome redox reactions and the balance between the reduced and oxidized cytochromes. We demonstrate that the temperature dependence of native redox reaction rates can be well described by the thermal activation law with an apparent energy of 32.5 kJ/mol, showing that the redox reaction rate is ∼10(15) times slower at liquid nitrogen temperature than at room temperature. PMID:26636934

  12. Intracellular Redox State as Target for Anti-Influenza Therapy: Are Antioxidants Always Effective?

    PubMed Central

    Sgarbanti, Rossella; Amatore, Donatella; Celestino, Ignacio; Marcocci, Maria Elena; Fraternale, Alessandra; Ciriolo, Maria Rosa; Magnani, Mauro; Saladino, Raffaele; Garaci, Enrico; Palamara, Anna Teresa; Nencioni, Lucia

    2014-01-01

    Influenza virus infections represent a big issue for public health since effective treatments are still lacking. In particular, the emergence of strains resistant to drugs limits the effectiveness of anti-influenza agents. For this reason, many efforts have been dedicated to the identification of new therapeutic strategies aimed at targeting the virus-host cell interactions. Oxidative stress is a characteristic of some viral infections including influenza. Because antioxidants defend cells from damage caused by reactive oxygen species induced by different stimuli including pathogens, they represent interesting molecules to fight infectious diseases. However, most of the available studies have found that these would-be panaceas could actually exacerbate the diseases they claim to prevent, and have thus revealed "the dark side" of these molecules. This review article discusses the latest opportunities and drawbacks of the antioxidants used in anti-influenza therapy and new perspectives. PMID:25478883

  13. Redox state of plastoquinone pool regulates expression of Arabidopsis thaliana genes in response to elevated irradiance.

    PubMed

    Adamiec, Małgorzata; Drath, Maria; Jackowski, Grzegorz

    2008-01-01

    DNA microarray technology was applied to gain insight into the role of the redox state of PQ pool as a retrograde factor mediating differential expression of Arabidopsis nuclear genes during the acclimation to changing irradiance. DNA microarray chips containing probes corresponding to 24,000 Arabidopsis nuclear genes were screened with cRNA samples prepared from leaves of plants exposed for 5 h to low irradiance (control) vs. medium, high and excessive irradiances (MI, HI and EI, respectively). Six hundred and sixty three genes were differentially expressed as a result of an exposure to at least one elevated irradiance. Among 663 differentially expressed genes a total of 50 were reverted by DCMU--24 ones modulated at medium irradiance, 32 ones modulated at high irradiance and a single one modulated at excessive irradiance. We postulate that their expression is regulated by redox state of plastoquinone (PQ) pool. Thus the PQ-mediated redox regulation of expression of Arabidopsis nuclear genes is probably limited to the irradiance window representing non-stressing conditions. We found that the promoter regions of the PQ-regulated genes contained conserved elements, suggesting transcriptional control by a shared set of trans-acting factors which participate in signal transduction from the redox state of the PQ pool. PMID:18231654

  14. State of charge monitoring methods for vanadium redox flow battery control

    NASA Astrophysics Data System (ADS)

    Skyllas-Kazacos, Maria; Kazacos, Michael

    2011-10-01

    During operation of redox flow batteries, differential transfer of ions and electrolyte across the membrane and gassing side reactions during charging, can lead to an imbalance between the two half-cells that results in loss of capacity. This capacity loss can be corrected by either simple remixing of the two solutions, or by chemical or electrochemical rebalancing. In order to develop automated electrolyte management systems therefore, the state-of-charge of each half-cell electrolyte needs to be known. In this study, two state-of-charge monitoring methods are investigated for use in the vanadium redox flow battery. The first method utilizes conductivity measurements to independently measure the state-of-charge of each half-cell electrolyte. The second method is based on spectrophotometric principles and uses the different colours of the charged and discharged anolyte and catholyte to monitor system balance and state-of charge of each half-cell of the VRB during operation.

  15. Mitochondria: Redox Metabolism and Dysfunction

    PubMed Central

    Kang, Jia; Pervaiz, Shazib

    2012-01-01

    Mitochondria are the main intracellular location for fuel generation; however, they are not just power plants but involved in a range of other intracellular functions including regulation of redox homeostasis and cell fate. Dysfunction of mitochondria will result in oxidative stress which is one of the underlying causal factors for a variety of diseases including neurodegenerative diseases, diabetes, cardiovascular diseases, and cancer. In this paper, generation of reactive oxygen/nitrogen species (ROS/RNS) in the mitochondria, redox regulatory roles of certain mitochondrial proteins, and the impact on cell fate will be discussed. The current state of our understanding in mitochondrial dysfunction in pathological states and how we could target them for therapeutic purpose will also be briefly reviewed. PMID:22593827

  16. Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis.

    PubMed

    Iyer, Smita S; Ramirez, Allan M; Ritzenthaler, Jeffrey D; Torres-Gonzalez, Edilson; Roser-Page, Susanne; Mora, Ana L; Brigham, Kenneth L; Jones, Dean P; Roman, Jesse; Rojas, Mauricio

    2009-01-01

    Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis. PMID:18931052

  17. Cytochrome redox states and respiratory control in mouse and beef heart mitochondria at steady-state levels of hypoxia.

    PubMed

    Harrison, David K; Fasching, Mario; Fontana-Ayoub, Mona; Gnaiger, Erich

    2015-11-15

    Mitochondrial control of cellular redox states is a fundamental component of cell signaling in the coordination of core energy metabolism and homeostasis during normoxia and hypoxia. We investigated the relationship between cytochrome redox states and mitochondrial oxygen consumption at steady-state levels of hypoxia in mitochondria isolated from beef and mouse heart (BHImt, MHImt), comparing two species with different cardiac dynamics and local oxygen demands. A low-noise, rapid spectrophotometric system using visible light for the measurement of cytochrome redox states was combined with high-resolution respirometry. Monophasic hyperbolic relationships were observed between oxygen consumption, JO2, and oxygen partial pressure, Po2, within the range <1.1 kPa (8.3 mmHg; 13 μM). P50j (Po2 at 0.5·Jmax) was 0.015 ± 0.0004 and 0.021 ± 0.003 kPa (0.11 and 0.16 mmHg) for BHImt and MHImt, respectively. Maximum oxygen consumption, Jmax, was measured at saturating ADP levels (OXPHOS capacity) with Complex I-linked substrate supply. Redox states of cytochromes aa3 and c were biphasic hyperbolic functions of Po2. The relationship between cytochrome oxidation state and oxygen consumption revealed a separation of distinct phases from mild to severe and deep hypoxia. When cytochrome c oxidation increased from fully reduced to 45% oxidized at 0.1 Jmax, Po2 was as low as 0.002 kPa (0.02 μM), and trace amounts of oxygen are sufficient to partially oxidize the cytochromes. At higher Po2 under severe hypoxia, respiration increases steeply, whereas redox changes are small. Under mild hypoxia, the steep slope of oxidation of cytochrome c when flux remains more stable represents a cushioning mechanism that helps to maintain respiration high at the onset of hypoxia. PMID:26251509

  18. Can we predict the intracellular metabolic state of a cell based on extracellular metabolite data?

    PubMed

    Granucci, Ninna; Pinu, Farhana R; Han, Ting-Li; Villas-Boas, Silas G

    2015-12-01

    The analysis of extracellular metabolites presents many technical advantages over the analysis of intracellular compounds, which made this approach very popular in recent years as a high-throughput tool to assess the metabolic state of microbial cells. However, very little effort has been made to determine the actual relationship between intracellular and extracellular metabolite levels. The secretion of intracellular metabolites has been traditionally interpreted as a consequence of an intracellular metabolic overflow, which is based on the premise that for a metabolite to be secreted, it must be over-produced inside the cell. Therefore, we expect to find a secreted metabolite at increased levels inside the cells. Here we present a time-series metabolomics study of Saccharomyces cerevisiae growing on a glucose-limited chemostat with parallel measurements of intra- and extracellular metabolites. Although most of the extracellular metabolites were also detected in the intracellular samples and showed a typical metabolic overflow behaviour, we demonstrate that the secretion of many metabolites could not be explained by the metabolic overflow theory. PMID:26400772

  19. Probing redox states in the ancient and modern crust and possible biosphere-lithosphere interactions

    NASA Astrophysics Data System (ADS)

    Trail, D.

    2015-12-01

    The oxidation states of modern-day terrestrial environments are broadly constrained, though we are at the earliest stages of directly quantifying redox states during the first 700 million years. Redox states are constrainable through a combination of high temperature laboratory experiments, analyses of detrital Hadean zircon, and younger well-studied zircons and their host rocks. The redox state of the solid earth and the biosphere are believed to be interwoven: how might we exploit this to probe for the existence of possible biosphere-lithosphere interactions on the early Earth? Some insight comes from the investigation of "modern-day" zircon-bearing rocks and the new application of techniques that allow us to directly probe element valence as a proxy for magma redox state (XANES). Other insights are possible through the study of young ~400 Ma (S)edimentary- and (I)gneous-type granitoids from Lachlan Fold Belt (LFB), where the magma chemical properties in the former may be influenced by the assimilation of sedimentary material containing organic matter. We observe that zircons from LFB S-type granitoids formed under more reducing conditions when compared to LFB zircon formed in I-type granitoids. This observation, while reflecting 9 granitoids and 289 analyses of zircons where over 400 different plutons have been identified, is consistent with the incorporation of (reduced) organic matter in the former and highlights one possible manner in which life may influence the composition of igneous minerals. The chemical properties of rocks or igneous minerals such as zircon may extend the search for ancient biological activity to the earliest period of known igneous activity, which dates back to ~4.4 billion years ago. If organic matter was incorporated into Hadean sediments that were then buried and melted, then these biological remnants could imprint a chemical signature within the subsequent melt and the resulting crystal assemblage, including zircon.

  20. Redox states of Desulfovibrio vulgaris DsrC, a key protein in dissimilatory sulfite reduction

    SciTech Connect

    Venceslau, Sofia S.; Cort, John R.; Baker, Erin Shammel; Chu, Rosalie K.; Robinson, Errol W.; Dahl, Christiane; Saraiva, Ligia M.; Pereira, Ines Ac

    2013-11-29

    Dissimilatory reduction of sulfite is carried out by the siroheme enzyme DsrAB, with the involvement of the protein DsrC having two conserved cysteine residues. Here, we report a study of the distribution of DsrC in cell extracts, a cysteine-labelling gel-shift assay to monitor its redox state and behaviour, and procedures to produce the different redox forms. We show that, in the model sulfate reducer Desulfovibrio vulgaris, the majority of DsrC is not associated with DsrAB and is thus free to interact with other proteins. In addition, we successfully produced DsrC with an intramolecular disulfide bond (oxidized state) by treatment with arginine.

  1. The Redox State of Transglutaminase 2 Controls Arterial Remodeling

    PubMed Central

    van den Akker, Jeroen; VanBavel, Ed; van Geel, Remon; Matlung, Hanke L.; Guvenc Tuna, Bilge; Janssen, George M. C.; van Veelen, Peter A.; Boelens, Wilbert C.; De Mey, Jo G. R.; Bakker, Erik N. T. P.

    2011-01-01

    While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall. PMID:21901120

  2. Optical imaging of mitochondrial redox state in rodent model of retinitis pigmentosa

    PubMed Central

    Ghanian, Zahra; Sepehr, Reyhaneh; Schmitt, Heather; Eells, Janis; Ranji, Mahsa

    2013-01-01

    Abstract. Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11±0.03 in the SD normal and 0.841±0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP. PMID:23291617

  3. Surface fluorescence studies of tissue mitochondrial redox state in isolated perfused rat lungs.

    PubMed

    Staniszewski, Kevin; Audi, Said H; Sepehr, Reyhaneh; Jacobs, Elizabeth R; Ranji, Mahsa

    2013-04-01

    We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time. PMID:23238793

  4. Optical imaging of mitochondrial redox state in rodent model of retinitis pigmentosa

    NASA Astrophysics Data System (ADS)

    Maleki, Sepideh; Gopalakrishnan, Sandeep; Ghanian, Zahra; Sepehr, Reyhaneh; Schmitt, Heather; Eells, Janis; Ranji, Mahsa

    2013-01-01

    Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11±0.03 in the SD normal and 0.841±0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP.

  5. Redox state and water content in the upper mantle: Linkages to the atmosphere, hydrosphere and continents

    NASA Astrophysics Data System (ADS)

    Li, Zhengxue

    Geochemical and petrologic tools were deployed to investigate the redox state and water content of the earth's upper mantle. Study results are discussed in the context of their linkages to the atmospheric oxygen level, hydrospheric water budget and lithospheric evolution of continents. Because the partitioning of V is redox-sensitive and otherwise similar to that of Sc which is not redox sensitive, the V/Sc ratios of basalts of different ages act as a natural recorder of the redox states of the upper mantle. Through a comparison between global mid-ocean ridge basalts and Archean basalts, the fO2 of the upper mantle was inferred to have changed by no more than 0.3 log units since Archean. Combined with results from a thermodynamic model simulating the redox reactions of volcanic gases, this observation argues against the idea that the increase in oxygen in the atmosphere ˜2.3 billion years ago was caused by redox transition in the upper mantle. Through a geochemical and petrologic study at the Feather River Ophiolite (in northern California), global water recycling rates at subduction zones were estimated based on reconstructed serpentinization depths for the oceanic lithospheric mantle. Within uncertainties, the estimated water recycling rates roughly match global volcanic dewatering rates, which suggest the hydrospheric water storage is current at steady-state. Based on water contents measured in mantle xenoliths from the Colorado Plateau and vicinity, the idea that the lithospheric mantle beneath the western North America was rehydrated by the dewatering of the flat-subducting Farallon slab is confirmed. As predicted by an updated flow law for olivine aggregates, hydration might have weakened the basal lithosphere beneath the Colorado Plateau and thus induced lithospheric thinning by ˜15 km as a result of basal erosion. Extrapolation of the flow law to thick, cratonic lithosphere further suggests lithospheric thinning of much larger extents can occur if enough

  6. Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt.

    PubMed

    Murata, Hiroaki; Ihara, Yoshito; Nakamura, Hajime; Yodoi, Junji; Sumikawa, Koji; Kondo, Takahito

    2003-12-12

    Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt. PMID:14522978

  7. Intermediate and stable redox states of cytochrome c studied by low temperature resonance Raman spectroscopy.

    PubMed Central

    Cartling, B

    1983-01-01

    Stabilized intermediate redox states of cytochrome c are generated by radiolytic reduction of initially oxidized enzyme in glass matrices at liquid nitrogen temperature. In the intermediate states the heme group is reduced by hydrated electrons, whereas the protein conformation is restrained close to its oxidized form by the low-temperature glass matrix. The intermediate and stable redox states of cytochrome c at neutral and alkaline pH are studied by low-temperature resonance Raman spectroscopy using excitations in resonance with the B (Soret) and Q1 (beta) optical transitions. The assignments of the cytochrome c resonance Raman bands are discussed. The observed spectral characteristics of the intermediate states as well as of the alkaline transition in the oxidized state are interpreted in terms of oxidation-state marker modes, spin-state marker modes, heme iron--axial ligand stretching modes, totally symmetric in-plane porphyrin modes, nontotally symmetric in-plane modes, and out-of-plane modes. PMID:6311300

  8. Does light scattering affect the OCT quantitation of redox state of cytochrome oxidase in bone tissue?

    NASA Astrophysics Data System (ADS)

    Xu, Xiangqun; Wang, Ruikang K.; El Haj, Alicia

    2002-06-01

    In our previous report, we have presented the possibility of optical coherence tomography (OCT) to monitor the redox state of mitochondria enzyme Cytochrome oxidase (CytOx) in bone tissue. The previous results showed that reduction of the enzyme in periosteal tissue leads to a change in attenuation coefficient of 1.68 +/- 0.67mm-1 by OCT measurements. The new results from cultured cells fixed in 300 (mu) l agarose plug showed the difference in attenuation coefficient is 0.26+-0.10 mm-1 (n = 9) for 7x106 astrocytoma cells and 0.28+-0.13 mm-1 (n = 7) for 20x106 astrocytoma cells in agarose plug, respectively between cells with oxidised and reduced enzyme at 820nm. A decrease in attenuation coefficient of 0.35+-0.09 mm-1 (n = 4) for 10 million SKMES cells in agarose was also observed with the redox shift of CytOx. The absorption coefficient of the oxidized-reduced form of CytOx is measured approximately 8.4+-1.5x10-3/mm (n=3) and 8.2+-1.0x10-3/mm (n=3) at 820nm for astrocytoma cells and rat periosteum respectively by means of a biochemical assay. Thereby it can be seen that the change in attenuation coefficient of cultured cells with redox shift of CytOx mainly results from the scattering change.

  9. Three Redox States of Trypanosoma brucei Alternative Oxidase Identified by Infrared Spectroscopy and Electrochemistry

    PubMed Central

    Maréchal, Amandine; Kido, Yasutoshi; Kita, Kiyoshi; Moore, Anthony L.; Rich, Peter R.

    2009-01-01

    Electrochemistry coupled with Fourier transform infrared (IR) spectroscopy was used to investigate the redox properties of recombinant alternative ubiquinol oxidase from Trypanosoma brucei, the organism responsible for African sleeping sickness. Stepwise reduction of the fully oxidized resting state of recombinant alternative ubiquinol oxidase revealed two distinct IR redox difference spectra. The first of these, signal 1, titrates in the reductive direction as an n = 2 Nernstian component with an apparent midpoint potential of 80 mV at pH 7.0. However, reoxidation of signal 1 in the same potential range under anaerobic conditions did not occur and only began with potentials in excess of 500 mV. Reoxidation by introduction of oxygen was also unsuccessful. Signal 1 contained clear features that can be assigned to protonation of at least one carboxylate group, further perturbations of carboxylic and histidine residues, bound ubiquinone, and a negative band at 1554 cm−1 that might arise from a radical in the fully oxidized protein. A second distinct IR redox difference spectrum, signal 2, appeared more slowly once signal 1 had been reduced. This component could be reoxidized with potentials above 100 mV. In addition, when both signals 1 and 2 were reduced, introduction of oxygen caused rapid oxidation of both components. These data are interpreted in terms of the possible active site structure and mechanism of oxygen reduction to water. PMID:19767647

  10. Paramagnetic Molecular Grippers: The Elements of Six-State Redox Switches.

    PubMed

    Milić, Jovana; Zalibera, Michal; Pochorovski, Igor; Trapp, Nils; Nomrowski, Julia; Neshchadin, Dmytro; Ruhlmann, Laurent; Boudon, Corinne; Wenger, Oliver S; Savitsky, Anton; Lubitz, Wolfgang; Gescheidt, Georg; Diederich, François

    2016-07-01

    The development of semiquinone-based resorcin[4]arene cavitands expands the toolbox of switchable molecular grippers by introducing the first paramagnetic representatives. The semiquinone (SQ) states were generated electrochemically, chemically, and photochemically. We analyzed their electronic, conformational, and binding properties by cyclic voltammetry, ultraviolet/visible (UV/vis) spectroelectrochemistry, electron paramagnetic resonance (EPR) and transient absorption spectroscopy, in conjunction with density functional theory (DFT) calculations. The utility of UV/vis spectroelectrochemistry and EPR spectroscopy in evaluating the conformational features of resorcin[4]arene cavitands is demonstrated. Guest binding properties were found to be enhanced in the SQ state as compared to the quinone (Q) or the hydroquinone (HQ) states of the cavitands. Thus, these paramagnetic SQ intermediates open the way to six-state redox switches provided by two conformations (open and closed) in three redox states (Q, SQ, and HQ) possessing distinct binding ability. The switchable magnetic properties of these molecular grippers and their responsiveness to electrical stimuli has the potential for development of efficient molecular devices. PMID:27300355

  11. The redox state of arc mantle using Zn/Fe systematics.

    PubMed

    Lee, Cin-Ty A; Luffi, Peter; Le Roux, Véronique; Dasgupta, Rajdeep; Albaréde, Francis; Leeman, William P

    2010-12-01

    Many arc lavas are more oxidized than mid-ocean-ridge basalts and subduction introduces oxidized components into the mantle. As a consequence, the sub-arc mantle wedge is widely believed to be oxidized. The Fe oxidation state of sub-arc mantle is, however, difficult to determine directly, and debate persists as to whether this oxidation is intrinsic to the mantle source. Here we show that Zn/Fe(T) (where Fe(T) = Fe(2+) + Fe(3+)) is redox-sensitive and retains a memory of the valence state of Fe in primary arc basalts and their mantle sources. During melting of mantle peridotite, Fe(2+) and Zn behave similarly, but because Fe(3+) is more incompatible than Fe(2+), melts generated in oxidized environments have low Zn/Fe(T). Primitive arc magmas have identical Zn/Fe(T) to mid-ocean-ridge basalts, suggesting that primary mantle melts in arcs and ridges have similar Fe oxidation states. The constancy of Zn/Fe(T) during early differentiation involving olivine requires that Fe(3+)/Fe(T) remains low in the magma. Only after progressive fractionation does Fe(3+)/Fe(T) increase and stabilize magnetite as a fractionating phase. These results suggest that subduction of oxidized crustal material may not significantly alter the redox state of the mantle wedge. Thus, the higher oxidation states of arc lavas must be in part a consequence of shallow-level differentiation processes, though such processes remain poorly understood. PMID:21124454

  12. Perturbation of Human Coronary Artery Endothelial Cell Redox State and NADPH Generation by Methylglyoxal

    PubMed Central

    Davies, Michael J.

    2014-01-01

    Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH). We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC) were incubated with high glucose (25 mM, 24 h, 37°C), or methylglyoxal (MGO), glyoxal, or glycolaldehyde (100–500 µM, 1 h, 37°C), before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05) decreased total thiols (∼35%), further experiments with MGO showed significant losses of GSH (∼40%) and NADPH (∼10%); these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10%) NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE) formed; lower levels of Nε-(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine (CML) were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis. PMID:24466151

  13. Optical cryo-imaging of kidney mitochondrial redox state in diabetic mice models

    NASA Astrophysics Data System (ADS)

    Maleki, S.; Sepehr, R.; Staniszewski, K.; Sheibani, N.; Sorenson, C. M.; Ranji, M.

    2012-03-01

    Oxidative stress (OS), which increases during diabetes, exacerbates the development and progression of diabetes complications including renal vascular and proximal tubule cell dysfunction. The objective of this study was to investigate the changes in the metabolic state of the tissue in diabetic mice kidneys using fluorescence imaging. Mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH-2 (Flavin Adenine Dinucleotide) are autofluorescent and can be monitored without exogenous labels by optical techniques. The ratio of the fluorescence intensity of these fluorophores, (NADH/FAD), called the NADH redox ratio (RR), is a marker of metabolic state of a tissue. We examined mitochondrial redox states of kidneys from diabetic mice, Akita/+ and its control wild type (WT) for a group of 8- and 12-week-old mice. Average intensity and histogram of maximum projected images of FAD, NADH, and NADH RR were calculated for each kidney. Our results indicated a 17% decrease in the mean NADH RR of the kidney from 8-week-old mice compared with WT mice and, a 30% decrease in the mean NADH RR of kidney from12-week-old mice compared with WT mice. These results indicated an increase in OS in diabetic animals and its progression over time. Thus, NADH RR can be used as a hallmark of OS in diabetic kidney allowing temporal identification of oxidative state.

  14. Chromium basalts - Experimental determination of redox states and partitioning among synthetic silicate phases

    NASA Technical Reports Server (NTRS)

    Schreiber, H. D.; Haskin, L. A.

    1976-01-01

    Experiments were performed on silicate compositions in the forsterite-anorthite-silica and forsterite-anorthite-diopside systems to determine the relative amounts of Cr(II), Cr(III), and Cr(VI) over a wide range of oxygen partial pressures from 10 to the -10th to 1 atm at 1500 and 1550 C. Redox states were measured by visible absorption spectroscopy and electron paramagnetic resonance spectroscopy and titration. It was found that Cr is present almost exclusively as Cr(III) in terrestrial basaltic liquids and as a mixture of Cr(III) and Cr(II) in lunar basaltic liquids.

  15. In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD(+)/NADH redox state.

    PubMed

    Zhao, Yuzheng; Wang, Aoxue; Zou, Yejun; Su, Ni; Loscalzo, Joseph; Yang, Yi

    2016-08-01

    NADH and its oxidized form NAD(+) have a central role in energy metabolism, and their concentrations are often considered to be among the most important readouts of metabolic state. Here, we present a detailed protocol to image and monitor NAD(+)/NADH redox state in living cells and in vivo using a highly responsive, genetically encoded fluorescent sensor known as SoNar (sensor of NAD(H) redox). The chimeric SoNar protein was initially developed by inserting circularly permuted yellow fluorescent protein (cpYFP) into the NADH-binding domain of Rex protein from Thermus aquaticus (T-Rex). It functions by binding to either NAD(+) or NADH, thus inducing protein conformational changes that affect its fluorescent properties. We first describe steps for how to establish SoNar-expressing cells, and then discuss how to use the system to quantify the intracellular redox state. This approach is sensitive, accurate, simple and able to report subtle perturbations of various pathways of energy metabolism in real time. We also detail the application of SoNar to high-throughput chemical screening of candidate compounds targeting cell metabolism in a microplate-reader-based assay, along with in vivo fluorescence imaging of tumor xenografts expressing SoNar in mice. Typically, the approximate time frame for fluorescence imaging of SoNar is 30 min for living cells and 60 min for living mice. For high-throughput chemical screening in a 384-well-plate assay, the whole procedure generally takes no longer than 60 min to assess the effects of 380 compounds on cell metabolism. PMID:27362337

  16. Heterogeneity of intracellular polymer storage states in enhanced biological phosphorus removal (EBPR)--observation and modeling.

    PubMed

    Bucci, Vanni; Majed, Nehreen; Hellweger, Ferdi L; Gu, April Z

    2012-03-20

    A number of agent-based models (ABMs) for biological wastewater treatment processes have been developed, but their skill in predicting heterogeneity of intracellular storage states has not been tested against observations due to the lack of analytical methods for measuring single-cell intracellular properties. Further, several mechanisms can produce and maintain heterogeneity (e.g., different histories, uneven division) and their relative importance has not been explored. This article presents an ABM for the enhanced biological phosphorus removal (EBPR) treatment process that resolves heterogeneity in three intracellular polymer storage compounds (i.e., polyphosphate, polyhydroxybutyrate, and glycogen) in three functional microbial populations (i.e., polyphosphate-accumulating, glycogen-accumulating, and ordinary heterotrophic organisms). Model predicted distributions were compared to those based on single-cell estimates obtained using a Raman microscopy method for a laboratory-scale sequencing batch reactor (SBR) system. The model can reproduce many features of the observed heterogeneity. Two methods for introducing heterogeneity were evaluated. First, biological variability in individual cell behavior was simulated by randomizing model parameters (e.g., maximum acetate uptake rate) at division. This method produced the best fit to the data. An optimization algorithm was used to determine the best variability (i.e., coefficient of variance) for each parameter, which suggests large variability in acetate uptake. Second, biological variability in individual cell states was simulated by randomizing state variables (e.g., internal nutrient) at division, which was not able to maintain heterogeneity because the memory in the internal states is too short. These results demonstrate the ability of ABM to predict heterogeneity and provide insights into the factors that contribute to it. Comparison of the ABM with an equivalent population-level model illustrates the effect

  17. Cytochrome b5 reductase, a plasma membrane redox enzyme, protects neuronal cells against metabolic and oxidative stress through maintaining redox state and bioenergetics.

    PubMed

    Hyun, Dong-Hoon; Lee, Ga-Hyun

    2015-12-01

    The plasma membrane redox system (PMRS) containing NADH-dependent reductases is known to be involved in the maintenance of redox state and bioenergetics. Neuronal cells are very vulnerable to oxidative stress and altered energy metabolism linked to mitochondrial dysfunction. However, the role of the PMRS in these pathways is far from clear. In this study, in order to investigate how cytochrome b5 reductase (b5R), one of the PM redox enzymes, regulates cellular response under stressed conditions, human neuroblastoma cells transfected with b5R were used for viability and mitochondrial functional assays. Cells transfected with b5R exhibited significantly higher levels of the NAD(+)/NADH ratio, consistent with increased levels of b5R activity. Overexpression of b5R made cells more resistant to H2O2 (oxidative stress), 2-deoxyglucose (metabolic stress), rotenone and antimycin A (energetic stress), and lactacystin (proteotoxic stress), but did not protect cells against H2O2 and serum withdrawal. Overexpression of b5R induced higher mitochondrial functions such as ATP production rate, oxygen consumption rate, and activities of complexes I and II, without formation of further reactive oxygen species, consistent with lower levels of oxidative/nitrative damage and resistance to apoptotic cell death. In conclusion, higher NAD(+)/NADH ratio and consequent more efficient mitochondrial functions are induced by the PMRS, enabling them to maintain redox state and energy metabolism under conditions of some energetic stresses. This suggests that b5R can be a target for therapeutic intervention for aging and neurodegenerative diseases. PMID:26611738

  18. Influence of the Phase State of Self-Assembling Redox Mediators on their Electrochemical Activity

    PubMed Central

    Muller, John P. E.; Aytar, Burcu S.; Kondo, Yukishige; Lynn, David M.; Abbott, Nicholas L.

