Importance Of Redox Regulation Research Articles

Antioxidant Responses and Redox Regulation Within Plant-Beneficial Microbe Interaction

The increase in extreme climate events associated with global warming is a great menace to crop productivity nowadays. In addition to abiotic stresses, warmer conditions favor the spread of infectious diseases affecting plant performance. Within this context, beneficial microbes constitute a sustainable alternative for the mitigation of the effects of climate change on plant growth and productivity. Used as biostimulants to improve plant growth, they also increase plant resistance to abiotic and biotic stresses through the generation of a primed status in the plant, leading to a better and faster response to stress. In this review, we have focused on the importance of a balanced redox status for the adequate performance of the plant and revisited the different antioxidant mechanisms supporting the biocontrol effect of beneficial microbes through the adjustment of the levels of reactive oxygen species (ROS). In addition, the different tools for the analysis of antioxidant responses and redox regulation have been evaluated. The importance of redox regulation in the activation of the immune responses through different mechanisms, such as transcriptional regulation, retrograde signaling, and post-translational modification of proteins, emerges as an important research goal for understanding the biocontrol activity of the beneficial microbes.

2H-Thiopyran-2-thione sulfine, a compound for converting H2S to HSOH/H2S2 and increasing intracellular sulfane sulfur levels

Reactive sulfane sulfur species such as persulfides (RSSH) and H2S2 are important redox regulators and closely linked to H2S signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert H2S to HSOH, and then to H2S2 in the presence of excess H2S. Meanwhile, the reaction product 2H-thiopyran-2-thione (TT) can be oxidized to reform TTS by biological oxidants. The reaction mechanism of TTS is studied experimentally and computationally. TTS can be conjugated to proteins to achieve specific delivery, and the combination of TTS and H2S leads to highly efficient protein persulfidation. When TTS is applied in conjunction with established H2S donors, the corresponding donors of H2S2 (or its equivalents) are obtained. Cell-based studies reveal that TTS can effectively increase intracellular sulfane sulfur levels and compensate for certain aspects of sulfide:quinone oxidoreductase (SQR) deficiency. These properties make TTS a conceptually new strategy for the design of donors of reactive sulfane sulfur species.

The Interaction and Effect of a Small MitoBlock Library as Inhibitor of ALR Protein-Protein Interaction Pathway.

MIA40 and ALR of the MIA pathway mediate the import of protein precursors that form disulfides into the mitochondrial intermembrane space. This import pathway is suggested to be a linear pathway in which MIA40 first binds to the precursor via a disulfide linkage and oxidizes it. Subsequently, ALR re-oxidizes MIA40 and then ALR transfers electrons to terminal electron acceptors. However, the precise mechanism by which ALR and MIA40 coordinate translocation is unknown. With a collection of small molecule modulators (MB-5 to MB-9 and MB-13) that inhibit ALR activity, we characterized the import mechanism in mitochondria. NMR studies show that most of the compounds bind to a similar region in ALR. Mechanistic studies with small molecules demonstrate that treatment with compound MB-6 locks the precursor in a state bound to MIA40, blocking re-oxidation of MIA40 by ALR. Thus, small molecules that target a similar region in ALR alter the dynamics of the MIA import pathway differently, resulting in a set of probes that are useful for studying the catalysis of the redox-regulated import pathway in model systems.

Construction of optical dual-mode sensing platform based on green emissive carbon quantum dots for effective detection of ClO- and cellular imaging

Hypochlorite (ClO-) is an important redox regulator in reactive oxygen species, which play a considerable role in oxidative stress and related diseases. Hence, accurate and sensitive monitoring of ClO- concentration was urgently needed in the fields of life sciences, food and environment. Bright green fluorescent carbon quantum dots (G-CQDs) were synthesized utilizing one-step hydrothermal method with citric acid and acriflavine precursors. Through TEM, FTIR, XPS and zeta potential characterization procedures, G-CQDs illustrated uniformly dispersed and significant number of –NH2 and –OH on the surface. Meanwhile, the fluorescence and colorimetric analysis exhibited wide linear range and low detection limit response to ClO-. The fluorescence changes of G-CQDs were identified via smartphone to realize mobile sensing of ClO-. Subsequently, G-CQDs was applied for visualization and quantitative detection of ClO- in drinking water samples with satisfactory recovery rate. More importantly, G-CQDs demonstrated good water solubility, optical stability and excellent biocompatibility, which offered a promising analysis approach in cell imaging and exogenous ClO- detection in living cells. G-CQDs illustrated bright prospect and great potential in practical application of ClO- associated disease prevention and early clinical diagnosis.

