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1
Tu, Benjamin P., and Jonathan S. Weissman. "Oxidative protein folding in eukaryotes." Journal of Cell Biology 164, no. 3 (February 2, 2004): 341–46. http://dx.doi.org/10.1083/jcb.200311055.
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The endoplasmic reticulum (ER) provides an environment that is highly optimized for oxidative protein folding. Rather than relying on small molecule oxidants like glutathione, it is now clear that disulfide formation is driven by a protein relay involving Ero1, a novel conserved FAD-dependent enzyme, and protein disulfide isomerase (PDI); Ero1 is oxidized by molecular oxygen and in turn acts as a specific oxidant of PDI, which then directly oxidizes disulfide bonds in folding proteins. While providing a robust driving force for disulfide formation, the use of molecular oxygen as the terminal electron acceptor can lead to oxidative stress through the production of reactive oxygen species and oxidized glutathione. How Ero1p distinguishes between the many different PDI-related proteins and how the cell minimizes the effects of oxidative damage from Ero1 remain important open questions.
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Darley-Usmar, V. M., A. Severn, V. J. O'Leary, and M. Rogers. "Treatment of macrophages with oxidized low-density lipoprotein increases their intracellular glutathione content." Biochemical Journal 278, no. 2 (September 1, 1991): 429–34. http://dx.doi.org/10.1042/bj2780429.
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Macrophages derived from the human monocyte cell line THP-1 or isolated from the peritoneum of C3H/HEJ mice were incubated with oxidized low-density lipoprotein (LDL) and the total glutathione content (oxidized plus reduced) was measured. An initial depletion of glutathione was followed by an increase, such that after a period of 24 h the glutathione content has approximately doubled. This response required the oxidation of the lipid phase of the LDL molecule, since both native LDL and acetylated LDL had little effect on glutathione levels. The response of the cells to oxidized LDL was dependent on the extent of oxidative modification of the protein. It was also found that 4-hydroxynonenal had a similar effect on THP-1 cells, and we suggest that this or other aldehydes present in oxidized LDL causes the induction of glutathione synthesis in response to an initial oxidative stress and consequent glutathione depletion. In addition, we found that both cell types possess transferases and peroxidases capable of detoxifying aldehydes and peroxides. However, treatment of cells with oxidized LDL or 4-hydroxynonenal for a period of 24 h had no effect on the activities of these enzymes.
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Iskusnykh, Igor Y., Anastasia A. Zakharova, and Dhruba Pathak. "Glutathione in Brain Disorders and Aging." Molecules 27, no. 1 (January 5, 2022): 324. http://dx.doi.org/10.3390/molecules27010324.
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Glutathione is a remarkably functional molecule with diverse features, which include being an antioxidant, a regulator of DNA synthesis and repair, a protector of thiol groups in proteins, a stabilizer of cell membranes, and a detoxifier of xenobiotics. Glutathione exists in two states—oxidized and reduced. Under normal physiological conditions of cellular homeostasis, glutathione remains primarily in its reduced form. However, many metabolic pathways involve oxidization of glutathione, resulting in an imbalance in cellular homeostasis. Impairment of glutathione function in the brain is linked to loss of neurons during the aging process or as the result of neurological diseases such as Huntington’s disease, Parkinson’s disease, stroke, and Alzheimer’s disease. The exact mechanisms through which glutathione regulates brain metabolism are not well understood. In this review, we will highlight the common signaling cascades that regulate glutathione in neurons and glia, its functions as a neuronal regulator in homeostasis and metabolism, and finally a mechanistic recapitulation of glutathione signaling. Together, these will put glutathione’s role in normal aging and neurological disorders development into perspective.
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Yang, Wei-Yu, Jueting Zheng, Xia-Guang Zhang, Li-Chuan Chen, Yu Si, Fei-Zhou Huang, and Wenjing Hong. "Charge transport through a water-assisted hydrogen bond in single-molecule glutathione disulfide junctions." Journal of Materials Chemistry C 8, no. 2 (2020): 481–86. http://dx.doi.org/10.1039/c9tc05686f.
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Wei, Yongfeng, Zhuoqun Su, Xiao-feng Kang, Yanli Guo, and Xiaoxue Mu. "Single-molecule transformation and analysis of glutathione oxidized and reduced in nanopore." Talanta 167 (May 2017): 526–31. http://dx.doi.org/10.1016/j.talanta.2017.02.059.
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Wróblewska, Joanna, Marcin Wróblewski, Iga Hołyńska-Iwan, Martyna Modrzejewska, Jarosław Nuszkiewicz, Weronika Wróblewska, and Alina Woźniak. "The Role of Glutathione in Selected Viral Diseases." Antioxidants 12, no. 7 (June 22, 2023): 1325. http://dx.doi.org/10.3390/antiox12071325.