    2014-01-01

    Self-assembling redox mediators have the potential to be broadly useful in a range of interfacial electrochemical contexts because the oxidation state and state of assembly of the mediator are closely coupled. In this paper, we report an investigation of the self-assembly of single- and double-tailed ferrocenyl amphiphiles (FTMA and BFDMA, respectively) at the surfaces of Pt electrodes and the impact of the dynamic assembled state of the amphiphiles on their rate of oxidation. We conclude that frozen aggregates of BFDMA adsorb to the surfaces of the Pt electrodes, and that slow dynamics of reorganization BFDMA within these aggregates limits the rate of electrooxidation of BFDMA. In contrast, FTMA, while forming assemblies on the surfaces of Pt electrodes, is characterized by fast reorganization dynamics and a corresponding rate of oxidation that is an order of magnitude greater than BFDMA. PMID:24882870

  19. Direct structural evidence of protein redox regulation obtained by in-cell NMR.

    PubMed

    Mercatelli, Eleonora; Barbieri, Letizia; Luchinat, Enrico; Banci, Lucia

    2016-02-01

    The redox properties of cellular environments are critical to many functional processes, and are strictly controlled in all living organisms. The glutathione-glutathione disulfide (GSH-GSSG) couple is the most abundant intracellular redox couple. A GSH redox potential can be calculated for each cellular compartment, which reflects the redox properties of that environment. This redox potential is often used to predict the redox state of a disulfide-containing protein, based on thermodynamic considerations. However, thiol-disulfide exchange reactions are often catalyzed by specific partners, and the distribution of the redox states of a protein may not correspond to the thermodynamic equilibrium with the GSH pool. Ideally, the protein redox state should be measured directly, bypassing the need to extrapolate from the GSH. Here, by in-cell NMR, we directly observe the redox state of three human proteins, Cox17, Mia40 and SOD1, in the cytoplasm of human and bacterial cells. We compare the observed distributions of redox states with those predicted by the GSH redox potential, and our results partially agree with the predictions. Discrepancies likely arise from the fact that the redox state of SOD1 is controlled by a specific partner, its copper chaperone (CCS), in a pathway which is not linked to the GSH redox potential. In principle, in-cell NMR allows determining whether redox proteins are at the equilibrium with GSH, or they are kinetically regulated. Such approach does not need assumptions on the redox potential of the environment, and provides a way to characterize each redox-regulating pathway separately. PMID:26589182

  20. Oxidative catalytic evolution of redox- and spin-states of a Fe-phthalocyanine studied by EPR

    NASA Astrophysics Data System (ADS)

    Bletsa, Eleni; Solakidou, Maria; Louloudi, Maria; Deligiannakis, Yiannis

    2016-04-01

    The catalytic-oxidative evolution of the redox/spin states of a Fe-phthalocyanine (Fe-Pc) catalyst was studied by electron paramagnetic resonance spectroscopy. Under oxidative catalytic conditions, Fe-Pc may evolve via multiple redox/spin conformations. Axial ligation of imidazole, O2 or t-Bu-OOH as oxidant, results in a complex multipath redox/spin landscape that was determined in detail herein. The high-spin conformations of Fe-Pc/imidazole evolve more slowly than the low-spin conformations. Catalytically active vs. inactive conformations were distinguished. A unified physicochemical catalytic reaction mechanism is discussed herein based on the distinct role of the various structural, spin and redox forms.

  1. Fruit ripening mutants reveal cell metabolism and redox state during ripening.

    PubMed

    Kumar, Vinay; Irfan, Mohammad; Ghosh, Sumit; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-03-01

    Ripening which leads to fruit senescence is an inimitable process characterized by vivid changes in color, texture, flavor, and aroma of the fleshy fruits. Our understanding of the mechanisms underlying the regulation of fruit ripening and senescence is far from complete. Molecular and biochemical studies on tomato (Solanum lycopersicum) ripening mutants such as ripening inhibitor (rin), nonripening (nor), and never ripe (Nr) have been useful in our understanding of fruit development and ripening. The MADS-box transcription factor RIN, a global regulator of fruit ripening, is vital for the broad aspects of ripening, in both ethylene-dependent and independent manners. Here, we have carried out microarray analysis to study the expression profiles of tomato genes during ripening of wild type and rin mutant fruits. Analysis of the differentially expressed genes revealed the role of RIN in regulation of several molecular and biochemical events during fruit ripening including fruit specialized metabolism and cellular redox state. The role of reactive oxygen species (ROS) during fruit ripening and senescence was further examined by determining the changes in ROS level during ripening of wild type and mutant fruits and by analyzing expression profiles of the genes involved in maintaining cellular redox state. Taken together, our findings suggest an important role of ROS during fruit ripening and senescence, and therefore, modulation of ROS level during ripening could be useful in achieving desired fruit quality. PMID:26008650

  2. Redox regulation of protein damage in plasma.

    PubMed

    Griffiths, Helen R; Dias, Irundika H K; Willetts, Rachel S; Devitt, Andrew

    2014-01-01

    The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation. In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites. PMID:24624332

  3. Robust Off- and Online Separation of Intracellularly Recorded Up and Down Cortical States

    PubMed Central

    Seamari, Yamina; Narváez, José A.; Vico, Francisco J.; Lobo, Daniel; Sanchez-Vives, Maria V.

    2007-01-01

    Background The neuronal cortical network generates slow (<1 Hz) spontaneous rhythmic activity that emerges from the recurrent connectivity. This activity occurs during slow wave sleep or anesthesia and also in cortical slices, consisting of alternating up (active, depolarized) and down (silent, hyperpolarized) states. The search for the underlying mechanisms and the possibility of analyzing network dynamics in vitro has been subject of numerous studies. This exposes the need for a detailed quantitative analysis of the membrane fluctuating behavior and computerized tools to automatically characterize the occurrence of up and down states. Methodology/Principal Findings Intracellular recordings from different areas of the cerebral cortex were obtained from both in vitro and in vivo preparations during slow oscillations. A method that separates up and down states recorded intracellularly is defined and analyzed here. The method exploits the crossover of moving averages, such that transitions between up and down membrane regimes can be anticipated based on recent and past voltage dynamics. We demonstrate experimentally the utility and performance of this method both offline and online, the online use allowing to trigger stimulation or other events in the desired period of the rhythm. This technique is compared with a histogram-based approach that separates the states by establishing one or two discriminating membrane potential levels. The robustness of the method presented here is tested on data that departs from highly regular alternating up and down states. Conclusions/Significance We define a simple method to detect cortical states that can be applied in real time for offline processing of large amounts of recorded data on conventional computers. Also, the online detection of up and down states will facilitate the study of cortical dynamics. An open-source MATLAB® toolbox, and Spike 2®-compatible version are made freely available. PMID:17849017

  4. Three-State Single-Molecule Naphthalenediimide Switch: Integration of a Pendant Redox Unit for Conductance Tuning.

    PubMed

    Li, Yonghai; Baghernejad, Masoud; Qusiy, Al-Galiby; Zsolt Manrique, David; Zhang, Guanxin; Hamill, Joseph; Fu, Yongchun; Broekmann, Peter; Hong, Wenjing; Wandlowski, Thomas; Zhang, Deqing; Lambert, Colin

    2015-11-01

    We studied charge transport through core-substituted naphthalenediimide (NDI) single-molecule junctions using the electrochemical STM-based break-junction technique in combination with DFT calculations. Conductance switching among three well-defined states was demonstrated by electrochemically controlling the redox state of the pendent diimide unit of the molecule in an ionic liquid. The electrical conductances of the dianion and neutral states differ by more than one order of magnitude. The potential-dependence of the charge-transport characteristics of the NDI molecules was confirmed by DFT calculations, which account for electrochemical double-layer effects on the conductance of the NDI junctions. This study suggests that integration of a pendant redox unit with strong coupling to a molecular backbone enables the tuning of charge transport through single-molecule devices by controlling their redox states. PMID:26403214

  5. Effect of the redox state of QB on electric field-induced charge recombination in Photosystem II.

    PubMed

    Hemelrijk, P W; van Gorkom, H J

    1996-05-01

    Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts ('blebs') by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate QB was observed and the earlier failure to detect it is explained. The influence of the QB/QB (-) oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states S2 and S3, but not in the state 'S4' (actually Z(+)S3). The presence of the oxidized secondary electron donor, tyrosine Z(+), appeared to prevent expression of the QB/QB (-) effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z(+) and the oxygen evolving complex. PMID:24271299

  6. Anr, the anaerobic global regulator, modulates the redox state and oxidative stress resistance in Pseudomonas extremaustralis.

    PubMed

    Tribelli, Paula M; Nikel, Pablo I; Oppezzo, Oscar J; López, Nancy I

    2013-02-01

    The role of Anr in oxidative stress resistance was investigated in Pseudomonas extremaustralis, a polyhydroxybutyrate-producing Antarctic bacterium. The absence of Anr caused increased sensitivity to hydrogen peroxide under low oxygen tension. This phenomenon was associated with a decrease in the redox ratio, higher oxygen consumption and higher reactive oxygen species production. Physiological responses of the mutant to the oxidized state included an increase in NADP(H) content, catalase activity and exopolysaccharide production. The wild-type strain showed a sharp decrease in the reduced thiol pool when exposed to hydrogen peroxide, not observed in the mutant strain. In silico analysis of the genome sequence of P. extremaustralis revealed putative Anr binding sites upstream from genes related to oxidative stress. Genes encoding several chaperones and cold shock proteins, a glutathione synthase, a sulfate transporter and a thiol peroxidase were identified as potential targets for Anr regulation. Our results suggest a novel role for Anr in oxidative stress resistance and in redox balance maintenance under conditions of restricted oxygen supply. PMID:23223440

  7. The role of iron redox state in the genotoxicity of ultrafine superparamagnetic iron oxide nanoparticles.

    PubMed

    Singh, Neenu; Jenkins, Gareth J S; Nelson, Bryant C; Marquis, Bryce J; Maffeis, Thierry G G; Brown, Andy P; Williams, Paul M; Wright, Chris J; Doak, Shareen H

    2012-01-01

    Ultrafine superparamagnetic iron oxide nanoparticles (USPION) hold great potential for revolutionising biomedical applications such as MRI, localised hyperthermia, and targeted drug delivery. Though evidence is increasing regarding the influence of nanoparticle physico-chemical features on toxicity, data however, is lacking that assesses a range of such characteristics in parallel. We show that iron redox state, a subtle though important physico-chemical feature of USPION, dramatically modifies the cellular uptake of these nanoparticles and influences their induction of DNA damage. Surface chemistry was also found to have an impact and evidence to support a potential mechanism of oxidative DNA damage behind the observed responses has been demonstrated. As human exposure to ferrofluids is predicted to increase through nanomedicine based therapeutics, these findings are important in guiding the fabrication of USPION to ensure they have characteristics that support biocompatibility. PMID:22027595

  8. Assay of the redox state of the tumor suppressor PTEN by mobility shift.

    PubMed

    Han, Seong-Jeong; Ahn, Younghee; Park, Iha; Zhang, Ying; Kim, Inyoung; Kim, Hyun Woo; Ku, Chang-Sub; Chay, Kee-Oh; Yang, Sung Yeul; Ahn, Bong Whan; Jang, Dong Il; Lee, Seung-Rock

    2015-05-01

    PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells. PMID:25637034

  9. Mutual Regulation of Epicardial Adipose Tissue and Myocardial Redox State by PPAR-γ/Adiponectin Signalling

    PubMed Central

    Antonopoulos, Alexios S.; Margaritis, Marios; Verheule, Sander; Recalde, Alice; Sanna, Fabio; Herdman, Laura; Psarros, Costas; Nasrallah, Hussein; Coutinho, Patricia; Akoumianakis, Ioannis; Brewer, Alison C.; Sayeed, Rana; Krasopoulos, George; Petrou, Mario; Tarun, Akansha; Tousoulis, Dimitris; Shah, Ajay M.; Casadei, Barbara; Channon, Keith M.

    2016-01-01

    Rationale: Adiponectin has anti-inflammatory effects in experimental models, but its role in the regulation of myocardial redox state in humans is unknown. Although adiponectin is released from epicardial adipose tissue (EpAT), it is unclear whether it exerts any paracrine effects on the human myocardium. Objective: To explore the cross talk between EpAT-derived adiponectin and myocardial redox state in the human heart. Methods and Results: EpAT and atrial myocardium were obtained from 306 patients undergoing coronary artery bypass grafting. Functional genetic polymorphisms that increase ADIPOQ expression (encoding adiponectin) led to reduced myocardial nicotinamide adenine dinucleotide phosphate oxidase–derived O2−, whereas circulating adiponectin and ADIPOQ expression in EpAT were associated with elevated myocardial O2−. In human atrial tissue, we demonstrated that adiponectin suppresses myocardial nicotinamide adenine dinucleotide phosphate oxidase activity, by preventing AMP kinase–mediated translocation of Rac1 and p47phox from the cytosol to the membranes. Induction of O2− production in H9C2 cardiac myocytes led to the release of a transferable factor able to induce peroxisome proliferator-activated receptor-γ–mediated upregulation of ADIPOQ expression in cocultured EpAT. Using a NOX2 transgenic mouse and a pig model of rapid atrial pacing, we found that oxidation products (such as 4-hydroxynonenal) released from the heart trigger peroxisome proliferator-activated receptor-γ–mediated upregulation of ADIPOQ in EpAT. Conclusions: We demonstrate for the first time in humans that adiponectin directly decreases myocardial nicotinamide adenine dinucleotide phosphate oxidase activity via endocrine or paracrine effects. Adiponectin expression in EpAT is controlled by paracrine effects of oxidation products released from the heart. These effects constitute a novel defense mechanism of the heart against myocardial oxidative stress. PMID:26838789

  10. The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae

    PubMed Central

    2013-01-01

    Background Pretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium. Results By cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(±0.005) to 0.08(±0.002) and the anabolic reduction charge decreased from 0.46(±0.11) to 0.27(±0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(±0.002) to 0.85(±0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group “Cell rescue, defense and virulence” was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was

  11. Co-variation of nitrogen isotopes and redox states through glacial-interglacial cycles in the Black Sea

    NASA Astrophysics Data System (ADS)

    Quan, Tracy M.; Wright, James D.; Falkowski, Paul G.

    2013-07-01

    In all aquatic environments, nitrogen cycling within the water column is strongly influenced by oxygen. We hypothesize that the nitrogen isotopic composition (δ15N) of organic matter deposited in the sediments is a proxy for the redox state of the water column at the time of deposition. We tested the hypothesis by measuring the bulk sedimentary δ15N values in a drill core from the Black Sea, a basin that alternates between oxic, less saline conditions and anoxic, marine conditions on glacial-interglacial time scales. We reconstructed these changes in Black Sea redox conditions using sedimentary δ15N, total organic carbon (TOC), total nitrogen (TN), redox-sensitive metals, and micropaleontological data from a deep-sea core (DSDP Site 380). The sedimentary data reveal that during the transitions between oxic and anoxic conditions, δ15N values increased relative to the preceding and succeeding quasi-steady-state oxic and anoxic periods. The results indicate that the reciprocal transitional states from anoxic to oxic conditions were accompanied by intense denitrification; during the quasi-stable oxic and anoxic states (characterized by glacial fresh water and interglacial marine conditions) nitrification and complete nitrate utilization, respectively, dominate the nitrogen cycle. While other factors may influence the δ15N record, our results support the hypothesis that the variations in nitrogen isotopic composition of organic matter are strongly influenced by changes in redox state in the Black Sea subphotic zone on glacial-interglacial time scales, and can be explained by a relatively simple model describing the effects of oxygen on the microbial processes that drive the nitrogen cycle in marine ecosystems. Our model suggests that the nitrogen isotopic composition of marine sediments, on geological time scales, can be used to reconstruct the redox state of the overlying water column.

  12. Obesity-Associated Oxidative Stress: Strategies Finalized to Improve Redox State

    PubMed Central

    Savini, Isabella; Catani, Maria Valeria; Evangelista, Daniela; Gasperi, Valeria; Avigliano, Luciana

    2013-01-01

    Obesity represents a major risk factor for a plethora of severe diseases, including diabetes, cardiovascular disease, non-alcoholic fatty liver disease, and cancer. It is often accompanied by an increased risk of mortality and, in the case of non-fatal health problems, the quality of life is impaired because of associated conditions, including sleep apnea, respiratory problems, osteoarthritis, and infertility. Recent evidence suggests that oxidative stress may be the mechanistic link between obesity and related complications. In obese patients, antioxidant defenses are lower than normal weight counterparts and their levels inversely correlate with central adiposity; obesity is also characterized by enhanced levels of reactive oxygen or nitrogen species. Inadequacy of antioxidant defenses probably relies on different factors: obese individuals may have a lower intake of antioxidant- and phytochemical-rich foods, such as fruits, vegetables, and legumes; otherwise, consumption of antioxidant nutrients is normal, but obese individuals may have an increased utilization of these molecules, likewise to that reported in diabetic patients and smokers. Also inadequate physical activity may account for a decreased antioxidant state. In this review, we describe current concepts in the meaning of obesity as a state of chronic oxidative stress and the potential interventions to improve redox balance. PMID:23698776

  13. High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool1[W][OA

    PubMed Central

    Lepetit, Bernard; Sturm, Sabine; Rogato, Alessandra; Gruber, Ansgar; Sachse, Matthias; Falciatore, Angela; Kroth, Peter G.; Lavaud, Johann

    2013-01-01

    In diatoms, the process of energy-dependent chlorophyll fluorescence quenching (qE) has an important role in photoprotection. Three components are essential for qE: (1) the light-dependent generation of a transthylakoidal proton gradient; (2) the deepoxidation of the xanthophyll diadinoxanthin (Dd) into diatoxanthin (Dt); and (3) specific nucleus-encoded antenna proteins, called Light Harvesting Complex Protein X (LHCX). We used the model diatom Phaeodactylum tricornutum to investigate the concerted light acclimation response of the qE key components LHCX, proton gradient, and xanthophyll cycle pigments (Dd+Dt) and to identify the intracellular light-responsive trigger. At high-light exposure, the up-regulation of three of the LHCX genes and the de novo synthesis of Dd+Dt led to a pronounced rise of qE. By inhibiting either the conversion of Dd to Dt or the translation of LHCX genes, qE amplification was abolished and the diatom cells suffered from stronger photoinhibition. Artificial modification of the redox state of the plastoquinone (PQ) pool via 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone resulted in a disturbance of Dd+Dt synthesis in an opposite way. Moreover, we could increase the transcription of two of the four LHCX genes under low-light conditions by reducing the PQ pool using 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone. Altogether, our results underline the central role of the redox state of the PQ pool in the light acclimation of diatoms. Additionally, they emphasize strong evidence for the existence of a plastid-to-nucleus retrograde signaling mechanism in an organism with plastids that derived from secondary endosymbiosis. PMID:23209128

  14. Influence Of The Redox State On The Electrical Conductivity Of Basaltic Melts

    NASA Astrophysics Data System (ADS)

    Pommier, A.; Gaillard, F.; Pichavant, M.

    2007-12-01

    The electrical conductivity is an efficient probe of mass transfer processes within silicate melts and magmas. Previous studies have established that the electrical conductivity is sensitive to parameters such as temperature, melt composition and pressure. In contrast to what is known for Fe-bearing minerals, little attention has been given to the influence of redox state on the electrical conductivity of melts. Experiments were performed on tephritic and basaltic compositions respectively from Mt. Vesuvius and Pu'u 'O'o. Measurements were carried out on cylindrical glass samples (OD: 6 mm, ID: 1 mm, L: 8 mm) drilled from glass obtained by fusing each rock sample at 1400°C in air. A two-electrode configuration was adopted, with the electrical impedance being radially measured. A Pt wire was used as the internal electrode whereas a Pt tube served as the external electrode. Experiments were conducted at 1 atm in a vertical furnace between 1200°C and 1300°C, both in air and in a CO-CO2 atmosphere at a fO2 corresponding to NNO+1. Both reduction and oxidation experiments were performed. In reduction experiments (pure CO2 then CO-CO2 gas mixture), electrical conductivities progressively increase with time. The reverse is observed in oxidation experiments (CO-CO2 gas mixture then pure CO2). These variations of electrical conductivities are correlated with modifications of the Fe2+/Fe3+ ratio in the melt, and are consistent with the respective structural roles of Fe2+ and Fe3+. In both types of experiments, a minimum of about 400 mn is necessary before a plateau is reached, interpreted to reflect the kinetics of attainment of the equilibrium Fe2+/Fe3+ ratio in the melt. Differences between plateau and initial values are typically of a few ohms, much higher than the sensitivity of our measurements (better than 0.1 ohm). When increasing temperature, the time required for reaching plateau values decreases. At NNO+1, the electrical activation energy (Ea) was determined for

  15. Redox State of Iron in Lunar Glasses using X-ray Absorption Spectroscopy and Multivariate Analysis

    NASA Astrophysics Data System (ADS)

    Dyar, M. D.; McCanta, M. C.; Lanzirotti, A.; Sutton, S. R.; Carey, C. J.; Mahadevan, S.; Rutherford, M. J.

    2014-12-01

    The oxidation state of igneous materials on a planet is a critically-important variable in understanding magma evolution on bodies in our solar system. However, direct and indirect methods for quantifying redox states are challenging, especially across the broad spectrum of silicate glass compositions found on airless bodies. On the Moon, early Mössbauer studies of bulk samples suggested the presence of significant Fe3+ (>10%) in lunar glasses (green, orange, brown); lunar analog glasses synthesized at fO2 <10-11 have similar Fe3+. All these Mössbauer spectra are challenging to interpret due to the presence of multiple coordination environments in the glasses. X-ray absorption spectroscopy (XAS) allows pico- and nano-scale interrogation of primitive planetary materials using the pre-edge, main edge, and EXAFS regions of absorption edge spectra. Current uses of XAS require availability of standards with compositions similar to those of unknowns and complex procedures for curve-fitting of pre-edge features that produce results with poorly constrained accuracy. A new approach to accurate and quantitative redox measurements with XAS is to couple use of spectra from synthetic glass standards covering a broad compositional range with multivariate analysis (MVA) techniques. Mössbauer and XAS spectra from a suite of 33 synthetic glass standards covering a wide range of compositions and fO2(Dyar et al., this meeting) were used to develop a MVA model that utilizes valuable predictive information not only in the major spectral peaks/features, but in all channels of the XAS region. Algorithms for multivariate analysis t were used to "learn" the characteristics of a data set as a function of varying spectral characteristics. These models were applied to the study of lunar glasses, which provide a challenging test case for these newly-developed techniques due to their very low fO2. Application of the new XAS calibration model to Apollo 15 green (15426, 15427 and 15425

  16. A symmetric organic-based nonaqueous redox flow battery and its state of charge diagnostics by FTIR

    DOE PAGESBeta

    Duan, Wentao; Vemuri, Rama Ses; Milshtein, Jarrod D.; Laramie, Sydney; Dmello, Rylan D.; Huang, Jinhua; Zhang, Lu; Hu, Dehong; Vijayakumar, M.; Wang, Wei; et al

    2016-03-10

    Redox flow batteries have shown outstanding promise for grid-scale energy storage to promote utilization of renewable energy and improve grid stability. Nonaqueous battery systems can potentially achieve high energy density because of their broad voltage window. In this paper, we report a new organic redox-active material for use in a nonaqueous redox flow battery, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) that has high solubility (>2.6 M) in organic solvents. PTIO exhibits electrochemically reversible disproportionation reactions and thus can serve as both anolyte and catholyte redox materials in a symmetric flow cell. The PTIO flow battery has a moderate cell voltage of ~1.7 V andmore » shows good cyclability under both cyclic voltammetry and flow cell conditions. Moreover, we demonstrate that FTIR can offer accurate estimation of the PTIO concentration in electrolytes and determine the state of charge of the PTIO flow cell, which suggests FTIR potentially as a powerful online battery status sensor. In conclusion, this study is expected to inspire more insights in this under-addressed area of state of charge analysis aiming at operational safety and reliability of flow batteries.« less

  17. Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+-ATPase.

    PubMed

    Ibarra, F R; Cheng, S X Jun; Agrén, M; Svensson, L-B; Aizman, O; Aperia, A

    2002-06-01

    The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on alpha-adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+-adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+-ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the alpha-adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+-ATPase on the Ser23 residue. The level of PKC induced Na+,K+-ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+-ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+-ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+-ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+-ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+-ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+-ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis. PMID:12028137

  18. Modeling the Time-dependent Changes in Electrical Conductivity of Basaltic Melts With Redox State

    NASA Astrophysics Data System (ADS)

    Pommier, A.; Gaillard, F.; Pichavant, M.

    2008-12-01

    The electrical conductivity σ is an efficient probe of mass transfer processes within silicate melts and magmas. Little attention has been given to the influence of redox state (fO2) on the melts conductivity. We present an experimental setup allowing electrical conductivity measurements for basaltic melts under variable fO2. We demonstrate a significant dependence of σ with fO2, allowing to characterize in situ the mechanisms and kinetics of redox changes in the melt. Experiments were conducted on basalts from Pu'u 'O'o, Hawaii, and Mt.Vesuvius, Italy. Measurements were performed cylindrical glass samples (OD: 6mm, ID: 1mm, L: 8mm) using an impedance spectrometer. Experiments were conducted in a 1atm vertical furnace, from 1200°C to 1400°C. Variable gas atmosphere (air, CO2 or CO-CO2 gas mixtures) were used, imposing ΔNNO from -1 to +7. Electrical conductivities were determined for the two melts at constant fO2, different T (constant fO2) and constant T, different fO2 (variable fO2) obtained by changing the gas composition. Isothermal reduction and oxidation cycles were performed. Glasses quenched from different T and fO2 conditions were analyzed by electron microprobe, the FeO concentration was determined by wet chemistry. In constant fO2 experiments, a small but detectable effect of fO2 on σ is evidenced. At 1300°C, the difference in the Kilauea sample conductivity between reduced (ΔNNO=-1) and oxidized (ΔNNO=+7) fO2 is <1(ohm.m)-1, the sample being more conductive when reduced. The temperature dependence of σ was fitted using Arrhenian equations, the activation energy Ea being 100kJ/mol. Sodium was identified as the main charge carrier in the melts. The fO2-effect on σ can thus be attributed to the influence of the Fe2+/Fe3+ ratio on sodium mobility. The fO2-dependence of σ was included in the model of Pommier et al.(2008), allowing the conductivity of natural melts to be calculated as a function of T, P, H2O, and fO2. Variable fO2 experiments

  19. Effects of Protonation State on a Tyrosine-Histidine Bioinspired Redox Mediator

    SciTech Connect

    Moore, Gary F.; Hambourger, Michael; Kodis, Gerdenis; Michl, Weston; Gust, Devens; Moore, Thomas A.; Moore, Ana L.

    2010-11-18

    The conversion of tyrosine to the corresponding tyrosyl radical in photosytem II (PSII) is an example of proton-coupled electron transfer. Although the tyrosine moiety (TyrZ) is known to function as a redox mediator between the photo-oxidized primary donor (P680 •+) and the Mn-containing oxygen-evolving complex, the protonation states involved in the course of the reaction remain an active area of investigation. Herein, we report on the optical, structural, and electrochemical properties of tyrosine-histidine constructs, which model the function of their naturally occurring counterparts in PSII. Electrochemical studies show that the phenoxyl/phenol couple of the model is chemically reversible and thermodynamically capable of water oxidation. Studies under acidic and basic conditions provide clear evidence that an ionizable proton controls the electrochemical potential of the tyrosine-histidine mimic and that an exogenous base or acid can be used to generate a low-potential or high-potential mediator, respectively. The phenoxyl/phenoxide couple associated with the low-potential mediator is thermodynamically incapable of water oxidation, whereas the relay associated with the high-potential mediator is thermodynamically incapable of reducing an attached photoexcited porphyrin. These studies provide insight regarding the mechanistic role of the tyrosine-histidine complex in water oxidation and strategies for making use of hydrogen bonds to affect the coupling between proton and electron transfer in artificial photosynthetic systems.

  20. Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

    PubMed Central

    Paes, Lisvane Silva; Suárez Mantilla, Brian; Zimbres, Flávia Menezes; Pral, Elisabeth Mieko Furusho; Diogo de Melo, Patrícia; Tahara, Erich B.; Kowaltowski, Alicia J.; Elias, Maria Carolina; Silber, Ariel Mariano

    2013-01-01

    Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ1-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle. PMID:23894476

  1. Changes in the redox state of sediments following the 2010 BP blowout

    NASA Astrophysics Data System (ADS)

    Hastings, D. W.; Brooks, G.; Hollander, D. J.; Larson, R. A.; Morford, J. L.; Romero, I.; Hammaker, S.; Hogan, A.; Roeder, T. K.