Selenium, Selenoproteins and 10-year Cardiovascular Risk: Results from the ATTICA Study.

Selenium (Se) is an essential trace element that is involved in several pathophysiological functions. The relationship of Se with cardiovascular disease remains inconclusive, especially regarding the role of different selenospecies. The present study assessed the levels of Se distribution in plasma selenoproteins, namely glutathione peroxidase 3 (GPx3), selenoprotein P (SelP) and selenoalbumin (SeAlb) and total Se in selenoproteins in relation to 10-year cardiovascular risk in the ATTICA prospective study. A sub-sample from the ATTICA Study's database, consisting of 278 subjects (114 women and 164 men) with data on Se and selenoproteins levels, was considered. SeGPx3, SelP, and SeAlb in human plasma were simultaneously determined by high-performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) at baseline. The duration of the follow-up was 8.74 ±2.36 years (mean± standard deviation) and cardiovascular outcomes were recorded. Cox proportional hazards models were applied with total Se or selenoprotein Se as independent variables adjusted for several covariates. Total Se in selenoproteins was positively related to 10-year relative risk of cardiovascular disease (Hazard Ratios of 3rd vs 2nd tertile 10.02, 95% CI:1.15, 92.34). Subjects with high Se but low SeGPx3, as identified by discordant percentiles in the distribution of SeGPx3 and Se, had a higher cardiovascular risk. The differentiated effects of circulating selenoproteins on cardiovascular disease risk in the present study, suggest the importance of redox regulation by specific selenoproteins.

Regulation of hyperoxia-induced neonatal lung injury via post-translational cysteine redox modifications

Preterm infants and patients with lung disease often have excess fluid in the lungs and are frequently treated with oxygen, however long-term exposure to hyperoxia results in irreversible lung injury. Although the adverse effects of hyperoxia are mediated by reactive oxygen species, the full extent of the impact of hyperoxia on redox-dependent regulation in the lung is unclear. In this study, neonatal mice overexpressing the beta-subunit of the epithelial sodium channel (β-ENaC) encoded by Scnn1b and their wild type (WT; C57Bl6) littermates were utilized to study the pathogenesis of high fraction inspired oxygen (FiO2)-induced lung injury. Results showed that O2-induced lung injury in transgenic Scnn1b mice is attenuated following chronic O2 exposure. To test the hypothesis that reversible cysteine-redox-modifications of proteins play an important role in O2-induced lung injury, we performed proteome-wide profiling of protein S-glutathionylation (SSG) in both WT and Scnn1b overexpressing mice maintained at 21% O2 (normoxia) or FiO2 85% (hyperoxia) from birth to 11–15 days postnatal. Over 7700 unique Cys sites with SSG modifications were identified and quantified, covering more than 3000 proteins in the lung. In both mouse models, hyperoxia resulted in a significant alteration of the SSG levels of Cys sites belonging to a diverse range of proteins. In addition, substantial SSG changes were observed in the Scnn1b overexpressing mice exposed to hyperoxia, suggesting that ENaC plays a critically important role in cellular regulation. Hyperoxia-induced SSG changes were further supported by the results observed for thiol total oxidation, the overall level of reversible oxidation on protein cysteine residues. Differential analyses reveal that Scnn1b overexpression may protect against hyperoxia-induced lung injury via modulation of specific processes such as cell adhesion, blood coagulation, and proteolysis. This study provides a landscape view of protein oxidation in the lung and highlights the importance of redox regulation in O2-induced lung injury.