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During inflammatory processes, immunocompetent cells are exposed to substantial amounts of free radicals and toxic compounds. Glutathione is a cysteine-containing tripeptide that is an important and ubiquitous antioxidant molecule produced in human organs. The intracellular content of GSH regulates the detoxifying capacity of cells, as well as the inflammatory and immune response. GSH is particularly important in the liver, where it serves as the major non-protein thiol involved in cellular antioxidant defense. There are numerous causes of hepatitis. The inflammation of the liver can be caused by a variety of infectious viruses. The relationship between oxidative stress and the hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis E virus (HEV) infection is not fully known. The aim of this study was to examine the relationship between hepatotropic viruses and glutathione status, including reduced glutathione (GSH) and oxidized glutathione (GSSG), as well as antioxidant enzymes, e.g., glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) in liver diseases.
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Ivanov, V. V., Ye V. Shakhristova, Ye A. Stepovaya, and V. V. Novitsky. "Effect of alloxan on glutathione system and oxidative protein modification in adipocytes of rats at experimental diabetes." Bulletin of Siberian Medicine 10, no. 3 (June 28, 2011): 44–47. http://dx.doi.org/10.20538/1682-0363-2011-3-44-47.
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There was carried out a research of the development of oxidative stress, the condition of glutathione dependant system of antioxidant protection in adipocytes of epididymal adipose tissue of rats when injecting alloxan. The development of oxidative stress in adipocytes was characterized by the increase of lipids hydroperoxide concentration, products reacting with thiobarbituric acid, and the increase of carbonylderived protein. Redox-condition in adipocytes was considerably changing that was specified by the decrease of the content of the reduced form of glutathione and tendency to the increase of glutathione disulfide content, decrease of ratio between reduced and oxidized forms of threepeptide. Damage of protein molecule at oxidative stress may lead to the abnormality of transduction of insulinic signal and appearance of insulin resistance in adipose tissue.
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Ewald, S. J., and P. H. Refling. "Co-immunoprecipitation of the Ly-5 molecule and an endogenous protease: a proteolytic system requiring a reducing agent and Ca2+1." Journal of Immunology 134, no. 4 (April 1, 1985): 2513–19. http://dx.doi.org/10.4049/jimmunol.134.4.2513.
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Abstract Sodium [3H]borohydride- and [35S]methionine-labeled Ly-5 molecules from mouse thymocytes and T lymphoma cells were isolated with specific antibody and Staphylococcus aureus Cowan I (SaCI) strain; after extensive washing of the complexes, elution with Laemmli's reducing buffer (0.05 M Tris [pH 6.8 or 6.0], 4% sodium dodecyl sulfate [SDS], and 2% 2-mercaptoethanol [2-ME]) resulted in partial breakdown of the isolated Ly-5 molecules from a Mr = 175,000 to 150,000. Other proteins present during the elution step showed no evidence of proteolysis. 2-ME and SDS were required for proteolysis; although addition of exogenous Ca2+ during elution was not necessary, both EDTA and EGTA inhibited breakdown of the molecule that could be overcome by excess Ca2+. Of a variety of protease inhibitors and thiol-reactive agents tested, only TAME and oxidized glutathione blocked proteolysis almost completely. SaCI, serum, and contaminating antibodies were ruled out as the source of the proteolytic activity. More stringent preclearing and washing conditions did not eliminate endogenous proteolysis of the Ly-5 molecule. The endogenous proteolytic fragment had a Mr distinct from the tryptic fragment of the Ly-5 molecule. We conclude that the Ly-5 molecule may be autolytic or tightly associated with a distinct cellular protease.
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Morgan, Bruce. "Reassessing cellular glutathione homoeostasis: novel insights revealed by genetically encoded redox probes." Biochemical Society Transactions 42, no. 4 (August 1, 2014): 979–84. http://dx.doi.org/10.1042/bst20140101.
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Glutathione is the most abundant small molecule thiol in nearly all eukaryotes. Whole-cell levels of oxidized (GSSG) and reduced (GSH) glutathione are variable and responsive to genetic and chemical manipulations, which has led to their relative levels being widely used as a marker of the ‘cellular redox state’ and to indicate the level of ‘oxidative stress’ experienced by cells, tissues and organisms. However, the applicability of glutathione as a marker for a generalized ‘cellular redox state’ is questionable, especially in the light of recent observations in yeast cells. In yeast, whole-cell GSSG changes are almost completely dependent upon the activity of an ABC-C (ATP-binding cassette-C) transporter, Ycf1 (yeast cadmium factor 1), which mediates sequestration of GSSG to the vacuole. In the absence of Ycf1 whole-cell GSSG content is strongly decreased and extremely robust to perturbation. These observations are consistent with highly specific redox-sensitive GFP probe-based measurements of the cytosolic glutathione pool and indicate that cytosolic GSSG reductive systems are easily able to reduce nearly all GSSG formed, even following treatment with large concentrations of oxidant. In the present paper, I discuss the consequences of these new findings for our understanding of glutathione homoeostasis in the eukaryotic cell.
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Abdillah, Ariq, Prasad M. Sonawane, Donghyeon Kim, Dooronbek Mametov, Shingo Shimodaira, Yunseon Park, and David G. Churchill. "Discussions of Fluorescence in Selenium Chemistry: Recently Reported Probes, Particles, and a Clearer Biological Knowledge." Molecules 26, no. 3 (January 28, 2021): 692. http://dx.doi.org/10.3390/molecules26030692.