    2012-12-01

    We have collected multi-core sediment cores from over 40 sites along the NE Gulf of Mexico continental slope following the 2010 Deepwater Horizon oil spill. We present the geochemical results from four select sites collected on August 2010, December 2010, February 2011, September 2011, and August 2012. Cores were extruded at 2 mm intervals, and sediments were analyzed for TOC, 13C, carbonate, short-lived radioisotopes (Pb-210, Cs-137, Be-7, Th-234) and grain size. Cores reveal a well-defined, internally stratified dark brown layer in the top 1-6 cm, with finer grain size than underlying sediments. Samples were digested at high temperature and pressure in concentrated nitric acid to dissolve both the oil and authigenic fractions, but not the detrital component. Samples were subsequently analyzed by ICP-MS. Although the Macondo crude oil is slightly enriched in Ni, V, and Co, with concentrations of 2.8, 0.9, and 0.08ppm, respectively, no significant enrichment of these metals is observed in Gulf of Mexico sediments. Sediment mass accumulation rates following the event range from 0.6 - 20 g/cm2/yr, which are one to two orders of magnitude higher than pre-spill rates. Organic and inorganic carbon deposition rates from the 2010 and February 2011 cores are also elevated one to two orders of magnitude. 13C signatures of this recent deposited material are slightly depleted relative to pre-oil event material. Large sedimentation rates, depleted 13C values and lack of bioturbation on the surface of the deep sediments studied supports the hypothesis of a large sea-snow-like blizzard event during the oil-spill in 2010. Bacterially mediated oxidation of organic matter is reflected in a well-established sequence of oxidation-reduction reactions. We exploit redox sensitive trace elements including Mn, Fe, Re, U, Mo, and V to infer changes in the redox state of sediments following this large pulse of organic matter to the seafloor.

  2. Increased generation of intracellular reactive oxygen species initiates selective cytotoxicity against the MCF-7 cell line resultant from redox active combination therapy using copper-thiosemicarbazone complexes.

    PubMed

    Akladios, Fady N; Andrew, Scott D; Parkinson, Christopher J

    2016-06-01

    The combination of cytotoxic copper-thiosemicarbazone complexes with phenoxazines results in an up to 50-fold enhancement in the cytotoxic potential of the thiosemicarbazone against the MCF-7 human breast adenocarcinoma cell line over the effect attributable to drug additivity-allowing minimization of the more toxic copper-thiosemicarbazone component of the therapy. The combination of a benzophenoxazine with all classes of copper complex examined in this study proved more effective than combinations of the copper complexes with related isoelectronic azines. The combination approach results in rapid elevation of intracellular reactive oxygen levels followed by apoptotic cell death. Normal fibroblasts representative of non-cancerous cells (MRC-5) did not display a similar elevation of reactive oxygen levels when exposed to similar drug levels. The minimization of the copper-thiosemicarbazone component of the therapy results in an enhanced safety profile against normal fibroblasts. PMID:26951232

  3. Crocin Effects on Human Myeloma Cells Regarding Intracellular Redox State, DNA Fragmentation, and Apoptosis or Necrosis Profile

    PubMed Central

    Rezaee, Ramin; Jamialahmadi, Khadijeh; Riahi Zanjani, Bamdad; Mahmoudi, Mahmoud; Abnous, Khalil; Zamani Taghizadeh Rabe, Shahrzad; Tabasi, Nafiseh; Zali, Marjan; Rezaee, Marjan; Amin, Bahareh; Karimi, Gholamreza

    2014-01-01

    Background: Well-documented studies reported several pharmacological properties for crocin, the active compound of Crocus sativus, such as its antitumor, radical scavenging, antidepressant, and memory-enhancing effects. Objectives: We aimed to evaluate the possible cytotoxic activity of crocin on B lymphocytes in human myeloma (U266 cell line) after 24- and 48-hour treatment. Materials and Methods: For this purpose, cell viability was determined by the colorimetric MTT assay and cell death pattern was evaluated using Annexin V-FITC/propidium iodide (PI) apoptosis detection kit. ROS (reactive oxygen species) production and DNA fragmentation were assessed using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) kit and PI staining, respectively. Results: The highest concentration of crocin significantly decreased ROS production after 48 hours of treatment. However, crocin had no effect on the expression level of HSP (Heat shock protein). Additionally, its administration caused a mild decline in cell viability and a mild increase in the population of DNA fragmented cells as well as apoptosis. Conclusions: In our study, no prominent effect was seen; therefore, in order to have a better perspective of crocin activity against cancerous cell lines, further studies are highly recommended. PMID:25625054

  4. Proteomics links the redox state to calcium signaling during bleaching of the scleractinian coral Acropora microphthalma on exposure to high solar irradiance and thermal stress.

    PubMed

    Weston, Andrew J; Dunlap, Walter C; Beltran, Victor H; Starcevic, Antonio; Hranueli, Daslav; Ward, Malcolm; Long, Paul F

    2015-03-01

    Shipboard experiments were each performed over a 2 day period to examine the proteomic response of the symbiotic coral Acropora microphthalma exposed to acute conditions of high temperature/low light or high light/low temperature stress. During these treatments, corals had noticeably bleached. The photosynthetic performance of residual algal endosymbionts was severely impaired but showed signs of recovery in both treatments by the end of the second day. Changes in the coral proteome were determined daily and, using recently available annotated genome sequences, the individual contributions of the coral host and algal endosymbionts could be extracted from these data. Quantitative changes in proteins relevant to redox state and calcium metabolism are presented. Notably, expression of common antioxidant proteins was not detected from the coral host but present in the algal endosymbiont proteome. Possible roles for elevated carbonic anhydrase in the coral host are considered: to restore intracellular pH diminished by loss of photosynthetic activity, to indirectly limit intracellular calcium influx linked with enhanced calmodulin expression to impede late-stage symbiont exocytosis, or to enhance inorganic carbon transport to improve the photosynthetic performance of algal symbionts that remain in hospite. Protein effectors of calcium-dependent exocytosis were present in both symbiotic partners. No caspase-family proteins associated with host cell apoptosis, with exception of the autophagy chaperone HSP70, were detected, suggesting that algal loss and photosynthetic dysfunction under these experimental conditions were not due to host-mediated phytosymbiont destruction. Instead, bleaching occurred by symbiont exocytosis and loss of light-harvesting pigments of algae that remain in hospite. These proteomic data are, therefore, consistent with our premise that coral endosymbionts can mediate their own retention or departure from the coral host, which may manifest as

  5. Proteomics Links the Redox State to Calcium Signaling During Bleaching of the Scleractinian Coral Acropora microphthalma on Exposure to High Solar Irradiance and Thermal Stress

    PubMed Central

    Weston, Andrew J.; Dunlap, Walter C.; Beltran, Victor H.; Starcevic, Antonio; Hranueli, Daslav; Ward, Malcolm; Long, Paul F.

    2015-01-01

    Shipboard experiments were each performed over a 2 day period to examine the proteomic response of the symbiotic coral Acropora microphthalma exposed to acute conditions of high temperature/low light or high light/low temperature stress. During these treatments, corals had noticeably bleached. The photosynthetic performance of residual algal endosymbionts was severely impaired but showed signs of recovery in both treatments by the end of the second day. Changes in the coral proteome were determined daily and, using recently available annotated genome sequences, the individual contributions of the coral host and algal endosymbionts could be extracted from these data. Quantitative changes in proteins relevant to redox state and calcium metabolism are presented. Notably, expression of common antioxidant proteins was not detected from the coral host but present in the algal endosymbiont proteome. Possible roles for elevated carbonic anhydrase in the coral host are considered: to restore intracellular pH diminished by loss of photosynthetic activity, to indirectly limit intracellular calcium influx linked with enhanced calmodulin expression to impede late-stage symbiont exocytosis, or to enhance inorganic carbon transport to improve the photosynthetic performance of algal symbionts that remain in hospite. Protein effectors of calcium-dependent exocytosis were present in both symbiotic partners. No caspase-family proteins associated with host cell apoptosis, with exception of the autophagy chaperone HSP70, were detected, suggesting that algal loss and photosynthetic dysfunction under these experimental conditions were not due to host-mediated phytosymbiont destruction. Instead, bleaching occurred by symbiont exocytosis and loss of light-harvesting pigments of algae that remain in hospite. These proteomic data are, therefore, consistent with our premise that coral endosymbionts can mediate their own retention or departure from the coral host, which may manifest as

  6. Does the Cerebrospinal Fluid Reflect Altered Redox State But Not Neurotrophic Support Loss in Parkinson's Disease?

    PubMed

    Martín de Pablos, Angel; García-Moreno, José-Manuel; Fernández, Emilio

    2015-10-10

    Alteration in neurotrophic factors support and antioxidant defenses in the central nervous system (CNS) along with deficit of ferritin have been associated with idiopathic Parkinson's disease (PD). The objectives were to analyze in the cerebrospinal fluid (CSF) of patients with PD and controls the following: (i) the levels of the neuroprotectant factors glial cell line-derived neurotrophic factor, persephin, neurturin, and brain-derived neurotrophic factor, (ii) the levels of transforming growth factor-β1 (TGFβ1) and transforming growth factor-β2 (TGFβ2), proinflammatory factors, (iii) the activity of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), catalase, superoxide dismutases (SODs), and peroxiredoxins (PRDxs), and (iv) ferritin levels. The study revealed that, among neurotrophic factors, only TGFβ1 levels were found to be enhanced in patients with PD (early, p < 0.05; advanced, p < 0.02). Regarding antioxidant enzymes, the activity of GPx, catalase, and PRDxs, all hydrogen peroxide scavengers, was found to be significantly reduced in patients (GPx, p < 0.001; catalase, p < 0.01; PRDxs, p < 0.01, one-way analysis of variance). Finally, ferritin content in CSF was significantly diminished over time in patients (early, p < 0.01, -49%; advanced, p < 0.001, -80.7%). Our observations lead to the hypothesis that parkinsonian patients suffer from a serious disturbance of redox state in the CNS, as evaluated through the CSF, characterized by reduced hydrogen peroxide scavenging and iron storage. PMID:26263410

  7. No Evidence of Exogenous Origin for the Abnormal Glutathione Redox State in Schizophrenia

    PubMed Central

    Ballesteros, Alejandro; Jiang, Pan; Summerfelt, Ann; Du, Xiaoming; Chiappelli, Joshua; O’Donnell, Patricio; Kochunov, Peter; Hong, L. Elliot

    2013-01-01

    Schizophrenia has been associated with low glutathione (GSH), one of the most important substrates for natural defense against oxidative stress. This abnormality is often attributed to genetic or other pathological causes. However, low GSH in schizophrenia could also be due to insufficient antioxidant consumption or other exogenous factors. We evaluated GSH in relation to diet, smoking, and medication status in schizophrenia patients. We recruited 54 participants (29 schizophrenia patients and 25 normal controls). The Antioxidant Dietary Source Questions was used to estimate the total antioxidant capacity (TAC) from participants’ diet. GSH and the oxidized form of glutathione (GSSG) were assayed. We found that GSH was significantly lower (p < 0.001) while %GSSG was 2 to 5 fold higher (p = 0.023) in patients compared with controls. No evidence for lower TAC dietary intake was found in schizophrenia patients compared with controls; rather nominally higher TAC level was found in the patients diet (p=0.02). Analysis of consumption of individual food categories also failed to find evidence of reduced dietary antioxidant intake in schizophrenia patients. Smoking and medications did not significantly predict the GSH deficit either. However, there was a significant smoking by diagnosis interaction on GSH (p=0.026) such that smoking was associated with higher GSH level in controls while smoking in patients was not associated with this effect. Schizophrenia patients may have an impaired upregulation of GSH synthesis that normally occurs due to smoking-induced antioxidative response. Lower GSH was independently present in patients on clozapine (p = 0.005) and patients on other antipsychotics (p < 0.001) compared with controls. In conclusion, none of the exogenous sources played a major role in explaining abnormalities in the glutathione pathway in patients. The state of abnormal glutathione redox may therefore be a part of schizophrenia pathophysiology. PMID:23466187

  8. Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration

    PubMed Central

    Zhang, Dongyun; Wang, Yulei; Liang, Yuguang; Zhang, Min; Wei, Jinlong; Zheng, Xiao; Li, Fei; Meng, Yan; Zhu, Nina Wu; Li, Jingxia; Wu, Xue-Ru; Huang, Chuanshu

    2014-01-01

    ABSTRACT Cell migration is a dynamic process that is central to a variety of physiological functions as well as disease pathogenesis. The modulation of cell migration by p27 (officially known as CDKN1B) has been reported, but the exact mechanism(s) whereby p27 interacts with downstream effectors that control cell migration have not been elucidated. By systematically comparing p27+/+ mouse embryonic fibroblasts (MEFs) with genetically ablated p27−/− MEFs using wound-healing, transwell and time-lapse microscopic analyses, we provide direct evidence that p27 inhibits both directional and random cell migration. Identical results were obtained with normal and cancer epithelial cells using complementary knockdown and overexpression approaches. Additional studies revealed that overexpression of manganese superoxide dismutase (MnSOD, officially known as SOD2) and reduced intracellular oxidation played a key role in increased cell migration in p27-deficient cells. Furthermore, we identified signal transducer and activator of transcription 3 (STAT3) as the transcription factor responsible for p27-regulated MnSOD expression, which was further mediated by ERK- and ATF1-dependent transactivation of the cAMP response element (CRE) within the Stat3 promoter. Collectively, our data strongly indicate that p27 plays a crucial negative role in cell migration by inhibiting MnSOD expression in a STAT3-dependent manner. PMID:24727615

  9. Faradic redox active material of Cu7S4 nanowires with a high conductance for flexible solid state supercapacitors

    NASA Astrophysics Data System (ADS)

    Javed, Muhammad Sufyan; Dai, Shuge; Wang, Mingjun; Xi, Yi; Lang, Qiang; Guo, Donglin; Hu, Chenguo

    2015-08-01

    The exploration of high Faradic redox active materials with the advantages of low cost and low toxicity has been attracting great attention for producing high energy storage supercapacitors. Here, the high Faradic redox active material of Cu7S4-NWs coated on a carbon fiber fabric (CFF) is directly used as a binder-free electrode for a high performance flexible solid state supercapacitor. The Cu7S4-NW-CFF supercapacitor exhibits excellent electrochemical performance such as a high specific capacitance of 400 F g-1 at the scan rate of 10 mV s-1 and a high energy density of 35 Wh kg-1 at a power density of 200 W kg-1, with the advantages of a light weight, high flexibility and long term cycling stability by retaining 95% after 5000 charge-discharge cycles at a constant current of 10 mA. The high Faradic redox activity and high conductance behavior of the Cu7S4-NWs result in a high pseudocapacitive performance with a relatively high specific energy and specific power. Such a new type of pseudocapacitive material of Cu7S4-NWs with its low cost is very promising for actual application in supercapacitors.The exploration of high Faradic redox active materials with the advantages of low cost and low toxicity has been attracting great attention for producing high energy storage supercapacitors. Here, the high Faradic redox active material of Cu7S4-NWs coated on a carbon fiber fabric (CFF) is directly used as a binder-free electrode for a high performance flexible solid state supercapacitor. The Cu7S4-NW-CFF supercapacitor exhibits excellent electrochemical performance such as a high specific capacitance of 400 F g-1 at the scan rate of 10 mV s-1 and a high energy density of 35 Wh kg-1 at a power density of 200 W kg-1, with the advantages of a light weight, high flexibility and long term cycling stability by retaining 95% after 5000 charge-discharge cycles at a constant current of 10 mA. The high Faradic redox activity and high conductance behavior of the Cu7S4-NWs result in

  10. Differential recruitment of PKC isoforms in HeLa cells during redox stress

    PubMed Central

    Rimessi, Alessandro; Rizzuto, Rosario; Pinton, Paolo

    2007-01-01

    The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation. PMID:18229448

  11. Untargeted Metabolomics Analysis Reveals a Link between ETHE1-Mediated Disruptive Redox State and Altered Metabolic Regulation.

    PubMed

    Sahebekhtiari, Navid; Nielsen, Camilla Bak; Johannsen, Mogens; Palmfeldt, Johan

    2016-05-01

    Defects in the gene encoding the persulfide dioxygenase ETHE1 are known to cause the severe inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of known clinical characteristics, the molecular mechanisms underlying the ETHE1 deficiency are still obscure. Herein, to further analyze the molecular phenotype of the disease, we applied an untargeted metabolomics approach on cultivated fibroblasts of EE patients for pinpointing alterations in metabolite levels. Metabolites, as direct signatures of biochemical functions, can decipher biochemical pathways involved in the cellular phenotype of patient cells. Using liquid chromatography-mass spectrometry-based untargeted metabolomics, we identified 18 metabolites that have altered levels in fibroblasts from EE patients. Our data demonstrate disrupted redox state in EE patient cells, which is reflected by significantly decreased level of reduced glutathione. Furthermore, the down-regulation of several intermediate metabolites such as the redox cofactors NAD(+) and NADH as well as Krebs cycle intermediates revealed clear alteration in metabolic regulation. Pantothenic acid and several amino acids exhibited decreased levels, whereas the β-citrylglutamate with a putative role in brain development had an increased level in the EE patient cells. These observations indicate the severe impact of ETHE1 deficiency on cellular physiology and redox state, meanwhile suggesting targets for experimental studies on novel treatment options for the devastating metabolic disorder. PMID:27074420

  12. Vitamin C in Health and Disease: Its Role in the Metabolism of Cells and Redox State in the Brain.

    PubMed

    Figueroa-Méndez, Rodrigo; Rivas-Arancibia, Selva

    2015-01-01

    Ever since Linus Pauling published his studies, the effects of vitamin C have been surrounded by contradictory results. This may be because its effects depend on a number of factors such as the redox state of the body, the dose used, and also on the tissue metabolism. This review deals with vitamin C pharmacokinetics and its participation in neurophysiological processes, as well as its role in the maintenance of redox balance. The distribution and the concentration of vitamin C in the organs depend on the ascorbate requirements of each and on the tissue distribution of sodium-dependent vitamin C transporter 1 and 2 (SVCT1 and SVCT2). This determines the specific distribution pattern of vitamin C in the body. Vitamin C is involved in the physiology of the nervous system, including the support and the structure of the neurons, the processes of differentiation, maturation, and neuronal survival; the synthesis of catecholamine, and the modulation of neurotransmission. This antioxidant interacts with self-recycling mechanisms, including its participation in the endogenous antioxidant system. We conclude that the pharmacokinetic properties of ascorbate are related to the redox state and its functions and effects in tissues. PMID:26779027

  13. Assessing the effect of humic acid redox state on organic pollutant sorption by combined electrochemical reduction and sorption experiments.

    PubMed

    Aeschbacher, Michael; Brunner, Sibyl H; Schwarzenbach, René P; Sander, Michael

    2012-04-01

    Natural Organic Matter (NOM) is a major sorbent for organic pollutants in soils and sediments. While sorption under oxic conditions has been well investigated, possible changes in the sorption capacity of a given NOM induced by reduction have not yet been studied. Reduction of quinones to hydroquinones, the major redox active moieties in NOM, increases the number of H-donor moieties and thus may affect sorption. This work compares the sorption of four nonionic organic pollutants of different polarities (naphthalene, acetophenone, quinoline, and 2-naphthol), and of the organocation paraquat to unreduced and electrochemically reduced Leonardite Humic Acid (LHA). The redox states of reduced and unreduced LHA in all sorption experiments were stable, as demonstrated by a spectrophotometric 2,6-dichlorophenol indophenol reduction assay. The sorption isotherms of the nonionic pollutants were highly linear, while paraquat sorption was strongly concentration dependent. LHA reduction did not result in significant changes in the sorption of all tested compounds, not even of the cationic paraquat at pH 7, 9, and 11. This work provides the first evidence that changes in NOM redox state do not largely affect organic pollutant sorption, suggesting that current sorption models are applicable both to unreduced and to reduced soil and sediment NOM. PMID:22372874

  14. Vitamin C in Health and Disease: Its Role in the Metabolism of Cells and Redox State in the Brain

    PubMed Central

    Figueroa-Méndez, Rodrigo; Rivas-Arancibia, Selva

    2015-01-01

    Ever since Linus Pauling published his studies, the effects of vitamin C have been surrounded by contradictory results. This may be because its effects depend on a number of factors such as the redox state of the body, the dose used, and also on the tissue metabolism. This review deals with vitamin C pharmacokinetics and its participation in neurophysiological processes, as well as its role in the maintenance of redox balance. The distribution and the concentration of vitamin C in the organs depend on the ascorbate requirements of each and on the tissue distribution of sodium-dependent vitamin C transporter 1 and 2 (SVCT1 and SVCT2). This determines the specific distribution pattern of vitamin C in the body. Vitamin C is involved in the physiology of the nervous system, including the support and the structure of the neurons, the processes of differentiation, maturation, and neuronal survival; the synthesis of catecholamine, and the modulation of neurotransmission. This antioxidant interacts with self-recycling mechanisms, including its participation in the endogenous antioxidant system. We conclude that the pharmacokinetic properties of ascorbate are related to the redox state and its functions and effects in tissues. PMID:26779027

  15. Regulation of the unbalanced redox state in a Schizosaccharomyces pombe tert-butyl hydroperoxide-resistant mutant.

    PubMed

    Gazdag, Z; Kálmán, Nikoletta; Blaskó, Agnes; Virág, Eszter; Belágyi, J; Pesti, M

    2014-06-01

    The one-gene mutation in the tert-butyl hydroperoxide-resistant mutant hyd1-190 of the fission yeast Schizosaccharomyces pombe led to a 4-fold increase in resistance to t-BuOOH and decreased specific concentrations of superoxide and total thiols in comparison with the parental strain hyd+. It suggested an unbalanced redox state of the cells, which induced continuously increased specific activities of glutathione peroxidase, glutathione reductase and glutathione S-transferase and decreased activities of the antioxidant enzymes superoxide dismutases and glucose-6-phosphate dehydrogenase to regulate the redox balance of the mutation-induced permanent, low-level but tolerable internal stress. These results may contribute to the understanding of internal, oxidative stress-related human diseases. PMID:24873914

  16. Assignment of Individual Metal Redox States in a Metalloprotein By Crystallographic Refinement at Multiple X-Ray Wavelengths

    SciTech Connect

    Einsle, O.; Andrade, S.L.A.; Dobbek, H.; Meyer, J.; Rees, D.C.; /Gottingen U. /Bayreuth U. /DRDC, Grenoble /Caltech

    2007-07-09

    A method is presented to derive anomalous scattering contributions for individual atoms within a protein crystal by collecting several sets of diffraction data at energies spread along an X-ray absorption edge of the element in question. The method has been applied to a [2Fe:2S] ferredoxin model system with localized charges in the reduced state of the iron-sulfur cluster. The analysis shows that upon reduction the electron resides at the iron atom closer to the protein surface. The technique should be sufficiently sensitive for more complex clusters with noninteger redox states and is generally applicable given that crystals are available.

  17. Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs after Exposure to Hyperoxia.

    PubMed

    Sepehr, R; Audi, S H; Staniszewski, K S; Haworth, S T; Jacobs, E R; Ranji, M

    2013-10-16

    Recently we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide (FADH2,)) as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this study was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (>95% O2 for 48 hours) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia. PMID:25379360

  18. Cooling History and Redox State of NWA 8694 Chassignite: Comparison with Chassigny and NWA 2737

    NASA Technical Reports Server (NTRS)

    Mikouchi, T.; Takenouchi, A.; Zolensky, M. E.

    2016-01-01

    NWA 8694 is a new chassignite whose constituent minerals are more Fe-rich than those in the other known chassignites (Chassigny and NWA 2737), and may suggest a petrogenetic relationship to nakhlites. In this abstract we report mineralogy of NWA 8694 to infer its cooling rate and redox state, and discuss its thermal and shock history in comparison with other chassignites. NWA 8694 is a cumulate dunite of approximately 2 mm olivine with interstitial pyroxene and feldspar. Olivine is homogeneous (Fo(sub 55-56)), but Ca decreases at the approximately 50-100 micrometer rim (0.25-0.1 wt% CaO). Because the Ca-depleted rim is narrower than those in other chassignites (approximately 50 ?micrometer), NWA 8694 may have cooled slightly faster than the others (approximately 30 C/yr), but would be in the same order. Pyroxenes are low- and high-Ca pyroxenes, both exhibiting sub-micron exsolution textures (0.2-0.3 micrometer wide lamellae with the spacing of 0.8-1.8 micrometers). Although the low-Ca pyroxene host has an orthopyroxene composition (Wo approximately 2), the EBSD analysis suggests a pigeonite structure (P2(sub 1)/c), which is also reported from the Chassigny pyroxene. The size of exsolution texture is a bit smaller, but broadly similar to those in other chassignites, implying a similar fast cooling rate (35-43 C/yr). Feldspars are isotropic (plagioclase: clustered around An25Or10, K-feldspar: approximately An19Or78), suggestive of extensive shock metamorphism, consistent with undulatory extinction of olivine. Feldspar compositions are around the equilibrium isotherm of approximately 800 C. The olivine and chromite compositions give an equilibration temperature of 760-810 C and logfO2 of QFM+/-0.3. The inferred fast cooling rate and high fO2 of NWA 8694 are both similar to those of Chassigny and NWA 2737, and suggest a common formation condition (e.g., thick lava flow or shallow intrusion) under oxidizing condition. The Fe-rich mineral compositions of NWA 8694 may be

  19. Optical imaging of mitochondrial redox state in rodent models with 3-iodothyronamine

    PubMed Central

    Ghanian, Zahra; Maleki, Sepideh; Reiland, Hannah; Bütz, Daniel E; Chiellini, Grazia; Porter, Fariba-Assadi; Ranji, Mahsa

    2016-01-01

    This study used an optical technique to measure the effects of treating low (10 mg/kg) and high (25 mg/kg) doses of 3-iodothyronamine (T1AM) on the metabolism in the kidney and heart of mice. The ratio of two intrinsic fluorophores in tissue, (NADH/FAD), called the NADH redox ratio (NADH RR), is a marker of the metabolic state of the tissue. A cryofluorescence imaging instrument was used to provide a quantitative assessment of NADH RR in both kidneys and hearts in mice treated with 3-iodothyronamine. We compared those results to corresponding tissues in control mice. In the kidneys of mice treated with a high dose T1AM, the mean values of the maximum projection of NADH RR were 2.6 ± 0.6 compared to 3.20 ± 0.03 in control mice, indicating a 19% (± 0.4) significant increase in oxidative stress (OS) in the high dose-treated kidneys (P=0.047). However, kidneys treated with a low dose of T1AM showed no difference in NADH RR compared to the kidneys of control mice. Furthermore, low versus high dose treatment of T1AM showed different responses in the heart than in the kidneys. The mean value of the maximum projection of NADH RR in the heart changed from 3.0 ± 0.3 to 3.2 ± 0.6 for the low dose and the high dose T1AM-treated mice, respectively, as compared to 2.8 ± 0.7 in control mice. These values correspond to a 9% (±0.5) (P=0.045) and 14% (±0.5) (P=0.008) significant increase in NADH RR in the T1AM-treated hearts, indicating that the high dose T1AM-treated tissues have reduced OS compared to the low dose-treated tissues or the control tissues. These results suggest that while T1AM at a high dose increases oxidative response in kidneys, it has a protective effect in the heart and may exert its effect through alternative pathways at different doses and at tissue specific levels. PMID:24302559

  20. Myocardin-related Transcription Factor Regulates Nox4 Protein Expression: LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE.

    PubMed

    Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam; Dan, Qinghong; Knudsen, Teresa E T; Szeto, Stephen G; Yuen, Darren A; Szászi, Katalin; Pedersen, Stine F; Kapus, András

    2016-01-01

    findings uncover new MRTF- and TAZ/YAP-dependent mechanisms, which link cytoskeleton remodeling and redox state and impact epithelial plasticity and myofibroblast transition. PMID:26555261

  1. The Valence State of Silicon and Redox Dynamics in Aluminosilicate Melts

    NASA Astrophysics Data System (ADS)

    Cooper, R. F.; Pettersen, C.; Everman, R. L.