Aim32 is a dual-localized 2Fe-2S mitochondrial protein that functions in redox quality control

Yeast is a facultative anaerobe and uses diverse electron acceptors to maintain redox-regulated import of cysteine-rich precursors via the mitochondrial intermembrane space assembly (MIA) pathway. With the growing diversity of substrates utilizing the MIA pathway, understanding the capacity of the intermembrane space (IMS) to handle different types of stress is crucial. We used MS to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Altered inheritance of mitochondria 32 (Aim32), a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1-binding protein. Detailed localization studies showed that Aim32 resided in both the mitochondrial matrix and IMS. Aim32 interacted with additional proteins including redox protein Osm1 and protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or mutation of conserved cysteine residues that coordinate the Fe-S center in Aim32 resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased. Aim32 also bound to several mitochondrial proteins under nonreducing conditions, suggesting a function in maintaining the redox status of proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress such as elevated temperature and hydroxyurea, and under anaerobic conditions. These studies suggest that the Fe-S protein Aim32 has a potential role in general redox homeostasis in the matrix and IMS. Thus, Aim32 may be poised as a sensor or regulator in quality control for a broad range of mitochondrial proteins.

Chemical Reactivities of Two Widely Used Ruthenium-Based CO-Releasing Molecules with a Range of Biologically Important Reagents and Molecules.

Ruthenium-based CO-releasing molecules (CO-RMs), CORM-2 and CORM-3, have been widely used as surrogates of CO for studying its biological effects in vitro and in vivo with much success. However, several previous solution-phase and in vitro studies have revealed the ability of such CO-RMs to chemically modify proteins and reduce aromatic nitro groups due to their intrinsic chemical reactivity under certain conditions. In our own work of studying the cytoprotective effects of CO donors, we were in need of assessing chemical factors that could impact the interpretation of results from CO donors including CORM-2,3 in various in vitro assays. For this, we examined the effects of CORM-2,3 toward representative reagents commonly used in various bioassays including resazurin, tetrazolium salts, nitrites, and azide-based H2S probes. We have also examined the effect of CORM-2,3 on glutathione disulfide (GSSG), which is a very important redox regulator. Our studies show the ability of these CO-RMs to induce a number of chemical and/or spectroscopic changes for several commonly used biological reagents under near-physiological conditions. These reactions/spectroscopic changes cannot be duplicated with CO-deleted CO-RMs (iCORMs), which are often used as negative controls. Furthermore, both CORM-2 and -3 are capable of consuming and reducing GSSG in solution. We hope that the results described will help in the future design of control experiments using Ru-based CO-RMs.

Redox regulation of glutathione peroxidase by thioredoxin in longan fruit in relation to senescence and quality deterioration

Thioredoxins (Trxs) are important redox regulators in organisms. However, their involvement in fruit senescence and quality deterioration remains unclear. In this study, one Trx (DlTrx1) and one NADPH-dependent Trx reductase (DlNRT1) cDNAs, were cloned from longan fruit. The DlTrx1 could be effectively reduced by the DlNTR1. Expression of DlTrx1 and DlNTR1 were up-regulated during fruit senescence and quality deterioration. We further identified 33 potential Trx target proteins in longan, including one glutathione peroxidase (DlGpx). DlTrx1 could physically interact with DlGpx. DlTrx1 in combination with DlNTR1 effectively activated DlGpx activity by regulating its redox state. Cys90 in DlGPx could form a disulfide bond with either Cys42 or Cys71, which were the sites of redox modulation. Furthermore, DlGpx exhibited a higher ratio of disulfide bonds to sulfhydryl groups in senescent or deteriorative fruit. We propose that Trx-mediated redox regulation of DlGpx is involved in senescence or quality deterioration of harvested longan fruit.

Recent Advances in the Photoautotrophic Metabolism of Cyanobacteria: Biotechnological Implications.

Cyanobacteria constitute the only phylum of oxygen-evolving photosynthetic prokaryotes that shaped the oxygenic atmosphere of our planet. Over time, cyanobacteria have evolved as a widely diverse group of organisms that have colonized most aquatic and soil ecosystems of our planet and constitute a large proportion of the biomass that sustains the biosphere. Cyanobacteria synthesize a vast array of biologically active metabolites that are of great interest for human health and industry, and several model cyanobacteria can be genetically manipulated. Hence, cyanobacteria are regarded as promising microbial factories for the production of chemicals from highly abundant natural resources, e.g., solar energy, CO2, minerals, and waters, eventually coupled to wastewater treatment to save costs. In this review, we summarize new important discoveries on the plasticity of the photoautotrophic metabolism of cyanobacteria, emphasizing the coordinated partitioning of carbon and nitrogen towards growth or compound storage, and the importance of these processes for biotechnological perspectives. We also emphasize the importance of redox regulation (including glutathionylation) on these processes, a subject which has often been overlooked.