    2005-12-01

    Physicists have long been aware of the many valence states of Si and the roles these play in the kinetics of thermal oxidation of Si single crystals and the molecular structure of the amorphous oxide film (e.g., Borman et al., 1991). Similarly, the dynamics of oxidation and of vaporization of SiC are also affected by the presence of Si2+ in the amorphous silica surface film (e.g., Dunham et al., 1998; Mendybaev et al., 2002). Nevertheless, Si2+,4+ heterovalency is little considered in redox studies of silicate melts as reported in the petrology literature. We have performed experiments in which a liquid bronze (Cu,Sn) alloy was reacted with (1) a magnesium aluminosilicate melt and (2) a Zn2+-doped magnesium aluminosilicate melt, all done at a low oxygen fugacity (sufficient to keep the metal alloy from oxidizing in reaction with the gas environment). The driving potential for metal melt-silicate melt reaction has two components: (a) reduction of the silicate melt and oxidation of the metal alloy; (b) formation of a homogeneous silicate solution that incorporates ionic Cu and Sn. The reaction morphologies present compelling evidence that Si4+ in the silicate melt is reduced in part to Si2+, initially so as to incorporate Cu+,2+ into the melt; as the reaction proceeds, however, the Si2+ mobility becomes important in charge-compensation of the "inward" flux of Sn2+. Addition of Zn2+ to the starting silicate melt forces a spatially periodic variation in the silicate melt structure (as suggested by the chemistry) as the reaction proceeds. In separate experiments, reduction of an FeO-bearing calcium-magnesium aluminosilicate melt in a CO-rich environment creates a reaction morphology suggestive of reduction of Si4+ to facilitate the incorporation of carbonate ions into the melt. These experiments are perhaps exotic; nevertheless, they provoke the consideration of the potential role(s) played by silicon valence in any "self-buffering" process associated with the evolution

  2. Electrochemical stimulation of microbial reductive dechlorination of pentachlorophenol using solid-state redox mediator (humin) immobilization.

    PubMed

    Zhang, Dongdong; Zhang, Chunfang; Li, Zhiling; Suzuki, Daisuke; Komatsu, Daisuke D; Tsunogai, Urumu; Katayama, Arata

    2014-07-01

    Immobilized solid-phase humin on a graphite electrode set at -500 mV (vs. standard hydrogen electrode) significantly enhanced the microbial reductive dechlorination of pentachlorophenol as a stable solid-phase redox mediator in bioelectrochemical systems (BESs). Compared with the suspended system, the immobilized system dechlorinated PCP at a much higher efficiency, achieving 116 μmol Cl(-)g(-1) humin d(-1). Fluorescence microscopy showed a conspicuous growth of bacteria on the negatively poised immobilized humin. Electron balance analyses suggested that the electrons required for microbial dechlorination were supplied primarily from the humin-immobilized electrode. Microbial community analyses based on 16S rRNA genes showed that Dehalobacter and Desulfovibrio grew on the immobilized humin as potential dechlorinators. These findings extend the potential of BESs using immobilized solid-phase humin as the redox mediator for in situ bioremediation, given the wide distribution of humin and its efficiency and stability as a mediator. PMID:24859215

  3. Label-free capacitive diagnostics: exploiting local redox probe state occupancy.

    PubMed

    Lehr, Joshua; Hobnouse, George C; Fernandes, Flávio C Bedatty; Bueno, Paulo R; Davis, Jason J

    2014-03-01

    An electrode surface confined redox group contributes to a substantial potential-dependent interfacial charging that can be sensitively probed and frequency-resolved by impedance-derived capacitance spectroscopy. In utilizing the sensitivity of this charging fingerprint to redox group environment, one can seek to generate derived sensory configurations. Exemplified here through the generation of mixed molecular films comprising ferrocene and antibody receptors to two clinically important targets, the label-free methodology is able to report on human prostatic acid phosphatase (PAP), a tumor marker, with a limit of detection of 11 pM and C-reactive protein with a limit of detection of 28 pM. Both assays exhibit linear ranges encompassing those of clinical value. PMID:24491045

  4. Redox state of p63 and p73 core domains regulates sequence-specific DNA binding.

    PubMed

    Tichý, Vlastimil; Navrátilová, Lucie; Adámik, Matej; Fojta, Miroslav; Brázdová, Marie

    2013-04-19

    Cysteine oxidation and covalent modification of redox sensitive transcription factors including p53 are known, among others, as important events in cell response to oxidative stress. All p53 family proteins p53, p63 and p73 act as stress-responsive transcription factors. Oxidation of p53 central DNA binding domain destroys its structure and abolishes its sequence-specific binding by affecting zinc ion coordination at the protein-DNA interface. Proteins p63 and p73 can bind the same response elements as p53 but exhibit distinct functions. Moreover, all three proteins contain highly conserved cysteines in central DNA binding domain suitable for possible redox modulation. In this work we report for the first time the redox sensitivity of p63 and p73 core domains to a thiol oxidizing agent azodicarboxylic acid bis[dimethylamide] (diamide). Oxidation of both p63 and p73 abolished sequence-specific binding to p53 consensus sequence, depending on the agent concentration. In the presence of specific DNA all p53 family core domains were partially protected against loss of DNA binding activity due to diamide treatment. Furthermore, we detected conditional reversibility of core domain oxidation for all p53 family members and a role of zinc ions in this process. We showed that p63 and p73 proteins had greater ability to resist the diamide oxidation in comparison with p53. Our results show p63 and p73 as redox sensitive proteins with possible functionality in response of p53 family proteins to oxidative stress. PMID:23501101

  5. Definition of the redox states of cobalt-precorrinoids: investigation of the substrate and redox specificity of CbiL from Salmonella typhimurium.

    PubMed

    Spencer, P; Stolowich, N J; Sumner, L W; Scott, A I

    1998-10-20

    The enzyme CbiL from the facultative anaerobe Salmonella typhimurium exhibits a high degree of homology to CobI from the aerobe Pseudomonas denitrificans (29% identity; 51% conservation obtained by a Blastp search of the ncbi database). As CobI catalyzes the third methylation in the aerobic pathway to vitamin B12 it is proposed that CbiL catalyzes the analogous step in the anaerobic pathway. Potential metallo and metal-free substrates were characterized and their redox states defined by a combination of physicochemical techniques (MALDI-MS, NMR, UV/vis, IR, and EPR) and then used to investigate the function of CbiL. CbiL exhibited an absolute requirement for the presence of a metal ion (Co(II), Ni(II), or Zn(II)) within the tetrapyrrole substrate. CbiL had no preference for the redox state of its cobalt tetrapyrrole substrate, methylating both the reduced form, Co(II) 2, 7-dimethyl-dipyrrocorphin (Co(II)-precorrin-2), and the oxidized form, Co(III) 2,7-dimethyl-isobacterioclorin (Co(III)-factor-II). In contrast CbiL had a marked preference for the oxidized Ni(II) and Zn(II)-2,7-dimethyl-isobacteriochlorin (Ni(II) and Zn(II)-factor-II). Removal of the metal ion from a product of CbiL (Zn(II)-factor-III) allowed characterization by 13C NMR, identifying the tetrapyrrole as 2,7,20-trimethyl-isobacteriochlorin (factor-3), indicating that CbiL methylates at C20, the same site as that methylated by CobI. Competition experiments, utilizing isotopic labeling to distinguish otherwise identical mass substrates and products, revealed that oxidized Co(III) or Ni(II)-factor-II were equally good substrates, whereas Co(II)-precorrin-2 was much preferred over Ni(II)-precorrin-2. Excess Ni(II)-precorrin-2 did not decrease CbiL methylation of Co(II)-precorrin-2, implying that CbiL has a low affinity for Ni(II)-precorrin-2. These results are interpreted on the basis of tetrapyrrole ruffling occurring on the optimization of the metallo-N bond distances. The greater flexibility of the

  6. Mitoplasticity: adaptation biology of the mitochondrion to the cellular redox state in physiology and carcinogenesis.

    PubMed

    Jose, Caroline; Melser, Su; Benard, Giovanni; Rossignol, Rodrigue

    2013-03-01

    Adaptation and transformation biology of the mitochondrion to redox status is an emerging domain of physiology and pathophysiology. Mitochondrial adaptations occur in response to accidental changes in cellular energy demand or supply while mitochondrial transformations are a part of greater program of cell metamorphosis. The possible role of mitochondrial adaptations and transformations in pathogenesis remains unexplored, and it has become critical to decipher the stimuli and the underlying molecular pathways. Immediate activation of mitochondrial function was described during acute exercise, respiratory chain injury, Endoplasmic Reticulum stress, genotoxic stress, or environmental toxic insults. Delayed adaptations of mitochondrial form, composition, and functions were evidenced for persistent changes in redox status as observed in endurance training, in fibroblasts grown in presence of respiratory chain inhibitors or in absence of glucose, in the smooth muscle of patients with severe asthma, or in the skeletal muscle of patients with a mitochondrial disease. Besides, mitochondrial transformations were observed in the course of human cell differentiation, during immune response activation, or in cells undergoing carcinogenesis. Little is known on the signals and downstream pathways that govern mitochondrial adaptations and transformations. Few adaptative loops, including redox sensors, kinases, and transcription factors were deciphered, but their implication in physiology and pathology remains elusive. Mitoplasticity could play a protective role against aging, diabetes, cancer, or neurodegenerative diseases. Research on adaptation and transformation could allow the design of innovative therapies, notably in cancer. PMID:22989324

  7. The Redox State of SECIS Binding Protein 2 Controls Its Localization and Selenocysteine Incorporation Function

    PubMed Central

    Papp, Laura V.; Lu, Jun; Striebel, Frank; Kennedy, Derek; Holmgren, Arne; Khanna, Kum Kum

    2006-01-01

    Selenoproteins are central controllers of cellular redox homeostasis. Incorporation of selenocysteine (Sec) into selenoproteins employs a unique mechanism to decode the UGA stop codon. The process requires the Sec insertion sequence (SECIS) element, tRNASec, and protein factors including the SECIS binding protein 2 (SBP2). Here, we report the characterization of motifs within SBP2 that regulate its subcellular localization and function. We show that SBP2 shuttles between the nucleus and the cytoplasm via intrinsic, functional nuclear localization signal and nuclear export signal motifs and that its nuclear export is dependent on the CRM1 pathway. Oxidative stress induces nuclear accumulation of SBP2 via oxidation of cysteine residues within a redox-sensitive cysteine-rich domain. These modifications are efficiently reversed in vitro by human thioredoxin and glutaredoxin, suggesting that these antioxidant systems might regulate redox status of SBP2 in vivo. Depletion of SBP2 in cell lines using small interfering RNA results in a decrease in Sec incorporation, providing direct evidence for its requirement for selenoprotein synthesis. Furthermore, Sec incorporation is reduced substantially after treatment of cells with agents that cause oxidative stress, suggesting that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins. PMID:16782878

  8. West Valley glass product qualification durability studies, FY 1987--1988: Effects of composition, redox state, thermal history, and groundwater

    SciTech Connect

    Reimus, M.A.H.; Piepel, G.F.; Mellinger, G.B.; Bunnell, L.R.

    1988-11-01

    The product qualification subtask of the West Valley Support Task (WVST) at Pacific Northwest Laboratory (PNL) provides support for the waste form qualification efforts at West Valley Nuclear Services Co. Testing is being conducted to determine waste form chemical durability in support of these efforts. The effects of composition, ferrous/ferric ratio (redox state), thermal history, and groundwater are being investigated. Glasses were tested using modified Materials Characterization Center (MCC) -3 and MCC-1 test methods. Results obtained in fiscal years (FY) 1987 and 1988 are presented here. 13 refs., 27 figs., 36 tabs.

  9. The Intra- or Extracellular Redox State Was Not Affected by a High vs. Low Glycemic Response Diet in Mice

    PubMed Central

    Kleckner, Amber S.; Wong, Siu; Corkey, Barbara E.

    2015-01-01

    A low glycemic response (LGR) vs. high glycemic response (HGR) diet helps curtail the development of obesity and diabetes, though the mechanisms are unknown. We hypothesized that consumption of a HGR vs. a LGR diet would lead to a more oxidized circulating redox state and predicted that a HGR diet would increase fat accumulation, reduce insulin sensitivity, and impair metabolic acclimation to a high fat diet in a mouse model. Hence, male C57BL/6 mice consumed a HGR or LGR diet for 16 weeks and a subset of the mice subsequently consumed a high fat diet for 4 weeks. We found that body mass increased at a faster rate for those consuming the HGR diet. Percent body fat was greater and percent lean mass was lesser in the HGR group starting at 12 weeks. However, the groups did not differ in terms of glucose tolerance at week 14 and metabolic parameters (respiratory exchange ratio, heat production, activity) at weeks 4 or 15. Moreover, mice on either diet did not show differences in metabolic acclimation to the high fat leg of the study. At the termination of the study, the groups did not differ in terms of redox pairs (lactate/pyruvate and β-hydroxybutyrate/acetoacetate) or thioredoxin reductase activity in blood. Also, total and oxidized glutathione levels and lipid peroxidation were similar in blood and liver. Correlations between baseline measures, longitudinal parameters, environmental conditions, and terminal metrics revealed that individual mice have innate propensities to metabolic regulation that may be difficult to perturb with diet alone; for example, starting mass correlated negatively with energy expenditure 4 weeks into the study and total hepatic glutathione at the end of the study. In conclusion, these data suggest that the mechanism by which HGR carbohydrates contributes to obesity is not via prolonged oxidation of the circulating redox state. PMID:26030878

  10. Turnover control of photosystem II: Use of redox-active herbicides to form the S[sub 3] state

    SciTech Connect

    Bocarsly, J.R.; Brudvig, G.W. )

    1992-12-02

    The O[sub 2]-evolving center of photosystem II, which contains an active-site tetramanganese-oxo cluster, catalyzes the four-electron oxidation of two water molecules to dioxygen, with the concomitant production of four H[sup +] and four electrons. During catalytic turnover, the manganese-oxo cluster steps through five intermediate oxidation states, which are known as the S[sub i] states (i = 0-4). While methods have been found to manipulate the system into S[sub 1] and S[sub 2] in high yields, efficient production of the S[sub 3] state in good yield at high concentration has not yet been achieved. Previous methods have suffered from the requirement of low protein concentration so that actinic flashes are saturating; the use of temperature to control S-state advancement under continuous illumination, which can lead to S-state scrambling; or the use of herbicides that bind to the Q[sub B] site and restrict the system to one turnover. The authors describe here a method for the high-yield production of the S[sub 3] state in highly-concentrated samples of photosystem II, through the use of electron-accepting herbicides which bind to the Q[sub B] site. Redox-active herbicides can be used, in principle, to limit S-state cycling to any desired number of turnovers, given the appropriate herbicide. This work has fundamental methodological implications not only for the study of photosystem II but also for other multistate redox protein systems.

  11. REDOX state analysis of platinoid elements in simulated high-level radioactive waste glass by synchrotron radiation based EXAFS

    NASA Astrophysics Data System (ADS)

    Okamoto, Yoshihiro; Shiwaku, Hideaki; Nakada, Masami; Komamine, Satoshi; Ochi, Eiji; Akabori, Mitsuo

    2016-04-01

    Extended X-ray Absorption Fine Structure (EXAFS) analyses were performed to evaluate REDOX (REDuction and OXidation) state of platinoid elements in simulated high-level nuclear waste glass samples prepared under different conditions of temperature and atmosphere. At first, EXAFS functions were compared with those of standard materials such as RuO2. Then structural parameters were obtained from a curve fitting analysis. In addition, a fitting analysis used a linear combination of the two standard EXAFS functions of a given elements metal and oxide was applied to determine ratio of metal/oxide in the simulated glass. The redox state of Ru was successfully evaluated from the linear combination fitting results of EXAFS functions. The ratio of metal increased at more reducing atmosphere and at higher temperatures. Chemical form of rhodium oxide in the simulated glass samples was RhO2 unlike expected Rh2O3. It can be estimated rhodium behaves according with ruthenium when the chemical form is oxide.

  12. PSI photoinhibition is more related to electron transfer from PSII to PSI rather than PSI redox state in Psychotria rubra.

    PubMed

    Huang, Wei; Yang, Ying-Jie; Zhang, Jiao-Lin; Hu, Hong; Zhang, Shi-Bao

    2016-07-01

    Although it has been believed that wild-type plants are capable of protecting photosystem I (PSI) under high light, our previous study indicates that PSI is sensitive to high light in the shade-established tree species Psychotria rubra. However, the underlying physiological mechanisms are unclear. In this study, we examined the roles of electron transfer from PSII to PSI and PSI redox state in PSI photoinhibition in P. rubra by treatments with lincomycin (Lin), diuron (DCMU), and methyl viologen (MV). After exposure to 2000 μmol photons m(-2) s(-1) for 2 h, PSI activity decreased by 35, 29, 3, and 49 % in samples treated with H2O, Lin, DCMU, and MV, respectively. Meanwhile, the MV-treated samples showed higher P700 oxidation ratio than the H2O-treated samples, suggesting the PSI photoinhibition under high light was accompanied by high levels of P700 oxidation ratio. PSI photoinhibition was alleviated in the DCMU-treated samples but was accelerated in the MV-treated samples, suggesting that PSI photoinhibition in P. rubra was mainly controlled by electron transfer from PSII to PSI. Taking together, PSI photoinhibition is more related to electron transfer from PSII to PSI rather than PSI redox state in P. rubra, which is different from the mechanisms of PSI photoinhibition in Arabidopsis thaliana and cucumber. PMID:27236700

  13. New Approach in Translational Medicine: Effects of Electrolyzed Reduced Water (ERW) on NF-κB/iNOS Pathway in U937 Cell Line under Altered Redox State.

    PubMed

    Franceschelli, Sara; Gatta, Daniela Maria Pia; Pesce, Mirko; Ferrone, Alessio; Patruno, Antonia; de Lutiis, Maria Anna; Grilli, Alfredo; Felaco, Mario; Croce, Fausto; Speranza, Lorenza

    2016-01-01

    It is known that increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can exert harmful effects, altering the cellular redox state. Electrolyzed Reduced Water (ERW) produced near the cathode during water electrolysis exhibits high pH, high concentration of dissolved hydrogen and an extremely negative redox potential. Several findings indicate that ERW had the ability of a scavenger free radical, which results from hydrogen molecules with a high reducing ability and may participate in the redox regulation of cellular function. We investigated the effect of ERW on H₂O₂-induced U937 damage by evaluating the modulation of redox cellular state. Western blotting and spectrophotometrical analysis showed that ERW inhibited oxidative stress by restoring the antioxidant capacity of superoxide dismutase, catalase and glutathione peroxidase. Consequently, ERW restores the ability of the glutathione reductase to supply the cell of an important endogenous antioxidant, such as GSH, reversing the inhibitory effect of H₂O₂ on redox balance of U937 cells. Therefore, this means a reduction of cytotoxicity induced by peroxynitrite via a downregulation of the NF-κB/iNOS pathway and could be used as an antioxidant for preventive and therapeutic application. In conclusion, ERW can protect the cellular redox balance, reducing the risk of several diseases with altered cellular homeostasis such as inflammation. PMID:27598129

  14. Redox Regulation of Mitochondrial Function

    PubMed Central

    Handy, Diane E.

    2012-01-01

    Abstract Redox-dependent processes influence most cellular functions, such as differentiation, proliferation, and apoptosis. Mitochondria are at the center of these processes, as mitochondria both generate reactive oxygen species (ROS) that drive redox-sensitive events and respond to ROS-mediated changes in the cellular redox state. In this review, we examine the regulation of cellular ROS, their modes of production and removal, and the redox-sensitive targets that are modified by their flux. In particular, we focus on the actions of redox-sensitive targets that alter mitochondrial function and the role of these redox modifications on metabolism, mitochondrial biogenesis, receptor-mediated signaling, and apoptotic pathways. We also consider the role of mitochondria in modulating these pathways, and discuss how redox-dependent events may contribute to pathobiology by altering mitochondrial function. Antioxid. Redox Signal. 16, 1323–1367. PMID:22146081

  15. Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox State In Vivo

    PubMed Central

    SWARTZ, HAROLD M.; KHAN, NADEEM; KHRAMTSOV, VALERY V.

    2009-01-01

    The aim of this article is to provide an overview of how electron paramagnetic resonance (EPR) can be used to measure redox-related parameters in vivo. The values of this approach include that the measurements are made under fully physiological conditions, and some of the measurements cannot be made by other means. Three complementary approaches are used with in vivo EPR: the rate of reduction or reactions of nitroxides, spin trapping of free radicals, and measurements of thiols. All three approaches already have produced unique and useful information. The measurement of the rate of decrease of nitroxides technically is the simplest, but difficult to interpret because the measured parameter, reduction in the intensity of the nitroxide signal, can occur by several different mechanisms. In vivo spin trapping can provide direct evidence for the occurrence of specific free radicals in vivo and reflect relative changes, but accurate absolute quantification remains challenging. The measurement of thiols in vivo also appears likely to be useful, but its development as an in vivo technique is at an early stage. It seems likely that the use of in vivo EPR to measure redox processes will become an increasingly utilized and valuable tool. PMID:17678441

  16. Integration of carbohydrate metabolism and redox state controls dauer larva formation in Caenorhabditis elegans.

    PubMed

    Penkov, Sider; Kaptan, Damla; Erkut, Cihan; Sarov, Mihail; Mende, Fanny; Kurzchalia, Teymuras V

    2015-01-01

    Under adverse conditions, Caenorhabditis elegans enters a diapause stage called the dauer larva. External cues signal the nuclear hormone receptor DAF-12, the activity of which is regulated by its ligands: dafachronic acids (DAs). DAs are synthesized from cholesterol, with the last synthesis step requiring NADPH, and their absence stimulates dauer formation. Here we show that NADPH levels determine dauer formation in a regulatory mechanism involving key carbohydrate and redox metabolic enzymes. Elevated trehalose biosynthesis diverts glucose-6-phosphate from the pentose phosphate pathway, which is the major source of cellular NADPH. This enhances dauer formation due to the decrease in the DA level. Moreover, DAF-12, in cooperation with DAF-16/FoxO, induces negative feedback of DA synthesis via activation of the trehalose-producing enzymes TPS-1/2 and inhibition of the NADPH-producing enzyme IDH-1. Thus, the dauer developmental decision is controlled by integration of the metabolic flux of carbohydrates and cellular redox potential. PMID:26290173

  17. Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs After Exposure to Hyperoxia

    PubMed Central

    Audi, Said H.; Staniszewski, Kevin S.; Haworth, Steven T.; Jacobs, Elizabeth R.; Ranji, Mahsa; Zablocki, Clement J.

    2013-01-01

    in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia. PMID:25379360

  18. Controlling the Charge State and Redox Properties of Supported Polyoxometalates via Soft Landing of Mass Selected Ions

    SciTech Connect

    Gunaratne, Kalupathirannehelage Don D.; Johnson, Grant E.; Andersen, Amity; Du, Dan; Zhang, Weiying; Prabhakaran, Venkateshkumar; Lin, Yuehe; Laskin, Julia

    2014-12-04

    We investigate the controlled deposition of Keggin polyoxometalate (POM) anions, PMo12O403- and PMo12O402-, onto different self-assembled monolayer (SAM) surfaces via soft landing of mass-selected ions. Utilizing in situ infrared reflection absorption spectroscopy (IRRAS), ex situ cyclic voltammetry (CV) and electronic structure calculations, we examine the structure and charge retention of supported multiply-charged POM anions and characterize the redox properties of the modified surfaces. SAMs of alkylthiol (HSAM), perfluorinated alkylthiol (FSAM), and alkylthiol terminated with NH3+ functional groups (NH3+SAM) are chosen as model substrates for soft landing to examine the factors which influence the immobilization and charge retention of multiply charged anionic molecules. The distribution of charge states of POMs on different SAM surfaces are determined by comparing the IRRAS spectra with vibrational spectra calculated using density functional theory (DFT). In contrast to the results obtained previously for multiply charged cations, soft landed anions are found to retain charge on all three SAM surfaces. This charge retention is attributed to the substantial electron binding energy of the POM anions. Investigation of redox properties by CV reveals that, while surfaces prepared by soft landing exhibit similar features to those prepared by adsorption of POM from solution, the soft landed POM2- has a pronounced shift in oxidation potential compared to POM3- for one of the redox couples. These results demonstrate that ion soft landing is uniquely suited for precisely controlled preparation of substrates with specific electronic and chemical properties that cannot be achieved using conventional deposition techniques.

  19. Alteration of the Redox State with Reactive Oxygen Species for 5-Fluorouracil-Induced Oral Mucositis in Hamsters

    PubMed Central

    Wada-Takahashi, Satoko; Takahashi, Shun-suke; Lee, Masaichi Chang-il

    2013-01-01

    Oral mucositis is often induced in patients receiving cancer chemotherapy treatment. It has been reported that oral mucositis can reduce quality of life, as well as increasing the incidence of mortality. The participation of reactive oxygen species (ROS) in the pathogenesis of oral mucositis is well known, but no report has actually demonstrated the presence of ROS. Thus, the purpose of this study was thus to demonstrate the involvement of ROS and the alteration of the redox state in oral mucositis using an in vivo L-band electron spin resonance (ESR) technique. An oral mucositis animal model induced by treatment of 5-fluorouracil with 10% acetic acid in hamster cheek pouch was used. Lipid peroxidation was measured as the level of malondialdehyde determined by the thiobarbituric acid reaction. The rate constants of the signal decay of nitroxyl compounds using in vivo L-band ESR were calculated from the signal decay curves. Firstly, we established the oral mucositis animal model induced by treatment of 5-fluorouracil with acetic acid in hamster cheek pouch. An increased level of lipid peroxidation in oral mucositis was found by measuring malondialdehyde using isolated hamster cheek pouch ulcer. In addition, as a result of in vivo L-band ESR measurements using our model animals, the decay rate constants of carbamoyl-PROXYL, which is a reagent for detecting the redox balance in tissue, were decreased. These results suggest that a redox imbalance might occur by excessive generation of ROS at an early stage of oral mucositis and the consumption of large quantities of antioxidants including glutathione in the locality of oral mucositis. These findings support the presence of ROS involved in the pathogenesis of oral mucositis with anti-cancer therapy, and is useful for the development of novel therapies drugs for oral mucositis. PMID:24376587

  20. A reversible early oxidized redox state that precedes macromolecular ROS damage in aging nontransgenic and 3xTg-AD mouse neurons.

    PubMed

    Ghosh, Debolina; LeVault, Kelsey R; Barnett, Aaron J; Brewer, Gregory J

    2012-04-25

    The brain depends on redox electrons from nicotinamide adenine dinucleotide (reduced form; NADH) to produce ATP and oxyradicals (reactive oxygen species [ROS]). Because ROS damage and mitochondrial dysregulation are prominent in aging and Alzheimer's disease (AD) and their relationship to the redox state is unclear, we wanted to know whether an oxidative redox shift precedes these markers and leads to macromolecular damage in a mouse model of AD. We used the 3xTg-AD mouse model, which displays cognitive deficits beginning at 4 months. Hippocampal/cortical neurons were isolated across the age span and cultured in common nutrients to control for possible hormonal and vascular differences. We found an increase of NAD(P)H levels and redox state in nontransgenic (non-Tg) neurons until middle age, followed by a decline in old age. The 3xTg-AD neurons maintained much lower resting NAD(P)H and redox states after 4 months, but the NADH regenerating capacity continuously declined with age beginning at 2 months. These redox characteristics were partially reversible with nicotinamide, a biosynthetic precursor of NAD+. Nicotinamide also protected against glutamate excitotoxicity. Compared with non-Tg neurons, 3xTg-AD neurons had more mitochondria/neuron and lower glutathione (GSH) levels that preceded age-related increases in ROS levels. These GSH deficits were again reversible with nicotinamide in 3xTg-AD neurons. Surprisingly, low macromolecular ROS damage was only elevated after 4 months in the 3xTg-AD neurons if antioxidants were removed. The present data suggest that a more oxidized redox state and a lower antioxidant GSH defense can be dissociated from neuronal ROS damage, changes that precede the onset of cognitive deficits in the 3xTg-AD model. PMID:22539844

  1. The effects of temperature, pH and redox state on the stability of glutamic acid in hydrothermal fluids

    NASA Astrophysics Data System (ADS)

    Lee, Namhey; Foustoukos, Dionysis I.; Sverjensky, Dimitri A.; Cody, George D.; Hazen, Robert M.