Effect of smokeless tobacco use on salivary glutathione levels among chronic periodontitis patients before and after non-surgical periodontal therapy.

INTRODUCTIONSmokeless tobacco (SLT) jeopardizes periodontal health and also produces an imbalance between reactive oxygen species (ROS) and antioxidants (AO) such as glutathione. Glutathione is an important redox regulator in saliva and its maintenance is essential for periodontal health. Periodontitis patients have a reduced total AO capacity in whole saliva, and periodontal therapy restores the redox balance. Hence, the purpose of this study was to investigate the effects of smokeless tobacco use on saliva glutathione levels in patients with chronic periodontitis and to evaluate these effects after non-surgical periodontal therapy.METHODSThe study included 100 subjects in four groups; healthy, gingivitis, and chronic periodontitis (CP) patients with and without SLT use. Saliva samples were collected, and clinical periodontal parameters were recorded at baseline and at one month after non-surgical periodontal therapy. Glutathione levels were analyzed using spectrophotometry at 412 nm. Statistical analysis was carried out using paired t-test, chi-squared, and analysis of variance (ANOVA).RESULTSMean glutathione values in saliva were found to be lower in periodontitis patients compared to SLT users at baseline and at 1 month post non-surgical periodontal therapy (p<0.001) In addition, non-surgical therapy leads to a highly significant improvement in the glutathione levels in gingivitis, in the CP with and without ST groups (p<0.001).CONCLUSIONSSuccessful non-surgical periodontal therapy leads to considerable progress in the redox balance, thus regulating glutathione levels and reducing the effects of SLT on the periodontium. This emphasises the importance of non-surgical therapy, especially among SLT users.

Nitroso-oxidative stress and cardiovascular disease

Vascular oxidant stress accompanies the development of many vascular diseases. Promoted by inflammation and thrombosis, vascular oxidant stress decreases bioavailable nitric oxide and leads to endothelial dysfunction. In this presentation, I will discuss the role of vasoprotective mechanisms involving the selenoproteins, glutathione peroxidase-1 and glutathione peroxidase-3, and their beneficial actions in minimizing nitroso-oxidative stress in cardiovascular diseases. Using a combination of genetic, molecular, biochemical, animal, and human data, I will demonstrate the importance of redox regulation in modulating bioavailable nitric oxide in the vasculature and minimizing the consequences of oxidative stress for the preservation of endothelial and vascular function.

Osm1 facilitates the transfer of electrons from Erv1 to fumarate in the redox-regulated import pathway in the mitochondrial intermembrane space.

Prokaryotes have aerobic and anaerobic electron acceptors for oxidative folding of periplasmic proteins. The mitochondrial intermembrane space has an analogous pathway with the oxidoreductase Mia40 and sulfhydryl oxidase Erv1, termed the mitochondrial intermembrane space assembly (MIA) pathway. The aerobic electron acceptors include oxygen and cytochrome c, but an acceptor that can function under anaerobic conditions has not been identified. Here we show that the fumarate reductase Osm1, which facilitates electron transfer from fumarate to succinate, fills this gap as a new electron acceptor. In addition to microsomes, Osm1 localizes to the mitochondrial intermembrane space and assembles with Erv1 in a complex. In reconstitution studies with reduced Tim13, Mia40, and Erv1, the addition of Osm1 and fumarate completes the disulfide exchange pathway that results in Tim13 oxidation. From in vitro import assays, mitochondria lacking Osm1 display decreased import of MIA substrates, Cmc1 and Tim10. Comparative reconstitution assays support that the Osm1/fumarate couple accepts electrons with similar efficiency to cytochrome c and that the cell has strategies to coordinate expression of the terminal electron acceptors. Thus Osm1/fumarate is a new electron acceptor couple in the mitochondrial intermembrane space that seems to function in both aerobic and anaerobic conditions.