    2014-06-01

    Natural hydrothermal vent environments cover a wide range of physicochemical conditions involving temperature, pH and redox state. The stability of simple biomolecules such as amino acids in such environments is of interest in various fields of study from the origin of life to the metabolism of microbes at the present day. Numerous previous experimental studies have suggested that amino acids are unstable under hydrothermal conditions and decompose rapidly. However, previous studies have not effectively controlled the redox state of the hydrothermal fluids. Here we studied the stability of glutamate with and without reducing hydrothermal conditions imposed by 13 mM aqueous H2 at temperatures of 150, 200 and 250 °C and initial (25 °C) pH values of 6 and 10 in a flow-through hydrothermal reactor with reaction times from 3 to 36 min. We combined the experimental measurements with theoretical calculations to model the in situ aqueous speciation and pH values. As previously observed under hydrothermal conditions, the main reaction involves glutamate cyclizing to pyroglutamate through a simple dehydration reaction. However, the amounts of decomposition products of the glutamate detected, including succinate, formate, carbon dioxide and ammonia depend on the temperature, the pH and particularly the redox state of the fluid. In the absence of dissolved H2, glutamate decomposes in the sequence glutamate, glutaconate, α-hydroxyglutarate, ketoglutarate, formate and succinate, and ultimately to CO2 and micromolar quantities of H2(aq). Model speciation calculations indicate the CO2, formate and H2(aq) are not in metastable thermodynamic equilibrium. However, with 13 mM H2(aq) concentrations, the amounts of decomposition products are suppressed at all temperatures and pH values investigated. The small amounts of CO2 and formate present are calculated to be in metastable equilibrium with the H2. It is further proposed that there is a metastable equilibrium between glutamate

  2. Modulation of the matrix redox signaling by mitochondrial Ca2+

    PubMed Central

    Santo-Domingo, Jaime; Wiederkehr, Andreas; De Marchi, Umberto

    2015-01-01

    Mitochondria sense, shape and integrate signals, and thus function as central players in cellular signal transduction. Ca2+ waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca2+ transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance, the molecular nature of the proteins involved in mitochondrial Ca2+ transport has been revealed only recently. Mitochondrial Ca2+ promotes energy metabolism through the activation of matrix dehydrogenases and down-stream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)+ ratio, but at the same time will increase reactive oxygen species (ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state, which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redox-sensitive sensors, real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca2+ combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca2+ and redox signals and their impact on cell function. In this review, we describe mitochondrial Ca2+ handling, focusing on a number of newly identified proteins involved in mitochondrial Ca2+ uptake and release. We further discuss our recent findings, revealing how mitochondrial Ca2+ influences the matrix redox state. As a result, mitochondrial Ca2+ is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease. PMID:26629314

  3. Modulation of the matrix redox signaling by mitochondrial Ca(2.).

    PubMed

    Santo-Domingo, Jaime; Wiederkehr, Andreas; De Marchi, Umberto

    2015-11-26

    Mitochondria sense, shape and integrate signals, and thus function as central players in cellular signal transduction. Ca(2+) waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca(2+) transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance, the molecular nature of the proteins involved in mitochondrial Ca(2+) transport has been revealed only recently. Mitochondrial Ca(2+) promotes energy metabolism through the activation of matrix dehydrogenases and down-stream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)(+) ratio, but at the same time will increase reactive oxygen species (ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state, which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redox-sensitive sensors, real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca(2+) combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca(2+) and redox signals and their impact on cell function. In this review, we describe mitochondrial Ca(2+) handling, focusing on a number of newly identified proteins involved in mitochondrial Ca(2+) uptake and release. We further discuss our recent findings, revealing how mitochondrial Ca(2+) influences the matrix redox state. As a result, mitochondrial Ca(2+) is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease. PMID:26629314

  4. Evidence for transcriptional and post-translational regulation of sucrose synthase in pea nodules by the cellular redox state.

    PubMed

    Marino, Daniel; Hohnjec, Natalija; Küster, Helge; Moran, Jose F; González, Esther M; Arrese-Igor, Cesar

    2008-05-01

    Nitrogen fixation (NF) in legume nodules is very sensitive to environmental constraints. Nodule sucrose synthase (SS; EC 2.4.1.13) has been suggested to play a crucial role in those circumstances because its downregulation leads to an impaired glycolytic carbon flux and, therefore, a depletion of carbon substrates for bacteroids. In the present study, the likelihood of SS being regulated by oxidative signaling has been addressed by the in vivo supply of paraquat (PQ) to nodulated pea plants and the in vitro effects of oxidizing and reducing agents on nodule SS. PQ produced cellular redox imbalance leading to an inhibition of NF. This was preceded by the downregulation of SS gene expression, protein content, and activity. In vitro, oxidizing agents were able to inhibit SS activity and this inhibition was completely reversed by the addition of dithiothreitol. The overall results are consistent with a regulation model of nodule SS exerted by the cellular redox state at both the transcriptional and post-translational levels. The importance of such mechanisms for the regulation of NF in response to environmental stresses are discussed. PMID:18393622

  5. Evidence for Changes in Redox State During Crystallization of Allende Type B1 Inclusions

    NASA Astrophysics Data System (ADS)

    Simon, S. B.; Davis, A. M.; Grossman, L.

    1992-07-01

    /Eu, sharp increases in trivalent REE content. Because the only elements that consistently show spikes, Ti and V, are also the only elements in fassaite with multiple oxidation states at the range of Tfo(sub)2 conditions under which refractory inclusions crystallized (Beckett, 1986), we now believe that the spikes reflect redox reactions that increased the Ti^3+ and V^3+ contents of the residual liquids. Gradual decreases in Ti^3+/Ti^tot from core to rim in fassaite in TS33 and TS34 and before the spikes in TS23 indicate that the residual liquids did not re-equilibrate with the reducing, solar nebular gas throughout most of the interval of fassaite crystallization. Late reequilibration, perhaps by entry of the solar gas via cracks in the cooling CAI, would increase the Ti^3+/Ti^tot of the liquid. The Ti^3+-poor rims of fassaite crystals would then be out of equilibrium with the liquid. Resorption of the rims may have occurred, followed by deposition of relatively Ti^3+-rich fassaite. This is consistent with the observations of sharp, irregular low-Ti/high-Ti boundaries. Of the V species presumed to be present in the CAIs, V^3+ should be favored in fassaite over V^2+, based on its ionic radius. Increasing the V^3+ content of the liquid is a problem, however, because this requires oxidation of V^2+ when Ti is being reduced. Perhaps, prior to reequilibration, the internal fO(sub)2 of the inclusions became high enough to stabilize V^4+, which was reduced to V%3+. REFERENCES: Beckett J.R. (1986) Ph.D. Thesis, University of Chicago, 373 pp. Simon S.B. and Grossman L. (1991) Meteoritics 26, 395.

  6. Statins as Regulators of Redox State in the Vascular Endothelium: Beyond Lipid Lowering

    PubMed Central

    Margaritis, Marios; Channon, Keith M.

    2014-01-01

    Abstract Significance: Endothelial dysfunction and the imbalance between nitric oxide (NO) and reactive oxygen species production in the vascular endothelium are important early steps in atherogenesis, a major socioeconomic health problem. Statins have well-established roles in primary and secondary prevention of cardiovascular disease (CVD), due to both their lipid-lowering capacity and their pleiotropic properties. It is therefore important to understand the mechanisms by which statins can modify endothelial function and affect atherogenesis. Recent Advances: In the last decade, the concept of statin pleiotropy has been reinforced by a large number of cell culture, animal, and translational studies. Statins have been shown to suppress the activity of pro-oxidant enzymes (such as NADPH oxidase) and pro-inflammatory transcriptional pathways in the endothelium. At the same time, they enhance endothelial NO synthase expression and activity while they also improve its enzymatic coupling. This leads to increased NO bioavailability and improved endothelial function. Critical Issues: Despite significant recent advances, the exact mechanisms of statin pleitropy are still only partially understood. The vast majority of the published literature relies on animal studies, while the actual mechanistic studies in humans are limited. Future Directions: The success of statins as endothelium redox-modifying agents with a direct impact on clinical outcome highlights the importance of the endothelium as a therapeutic target in CVD. Better understanding of the mechanisms that underlie endothelial dysfunction could lead to the design of novel therapeutic strategies that target the vascular endothelium for the prevention and treatment of CVD. Antioxid. Redox Signal. 20, 1198–1215. PMID:24111702

  7. Mitochondrial redox state and Ca2+ sparks in permeabilized mammalian skeletal muscle.

    PubMed

    Isaeva, Elena V; Shkryl, Vyacheslav M; Shirokova, Natalia

    2005-06-15

    Intact skeletal muscle fibres from adult mammals exhibit neither spontaneous nor stimulated Ca(2+) sparks. Mechanical or chemical skinning procedures have been reported to unmask sparks. The present study investigates the mechanisms that determine the development of Ca(2+) spark activity in permeabilized fibres dissected from muscles with different metabolic capacity. Spontaneous Ca(2+) sparks were detected with fluo-3 and single photon confocal microscopy; mitochondrial redox potential was evaluated from mitochondrial NADH signals recorded with two-photon confocal microscopy, and Ca(2+) load of the sarcoplasmic reticulum (SR) was estimated from the amplitude of caffeine-induced Ca(2+) transients recorded with fura-2 and digital photometry. In three fibre types studied, there was a time lag between permeabilization and spark development. Under all experimental conditions, the delay was the longest in slow-twitch oxidative fibres, intermediate in fast-twitch glycolytic-oxidative fibres, and the shortest in fast-twitch glycolytic cells. The temporal evolution of Ca(2+) spark frequencies was bell-shaped, and the maximal spark frequency was reached slowly in mitochondria-rich oxidative cells but quickly in mitochondria-poor glycolytic fibres. The development of spontaneous Ca(2+) sparks did not correlate with the SR Ca(2+) content of the fibre, but did correlate with the redox potential of their mitochondria. Treatment of fibres with scavengers of reactive oxygen species (ROS), such as superoxide dismutase (SOD) and catalase, dramatically and reversibly reduced the spark frequency and also delayed their appearance. In contrast, incubation of fibres with 50 microm H(2)O(2) sped up the development of Ca(2+) sparks and increased their frequency. These results indicate that the appearance of Ca(2+) sparks in permeabilized skeletal muscle cells depends on the fibre's oxidative strength and that misbalance between mitochondrial ROS production and the fibre's ability to fight

  8. Real-time assays for monitoring the influence of sulfide and sulfane sulfur species on protein thiol redox states.

    PubMed

    Greiner, Romy; Dick, Tobias P

    2015-01-01

    Hydrogen sulfide (H2S) is known to induce persulfidation of protein thiols. However, the process of H2S-induced persulfidation is not fully understood as it requires an additional oxidant. There are several mechanistic possibilities and it is of interest to determine which pathway is kinetically most relevant. Here, we detail in vitro assays for the real-time monitoring of thiol redox states in two model proteins with oxidizable cysteines, PTEN, and roGFP2. These allow kinetic measurements of the response of defined protein thiols (or disulfides) to sulfide and sulfane sulfur species. The combination of these assays with cold cyanolysis reveals the role of intermediary sulfane sulfur species in H2S-induced protein thiol oxidation. PMID:25747475

  9. Direct determination of the redox status of cysteine residues in proteins in vivo

    SciTech Connect

    Hara, Satoshi; Tatenaka, Yuki; Ohuchi, Yuya; Hisabori, Toru

    2015-01-02

    Highlights: • A new DNA-maleimide which is cleaved by UV irradiation, DNA-PCMal, was developed. • DNA-PCMal can be used like DNA-Mal to analyze the redox state of cysteine residues. • It is useful for detecting the thiol redox status of a protein in vivo by Western blotting method. • Thus, DNA-PCMal can be a powerful tool for redox proteomics analysis. - Abstract: The redox states of proteins in cells are key factors in many cellular processes. To determine the redox status of cysteinyl thiol groups in proteins in vivo, we developed a new maleimide reagent, a photocleavable maleimide-conjugated single stranded DNA (DNA-PCMal). The DNA moiety of DNA-PCMal is easily removed by UV-irradiation, allowing DNA-PCMal to be used in Western blotting applications. Thereby the state of thiol groups in intracellular proteins can be directly evaluated. This new maleimide compound can provide information concerning redox proteins in vivo, which is important for our understanding of redox networks in the cell.

  10. Redox controls UPR to control redox.

    PubMed

    Eletto, Davide; Chevet, Eric; Argon, Yair; Appenzeller-Herzog, Christian

    2014-09-01

    In many physiological contexts, intracellular reduction-oxidation (redox) conditions and the unfolded protein response (UPR) are important for the control of cell life and death decisions. UPR is triggered by the disruption of endoplasmic reticulum (ER) homeostasis, also known as ER stress. Depending on the duration and severity of the disruption, this leads to cell adaptation or demise. In this Commentary, we review reductive and oxidative activation mechanisms of the UPR, which include direct interactions of dedicated protein disulfide isomerases with ER stress sensors, protein S-nitrosylation and ER Ca(2+) efflux that is promoted by reactive oxygen species. Furthermore, we discuss how cellular oxidant and antioxidant capacities are extensively remodeled downstream of UPR signals. Aside from activation of NADPH oxidases, mitogen-activated protein kinases and transcriptional antioxidant responses, such remodeling prominently relies on ER-mitochondrial crosstalk. Specific redox cues therefore operate both as triggers and effectors of ER stress, thus enabling amplification loops. We propose that redox-based amplification loops critically contribute to the switch from adaptive to fatal UPR. PMID:25107370

  11. Increasing tetrahydrobiopterin in cardiomyocytes adversely affects cardiac redox state and mitochondrial function independently of changes in NO production.

    PubMed

    Sethumadhavan, Savitha; Whitsett, Jennifer; Bennett, Brian; Ionova, Irina A; Pieper, Galen M; Vasquez-Vivar, Jeannette

    2016-04-01

    Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (<6 months) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous iron-nitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function. PMID:26826575

  12. Characterization of the particulate methane monooxygenase metal centers in multiple redox states by X-ray absorption spectroscopy.

    PubMed

    Lieberman, Raquel L; Kondapalli, Kalyan C; Shrestha, Deepak B; Hakemian, Amanda S; Smith, Stephen M; Telser, Joshua; Kuzelka, Jane; Gupta, Rajeev; Borovik, A S; Lippard, Stephen J; Hoffman, Brian M; Rosenzweig, Amy C; Stemmler, Timothy L

    2006-10-01

    The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions. PMID:16999437

  13. Real-Time Measurements of the Redox States of c-Type Cytochromes in Electroactive Biofilms: A Confocal Resonance Raman Microscopy Study

    PubMed Central

    Virdis, Bernardino; Millo, Diego; Donose, Bogdan C.; Batstone, Damien J.

    2014-01-01

    Confocal Resonance Raman Microscopy (CRRM) was used to probe variations of redox state of c-type cytochromes embedded in living mixed-culture electroactive biofilms exposed to different electrode polarizations, under potentiostatic and potentiodynamic conditions. In the absence of the metabolic substrate acetate, the redox state of cytochromes followed the application of reducing and oxidizing electrode potentials. Real-time monitoring of the redox state of cytochromes during cyclic voltammetry (CV) in a potential window where cytochromes reduction occurs, evidenced a measurable time delay between the oxidation of redox cofactors probed by CV at the electrode interface, and oxidation of distal cytochromes probed by CRRM. This delay was used to tentatively estimate the diffusivity of electrons through the biofilm. In the presence of acetate, the resonance Raman spectra of young (10 days, j = 208±49 µA cm−2) and mature (57 days, j = 267±73 µA cm−2) biofilms show that cytochromes remained oxidized homogeneously even at layers as far as 70 µm from the electrode, implying the existence of slow metabolic kinetics that do not result in the formation of a redox gradient inside the biofilm during anode respiration. However, old biofilms (80 days, j = 190±37 µA cm−2) with thickness above 100 µm were characterized by reduced catalytic activity compared to the previous developing stages. The cytochromes in these biofilm were mainly in the reduced redox state, showing that only aged mixed-culture biofilms accumulate electrons during anode respiration. These results differ substantially from recent observations in pure Geobacter sulfurreducens electroactive biofilms, in which accumulation of reduced cytochromes is already observed in thinner biofilms, thus suggesting different bottlenecks in current production for mixed-culture and G. sulfurreducens biofilms. PMID:24587123

  14. Probing the redox states at the surface of electroactive nanoporous NiO thin films.

    PubMed

    Marrani, Andrea G; Novelli, Vittoria; Sheehan, Stephen; Dowling, Denis P; Dini, Danilo

    2014-01-01

    Nanoporous NiO thin film electrodes were obtained via plasma-assisted microwave sintering and characterized by means of a combination of electrochemical techniques and X-ray photoelectron spectroscopy (XPS). The aim of this study is the elucidation of the nature of the surface changes introduced by the redox processes of this nanostructured material. NiO undergoes two distinct electrochemical processes of oxidation in aqueous electrolyte with the progress of NiO anodic polarization. These findings are consistent with the sequential formation of oxyhydroxide species at the surface, the chemical nature of which was assessed by XPS. Electronic relaxation effects in the Ni 2p spectra clearly indicated that the superficial oxyhydroxide species resulted to be β-NiOOH and γ-NiOOH. We also show for the first time spectral evidence of an electrochemically generated Ni(IV) species. This study has direct relevance for those applications in which NiO electrodes are utilized in aqueous electrolyte, namely catalytic water splitting or electrochromism, and may constitute a starting point for the comprehension of electronic phenomena at the NiO/organic electrolyte interface of cathodic dye-sensitized solar cells (p-DSCs). PMID:24325361

  15. Effects of Moderate Aerobic Exercise on Cognitive Abilities and Redox State Biomarkers in Older Adults.

    PubMed

    Alghadir, Ahmad H; Gabr, Sami A; Al-Eisa, Einas S

    2016-01-01

    We used a moderate aerobic exercise program for 24 weeks to measure the positive impact of physical activity on oxidative stress and inflammatory markers and its association with cognitive performance in healthy older adults. A total of 100 healthy subjects (65-95 Yrs) were randomly classified into two groups: control group (n = 50) and exercise group (n = 50). Cognitive functioning, physical activity score, MDA, 8-OHdG, TAC, and hs-CRP were assessed using LOTCA battery, prevalidated PA questionnaire, and immunoassay techniques. LOTCA 7-set scores of cognitive performance showed a significant correlation with physical activity status and the regulation of both oxidative stress free radicals and inflammatory markers in all older subjects following 24 weeks of moderate exercise. Physically active persons showed a higher cognitive performance along with reduction in the levels of MDA, 8-OHdG, and hs-CRP and increase in TAC activity compared with sedentary participants. Cognitive performance correlated positively with the increase in TAC activity and physical fitness scores and negatively with MDA, 8-OHdG, and hs-CRP, respectively. There was a significant improvement in motor praxis, vasomotor organization, thinking operations, and attention and concentration among older adults. In conclusion, moderate aerobic training for 24 weeks has a positive significant effect in improving cognitive functions via modulating redox and inflammatory status of older adults. PMID:27195073

  16. Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

    PubMed Central

    Sporty, Jennifer L.; Kabir, Md. Mohiuddin; Turteltaub, Kenneth W.; Ognibene, Ted; Lin, Su-Ju; Bench, Graham

    2009-01-01

    A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotin-amide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH3CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV–Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures. PMID:18763242

  17. Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae.

    PubMed

    Sporty, Jennifer L; Kabir, Md Mohiuddin; Turteltaub, Kenneth W; Ognibene, Ted; Lin, Su-Ju; Bench, Graham

    2008-10-01

    A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH(3)CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures. PMID:18763242

  18. Effects of Moderate Aerobic Exercise on Cognitive Abilities and Redox State Biomarkers in Older Adults

    PubMed Central

    Al-Eisa, Einas S.

    2016-01-01

    We used a moderate aerobic exercise program for 24 weeks to measure the positive impact of physical activity on oxidative stress and inflammatory markers and its association with cognitive performance in healthy older adults. A total of 100 healthy subjects (65–95 Yrs) were randomly classified into two groups: control group (n = 50) and exercise group (n = 50). Cognitive functioning, physical activity score, MDA, 8-OHdG, TAC, and hs-CRP were assessed using LOTCA battery, prevalidated PA questionnaire, and immunoassay techniques. LOTCA 7-set scores of cognitive performance showed a significant correlation with physical activity status and the regulation of both oxidative stress free radicals and inflammatory markers in all older subjects following 24 weeks of moderate exercise. Physically active persons showed a higher cognitive performance along with reduction in the levels of MDA, 8-OHdG, and hs-CRP and increase in TAC activity compared with sedentary participants. Cognitive performance correlated positively with the increase in TAC activity and physical fitness scores and negatively with MDA, 8-OHdG, and hs-CRP, respectively. There was a significant improvement in motor praxis, vasomotor organization, thinking operations, and attention and concentration among older adults. In conclusion, moderate aerobic training for 24 weeks has a positive significant effect in improving cognitive functions via modulating redox and inflammatory status of older adults. PMID:27195073

  19. [The role of oxidative protein modification and the gluthatione system in modulation of the redox status of breast epithelial cells].

    PubMed

    Stepovaya, E A; Shakhristova, E V; Ryazantseva, N V; Nosareva, O L; Yakushina, V D; Nosova, A I; Gulaya, V S; Stepanova, E A; Chil'chigashev, R I; Novitsky, V V

    2016-01-01

    The effects of the SH-group blocker N-ethylmaleimide (NEM) and thiol group protector 1,4-dithioerythritol (DTE) on the redox status of cells HBL-100 cells, oxidative modification of their proteins and the state of glutathione and thioredoxin systems have been investigated. Breast epithelial cells cultivated in the presence of NEM were characterized by decreased redox status, increased glutathione reductase activity, and increased concentrations of products of irreversible oxidative modification of protein and amino acids. Cultivation of HBL-100 cells in the presence of DTE resulted in a shift of the redox status towards reduction processes and increased reversible protein modification by glutathionylation. The proposed model of intracellular redox modulation may be used in the development of new therapeutic approaches to treat diseases accompanied by impaired redox homeostasis (e.g. oncologic, inflammatory, cardiovascular and neurodegenerative disease). PMID:26973189

  20. Redox chemistry of copper-amyloid-beta: the generation of hydroxyl radical in the presence of ascorbate is linked to redox-potentials and aggregation state.

    PubMed

    Guilloreau, Luc; Combalbert, Sarah; Sournia-Saquet, Alix; Mazarguil, Honoré; Faller, Peter

    2007-07-23

    Aggregation of the beta-amyloid peptide (Abeta) to amyloid plaques is a key event in Alzheimer's disease. According to the amyloid-cascade hypothesis, Abeta aggregates are toxic to neurons through the production of reactive oxygen species (ROS). Copper ions play an important role, because they are able to bind to Abeta and influence its aggregation properties. Moreover, Cu-Abeta is supposed to be directly involved in ROS production. To get a better understanding of these reactions, we measured the production of HO(.) and the redox potential of Cu-Abeta. The results were compared to other biological copper-peptide complexes in order to get an insight into the biological relevance. Cu-Abeta produced more HO(.) than the complex of copper with Asp-Ala-His-Lys (Cu-DAHK), but less than with Gly-His-Lys (Cu-GHK). Cyclic voltammetry revealed that the order for reduction potential is Cu-GHK>Cu-Abeta>Cu-DAHK, but for the oxidation potential the order is reversed. Thus, easier copper redox cycling correlated to higher HO(.) production. The copper complex of the form Abeta1-42 showed a HO(.) production five-times higher than that of the form Abeta1-40. Time-dependence and aggregation studies suggest that an aggregation intermediate is responsible for this increased HO(.) production. PMID:17577900

  1. Endoplasmic reticulum stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of Arabidopsis thaliana

    PubMed Central

    Turkan, Ismail

    2014-01-01

    Inefficient chaperone activity in endoplasmic reticulum (ER) causes accumulation of unfolded proteins and is called ER stress, which triggers the unfolded protein response. For proper oxidative protein folding, reactive oxygen species (ROS) such as H2O2 are produced in the ER. Although the role of ROS during abiotic stresses such as salinity is well documented, the role of ER-related ROS production and its signalling is not yet known. Moreover, how H2O2 production, redox regulation, and antioxidant defence are affected in salt-treated plants when ER protein-folding machinery is impaired needs to be elucidated. For this aim, changes in NADPH-oxidase-dependent ROS signalling and H2O2 content at sequential time intervals and after 48h of ER stress, induced by tunicamycin (Tm), salinity, and their combination were determined in Arabidopsis thaliana. The main root growth was inhibited by ER stress, while low levels of Tm caused an increase in lateral root density. Salt stress and Tm induced the expression of ER-stress-related genes (bZIP17, bZIP28, bZIP60, TIN1, BiP1, BiP3) and ERO1. Tm induced expression of RBOHD and RBOHF, which led to an early increase in H2O2 and triggered ROS signalling. This study is the first report that ER stress induces the antioxidant system and the Asada–Halliwell pathway of A. thaliana in a similar way to salinity. ER stress caused oxidative damage, as evident by increased H2O2 accumulation, lipid peroxidation, and protein oxidation. As a result, this study shows that ER stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of A. thaliana. PMID:24558072

  2. Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool.

    PubMed Central

    Escoubas, J M; Lomas, M; LaRoche, J; Falkowski, P G

    1995-01-01

    The eukaryotic green alga Dunaliella tertiolecta acclimates to decreased growth irradiance by increasing cellular levels of light-harvesting chlorophyll protein complex apoproteins associated with photosystem II (LHCIIs), whereas increased growth irradiance elicits the opposite response. Nuclear run-on transcription assays and measurements of cab mRNA stability established that light intensity-dependent changes in LHCII are controlled at the level of transcription. cab gene transcription in high-intensity light was partially enhanced by reducing plastoquinone with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), whereas it was repressed in low-intensity light by partially inhibiting the oxidation of plastoquinol with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Uncouplers of photosynthetic electron transport and inhibition of water splitting had no effect on LHCII levels. These results strongly implicate the redox state of the plastoquinone pool in the chloroplast as a photon-sensing system that is coupled to the light-intensity regulation of nuclear-encoded cab gene transcription. The accumulation of cellular chlorophyll at low-intensity light can be blocked with cytoplasmically directed phosphatase inhibitors, such as okadaic acid, microcystin L-R, and tautomycin. Gel mobility-shift assays revealed that cells grown in high-intensity light contained proteins that bind to the promoter region of a cab gene carrying sequences homologous to higher plant light-responsive elements. On the basis of these experimental results, we propose a model for a light intensity signaling system where cab gene expression is reversibly repressed by a phosphorylated factor coupled to the redox status of plastoquinone through a chloroplast protein kinase. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479759

  3. Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria.

    PubMed

    Morgan, J E; Wikström, M

    1991-01-29

    We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are

  4. Multi-State Transition Kinetics of Intracellular Signaling Molecules by Single-Molecule Imaging Analysis.

    PubMed

    Matsuoka, Satomi; Miyanaga, Yukihiro; Ueda, Masahiro

    2016-01-01

    The chemotactic signaling of eukaryotic cells is based on a chain of interactions between signaling molecules diffusing on the cell membrane and those shuttling between the membrane and cytoplasm. In this chapter, we describe methods to quantify lateral diffusion and reaction kinetics on the cell membrane. By the direct visualization and statistic analyses of molecular Brownian movement achieved by single-molecule imaging techniques, multiple states of membrane-bound molecules are successfully revealed with state transition kinetics. Using PTEN, a phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) 3'-phosphatase, in Dictyostelium discoideum undergoing chemotaxis as a model, each process of the analysis is described in detail. The identified multiple state kinetics provides an essential clue to elucidating the molecular mechanism of chemoattractant-induced dynamic redistribution of the signaling molecule asymmetrically on the cell membrane. Quantitative parameters for molecular reactions and diffusion complement a conventional view of the chemotactic signaling system, where changing a static network of molecules connected by causal relationships into a spatiotemporally dynamic one permits a mathematical description of stochastic migration of the cell along a shallow chemoattractant gradient. PMID:27271914

  5. Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation.

    PubMed

    Tsuge, Yota; Uematsu, Kimio; Yamamoto, Shogo; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

    2015-07-01

    Rapid sugar consumption is important for the microbial production of chemicals and fuels. Here, we show that overexpression of the NADH dehydrogenase gene (ndh) increased glucose consumption rate in Corynebacterium glutamicum under oxygen-deprived conditions through investigating the relationship between the glucose consumption rate and intracellular NADH/NAD(+) ratio in various mutant strains. The NADH/NAD(+) ratio was strongly repressed under oxygen deprivation when glucose consumption was accelerated by the addition of pyruvate or sodium hydrogen carbonate. Overexpression of the ndh gene in the wild-type strain under oxygen deprivation decreased the NADH/NAD(+) ratio from 0.32 to 0.13, whereas the glucose consumption rate increased by 27%. Similarly, in phosphoenolpyruvate carboxylase gene (ppc)- or malate dehydrogenase gene (mdh)-deficient strains, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.66 to 0.37 and 2.20 to 0.57, respectively, whereas the glucose consumption rate increased by 57 and 330%, respectively. However, in a lactate dehydrogenase gene (L-ldhA)-deficient strain, although the NADH/NAD(+) ratio decreased from 5.62 to 1.13, the glucose consumption rate was not markedly altered. In a tailored D-lactate-producing strain, which lacked ppc and L-ldhA genes, but expressed D-ldhA from Lactobacillus delbrueckii, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.77 to 0.56, and increased the glucose consumption rate by 50%. Overall, the glucose consumption rate was found to be inversely proportional to the NADH/NAD(+) ratio in C. glutamicum cultured under oxygen deprivation. These findings could provide an option to increase the productivity of chemicals and fuels under oxygen deprivation. PMID:25808520

  6. Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement. PMID:27299603

  7. NADP redox state and mitochondrial Ca2+ efflux: a controversial issue.

    PubMed

    Vercesi, A E; Pereira-da-Silva, L

    1984-01-01

    It has been shown that Ca2+ efflux from respiring liver mitochondria is promoted by the oxidized steady state of mitochondrial pyridine nucleotides (NAD(P)), whereas Ca2+ retention is favored by a more reduced state (Lehninger, A.L., Vercesi, A.E. and Bababunmi, E.A., Proceedings of the National Academy of Sciences USA, 75, 1690-1694, 1978). The physiological relevance and the molecular mechanism responsible for the process are controversial and are discussed here. PMID:6529616

  8. Quantitative measurement of redox potential in hypoxic cells using SERS nanosensors

    NASA Astrophysics Data System (ADS)

    Jiang, Jing; Auchinvole, Craig; Fisher, Kate; Campbell, Colin J.