The peroxisomal import receptor PEX5 functions as a stress sensor, retaining catalase in the cytosol in times of oxidative stress

Accumulating evidence indicates that peroxisome functioning, catalase localization, and cellular oxidative balance are intimately interconnected. Nevertheless, it remains largely unclear why modest increases in the cellular redox state especially interfere with the subcellular localization of catalase, the most abundant peroxisomal antioxidant enzyme. This study aimed at gaining more insight into this phenomenon. Therefore, we first established a simple and powerful approach to study peroxisomal protein import and protein-protein interactions in living cells in response to changes in redox state. By employing this approach, we confirm and extend previous observations that Cys-11 of human PEX5, the shuttling import receptor for peroxisomal matrix proteins containing a C-terminal peroxisomal targeting signal (PTS1), functions as a redox switch that modulates the protein's activity in response to intracellular oxidative stress. In addition, we show that oxidative stress affects the import of catalase, a non-canonical PTS1-containing protein, more than the import of a reporter protein containing a canonical PTS1. Furthermore, we demonstrate that changes in the local redox state do not affect PEX5-substrate binding and that human PEX5 does not oligomerize in cellulo, not even when the cells are exposed to oxidative stress. Finally, we present evidence that catalase retained in the cytosol can protect against H2O2-mediated redox changes in a manner that peroxisomally targeted catalase does not. Together, these findings lend credit to the idea that inefficient catalase import, when coupled with the role of PEX5 as a redox-regulated import receptor, constitutes a cellular defense mechanism to combat oxidative insults of extra-peroxisomal origin.

Regulatory Role of Redox Balance in Determination of Neural Precursor Cell Fate.

In 1990s, reports of discovery of a small group of cells capable of proliferation and contribution to formation of new neurons in the central nervous system (CNS) reversed a century-old concept on lack of neurogenesis in the adult mammalian brain. These cells are found in all stages of human life and contribute to normal cellular turnover of the CNS. Therefore, the identity of regulating factors that affect their proliferation and differentiation is a highly noteworthy issue for basic scientists and their clinician counterparts for therapeutic purposes. The cues for such control are embedded in developmental and environmental signaling through a highly regulated tempo-spatial expression of specific transcription factors. Novel findings indicate the importance of reactive oxygen species (ROS) in the regulation of this signaling system. The elusive nature of ROS signaling in many vital processes from cell proliferation to cell death creates a complex literature in this field. Here, we discuss the emerging thoughts on the importance of redox regulation of proliferation and maintenance in mammalian neural stem and progenitor cells under physiological and pathological conditions. The current knowledge on ROS-mediated changes in redox-sensitive proteins that govern the molecular mechanisms in proliferation and differentiation of these cells is reviewed.

Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1-101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.

Dietary Cocoa Improves Hepatic Mitochondrial Function in Obese Mice

Nonalcoholic fatty liver disease (NAFLD) is an important co‐morbidity of obesity. Previously, we found that dietary cocoa (Theobroma cacao) supplementation reduced hepatic lipid content and inflammation in obese mice. Here, we investigated the effect of dietary cocoa on hepatic mitochondrial biogenesis, oxidative stress, and antioxidant response. Male C57BL/6J were fed HF (60 % kcal from fat) diet for 8 wks, and then randomized to continue HF diet or to consume HF diet supplemented with 80 mg/g cocoa powder (HF‐HFC) for an additional 10 wks. We found cocoa treatment increases hepatic mitochondrial DNA copy number by 48.6% compared to HF‐fed mice, suggesting increased mitochondrial biogenesis. Using real‐time PCR, we found that mRNA levels of sirtuin (sirt)1, peroxisome proliferator‐activated receptor gamma coactivator 1a, and nuclear respiratory factor 1 were significantly increased in HF‐HFC compared with HF‐fed mice. These genes have been reported to promote mitochondrial biogenesis. We found that cocoa supplementation decreased hepatic lipid peroxidation by 57% compared to HF‐fed controls indicating reduced oxidative stress. Cocoa supplementation also increased the expression of Sirt3, an important mitochondrial redox regulator, as well as manganese superoxide dismutase and glutathione peroxidase (Gpx)1 was increased in cocoa supplemented mice HF‐HFC compared to HF‐fed controls. Cocoa also increased hepatic Gpx activity by 43% compared to HF‐fed controls. In summary, our results demonstrate that the beneficial effects of cocoa supplementation on NAFLD may be mediated by increases in mitochondrial biogenesis and related antioxidant response signaling.

Evaluation of glutathione level in gingival crevicular fluid in periodontal health, in chronic periodontitis and after nonsurgical periodontal therapy: A clinicobiochemical study.