    2014-09-01

    Hypoxia is considered to be a reductive disorder of cells that is caused either by a lack of oxygen or by the dysregulation of metabolic pathways and is thought to play a role in the pathology of diseases including stroke and cancer. One aspect of hypoxia that remains poorly investigated is the dysregulation of cellular redox potential and its role in controlling biological pathway activation. Since there is currently no way of quantitatively measuring the intracellular redox potential of hypoxic cells, this provided us with the motivation to develop optical nanosensors whose Surface-Enhanced Raman (SER) spectrum provides a quantitative measure of redox potential in hypoxic cells. Our nanosensors are made from organic reporter molecules that show oxidation-state-dependent changes in the Raman spectrum and are chemically adsorbed onto gold nanoshells. These nanosensors can be taken up by cells, and by collecting the SER spectrum we can calculate the localised intracellular redox potential from single hypoxic cells in a non-invasive, reversible way.Hypoxia is considered to be a reductive disorder of cells that is caused either by a lack of oxygen or by the dysregulation of metabolic pathways and is thought to play a role in the pathology of diseases including stroke and cancer. One aspect of hypoxia that remains poorly investigated is the dysregulation of cellular redox potential and its role in controlling biological pathway activation. Since there is currently no way of quantitatively measuring the intracellular redox potential of hypoxic cells, this provided us with the motivation to develop optical nanosensors whose Surface-Enhanced Raman (SER) spectrum provides a quantitative measure of redox potential in hypoxic cells. Our nanosensors are made from organic reporter molecules that show oxidation-state-dependent changes in the Raman spectrum and are chemically adsorbed onto gold nanoshells. These nanosensors can be taken up by cells, and by collecting the SER

  9. Fe and S redox states during serpentinite dehydration in subduction settings

    NASA Astrophysics Data System (ADS)

    Merkulova, Margarita; Munoz, Manuel; Vidal, Olivier; Brunet, Fabrice

    2016-04-01

    present highly oxidizing properties. At higher P-T conditions, higher amounts of water are released with minor oxygen release. In addition, sulfur is shown to be progressively reduced at temperature 450-500°C due to pyrite to pyrrhotite transition. The reaction of pyrite reduction was observed to happen with sequestration of Fe from silicates and a release of oxygen. Effectively, the presence of sulphides in serpentinites contribute additional oxygen to the fluid, whereas the release of S may be negligible. The detailed study of the evolution of redox conditions during serpentinite dehydration in subduction zones will help constraining, 1) the behavior and mobility, from slab to the upper mantle, of elements of economical interest, as well as 2) the global geochemical cycling of elements. References: 1. Hacker et al. (2003) J. Geophys. Res. 108, article number 2029. 2. Ulmer & Trommsdorff (1995) Science 268, 858-861. 3. Debret et al. (2014) EPSL 400, 206-218. 4. Alt et al. (2013) Lithos 178, 40-54. 5. Pokrovski & Dubrovinsky (2011) Science 331, 1052-1056.

  10. Redox effects on the excited-state lifetime in chlorosomes and bacteriochlorophyll c oligomers.

    PubMed Central

    van Noort, P I; Zhu, Y; LoBrutto, R; Blankenship, R E

    1997-01-01

    Oligomers of [E,E] BChl CF (8, 12-diethyl bacteriochlorophyll c esterified with farnesol (F)) and [Pr,E] BChl CF (analogously, M methyl, Pr propyl) in hexane and aqueous detergent or lipid micelles were studied by means of steady-state absorption, time-resolved fluorescence, and electron spin resonance spectroscopy. The maximum absorption wavelength, excited-state dynamics, and electron spin resonance (EPR) linewidths are similar to those of native and reconstituted chlorosomes of Chlorobium tepidum. The maximum absorption wavelength of oligomers of [E,E] BChl CF was consistently blue-shifted as compared to that of [Pr,E] BChl CF oligomers, which is ascribed to the formation of smaller oligomers with [E,E] BChl CF than [Pr,E] BChl CF. Time-resolved fluorescence measurements show an excited-state lifetime of 10 ps or less in nonreduced samples of native and reconstituted chlorosomes of Chlorobium tepidum. Under reduced conditions the excited-state lifetime increased to tens of picoseconds, and energy transfer to BChl a or long-wavelength absorbing BChl c was observed. Oligomers of [E,E] BChl CF and [Pr,E] BChl CF in aqueous detergent or lipid micelles show a similar short excited-state lifetime under nonreduced conditions and an increase up to several tens of picoseconds upon reduction. These results indicate rapid quenching of excitation energy in nonreduced samples of chlorosomes and aqueous BChl c oligomers. EPR spectroscopy shows that traces of oxidized BChl c radicals are present in nonreduced and absent in reduced samples of chlorosomes and BChl c oligomers. This suggests that the observed short excited-state lifetimes in nonreduced samples of chlorosomes and BChl c oligomers may be ascribed to excited-state quenching by BChl c radicals. The narrow EPR linewidth suggests that the BChl c are arranged in clusters of 16 and 6 molecules in chlorosomes of Chlorobium tepidum and Chloroflexus aurantiacus, respectively. PMID:8994616

  11. Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium

    PubMed Central

    Jozefczak, Marijke; Bohler, Sacha; Schat, Henk; Horemans, Nele; Guisez, Yves; Remans, Tony; Vangronsveld, Jaco; Cuypers, Ann

    2015-01-01

    Background and Aims Cadmium (Cd) is a non-essential trace element that elicits oxidative stress. Plants respond to Cd toxicity via increasing their Cd-chelating and antioxidative capacities. They predominantly chelate Cd via glutathione (GSH) and phytochelatins (PCs), while antioxidative defence is mainly based on the use and recycling of both GSH and ascorbate (AsA), complemented by superoxide dismutase (SOD) and catalase (CAT). In addition, both metabolites act as a substrate for the regeneration of other essential antioxidants, which neutralize and regulate reactive oxygen species (ROS). Together, these functions influence the concentration and cellular redox state of GSH and AsA. In this study, these two parameters were examined in plants of Arabidopsis thaliana exposed to sub-lethal Cd concentrations. Methods Wild-type plants and mutant arabidopsis plants containing 30–45 % of wild-type levels of GSH (cad2-1) or 40–50 % of AsA (vtc1-1), together with the double-mutant (cad2-1 vtc1-1) were cultivated in a hydroponic system and exposed to sub-lethal Cd concentrations. Cadmium detoxification was investigated at different levels including gene expression and metabolite concentrations. Key Results In comparison with wild-type plants, elevated basal thiol levels and enhanced PC synthesis upon exposure to Cd efficiently compensated AsA deficiency in vtc1-1 plants and contributed to decreased sensitivity towards Cd. Glutathione-deficient (cad2-1 and cad2-1 vtc1-1) mutants, however, showed a more oxidized GSH redox state, resulting in initial oxidative stress and a higher sensitivity to Cd. In order to cope with the Cd stress to which they were exposed, GSH-deficient mutants activated multiple alternative pathways. Conclusions Our observations indicate that GSH and AsA deficiency differentially alter plant GSH homeostasis, resulting in opposite Cd sensitivities relative to wild-type plants. Upon Cd exposure, GSH-deficient mutants were hampered in chelation. They

  12. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  13. Organ specific mapping of in vivo redox state in control and cigarette smoke-exposed mice using EPR/NMR co-imaging

    NASA Astrophysics Data System (ADS)

    Caia, George L.; Efimova, Olga V.; Velayutham, Murugesan; El-Mahdy, Mohamed A.; Abdelghany, Tamer M.; Kesselring, Eric; Petryakov, Sergey; Sun, Ziqi; Samouilov, Alexandre; Zweier, Jay L.

    2012-03-01

    In vivo mapping of alterations in redox status is important for understanding organ specific pathology and disease. While electron paramagnetic resonance imaging (EPRI) enables spatial mapping of free radicals, it does not provide anatomic visualization of the body. Proton MRI is well suited to provide anatomical visualization. We applied EPR/NMR co-imaging instrumentation to map and monitor the redox state of living mice under normal or oxidative stress conditions induced by secondhand cigarette smoke (SHS) exposure. A hybrid co-imaging instrument, EPRI (1.2 GHz)/proton MRI (16.18 MHz), suitable for whole-body co-imaging of mice was utilized with common magnet and gradients along with dual EPR/NMR resonators that enable co-imaging without sample movement. The metabolism of the nitroxide probe, 3-carbamoyl-proxyl (3-CP), was used to map the redox state of control and SHS-exposed mice. Co-imaging allowed precise 3D mapping of radical distribution and reduction in major organs such as the heart, lungs, liver, bladder and kidneys. Reductive metabolism was markedly decreased in SHS-exposed mice and EPR/NMR co-imaging allowed quantitative assessment of this throughout the body. Thus, in vivo EPR/NMR co-imaging enables in vivo organ specific mapping of free radical metabolism and redox stress and the alterations that occur in the pathogenesis of disease.

  14. Excited State Processes in Transition Metal Complexes, Redox Splitting in Soluble Polymers

    SciTech Connect

    Meyer, T.J.; Papanikolas, J.M.

    2002-08-08

    The photochemical and photophysical properties of polypyridyl complexes of Ru, Os, and Re have been investigated by transient absorption, emission, resonance raman and infrared spectroscopies. The latter technique has been especially useful in defining the acceptor ligand in metal-to-ligand charge transfer (MLCT) excited states and probing the details of excited state electronic and molecular structure. Derivatives of these complexes have been attached to soluble polystyrene polymers. In the resulting metal complex polymer composites it has been possible to demonstrate long range energy transfer and the existence of an antenna effect and create a mimic for the active site in the photosynthetic membrane.

  15. Energy conversion based on molecular excited states: Redox splitting in soluble polymers. Final report

    SciTech Connect

    Meyer, T.J.

    1995-12-31

    A general method was developed for preparing complexes of Ru(II) with three different bidentate ligands; it is being extended to monodentate ligands for more synthetic versatility. This method was used to prepare a series of complexes with pre-designed absorption properties, with the goal of ``black absorbers`` for use as antenna chromophores in a light-to-chemical energy conversion array. The energy gap law for nonradiative decay was studied for preparing near-IR luminophores with long excited state lifetimes. The problem of destructive dd excited states in Ru(II) polypyridyl complexes was focused on, with success in preparing an extremely photo-inert complex with monodentate pyridine ligands. Time-resolved resonance Raman and infrared spectroscopy were used to study subtle excited state properties of complexes of Ru(II), Os(II), and Re(I). Success was achieved in controlled immobilization of d{sup 6} chromophores and quenchers on styrenic polymers. Having perfected our synthetic technique, we have begun to optimize the ground and excited state properties such as chromophore density, dipole orientation, and lifetime.

  16. Host Coenzyme Q Redox State Is an Early Biomarker of Thermal Stress in the Coral Acropora millepora

    PubMed Central

    Motti, Cherie A.; Miller, David J.; van Oppen, Madeleine J. H.

    2015-01-01

    Bleaching episodes caused by increasing seawater temperatures may induce mass coral mortality and are regarded as one of the biggest threats to coral reef ecosystems worldwide. The current consensus is that this phenomenon results from enhanced production of harmful reactive oxygen species (ROS) that disrupt the symbiosis between corals and their endosymbiotic dinoflagellates, Symbiodinium. Here, the responses of two important antioxidant defence components, the host coenzyme Q (CoQ) and symbiont plastoquinone (PQ) pools, are investigated for the first time in colonies of the scleractinian coral, Acropora millepora, during experimentally-induced bleaching under ecologically relevant conditions. Liquid chromatography-mass spectrometry (LC-MS) was used to quantify the states of these two pools, together with physiological parameters assessing the general state of the symbiosis (including photosystem II photochemical efficiency, chlorophyll concentration and Symbiodinium cell densities). The results show that the responses of the two antioxidant systems occur on different timescales: (i) the redox state of the Symbiodinium PQ pool remained stable until twelve days into the experiment, after which there was an abrupt oxidative shift; (ii) by contrast, an oxidative shift of approximately 10% had occurred in the host CoQ pool after 6 days of thermal stress, prior to significant changes in any other physiological parameter measured. Host CoQ pool oxidation is thus an early biomarker of thermal stress in corals, and this antioxidant pool is likely to play a key role in quenching thermally-induced ROS in the coral-algal symbiosis. This study adds to a growing body of work that indicates host cellular responses may precede the bleaching process and symbiont dysfunction. PMID:26426118

  17. Alterations of oxygen uptake and the redox state of ubiquinone in rabbit sperm exposed to a variety of physiologic treatments.

    PubMed

    Killian, G J; Gelerinter, E; Chapman, D A

    1985-11-01

    The rate of TEMPONE reduction by electrons originating from ubiquinone in intact rabbit spermatozoa was observed for control, high ionic strength (HIS) medium-treated, and HIS-seminal plasma-treated (HIS-SP) samples. The presence of TEMPONE in the incubation medium had no effect on oxygen consumption, demonstrating the utility of TEMPONE as a nonperturbing probe of the ubiquinol redox state. The rate of TEMPONE reduction was significantly increased over control levels for sperm incubated in hypertonic medium and was correlated to a decrease in oxygen consumption and a relative increase in ATP in the total adenine nucleotide pool. This increase in TEMPONE reduction in HIS sperm was reversed by treatment of sperm with seminal plasma, but seminal plasma had no effect on oxygen consumption or relative amounts of ATP in the adenine nucleotide pool. These observations are consistent with state 3 respiration in control sperm and state 4 respiration in HIS- and HIS-SP-treated sperm. Arrhenius data were obtained for ejaculated and epididymal sperm subjected to a variety of treatments. Lines fitted to plots of Arrhenius data revealed that each treatment affected the activation energy and intercept relative to controls. Evidence is presented for a phase transition occurring at 13 degrees C based on changes in the rate of TEMPONE reduction by ubiquinol. It was noted that, above the phase transition, rate constants for the reaction were dependent upon both treatment and temperature, but below the transition the differential effects of treatment were no longer apparent. The present study has demonstrated that events taking place in the respiratory chain can be closely monitored by measuring oxygen uptake and TEMPONE reduction, and that these events are affected by alterations in the sperm environment. PMID:4084632

  18. Host Coenzyme Q Redox State Is an Early Biomarker of Thermal Stress in the Coral Acropora millepora.

    PubMed

    Lutz, Adrian; Raina, Jean-Baptiste; Motti, Cherie A; Miller, David J; van Oppen, Madeleine J H

    2015-01-01

    Bleaching episodes caused by increasing seawater temperatures may induce mass coral mortality and are regarded as one of the biggest threats to coral reef ecosystems worldwide. The current consensus is that this phenomenon results from enhanced production of harmful reactive oxygen species (ROS) that disrupt the symbiosis between corals and their endosymbiotic dinoflagellates, Symbiodinium. Here, the responses of two important antioxidant defence components, the host coenzyme Q (CoQ) and symbiont plastoquinone (PQ) pools, are investigated for the first time in colonies of the scleractinian coral, Acropora millepora, during experimentally-induced bleaching under ecologically relevant conditions. Liquid chromatography-mass spectrometry (LC-MS) was used to quantify the states of these two pools, together with physiological parameters assessing the general state of the symbiosis (including photosystem II photochemical efficiency, chlorophyll concentration and Symbiodinium cell densities). The results show that the responses of the two antioxidant systems occur on different timescales: (i) the redox state of the Symbiodinium PQ pool remained stable until twelve days into the experiment, after which there was an abrupt oxidative shift; (ii) by contrast, an oxidative shift of approximately 10% had occurred in the host CoQ pool after 6 days of thermal stress, prior to significant changes in any other physiological parameter measured. Host CoQ pool oxidation is thus an early biomarker of thermal stress in corals, and this antioxidant pool is likely to play a key role in quenching thermally-induced ROS in the coral-algal symbiosis. This study adds to a growing body of work that indicates host cellular responses may precede the bleaching process and symbiont dysfunction. PMID:26426118

  19. Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress.

    PubMed

    Sepehr, Reyhaneh; Staniszewski, Kevin; Maleki, Sepideh; Jacobs, Elizabeth R; Audi, Said; Ranji, Mahsa

    2012-04-01

    Ventilation with enhanced fractions of O(2) (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O(2)) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs. PMID:22559688

  20. Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress

    NASA Astrophysics Data System (ADS)

    Sepehr, Reyhaneh; Staniszewski, Kevin; Maleki, Sepideh; Jacobs, Elizabeth R.; Audi, Said; Ranji, Mahsa

    2012-04-01

    Ventilation with enhanced fractions of O2 (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O2) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs.

  1. Redox Control of Renal Function and Hypertension

    PubMed Central

    Whaley-Connell, Adam; Sowers, James R.

    2008-01-01

    Abstract Loss of redox homeostasis and formation of excessive free radicals play an important role in the pathogenesis of kidney disease and hypertension. Free radicals such as reactive oxygen species (ROS) are necessary in physiologic processes. However, loss of redox homeostasis contributes to proinflammatory and profibrotic pathways in the kidney, which in turn lead to reduced vascular compliance and proteinuria. The kidney is susceptible to the influence of various extracellular and intracellular cues, including the renin–angiotensin–aldosterone system (RAAS), hyperglycemia, lipid peroxidation, inflammatory cytokines, and growth factors. Redox control of kidney function is a dynamic process with reversible pro– and anti-free radical processes. The imbalance of redox homeostasis within the kidney is integral in hypertension and the progression of kidney disease. An emerging paradigm exists for renal redox contribution to hypertension. Antioxid. Redox Signal. 11, 2047–2089. PMID:18821850

  2. Absence of Mycobacterium intracellulare and Presence of Mycobacterium chimaera in Household Water and Biofilm Samples of Patients in the United States with Mycobacterium avium Complex Respiratory Disease

    PubMed Central

    Iakhiaeva, Elena; Williams, Myra D.; Brown-Elliott, Barbara A.; Vasireddy, Sruthi; Vasireddy, Ravikiran; Lande, Leah; Peterson, Donald D.; Sawicki, Janet; Kwait, Rebecca; Tichenor, Wellington S.; Turenne, Christine; Falkinham, Joseph O.

    2013-01-01

    Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC. PMID:23536397

  3. Investigation of multi-state charge-storage properties of redox-active organic molecules in silicon-molecular hybrid devices for DRAM and Flash applications

    NASA Astrophysics Data System (ADS)

    Gowda, Srivardhan Shivappa

    Molecular electronics has recently spawned a considerable amount of interest with several molecules possessing charge-conduction and charge-storage properties proposed for use in electronic devices. Hybrid silicon-molecular technology has the promise of augmenting the current silicon technology and provide for a transitional path to future molecule-only technology. The focus of this dissertation work has been on developing a class of hybrid silicon-molecular electronic devices for DRAM and Flash memory applications utilizing redox-active molecules. This work exploits the ability of molecules to store charges with single-electron precision at room temperature. The hybrid devices are fabricated by forming self-assembled monolayers of redox-active molecules on Si and oxide (SiO2 and HfO2) surfaces via formation of covalent linkages. The molecules possess discrete quantum states from which electrons can tunnel to the Si substrate at discrete applied voltages (oxidation process, cell write), leaving behind a positively charged layer of molecules. The reduction (erase) process, which is the process of electrons tunneling back from Si to the molecules, neutralizes the positively charged molecular monolayer. Hybrid silicon-molecular capacitor test structures were electrically characterized with an electrolyte gate using cyclic voltammetry (CyV) and impedance spectroscopy (CV) techniques. The redox voltages, kinetics (write/erase speeds) and charge-retention characteristics were found to be strongly dependent on the Si doping type and densities, and ambient light. It was also determined that the redox energy states in the molecules communicate with the valence band of the Si substrate. This allows tuning of write and read states by modulating minority carriers in n- and p-Si substrates. Ultra-thin dielectric tunnel barriers (SiO2, HfO2) were placed between the molecules and the Si substrate to augment charge-retention for Flash memory applications. The redox response was

  4. Oxidovanadium catechol complexes: radical versus non-radical states and redox series.

    PubMed

    Kundu, Suman; Maity, Suvendu; Weyhermüller, Thomas; Ghosh, Prasanta

    2013-07-01

    A new family of oxidovanadium complexes, [(L1(R))(VO)(L(R(')))] (R = H, R' = H, 1; R = H, R' = -CMe3, 2; R = H, R' = Me, 3; R = -CMe3, R' = H, 4 and R = -CMe3, R' = -CMe3, 5), incorporating tridentate L1(R)H ligands (L1(R)H = 2,4-di-R-6-{(2-(pyridin-2-yl)hydrazono)methyl}phenol) and substituted catechols (L(R('))H2) was substantiated. The V-Ophenolato (cis to V═O), V-OCAT (cis to V═O) and V-OCAT (trans to V═O) lengths span the ranges, 1.894(2)-1.910(2), 1.868(2)-1.887(2), and 2.120(2)-2.180(2) Å. The metrical oxidation states (MOS) of the catechols in 1-5 are fractional and vary from -1.43 to -1.60. The (51)V isotropic chemical shifts of solids and solutions of 1-5 are deshielded ((51)V CP MAS: -19.8 to +248.6; DMSO-d6: +173.9 to +414.55 ppm). The closed shell singlet (CSS) solutions of 1-5 are unstable due to open shell singlet (OSS) perturbations. The ground electronic states of 1-5 are defined by the resonance contribution of the catecholates (L(R('))CAT(2-)) and L(R('))SQ(-•) coordinated to the [VO](3+) and [VO](2+) ions. 1-5 are reversibly reducible by one electron at -(0.58-0.87) V, referenced vs ferrocenium/ferrocene, to VO(2+) complexes, [(L1(R-))(VO(2+))(L(R('))CAT(2-))](-) [1-5](-). 1-5 display another quasi-reversible or irreversible reduction wave at -(0.80-1.32) V due to the formation of hydrazone anion radical (L1(R2-•)) complexes, [(L1(R2-•))(VO(2+))(L(R('))CAT(2-))](2-), [1-5](2-), with S = 1 authenticated by the unrestricted density functional theory (DFT) calculations on 1(2-) and 3(2-) ions. Frozen glasses electron paramagnetic resonance (EPR) spectra of [1-5](-) ions [e.g., for 2, g|| = 1.948, g⊥ = 1.979, A|| = 164, A⊥ = 60] affirmed that [1-5](-) ions are the [VO](2+) complexes of L(R')CAT(2-). Spectro-electrochemical measurements and time-dependent DFT (TD DFT) calculations on 1, 3, 1(-), 3(-), and 1(2-) disclosed that the near infrared (NIR) absorption bands of 1-5 at 800 nm are due to the CSS-OSS metal to ligand charge

  5. Thioredoxins, Glutaredoxins, and Peroxiredoxins—Molecular Mechanisms and Health Significance: from Cofactors to Antioxidants to Redox Signaling

    PubMed Central

    Hanschmann, Eva-Maria; Godoy, José Rodrigo; Berndt, Carsten; Hudemann, Christoph

    2013-01-01

    Abstract Thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs) have been characterized as electron donors, guards of the intracellular redox state, and “antioxidants”. Today, these redox catalysts are increasingly recognized for their specific role in redox signaling. The number of publications published on the functions of these proteins continues to increase exponentially. The field is experiencing an exciting transformation, from looking at a general redox homeostasis and the pathological oxidative stress model to realizing redox changes as a part of localized, rapid, specific, and reversible redox-regulated signaling events. This review summarizes the almost 50 years of research on these proteins, focusing primarily on data from vertebrates and mammals. The role of Trx fold proteins in redox signaling is discussed by looking at reaction mechanisms, reversible oxidative post-translational modifications of proteins, and characterized interaction partners. On the basis of this analysis, the specific regulatory functions are exemplified for the cellular processes of apoptosis, proliferation, and iron metabolism. The importance of Trxs, Grxs, and Prxs for human health is addressed in the second part of this review, that is, their potential impact and functions in different cell types, tissues, and various pathological conditions. Antioxid. Redox Signal. 19, 1539–1605. PMID:23397885

  6. The Oxidation State of Komatiites and the Redox History of the Mantle

    NASA Astrophysics Data System (ADS)

    Nicklas, R. W.; Puchtel, I. S.; Ash, R. D.

    2015-12-01

    Oxygen fugacity (fO2) is an important intensive variable in magmatic systems. Previous studies argued that, at the level of resolution of ca. 1.0 ΔNNO log units, the mantle has been at a near-constant oxidation state since core formation [1,3]. Here, we revisit this hypothesis using the V partitioning between olivine or chromite and komatiite liquid as oxybarometers [1,2] by obtaining high-precision V abundance data for komatiite lava flows. Whole-rock samples collected across each lava flow were analyzed for V and other transition metal abundances using Standard Addition ICP-MS (SA ICP-MS); liquidus olivines and chromites were analyzed using Laser Ablation ICP-MS. Our external precision for V concentrations is 5% (2SD) for SA-ICP-MS, based on replicate analysis of standard reference materials. The V data, when plotted against wt.% MgO, define regression lines consistent with olivine control for V. Linear regressions through the V vs. MgO data for samples for each flow were used to determine V content of the emplaced lavas using known MgO contents. Calculated partition coefficients for V were used to determine the oxygen fugacity of each komatiite system using experimental calibrations of [1,2] with a precision of 0.10 - 0.05 ΔNNO log units (2SE). The calculated oxygen fugacities show a well-defined trend of increasing fO2 (>0.5 ΔNNO log units) over ~1.0 Ga of Earth's history, approaching that of modern mantle at 2.4 Ga, immediately before the Great Oxidation Event (GOE). An exception is the 3.55 Ga Schapenburg komatiite, which plots 0.5 log units above the trend, likely reflecting primordial mantle heterogeneity. Our data suggest that the mantle was becoming increasingly oxidized leading up to the GOE. A change in deep Earth buffering capacity could change the oxidation state of volcanic gases, triggering the rise in atmospheric O2 at 2.4 Ga. [1] Canil (1997) Nature 389. [2] Canil, 1999; [3] Li et al. (2004) EPSL 228. Oxygen fugacity (fO2) is an important

  7. A direct way of redox sensing.

    PubMed

    Benoit, Roger; Auer, Manfred

    2011-01-01

    The function and activity of many proteins can be regulated by changes in the intracellular redox potential. This regulation can involve posttranslational modifications mediated by redox-sensitive pathways. A more direct way to sense redox changes is through reversible covalent modification of cysteine residues of proteins by reactive oxygen species (ROS), e.g. H2O2, and reactive nitrogen species (RNS), e.g. NO. Known cysteine modifications include disulfide bonds, S-nitrosylation, S-glutathionylation, as well as sulphenic acid or sulphinic acid formation. Cysteine-based redox switches are difficult to predict because currently the knowledge of precise consensus sequences is limited. One recurrent feature of known redox switches is the close proximity of polar amino acids to the reactive cysteine, resulting in stabilization of the reactive thiolate anion form. There is growing evidence that intracellular thiol-based redox sensing and signaling mechanisms may also be involved in the regulation of RNA-binding proteins. Here, we discuss the concept of cysteine-based redox sensing and signaling, the potential importance of redox switches in RNA-binding proteins and open questions in the field. PMID:21220941

  8. High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization.

    PubMed

    Golden, Emily; Karton, Amir; Vrielink, Alice

    2014-12-01

    Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occurs via a hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Å resolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Å resolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density peak is observed within a covalent-bond distance of N5 of the flavin moiety, suggesting that a hydride-transfer event has occurred as a result of substrate oxidation trapping the flavin in the electron-rich reduced state. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom. High-level density-functional theory calculations were performed to correlate the crystallographic findings with the energetics of this unusual arrangement of the flavin moiety. These calculations suggest that strong hydrogen-bond interactions between Gly120 and the flavin N5 centre may play an important role in these structural features. PMID:25478834

  9. Synthesis and characterization of aluminum-α-diimine complexes over multiple redox states.

    PubMed

    Cole, Bren E; Wolbach, Jeffrey P; Dougherty, William G; Piro, Nicholas A; Kassel, W Scott; Graves, Christopher R

    2014-04-01

    The aluminum complexes (LMes(2-))AlCl(THF) (3) and (LDipp(-))AlCl2 (4) (LMes = N,N'-bis[2,4,6-trimethylphenyl]-2,3-dimethyl-1,4-diazabutadiene, LDipp = N,N'-bis[2,6-diisopropylphenyl]-2,3-dimethyl-1,4-diazabutadiene) were prepared by direct reduction of the ligands with sodium metal followed by salt metathesis with AlCl3. The (LMes(-))AlCl2 (5) complex was prepared through one-electron oxidative functionalization of 3 with either AgCl or CuCl. Complex 3 was characterized using (1)H and (13)C NMR spectoscopies. Single-crystal X-ray diffraction analysis of the complexes revealed that 3-5 are all four-coordinate, with 3 exhibiting a trigonal pyramidal geometry, while 4 and 5 exist between trigonal pyramidal and tetrahedral. Notable in the LMes complexes 3 and 5 is a systematic lengthening of the C-Nimido bonds and shortening of the C-C bond in the N-C-C-N backbone with increased electron density on the ligand. The geometries of the complexes 3 and 5 were optimized using DFT, which showed primarily ligand-based frontier orbitals, supporting the analysis of the solid-state structural data. The complexes 3-5 were also characterized by electrochemistry. The cyclic voltamogram of complex 3 showed an oxidation processes at -0.94 and -0.03 V versus ferrocene, while complexes 4 and 5 exhibit both reduction (-1.37 and -1.34 V, respectively) and oxidation (-0.62 and -0.73 V, respectively) features. PMID:24660986

  10. Electronegativity and redox reactions.

    PubMed

    Miranda-Quintana, Ramón Alain; Martínez González, Marco; Ayers, Paul W

    2016-08-10

    Using the maximum hardness principle, we show that the oxidation potential of a molecule increases as its electronegativity increases and also increases as its electronegativity in its oxidized state increases. This insight can be used to construct a linear free energy relation for the oxidation potential, which we train on a set of 31 organic redox couples and test on a set of 10 different redox reactions. Better results are obtained when the electronegativity of the oxidized/reduced reagents are adjusted to account for the reagents' interaction with their chemical environment. PMID:27451962

  11. Redox Redone.

    ERIC Educational Resources Information Center

    Petty, John T.

    1996-01-01

    Presents an extension of the change in oxidation number method that is used for balancing skeletal redox reactions in aqueous solutions. Retains most of the simplicity of the change in oxidation number method but provides the additional step-by-step process necessary for the beginner to balance an equation. (JRH)

  12. Effects of chronic elevated ozone concentration on the redox state and fruit yield of red pepper plant Capsicum baccatum.

    PubMed

    Bortolin, Rafael Calixto; Caregnato, Fernanda Freitas; Divan, Armando Molina; Reginatto, Flávio Henrique; Gelain, Daniel Pens; Moreira, José Cláudio Fonseca

    2014-02-01

    Ozone (O3) is one of the most harmful air pollutants to crops, contributing to high losses on crop yield. Tropospheric O3 background concentrations have increased since pre-industrial times reaching phytotoxic concentrations in many world regions. Capsicum peppers are the second most traded spice in the world, but few studies concerning the O3 effects in this genus are known. Thereby, the aim of this work was to evaluate the effects of chronic exposure to elevated O3 concentrations in red pepper plant Capsicum baccatum L. var. pendulum with especial considerations on the leaf redox state and fruit yield. Fifteen C. baccatum plants were exposed to O3 in open-top chambers during fruit ripening (62 days) at a mean concentration of 171.6 µg/m(3) from 10:00 am to 4:00 pm. We found that O3 treated plants significantly decreased the amount and the total weight of fruits, which were probably a consequence of the changes on leaf oxidative status induced by ozone exposure. Ozone exposed plants increased the reactive oxygen species (ROS) levels on the leaves, which may be associated with the observed decrease on the activity of enzymatic antioxidant defense system, as well with lower levels of polyphenol and reduced thiol groups. Enhanced ROS production and the direct O3 reaction lead to biomacromolecules damages as seen in the diminished chlorophyll content and in the elevated lipid peroxidation and protein carbonylation levels. Through a correlation analysis it was possible to observe that polyphenols content was more important to protect pepper plants against oxidative damages to lipids than to proteins. PMID:24238720

  13. The responses of cytochrome redox state and energy metabolism to dehydration support a role for cytoplasmic viscosity in desiccation tolerance

    PubMed

    Leprince; Hoekstra

    1998-12-01

    To characterize the depression of metabolism in anhydrobiotes, the redox state of cytochromes and energy metabolism were studied during dehydration of soaked cowpea (Vigna unguiculata) cotyledons and pollens of Typha latifolia and Impatiens glandulifera. Between water contents (WC) of 1.0 and 0.6 g H2O/g dry weight (g/g), viscosity as measured by electron spin resonance spectroscopy increased from 0.15 to 0.27 poise. This initial water loss was accompanied by a 50% decrease in respiration rates, whereas the adenylate energy charge remained constant at 0.8, and cytochrome c oxidase (COX) remained fully oxidized. From WC of 0.6 to 0.2 g/g, viscosity increased exponentially. The adenylate energy charge declined to 0.4 in seeds and 0.2 in pollen, whereas COX became progressively reduced. At WC of less than 0.2 g/g, COX remained fully reduced, whereas respiration ceased. When dried under N2, COX remained 63% reduced in cotyledons until WC was 0.7 g/g and was fully reduced at 0.2 g/g. During drying under pure O2, the pattern of COX reduction was similar to that of air-dried tissues, although the maximum reduction was 70% in dried tissues. Thus, at WC of less than 0.6 g/g, the reduction of COX probably originates from a decreased O2 availability as a result of the increased viscosity and impeded diffusion. We suggest that viscosity is a valuable parameter to characterize the relation between desiccation and decrease in metabolism. The implications for desiccation tolerance are discussed. PMID:9847099

  14. Regulation of hypoxia-inducible factor-α isoforms and redox state by carotid body neural activity in rats

    PubMed Central

    Peng, Ying-Jie; Yuan, Guoxiang; Khan, Shakil; Nanduri, Jayasri; Makarenko, Vladislav V; Reddy, Vaddi Damodara; Vasavda, Chirag; Kumar, Ganesh K; Semenza, Gregg L; Prabhakar, Nanduri R

    2014-01-01

    Previous studies reported that chronic intermittent hypoxia (CIH) results in an imbalanced expression of hypoxia-inducible factor-α (HIF-α) isoforms and oxidative stress in rodents, which may be due either to the direct effect of CIH or indirectly via hitherto uncharacterized mechanism(s). As neural activity is a potent regulator of gene transcription, we hypothesized that carotid body (CB) neural activity contributes to CIH-induced HIF-α isoform expression and oxidative stress in the chemoreflex pathway. Experiments were performed on adult rats exposed to CIH for 10 days. Rats exposed to CIH exhibited: increased HIF-1α and decreased HIF-2α expression; increased NADPH oxidase 2 and decreased superoxide dismutase 2 expression; and oxidative stress in the nucleus tractus solitarius and rostral ventrolateral medulla as well as in the adrenal medulla (AM), a major end organ of the sympathetic nervous system. Selective ablation of the CB abolished these effects. In the AM, sympathetic activation by the CB chemoreflex mediates CIH-induced HIF-α isoform imbalance via muscarinic acetylcholine receptor-mediated Ca2+ influx, and the resultant activation of mammalian target of rapamycin pathway and calpain proteases. Rats exposed to CIH presented with hypertension, elevated sympathetic activity and increased circulating catecholamines. Selective ablation of either the CB (afferent pathway) or sympathetic innervation to the AM (efferent pathway) abolished these effects. These observations uncover CB neural activity-dependent regulation of HIF-α isoforms and the redox state by CIH in the central and peripheral nervous systems associated with the chemoreflex. PMID:24973414

  15. In vivo simultaneous cortical and intracortical monitoring of cerebral blood flow and mitochondrial redox state in experimental animals

    NASA Astrophysics Data System (ADS)

    Barbiro-Michaely, E.; Zuckerman, T.; Zarchin, N.; Rinkevich, S.; Knoller, N.; Hadani, M.; Mayevsky, A.

    2003-07-01

    Monitoring of intra-mitochondrial NADH redox state is a common in-vivo technique in experimental animals and is rare in clinical studies. The combination of NADH monitoring with the Laser Doppler flowmetry for cerebral blood flow monitoring was described in various publications. Until now, very small effort was made to monitor NADH and CBF inside the cortex of experimental animals. The significance of this monitoring is in its application to experimental models of Parkinson"s disease or to clinical monitoring situations in the intensive care unit, when ICP is monitored. Here we compared the responses of the gerbil or rat brain to oxygen deficiency, monitored on the brain surface and in different depths. After the animals were anesthetized, the two common carotid arteries (gerbil) were isolated and prepared for following occlusion. The brain was exposed and two optical probes were located on its surface. Ischemia was induced by occluding the two carotid arteries, and anoxia was preformed by inhalation of pure N2. After recovery, one of the probes was inserted into the cortex (0.5-3mm) and a second ischemia or anoxia was preformed. The results showed that: 1. It is possible to monitor both CBF and NADH on the brain surface simultaneously with intracortical location. 2. The responses of the brain to ischemia or anoxia was smaller inside the cortex comparing to brain surface. 3. Negative correlation was found between CBF and NADH in both locations and models. In conclusion, this new model of simultaneously monitoring of CBF and NADH in different cerebral locations can shed light on various pathophysiological situations.

  16. In-vitro monitoring of redox state of cytochrome oxidase in bone by optical coherence quantitation based on low-coherence interferometry

    NASA Astrophysics Data System (ADS)

    Xu, Xiangqun; Wang, Ruikang K.; El Haj, Alicia J.

    2001-05-01

    We present optical coherence quantitation technique to monitor the redox state of mitochondria enzyme Cytochrome oxidase (CytOx) in bone tissue by the use of optical coherence tomography (OCT) system. Superluminescent diode (SLD) with its peak emission wavelength ((lambda) = 820nm) on the absorption band of oxidized form of CytOx was used in the experiments. The reflectance returning from the liquid phantoms (naphthol green B with intralipid) and bone tissue specimens (periosteum of calvaria from newborn rats) as a function of penetration depth was used to quantify the absorption changes of the sample. Absorption coefficients of naphthol green B were accurately quantified by the linear relationship between attenuation coefficients from the slopes of the reflected signals and naphthol green B concentration. The results show that the attenuation coefficient decreases in periosteums as CytOx is reduced by sodium dithionite, demonstrating the feasibility of this method to quantify the redox state of tissues studied. A 70% +/- 7% (n=4) reduction of attenuation coefficients in periosteums was clearly observed with redox change of CytOx after 5 min reduction. In addition, the results demonstrate that the OCT system is also capable of imaging the calvaria tomographically with a resolution at 9 microns, which could only be previously obtained by the conventional excisional biopsy.

  17. Chromium isotopes in carbonates — A tracer for climate change and for reconstructing the redox state of ancient seawater

    NASA Astrophysics Data System (ADS)

    Frei, R.; Gaucher, C.; Døssing, L. N.; Sial, A. N.

    2011-12-01

    powerful tool for reconstructing the redox state of ancient seawater since positive values indicate that, at least locally, Neoproterozoic shallow ocean waters were sufficiently oxidized to fractionate chromium and/or that oxygen levels of the atmosphere were sufficient to transform Cr(III) into the more mobile hexavalent Cr(VI) formed during weathering processes on land. The fact that 87Sr/ 86Sr values, despite δ 13C fluctuations, remain low (indicative of a strong hydrothermal input into the basin at his time) implies that CO 2 limitation was the cause of negative δ 13C and δ 53Cr excursions in otherwise nutrient rich late Neoproterozoic basins, and that glaciation is only one more consequence of a tectonically driven, biologically mediated system. In such a scenario, glaciation acts as an amplifier of δ 53Cr signals. These signals in marine carbonates are a sensitive tracer for redox processes in the ocean and/or on land and have the potential to contribute significantly, in combination with the other commonly used isotopic tracers, to the reconstruction of climatic changes, particularly those that are associated with major glaciation periods in Earth's history.

  18. Chromium Isotopes in Carbonates - a Tracer for Climate Change and for Reconstructing the Redox state of Ancient Seawater

    NASA Astrophysics Data System (ADS)

    Frei, R.; Gaucher, C.; Dossing, L. N.; Sial, A. N.

    2011-12-01

    the redox state of ancient seawater since positive values indicate that, at least locally, Neoproterozoic shallow ocean waters were sufficiently oxidized to fractionate chromium and/or that oxygen levels of the atmosphere were sufficient to transform Cr(III) into the more mobile hexavalent Cr(VI) formed during weathering processes on land. The fact that 87Sr/86Sr values, despite δ13C fluctuations, remain low (indicative of a strong hydrothermal input into the basin at his time) implies that CO2 limitation was the cause of negative δ13C and δ53Cr excursions in otherwise nutrient rich late Vendian basins, and that glaciation is only one more consequence of a tectonically driven, biologically mediated system. In such a scenario, glaciation acts as an amplifier of δ53Cr signals. These signals in marine carbonates are a sensitive tracer for redox processes in the ocean and/or on land and have the potential to contribute significantly to the reconstruction of climatic changes, particularly those that are associated with major glaciation periods in Earth's history.

  19. Optical Cryoimaging Reveals a Heterogeneous Distribution of Mitochondrial Redox State in ex vivo Guinea Pig Hearts and Its Alteration During Ischemia and Reperfusion.

    PubMed

    Ranji, Mahsa; Motlagh, Mohammad Masoudi; Salehpour, Fahimeh; Sepehr, Reyhaneh; Heisner, James S; Dash, Ranjan K; Camara, Amadou K S

    2016-01-01

    Oxidation of substrates to generate ATP in mitochondria is mediated by redox reactions of NADH and FADH2. Cardiac ischemia and reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation. We hypothesize that IR alters the metabolic heterogeneity of mitochondrial redox state of the heart that is only evident in the 3-D optical cryoimaging of the perfused heart before, during, and after IR. The study involved four groups of hearts: time control (TC: heart perfusion without IR), global ischemia (Isch), global ischemia followed by reperfusion (IR) and TC with PCP (a mitochondrial uncoupler) perfusion. Mitochondrial NADH and FAD autofluorescence signals were recorded spectrofluorometrically online in guinea pig ex vivo-perfused hearts in the Langendorff mode. At the end of each specified protocol, hearts were rapidly removed and snap frozen in liquid N2 for later 3-D optical cryoimaging of the mitochondrial NADH, FAD, and NADH/FAD redox ratio (RR). The TC hearts revealed a heterogeneous spatial distribution of NADH, FAD, and RR. Ischemia and IR altered the spatial distribution and caused an overall increase and decrease in the RR by 55% and 64%, respectively. Uncoupling with PCP resulted in the lowest level of the RR (73% oxidation) compared with TC. The 3-D optical cryoimaging of the heart provides novel insights into the heterogeneous distribution of mitochondrial NADH, FAD, RR, and metabolism from the base to the apex during ischemia and IR. This 3-D information of the mitochondrial redox state in the normal and ischemic heart was not apparent in the dynamic spectrofluorometric data. PMID:27574574

  20. Immobilized ferrocenium in tetraurea calix[4]arene heterodimers: Self assembly on gold, electrochemical responses, and detection of redox states by a tip: Towards molecular information storage

    NASA Astrophysics Data System (ADS)

    Xu, Songbo

    Calix[4]arene derivatives attached with sulfur functions were allowed to bind on metal surfaces. While they exist as single molecules in polar solvents, calix[4]arenes of this type form dimeric capsules in aprotic, apolar solvents. A solvent molecule is usually included in such a capsule, if no guest with a higher affinity is present. In the presence of an equimolar amount of a tetratosylurea functionalized calix[4]arene, the heterodimers were formed, inclusive guest molecules. Self-assembled monolayers (SAMs) were formed using the single calix[4]arenes with sulfur attachments and the heterodimeric capsules. Chloroform, dichloromethane and ferrocenium cations were used as guests in these immobilized heterodimeric capsules. The particular supramolecular architecture of the heterodimers should ensure after immobilization that decomposition of the capsules and release of the guest is impossible or at least hindered. The self-assembly process, and the inclusion of the various guests, such as dichloromethane or ferrocenium, were investigated with a variety of methods, including classical surface plasmon spectroscopy, gold nanoparticle absorption spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The stability of the ferrocenium filled SAMs in electrolyte solutions and the film quality was tested by cyclic voltammetry. The electrochemical response of the ferrocenium encapsulated in the heterodimer capsules both in SAMs on a gold disk electrode and in solution was investigated with cyclic voltammetry. The stability of the electrochemically active monolayers was tested by a heat treatment and multiple cycling. Reversible redox reactions for immobilized encapsulated ferrocenium on gold surfaces was demonstrated. The redox states of immobilized encapsulated ferrocenium (filled in calix[4]arene heterodimers) on gold surface were recognized by a tip of a scanning electrochemical microscopy (SECM). The SECM tip can be also used to monitor or change the

  1. Optical Cryoimaging Reveals a Heterogeneous Distribution of Mitochondrial Redox State in ex vivo Guinea Pig Hearts and Its Alteration During Ischemia and Reperfusion

    PubMed Central

    Motlagh, Mohammad Masoudi; Salehpour, Fahimeh; Sepehr, Reyhaneh; Heisner, James S.; Dash, Ranjan K.; Camara, Amadou K. S.

    2016-01-01

    Oxidation of substrates to generate ATP in mitochondria is mediated by redox reactions of NADH and FADH2. Cardiac ischemia and reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation. We hypothesize that IR alters the metabolic heterogeneity of mitochondrial redox state of the heart that is only evident in the 3-D optical cryoimaging of the perfused heart before, during, and after IR. The study involved four groups of hearts: time control (TC: heart perfusion without IR), global ischemia (Isch), global ischemia followed by reperfusion (IR) and TC with PCP (a mitochondrial uncoupler) perfusion. Mitochondrial NADH and FAD autofluorescence signals were recorded spectrofluorometrically online in guinea pig ex vivo-perfused hearts in the Langendorff mode. At the end of each specified protocol, hearts were rapidly removed and snap frozen in liquid N2 for later 3-D optical cryoimaging of the mitochondrial NADH, FAD, and NADH/FAD redox ratio (RR). The TC hearts revealed a heterogeneous spatial distribution of NADH, FAD, and RR. Ischemia and IR altered the spatial distribution and caused an overall increase and decrease in the RR by 55% and 64%, respectively. Uncoupling with PCP resulted in the lowest level of the RR (73% oxidation) compared with TC. The 3-D optical cryoimaging of the heart provides novel insights into the heterogeneous distribution of mitochondrial NADH, FAD, RR, and metabolism from the base to the apex during ischemia and IR. This 3-D information of the mitochondrial redox state in the normal and ischemic heart was not apparent in the dynamic spectrofluorometric data. PMID:27574574

  2. Expression, purification, crystallization and X-ray crystallographic studies of different redox states of the active site of thioredoxin 1 from the whiteleg shrimp Litopenaeus vannamei

    PubMed Central

    Campos-Acevedo, Adam A.; Garcia-Orozco, Karina D.; Sotelo-Mundo, Rogerio R.; Rudiño-Piñera, Enrique

    2013-01-01

    Thioredoxin (Trx) is a 12 kDa cellular redox protein that belongs to a family of small redox proteins which undergo reversible oxidation to produce a cystine disulfide bond through the transfer of reducing equivalents from the catalytic site cysteine residues (Cys32 and Cys35) to a disulfide substrate. In this study, crystals of thioredoxin 1 from the Pacific whiteleg shrimp Litopenaeus vannamei (LvTrx) were successfully obtained. One data set was collected from each of four crystals at 100 K and the three-dimensional structures of the catalytic cysteines in different redox states were determined: reduced and oxidized forms at 2.00 Å resolution using data collected at a synchrotron-radiation source and two partially reduced structures at 1.54 and 1.88 Å resolution using data collected using an in-house source. All of the crystals belonged to space group P3212, with unit-cell parameters a = 57.5 (4), b = 57.5 (4), c = 118.1 (8) Å. The asymmetric unit contains two subunits of LvTrx, with a Matthews coefficient (V M) of 2.31 Å3 Da−1 and a solvent content of 46%. Initial phases were determined by molecular replacement using the crystallographic model of Trx from Drosophila melanogaster as a template. In the present work, LvTrx was overexpressed in Escherichia coli, purified and crystallized. Structural analysis of the different redox states at the Trx active site highlights its reactivity and corroborates the existence of a dimer in the crystal. In the crystallographic structures the dimer is stabilized by several interactions, including a disulfide bridge between Cys73 of each LvTrx monomer, a hydrogen bond between the side chain of Asp60 of each monomer and several hydrophobic interactions, with a noncrystallographic twofold axis. PMID:23695560

  3. Redox signaling in cardiovascular health and disease

    PubMed Central

    Madamanchi, Nageswara R.; Runge, Marschall S.

    2013-01-01

    Spatiotemporal regulation of the activity of a vast array of intracellular proteins and signaling pathways by reactive oxygen species (ROS) governs normal cardiovascular function. However, data from experimental and animal studies strongly support that dysregulated redox signaling, resulting from hyper-activation of various cellular oxidases or mitochondrial dysfunction, is integral to the pathogenesis and progression of cardiovascular disease (CVD). In this review, we address how redox signaling modulates the protein function, the various sources of increased oxidative stress in CVD, and the labyrinth of redox-sensitive molecular mechanisms involved in the development of atherosclerosis, hypertension, cardiac hypertrophy and heart failure, and ischemia–reperfusion injury. Advances in redox biology and pharmacology for inhibiting ROS production in specific cell types and subcellular organelles combined with the development of nanotechnology-based new in vivo imaging systems and targeted drug delivery mechanisms may enable fine-tuning of redox signaling for the treatment and prevention of CVD. PMID:23583330

  4. Soluble Iron as an In Situ Indicator of the Redox State of Humic Substances in Arctic Soil: Implications for Seasonal Regeneration of Oxidized Terminal Electron Acceptors

    NASA Astrophysics Data System (ADS)

    Lipson, D.; Zlamal, J. E.; Srinivas, A. J.; Raab, T. K.

    2014-12-01

    Ferric iron (Fe(III)) and humic substances (HS) are important terminal electron acceptors for anaerobic respiration in wet tundra soils of the Arctic Coastal Plain near Barrow, Alaska. These soils are rich in both solid phase Fe minerals (including oxides such as ferrihydrite and goethite and other minerals with reduced or mixed valence such as siderite and magnetite) and soluble Fe, chelated by siderophores and other small organic molecules. This latter pool may also include nanocolloidal Fe: extremely fine-grained minerals that pass through a 0.2 micron filter. Both the solid phase and aqueous Fe pools undergo seasonal changes in redox state as a result of biological reduction by Fe-reducing microorganisms and oxidation by a variety of potential mechanisms, both abiotic and biotic. These redox cycles of solid and aqueous pools are not in phase: solid phase Fe became progressively more reduced from mid- to late summer, while aqueous phase Fe became reduced over the first half of the summer. It is well-known that HS interact with Fe, and that HS can act as electron shuttles in the reduction of Fe oxides. In other ecosystems chelated Fe(III) has been incubated with soil samples and the resulting Fe(II) produced is used as an indicator of the reducing power of HS. In these Fe-rich Arctic soils, HS are continuously in contact with chelated Fe, and therefore we interpret the redox state of this pool as an indicator of HS redox status. To verify this we conducted redox titrations of extracted HS with both reduced and oxidized Fe chelates and showed that chelated Fe could interact with HS both as electron acceptor and donator. In a field experiment, the addition of oxidized humic acids to soils resulted in an immediate oxidation of the aqueous Fe pool within 24 hours, which we attribute to abiotic oxidation of Fe by HS, followed by a slow reduction of this pool over the next week, presumably due to biological Fe reduction of the HS/aqueous Fe pool. At the end of summer

  5. Intrinsic oxygen fugacity measurements on seven chondrites, a pallasite, and a tektite and the redox state of meteorite parent bodies

    USGS Publications Warehouse

    Brett, R.; Sato, M.

    1984-01-01

    Intrinsic oxygen-fugacity (fO2) measurements were made on five ordinary chondrites, a carbonaceous chondrite, an enstatite chondrite, a pallasite, and a tektite. Results are of the form of linear log fO2 - 1 T plots. Except for the enstatite chondrite, measured results agree well with calculated estimates by others. The tektite produced fO2 values well below the range measured for terrestrial and lunar rocks. The lowpressure atmospheric regime that is reported to follow large terrestrial explosions, coupled with a very high temperature, could produce glass with fO2 in the range measured. The meteorite Salta (pallasite) has low fO2 and lies close to Hvittis (E6). Unlike the other samples, results for Salta do not parallel the iron-wu??stite buffer, but are close to the fayalite-quartz-iron buffer in slope. Minor reduction by graphite appears to have taken place during metamorphism of ordinary chondrites. fO2 values of unequilibrated chondrites show large scatter during early heating suggesting that the constituent phases were exposed to a range of fO2 conditions. The samples equilibrated with respect to fO2 in relatively short time on heating. Equilibration with respect to fO2 in ordinary chondrites takes place between grades 3 and 4 of metamorphism. Application of P - T - fO2 relations in the system C-CO-CO2 indicates that the ordinary chondrites were metamorphosed at pressures of 3-20 bars, as it appears that they lay on the graphite surface. A steep positive thermal gradient in a meteorite parent body lying at the graphite surface will produce thin reduced exterior, an oxidized near-surface layer, and an interior that is increasingly reduced with depth; a shallow thermal gradient will produce the reverse. A body heated by accretion on the outside will have a reduced exterior and oxidized interior. Meteorites from the same parent body clearly are not required to have similar redox states. ?? 1984.

  6. Redox state of deep off-craton lithospheric mantle: new data from garnet and spinel peridotites from Vitim, southern Siberia

    NASA Astrophysics Data System (ADS)

    Goncharov, A. G.; Ionov, D. A.

    2012-11-01

    Oxygen fugacity ( fO2) affects melting, metasomatism, speciation of C-O-H fluids and carbon-rich phases in the upper mantle. fO2 of deep off-craton mantle is poorly known because garnet-peridotite xenoliths are rare in alkali basalts. We examine the redox and thermal state of the lithospheric mantle between the Siberian and North China cratons using new Fe3+/ΣFe ratios in garnet and spinel obtained by Mössbauer spectroscopy, major element data and P- T estimates for 22 peridotite xenoliths as well as published data for 15 xenoliths from Vitim, Russia. Shallow spinel-facies mantle is more oxidized than deep garnet peridotites (average, -0.1 vs. -2.5 Δlog fO2(FMQ)). For intermediate garnet-spinel peridotites, fO2 estimates from spinel-based oxybarometers are 1.5-3.2 Δlog fO2(FMQ) lower than those from garnet-based oxybarometers. These rocks may be out of phase and chemical inter-mineral equilibrium because the spinel-garnet reaction and concomitant changes in mineral chemistry do not keep up with P- T changes (e.g., lithospheric heating by recent volcanism) due to slow diffusion of trivalent cations and because gar-, gar-spl and spl-facies rocks may coexist on centimeter-meter scale. The spinel-based fO2 estimates may not be correct while garnet-based fO2 values provide conditions before the heating. The T (780-1,100 °C) and fO2 ranges of the Vitim xenoliths overlap those of coarse garnet and spinel cratonic peridotites. However, because of a higher geothermal gradient, the deepest Vitim garnet peridotites are more reduced (by 0.5-2.0 Δlog fO2(FMQ)) than cratonic garnet peridotites at similar depths, and the "water maximum" conditions (>80 % H2O) in the off-craton mantle exist in a more shallow and narrow depth range (60-85 km) than in cratonic roots (100-170 km). The base of the off-craton lithospheric mantle (≥90 km) at 2.5 GPa and 1,150 °C has fO2 of -3.0 ∆log fO2(FMQ), with dominant CH4 and H2O and minor H2 in the fluid. Melting near the base of off

  7. Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.