Context:Periodontitis is predominantly due to exaggerated host response to pathogenic microorganisms and their products which causes an imbalance between the reactive oxygen species-antioxidant in gingival crevicular fluid (GCF). Glutathione is an important redox regulator in GCF and maintenance of stable reduced glutathione (GSH):oxidized glutathione (GSSG) ratio is essential for periodontal health.Aims:The present study was undertaken to evaluate and compare the level of glutathione and redox balance (GSH: GSSG ratio) in GCF of chronic periodontitis patients, periodontally healthy controls and also to evaluate the effect of nonsurgical periodontal therapy on the level of glutathione and redox balance during 3 months postoperative visit.Study Design:Baseline GCF samples were collected from 20 chronic periodontitis patients and 20 periodontally healthy subjects for GSH and GSSG levels estimation. Periodontitis patients were recalled 3 months postnonsurgical periodontal therapy to re-sample GCF.Materials and Methods:GSH and GSSG levels were measured by high-performance liquid chromatography. The values were statistically analyzed by Paired t-test.Results:The mean GSH and GSSG values in GCF were found to be significantly lower in periodontitis patients pre- and 3 months post-nonsurgical periodontal therapy, compared with those in the control group subjects. In addition, the successful nonsurgical therapy even though leading to a significant improvement in the GSH and GSSG levels, does not restore glutathione concentration to the levels seen in healthy subjects.Conclusion:Successful nonsurgical periodontal therapy leads to significant improvement in the redox balance (GSH: GSSG ratio) in chronic periodontitis patients.

Abstract 336: The Imbalanced Redox Homeostasis in Senescent Endothelial Cells is Due to Dysregulated Thioredoxin-1 and NADPH-Oxidase 4

Senescent endothelial cells are not only present in aged vessels, but also in atherosclerotic lesions, where they contribute to endothelial dysfunction. One hallmark of cellular senescence is an imbalanced redox homeostasis. However, the underlying mechanisms how this imbalance occurs are not completely understood. Thioredoxin-1 (Trx-1) is one of the most important redox regulators in primary human endothelial cells. It has previously been shown that Trx-1, in addition to its oxidoreductase activity has anti-apoptotic properties. Cellular generators of reactive oxygen species (ROS) are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate whether and how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells, an ex vivo model mirroring the situation in aged or diseased vessels. We treated primary human endothelial cells for two weeks with hydrogen peroxide to generate stress-induced premature senescence in these cells. Senescence induction was confirmed by an increase in senescence-associated beta-Galactosidase and nuclear p21, two established senescence markers. Moreover, cytosolic and mitochondrial reactive oxygen species formation were enhanced. Concomitantly, we observed decreased Trx-1 levels and elevated NOX4 levels, which could explain the imbalance in the cellular redox homeostasis. Moreover, the lysosomal protease Cathepsin D, known to degrade Trx-1 under short-term oxidative stress, was over-activated, providing a mechanistic basis for reduced Trx-1 protein levels. Inhibition of “over-active” Cathepsin D by the specific, cell-permeable inhibitor pepstatin A counteracted the increase in nuclear p21, ROS formation and degradation of Trx-1, thus leading to blockade of stress-induced premature senscence by stabilizing the cellular redox homeostasis. Moreover, aortic Trx-1 levels negatively correlated with NOX4 expression in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium and their wildtype littermates. Our data show that loss of Trx-1 and upregulation of NOX4 may importantly contribute to the imbalanced redox homeostasis in senescent endothelial cells ex vivo and in vivo.

Glucan, Water Dikinase Exerts Little Control over Starch Degradation in Arabidopsis Leaves at Night.

The first step on the pathway of starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase (GWD). It has been suggested that GWD is important for the control of starch degradation, because its transcript levels undergo strong diel fluctuations, its activity is subject to redox regulation in vitro, and starch degradation is strongly decreased in gwd mutant plants. To test this suggestion, we analyzed changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts were strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD. We found that GWD protein levels do not vary over the diel cycle and that the protein has a half-life of 2 d. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. Surprisingly, this degree of reduction also inhibits starch synthesis in the light. To discover the importance of redox regulation, we generated transgenic plants expressing constitutively active GWD. These plants retained normal control of degradation. We conclude that GWD exerts only a low level of control over starch degradation in Arabidopsis leaves.