    PubMed

    Carroll, Dustin; Howard, Diana; Zhu, Haining; Paumi, Christian M; Vore, Mary; Bondada, Subbarao; Liang, Ying; Wang, Chi; St Clair, Daret K

    2016-08-01

    Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases. PMID:27212018

  8. Redox Dysregulation in Vascular Pathobiology.

    PubMed

    Loscalzo, Joseph

    2014-10-01

    Oxidation-reduction (redox) reactions comprise a subset of fundamental biochemical reactions found throughout biological systems. While redox reactions are involved in many normal cellular functions, excess oxidative potential, or oxidative stress, can lead to cellular dysfunction and injury. Multiple protective antioxidant systems have evolved to guard against the adverse consequences of oxidant stress and injury. These systems include low-molecular-weight antioxidants, such as the glutathione-glutathione disulfide redox couple; the thiol proteome, whose various oxidation states can serve as a global redox buffer; and antioxidant enzymes, such as the superoxide dismutases, catalase, peroxidredoxins, and the glutathione peroxidases. One example of an essential antioxidant enzyme whose deficiency contributes to pathobiology in the vasculature is glutathione peroxidase-3 (GPx-3), the principal antioxidant enzyme in the extracellular compartment. This enzyme catalyzes the reduction of hydrogen and lipid peroxides to water and lipid alcohols, respectively, and does so using reducing equivalents provided by glutathione. As a selenoprotein, it requires unique translational machinery for its expression, as well as adequate selenium stores; its primary site of synthesis is the renal tubule, although all nucleated cells can express low levels of the enzyme. We have previously demonstrated that a deficiency of GPx-3 leads to enhanced platelet activation, and is an independent risk factor for acute ischemic stroke in the young. We recently developed a GPx-3-deficient mouse model, and demonstrated endothelial dysfunction as well as increased platelet-dependent thrombosis in an acute ischemic stroke model. Importantly, platelet inhibitors or small-molecule superoxide and hydrogen peroxide scavengers greatly attenuated the size of the ischemic stroke and its functional consequences in this model. These data support the importance of GPx-3as a key antioxidant enzyme that

  9. Closed-state inactivation involving an internal gate in Kv4.1 channels modulates pore blockade by intracellular quaternary ammonium ions

    PubMed Central

    Fineberg, Jeffrey D.; Szanto, Tibor G.; Panyi, Gyorgy; Covarrubias, Manuel

    2016-01-01

    Voltage-gated K+ (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. Here, we hypothesize that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, we deduced the state of the Kv4.1 channel intracellular gate by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. We found that inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. We conclude that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate. PMID:27502553

  10. Closed-state inactivation involving an internal gate in Kv4.1 channels modulates pore blockade by intracellular quaternary ammonium ions.

    PubMed

    Fineberg, Jeffrey D; Szanto, Tibor G; Panyi, Gyorgy; Covarrubias, Manuel

    2016-01-01

    Voltage-gated K(+) (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. Here, we hypothesize that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, we deduced the state of the Kv4.1 channel intracellular gate by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. We found that inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. We conclude that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate. PMID:27502553

  11. Differential reconstitution of mitochondrial respiratory chain activity and plasma redox state by cysteine and ornithine in a model of cancer cachexia.

    PubMed

    Ushmorov, A; Hack, V; Dröge, W

    1999-07-15

    The mechanism of wasting, as it occurs in malignant diseases and various etiologically unrelated conditions, is still poorly understood. We have, therefore, studied putative cause/effect relationships in a murine model of cancer cachexia, C57BL/6 mice bearing the fibrosarcoma MCA-105. The plasma of these mice showed decreased albumin and increased glutamate levels, which are typically found in practically all catabolic conditions. Skeletal muscles from tumor-bearing mice were found to have an abnormally low mitochondrial respiratory chain activity (mito.RCA) and significantly decreased glutathione (GSH) levels. The decrease in mito.RCA was correlated with an increase in the i.m. GSH disulfide/GSH ratio, the plasma cystine/thiol ratio, and the GSH disulfide/GSH ratio in the bile. This is indicative of a generalized shift in the redox state extending through different body fluids. Treatment of tumor-bearing mice with ornithine, a precursor of the radical scavenger spermine, reversed both the decrease in mito.RCA and the change in the redox state, whereas treatment with cysteine, a GSH precursor, normalized only the redox state. Treatment of normal mice with difluoromethyl-ornithine, a specific inhibitor of ornithine decarboxylase and spermine biosynthesis, inhibited the mito.RCA in the skeletal muscle tissue, thus illustrating the importance of the putrescine/spermine pathway in the maintenance of mito.RCA. Ornithine, cysteine, and N-acetyl-cysteine (NAC) also reconstituted the abnormally low concentrations of the GSH precursor glutamate in the skeletal muscle tissue of tumor-bearing mice. Higher doses, however, enhanced tumor growth and increased the plasma glucose level in normal mice. In the latter, cysteine and NAC also decreased i.m. catalase and GSH peroxidase activities. Taken together, our studies on the effects of ornithine, cysteine, and NAC illuminate some of the mechanistic pathways involved in cachexia and suggest targets for therapeutic intervention

  12. Solid-State Redox Switching of Magnetic Exchange and Electronic Conductivity in a Benzoquinoid-Bridged Mn(II) Chain Compound.

    PubMed

    Jeon, Ie-Rang; Sun, Lei; Negru, Bogdan; Van Duyne, Richard P; Dincă, Mircea; Harris, T David

    2016-05-25

    We demonstrate that incorporation of a redox-active benzoquinoid ligand into a one-dimensional chain compound can give rise to a material that exhibits simultaneous solid-state redox switching of optical, magnetic, and electronic properties. Metalation of the ligand 4,5-bis(pyridine-2-carboxamido)-1,2-catechol ((N,O)LH4) with Mn(III) affords the chain compound Mn((N,O)L)(DMSO). Structural and spectroscopic analysis of this compound show the presence of Mn(II) centers bridged by (N,O)L(2-) ligands, resulting partially from a spontaneous ligand-to-metal electron transfer. Upon soaking in a solution of the reductant Cp2Co, Mn((N,O)L)(DMSO) undergoes a ligand-centered solid-state reduction to [Mn((N,O)L)](-), as revealed by a suite of techniques, including Raman and X-ray absorption spectroscopy. The ligand-based reduction engenders a dramatic modulation of the physical properties of the chain compound. An electrochromic response, evidenced by a color change from dark green to dark purple is accompanied by a nearly 40-fold increase in magnetic coupling strength, from J = -0.38(1) to -15.6(2) cm(-1), and a 10,000-fold increase in electronic conductivity, from σ = 2.33(1) × 10(-12) S/cm (Ea = 0.64(1) eV) to 8.61(1) × 10(-8) S/cm (Ea = 0.39(1) eV). Importantly, the chemical reduction is reversible: treatment of the reduced compound with [Cp2Fe](+) regenerates the oxidized chain. Taken together, these results highlight the ability of benzoquinoid ligands to facilitate solid-state ligand-based redox reactions in nonporous coordination solids, giving rise to reversible switching of optical properties, magnetic exchange interactions, and electronic conductivity. PMID:27116424

  13. Intracellular microlasers

    NASA Astrophysics Data System (ADS)

    Humar, Matjaž; Hyun Yun, Seok

    2015-09-01

    Optical microresonators, which confine light within a small cavity, are widely exploited for various applications ranging from the realization of lasers and nonlinear devices to biochemical and optomechanical sensing. Here we use microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explore two distinct types of microresonator—soft and hard—that support whispering-gallery modes. Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (˜500 pN μm-2) and its dynamic fluctuations at a sensitivity of 20 pN μm-2 (20 Pa). In a second form, whispering-gallery modes within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes.

  14. Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation

    NASA Astrophysics Data System (ADS)

    Wierer, S.; Elgass, K.; Bieker, S.; Zentgraf, U.; Meixner, A. J.; Schleifenbaum, F.

    2011-02-01

    The analysis of molecular processes in living (plant) cells such as signal transduction, DNA replication, carbon metabolism and senescence has been revolutionized by the use of green fluorescent protein (GFP) and its variants as specific cellular markers. Many cell biological processes are accompanied by changes in the intracellular redox potential. To monitor the redox potential, a redox-sensitive mutant of GFP (roGFP) was created, which shows changes in its optical properties in response to changes in the redox state of its surrounding medium. For a quantitative analysis in living systems, it is essential to know the optical properties of roGFP in vitro. Therefore, we applied spectrally resolved fluorescence spectroscopy on purified roGFP exposed to different redox potentials to determine shifts in both the absorption and the emission spectra of roGFP. Based on these in vitro findings, we introduce a new approach using one-wavelength excitation to use roGFP for the in vivo analysis of cell biological processes. We demonstrate the ability this technique by investigating chloroplast-located Grx1-roGFP2 expressing Arabidopsis thaliana cells as example for dynamically moving intracellular compartments. This is not possible with the two-wavelength excitation technique established so far, which hampers a quantitative analysis of highly mobile samples due to the time delay between the two measurements and the consequential displacement of the investigated area.

  15. 1H- and 13C-NMR investigation of redox-state-dependent and temperature-dependent conformation changes in horse cytochrome c.

    PubMed

    Turner, D L; Williams, R J

    1993-02-01

    The redox-state dependent changes in chemical shift, which have been measured for almost 100 CHn groups in the 13C-NMR spectra of horse cytochrome c [Santos, H., and Turner, D. L. (1992) Eur. J. Biochem. 206, 721-728], have been used to investigate the nature of the redox-related change in conformation. Apart from the haem and its axial ligands, the shifts are found to be dominated by the electron-nuclear dipolar coupling in the oxidised form, as was the case in 1H-NMR studies. These pseudocontact shifts are well described by using an empirically determined magnetic susceptibility tensor in conjunction with atomic coordinates for the horse cytochrome c. The groups which fit least well are located in the vicinity of Trp59. Comparison between 1H and 13C shifts and their temperature dependence shows that the differences from expectation based on a single structure for both oxidation states are caused largely by changes in the diamagnetic contribution to the chemical shifts. Since these are different for 1H and 13C resonances they indicate, independently from crystal structure data, some redox-related movement of the protein under the haem. The significance of these results for understanding electron transfer pathways is discussed. Finally, the temperature dependence of the pseudocontact shifts in the range 30-50 degrees C is shown to be anomalous. Approximately half of the anomalous effect may be attributed to Zeeman mixing of the electronic wavefunctions with a spin-orbit coupling constant lambda = 241 cm-1, while the other half is attributed to thermal expansion of the protein. PMID:8382154

  16. Oxygen Evolution Reaction Dynamics, Faradaic Charge Efficiency, and the Active Metal Redox States of Ni-Fe Oxide Water Splitting Electrocatalysts.

    PubMed

    Görlin, Mikaela; Chernev, Petko; Ferreira de Araújo, Jorge; Reier, Tobias; Dresp, Sören; Paul, Benjamin; Krähnert, Ralph; Dau, Holger; Strasser, Peter

    2016-05-01

    Mixed Ni-Fe oxides are attractive anode catalysts for efficient water splitting in solar fuels reactors. Because of conflicting past reports, the catalytically active metal redox state of the catalyst has remained under debate. Here, we report an in operando quantitative deconvolution of the charge injected into the nanostructured Ni-Fe oxyhydroxide OER catalysts or into reaction product molecules. To achieve this, we explore the oxygen evolution reaction dynamics and the individual faradaic charge efficiencies using operando differential electrochemical mass spectrometry (DEMS). We further use X-ray absorption spectroscopy (XAS) under OER conditions at the Ni and Fe K-edges of the electrocatalysts to evaluate oxidation states and local atomic structure motifs. DEMS and XAS data consistently reveal that up to 75% of the Ni centers increase their oxidation state from +2 to +3, while up to 25% arrive in the +4 state for the NiOOH catalyst under OER catalysis. The Fe centers consistently remain in the +3 state, regardless of potential and composition. For mixed Ni100-xFex catalysts, where x exceeds 9 atomic %, the faradaic efficiency of O2 sharply increases from ∼30% to 90%, suggesting that Ni atoms largely remain in the oxidation state +2 under catalytic conditions. To reconcile the apparent low level of oxidized Ni in mixed Ni-Fe catalysts, we hypothesize that a kinetic competition between the (i) metal oxidation process and the (ii) metal reduction step during O2 release may account for an insignificant accumulation of detectable high-valent metal states if the reaction rate of process (ii) outweighs that of (i). We conclude that a discussion of the superior catalytic OER activity of Ni-FeOOH electrocatalysts in terms of surface catalysis and redox-inactive metal sites likely represents an oversimplification that fails to capture essential aspects of the synergisms at highly active Ni-Fe sites. PMID:27031737

  17. Non-equilibrium thermodynamics of thiol/disulfide redox systems: A perspective on redox systems biology

    PubMed Central

    Kemp, Melissa; Go, Young-Mi; Jones, Dean P.

    2008-01-01

    Understanding the dynamics of redox elements in biologic systems remains a major challenge for redox signaling and oxidative stress research. Central redox elements include evolutionarily conserved subsets of cysteines and methionines of proteins which function as sulfur switches and labile reactive oxygen species (ROS) and reactive nitrogen species (RNS) which function in redox signaling. The sulfur switches depend upon redox environments in which rates of oxidation are balanced with rates of reduction through the thioredoxins, glutathione/glutathione disulfide and cysteine/cystine redox couples. These central couples, which we term redox control nodes, are maintained at stable but non-equilibrium steady states, are largely independently regulated in different subcellular compartments and are quasi-independent from each other within compartments. Disruption of the redox control nodes can differentially affect sulfur switches, thereby creating a diversity of oxidative stress responses. Systems biology provides approaches to address the complexity of these responses. In the present review, we summarize thiol/disulfide pathway, redox potential and rate information as a basis for kinetic modeling of sulfur switches. The summary identifies gaps in knowledge especially related to redox communication between compartments, definition of redox pathways and discrimination between types of sulfur switches. A formulation for kinetic modeling of GSH/GSSG redox control indicates that systems biology could encourage novel therapeutic approaches to protect against oxidative stress by identifying specific redox-sensitive sites which could be targeted for intervention. PMID:18155672

  18. Monitoring Changes in the Redox State of Myoglobin in Cardiomyocytes by Raman Spectroscopy Enables the Protective Effect of NO Donors to Be Evaluated.

    PubMed

    Almohammedi, Abdullah; Kapetanaki, Sofia M; Hudson, Andrew J; Storey, Nina M

    2015-10-20

    Raman microspectroscopy has been used to monitor changes in the redox and ligand-coordination states of the heme complex in myoglobin during the preconditioning of ex vivo cardiomyocytes with pharmacological drugs that release nitric oxide (NO). These chemical agents are known to confer protection on heart tissue against ischemia-reperfusion injury. Subsequent changes in the redox and ligand-coordination states during experimental simulations of ischemia and reperfusion have also been monitored. We found that these measurements, in real time, could be used to evaluate the preconditioning treatment of cardiomyocytes and to predict the likelihood of cell survival following a potentially lethal period of ischemia. Evaluation of the preconditioning treatment was done at the single-cell level. The binding of NO to myoglobin, giving a 6-coordinate ferrous-heme complex, was inferred from the measured Raman bands of a cardiomyocyte by comparison to pure solution of the protein in the presence of NO. A key change in the Raman spectrum was observed after perfusion of the NO-donor was completed, where, if the preconditioning treatment was successful, the bands corresponding to the nitrosyl complex were replaced by bands corresponding to metmyoglobin, Mb(III). An observation of Mb(III) bands in the Raman spectrum was made for all of the cardiomyocytes that recovered contractile function, whereas the absence of Mb(III) bands always indicated that the cardiomyocyte would be unable to recover contractile function following the simulated conditions of ischemia and reperfusion in these experiments. PMID:26407187

  19. Cephalosporin-induced alteration in hepatic glutathione redox state. A potential mechanism for inhibition of hepatic reduction of vitamin K1,2,3-epoxide in the rat.

    PubMed Central

    Mitchell, M C; Mallat, A; Lipsky, J J

    1990-01-01

    Hypoprothrombinemia is a serious adverse effect of antimicrobial therapy that occurs after administration of some second- and third-generation cephalosporins which contain the methyltetrazole-thiol (MTT) group. Previous studies have shown that in vitro MTT directly inhibits microsomal gamma-carboxylation of a synthetic pentapeptide. Since MTT is a thiocarbamide, a type of compound that can increase oxidation of glutathione, the present studies were carried out to determine whether alterations in hepatic glutathione redox state might interfere with vitamin K metabolism. Dose-related increases in biliary efflux and hepatic concentration of oxidized glutathione (GSSG) occurred after intravenous administration of MTT or MTT-containing antibiotics to rats. This finding suggested that these compounds could alter the hepatic glutathione redox state in vivo. Microsomal reduction of vitamin K epoxide occurred in the presence of 100 microM dithiothreitol (DTT), but was inhibited by preincubation with GSSG at concentrations as low as 10 microM. At higher concentrations of DTT (1.0 mM) inhibition by GSSG persisted, but higher concentrations were required, suggesting that the thiol/disulfide ratio, rather than the absolute concentration of GSSG was important. By contrast, GSSG did not effect microsomal gamma-carboxylation of a pentapeptide, using either vitamin K1 or its hydroquinone as a cofactor. These findings suggest a novel mechanism for the hypoprothrombinemia occurring after administration of MTT-containing antibiotics. PMID:1978724

  20. Liquid and solid-state Cl- -sensitive microelectrodes. Characteristics and application to intracellular Cl- activity in Balanus photoreceptor

    PubMed Central

    1977-01-01

    When intracellular chloride activity (aiCl) was monitored with chloride- sensitive liquid ion exchanges (CLIX) microelectrodes in Balanus photoreceptors, replacement of extracellular chloride (Cl0) by methanesulfonate or glutamate was followed by a rapid but incomplete loss of aiCl. When propionate was used as the extracellular anion substitute, CLIX electrodes detected an apparent gain in aiCl, while a newly designed Ag-AgCl wire-in glass microelectrode showed a loss of aiCl under the same conditions. This discrepancy in Cl- washout when propionate replaced Cl0 is explained by the differences in selectivity of CLIX and Ag-AgCl electrodes for native intracellular anions and for the extracellular anion substitute which also replaces Cli and interferes in the determination of aiCl. Both electrodes indicate that ECl approximately Em when the cells are bathed in normal barnacle saline, and both electrodes showed the rate of Cl washout (tau approximately 5 min) to be independent of Cli when Cl0 was replaced by glutamate. Details of Ag-AgCl microelectrode construction are presented. These electrodes were tested and found to be insensitive to the organic anion substitutes used in this study. Selectivity data of CLIX electrodes for several anions of biological interest are described. PMID:21223

  1. Intracellular microlasers

    PubMed Central

    Humar, Matjaž; Yun, Seok Hyun

    2015-01-01

    Optical microresonators1 which confine light within a small cavity are widely exploited for various applications ranging from the realization of lasers2 and nonlinear devices3, 4, 5 to biochemical and optomechanical sensing6, 7, 8, 9, 10, 11. Here we employ microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explored two distinct types of microresonators: soft and hard, that support whispering-gallery modes (WGM). Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (~500 pN/μm2) and its dynamic fluctuations at a sensitivity of 20 pN/μm2 (20 Pa). In a second form, WGMs within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes. PMID:26417383

  2. The autolysis of human HtrA1 is governed by the redox state of its N-terminal domain.

    PubMed

    Risør, Michael W; Poulsen, Ebbe Toftgaard; Thomsen, Line R; Dyrlund, Thomas F; Nielsen, Tania A; Nielsen, Niels Chr; Sanggaard, Kristian W; Enghild, Jan J

    2014-06-17

    Human HtrA1 (high-temperature requirement protein A1) belongs to a conserved family of serine proteases involved in protein quality control and cell fate. The homotrimeric ubiquitously expressed protease has chymotrypsin-like specificity and primarily targets hydrophobic stretches in selected or misfolded substrate proteins. In addition, the enzyme is capable of exerting autolytic activity by removing the N-terminal insulin-like growth factor binding protein (IGFBP)/Kazal-like tandem motif without affecting the protease activity. In this study, we have addressed the mechanism governing the autolytic activity and find that it depends on the integrity of the disulfide bonds in the N-terminal IGFBP/Kazal-like domain. The specificity of the autolytic cleavage reveals a strong preference for cysteine in the P1 position of HtrA1, explaining the lack of autolysis prior to disulfide reduction. Significantly, the disulfides were reduced by thioredoxin, suggesting that autolysis of HtrA1 in vivo is linked to the endogenous redox balance and that the N-terminal domain acts as a redox-sensing switch. PMID:24846539

  3. How Innocent are Potentially Redox Non-Innocent Ligands? Electronic Structure and Metal Oxidation States in Iron-PNN Complexes as a Representative Case Study.

    PubMed

    Butschke, Burkhard; Fillman, Kathlyn L; Bendikov, Tatyana; Shimon, Linda J W; Diskin-Posner, Yael; Leitus, Gregory; Gorelsky, Serge I; Neidig, Michael L; Milstein, David

    2015-05-18

    Herein we present a series of new α-iminopyridine-based iron-PNN pincer complexes [FeBr2LPNN] (1), [Fe(CO)2LPNN] (2), [Fe(CO)2LPNN](BF4) (3), [Fe(F)(CO)2LPNN](BF4) (4), and [Fe(H)(CO)2LPNN](BF4) (5) with formal oxidation states ranging from Fe(0) to Fe(II) (LPNN = 2-[(di-tert-butylphosphino)methyl]-6-[1-(2,4,6-mesitylimino)ethyl]pyridine). The complexes were characterized by a variety of methods including (1)H, (13)C, (15)N, and (31)P NMR, IR, Mössbauer, and X-ray photoelectron spectroscopy (XPS) as well as electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopy, SQUID magnetometry, and X-ray crystallography, focusing on the assignment of the metal oxidation states. Ligand structural features suggest that the α-iminopyridine ligand behaves as a redox non-innocent ligand in some of these complexes, particularly in [Fe(CO)2LPNN] (2), in which it appears to adopt the monoanionic form. In addition, the NMR spectroscopic features ((13)C, (15)N) indicate the accumulation of charge density on parts of the ligand for 2. However, a combination of spectroscopic measurements that more directly probe the iron oxidation state (e.g., XPS), density functional theory (DFT) calculations, and electronic absorption studies combined with time-dependent DFT calculations support the description of the metal atom in 2 as Fe(0). We conclude from our studies that ligand structural features, while useful in many assignments of ligand redox non-innocence, may not always accurately reflect the ligand charge state and, hence, the metal oxidation state. For complex 2, the ligand structural changes are interpreted in terms of strong back-donation from the metal center to the ligand as opposed to electron transfer. PMID:25918944

  4. Metabolic Control of Redox and Redox Control of Metabolism in Plants

    PubMed Central

    Fernie, Alisdair R.

    2014-01-01

    Abstract Significance: Reduction-oxidation (Redox) status operates as a major integrator of subcellular and extracellular metabolism and is simultaneously itself regulated by metabolic processes. Redox status not only dominates cellular metabolism due to the prominence of NAD(H) and NADP(H) couples in myriad metabolic reactions but also acts as an effective signal that informs the cell of the prevailing environmental conditions. After relay of this information, the cell is able to appropriately respond via a range of mechanisms, including directly affecting cellular functioning and reprogramming nuclear gene expression. Recent Advances: The facile accession of Arabidopsis knockout mutants alongside the adoption of broad-scale post-genomic approaches, which are able to provide transcriptomic-, proteomic-, and metabolomic-level information alongside traditional biochemical and emerging cell biological techniques, has dramatically advanced our understanding of redox status control. This review summarizes redox status control of metabolism and the metabolic control of redox status at both cellular and subcellular levels. Critical Issues: It is becoming apparent that plastid, mitochondria, and peroxisome functions influence a wide range of processes outside of the organelles themselves. While knowledge of the network of metabolic pathways and their intraorganellar redox status regulation has increased in the last years, little is known about the interorganellar redox signals coordinating these networks. A current challenge is, therefore, synthesizing our knowledge and planning experiments that tackle redox status regulation at both inter- and intracellular levels. Future Directions: Emerging tools are enabling ever-increasing spatiotemporal resolution of metabolism and imaging of redox status components. Broader application of these tools will likely greatly enhance our understanding of the interplay of redox status and metabolism as well as elucidating and

  5. Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state.

    PubMed

    Dotson, P Patrick; Karakashian, Alexander A; Nikolova-Karakashian, Mariana N

    2015-02-01

    Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys(617) to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state. PMID:25287744

  6. Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state

    PubMed Central

    Dotson, P. Patrick; Karakashian, Alexander A.; Nikolova-Karakashian, Mariana N.

    2015-01-01

    Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys617 to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state. PMID:25287744

  7. Redox-Active Metal-Organic Frameworks: Highly Stable Charge-Separated States through Strut/Guest-to-Strut Electron Transfer.

    PubMed

    Sikdar, Nivedita; Jayaramulu, Kolleboyina; Kiran, Venkayala; Rao, K Venkata; Sampath, Srinivasan; George, Subi J; Maji, Tapas Kumar

    2015-08-10

    Molecular organization of donor and acceptor chromophores in self-assembled materials is of paramount interest in the field of photovoltaics or mimicry of natural light-harvesting systems. With this in mind, a redox-active porous interpenetrated metal-organic framework (MOF), {[Cd(bpdc)(bpNDI)]⋅4.5 H2 O⋅DMF}n (1) has been constructed from a mixed chromophoric system. The μ-oxo-bridged secondary building unit, {Cd2 (μ-OCO)2 }, guides the parallel alignment of bpNDI (N,N'-di(4-pyridyl)-1,4,5,8-naphthalenediimide) acceptor linkers, which are tethered with bpdc (bpdcH2 =4,4'-biphenyldicarboxylic acid) linkers of another entangled net in the framework, resulting in photochromic behaviour through inter-net electron transfer. Encapsulation of electron-donating aromatic molecules in the electron-deficient channels of 1 leads to a perfect donor-acceptor co-facial organization, resulting in long-lived charge-separated states of bpNDI. Furthermore, 1 and guest encapsulated species are characterised through electrochemical studies for understanding of their redox properties. PMID:26206156

  8. Phosphorus Redox on the Early Earth: First Identification of Low-Oxidation State Phosphorus Compounds in Terrestrial Samples

    NASA Astrophysics Data System (ADS)

    Block, K. M.; Pasek, M. A.

    2008-12-01

    Phosphorus is one of the key elements in biochemical systems, playing an important role in metabolism as ATP and other coenzymes, in replication as DNA and RNA, and in cellular structure as phospholipids. The geochemical cycling of phosphorus on the Earth is usually confined to the rock cycle- redox reactions of phosphorus are never considered. However, it has been proposed that redox reactions of phosphorus were important on the early Earth (Pasek, PNAS 2008). Indeed, such a suggestion is buttressed by the discovery of condensed phosphate formation linked to the oxidation of reduced P compounds. However, prior to the present work, there has been no report of these P compounds in geologic samples. Here we report the first occurrence of reduced P in samples of fulgurites, the glassy material resulting from the fusion of sand, soil, or rock during a lightning strike. On average, lightning strikes the Earth's surface at a rate of approximately 65 times per second (Krider et al., J. Geophys. Res.,1968) exposing target areas to extreme energy dissipation and temperatures. Through electron microprobe analyses and NMR we have identified naturally formed metal droplets containing Fe and P within several fulgurite samples and Ca-phosphite compounds. These droplets are highly reduced compared to the original material and are not naturally present in the target area, rather they were formed through the rapid, intense heating and quenching experienced during fulgurite formation. This process provides a natural means to create localized environments with greater than normal abundances of reduced Fe and P, less commonly found on Earth's surface than their oxidized counterparts. In particular, small areas that receive repeated lightning strikes due to topography or local weather patterns (e.g. hilltops) could potentially house unique microhabitats with reduced elements available for biological use.

  9. Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae

    PubMed Central

    Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

    2015-01-01

    The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed. PMID:25628332

  10. Ediacaran Redox Fluctuations

    NASA Astrophysics Data System (ADS)

    Sahoo, S. K.; Jiang, G.; Planavsky, N. J.; Kendall, B.; Owens, J. D.; Anbar, A. D.; Lyons, T. W.

    2013-12-01

    Evidence for pervasive oxic conditions, and likely even deep ocean oxygenation has been documented at three intervals in the lower (ca. 632 Ma), middle (ca. 580 Ma) and upper (ca. 551 Ma) Ediacaran. The Doushantuo Formation in South China hosts large enrichments of redox-sensitive trace element (e.g., molybdenum, vanadium and uranium) in anoxic shales, which are indicative of a globally oxic ocean-atmosphere system. However, ocean redox conditions between these periods continue to be a topic of debate and remain elusive. We have found evidence for widespread anoxic conditions through much of the Ediacaran in the deep-water Wuhe section in South China. During most of the Ediacaran-early Cambrian in basinal sections is characterized by Fe speciation data and pyrite morphologies that indicate deposition under euxinic conditions with near-crustal enrichments of redox-sensitive element and positive pyrite-sulfur isotope values, which suggest low levels of marine sulfate and widespread euxinia. Our work reinforces an emerging view that the early Earth, including the Ediacaran, underwent numerous rises and falls in surface oxidation state, rather than a unidirectional rise as originally imagined. The Ediacaran ocean thus experienced repetitive expansion and contraction of marine chalcophilic trace-metal levels that may have had fundamental impact on the slow evolution of early animals and ecosystems. Further, this framework forces us to re-examine the relationship between Neoproterozoic oxygenation and metazoan diversification. Varying redox conditions through the Cryogenian and Ediacaran may help explain molecular clock and biomarker evidence for an early appearance and initial diversification of metazoans but with a delay in the appearance of most major metazoan crown groups until close to Ediacaran-Cambrian boundary.