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In this review from literature appearing over about the past 5 years, we focus on selected selenide reports and related chemistry; we aimed for a digestible, relevant, review intended to be usefully interconnected within the realm of fluorescence and selenium chemistry. Tellurium is mentioned where relevant. Topics include selenium in physics and surfaces, nanoscience, sensing and fluorescence, quantum dots and nanoparticles, Au and oxide nanoparticles quantum dot based, coatings and catalyst poisons, thin film, and aspects of solar energy conversion. Chemosensing is covered, whether small molecule or nanoparticle based, relating to metal ion analytes, H2S, as well as analyte sulfane (biothiols—including glutathione). We cover recent reports of probing and fluorescence when they deal with redox biology aspects. Selenium in therapeutics, medicinal chemistry and skeleton cores is covered. Selenium serves as a constituent for some small molecule sensors and probes. Typically, the selenium is part of the reactive, or active site of the probe; in other cases, it is featured as the analyte, either as a reduced or oxidized form of selenium. Free radicals and ROS are also mentioned; aggregation strategies are treated in some places. Also, the relationship between reduced selenium and oxidized selenium is developed.
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Chai, Yuh-Cherng, and John J. Mieyal. "Glutathione and Glutaredoxin—Key Players in Cellular Redox Homeostasis and Signaling." Antioxidants 12, no. 8 (August 3, 2023): 1553. http://dx.doi.org/10.3390/antiox12081553.
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This Special Issue of Antioxidants on Glutathione (GSH) and Glutaredoxin (Grx) was designed to collect review articles and original research studies focused on advancing the current understanding of the roles of the GSH/Grx system in cellular homeostasis and disease processes. The tripeptide glutathione (GSH) is the most abundant non-enzymatic antioxidant/nucleophilic molecule in cells. In addition to various metabolic reactions involving GSH and its oxidized counterpart GSSG, oxidative post-translational modification (PTM) of proteins has been a focal point of keen interest in the redox field over the last few decades. In particular, the S-glutathionylation of proteins (protein-SSG formation), i.e., mixed disulfides between GSH and protein thiols, has been studied extensively. This reversible PTM can act as a regulatory switch to interconvert inactive and active forms of proteins, thereby mediating cell signaling and redox homeostasis. The unique architecture of the GSH molecule enhances its relative abundance in cells and contributes to the glutathionyl specificity of the primary catalytic activity of the glutaredoxin enzymes, which play central roles in redox homeostasis and signaling, and in iron metabolism in eukaryotes and prokaryotes under physiological and pathophysiological conditions. The class-1 glutaredoxins are characterized as cytosolic GSH-dependent oxidoreductases that catalyze reversible protein S-glutathionylation specifically, thereby contributing to the regulation of redox signal transduction and/or the protection of protein thiols from irreversible oxidation. This Special Issue includes nine other articles: three original studies and six review papers. Together, these ten articles support the central theme that GSH/Grx is a unique system for regulating thiol-redox hemostasis and redox-signal transduction, and the dysregulation of the GSH/Grx system is implicated in the onset and progression of various diseases involving oxidative stress. Within this context, it is important to appreciate the complementary functions of the GSH/Grx and thioredoxin systems not only in thiol-disulfide regulation but also in reversible S-nitrosylation. Several potential clinical applications have emerged from a thorough understanding of the GSH/Grx redox regulatory system at the molecular level, and in various cell types in vitro and in vivo, including, among others, the concept that elevating Grx content/activity could serve as an anti-fibrotic intervention; and discovering small molecules that mimic the inhibitory effects of S-glutathionylation on dimer association could identify novel anti-viral agents that impact the key protease activities of the HIV and SARS-CoV-2 viruses. Thus, this Special Issue on Glutathione and Glutaredoxin has focused attention and advanced understanding of an important aspect of redox biology, as well as spawning questions worthy of future study.
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Alphey, Magnus S., Janine König, and Alan H. Fairlamb. "Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases." Biochemical Journal 414, no. 3 (August 27, 2008): 375–81. http://dx.doi.org/10.1042/bj20080889.
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TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite Km and Vmax values. The structure of the reduced enzyme at 2.1 Å (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 Å (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.
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Li, Ben, Chufan Wang, Peng Lu, Yumeng Ji, Xufeng Wang, Chaoyang Liu, Xiaohu Lu, Xiaohan Xu, and Xiaowei Wang. "IDH1 Promotes Foam Cell Formation by Aggravating Macrophage Ferroptosis." Biology 11, no. 10 (September 23, 2022): 1392. http://dx.doi.org/10.3390/biology11101392.
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A distinctive feature of ferroptosis is intracellular iron accumulation and the impairment of antioxidant capacity, resulting in a lethal accumulation of lipid peroxides leading to cell death. This study was conducted to determine whether inhibiting isocitrate dehydrogenase 1 (IDH1) may help to prevent foam cell formation by reducing oxidized low-density lipoprotein (ox-LDL)-induced ferroptosis in macrophages and activating nuclear factor erythroid 2-related factor 2 (NRF2). Gene expression profiling (GSE70126 and GSE70619) revealed 21 significantly different genes, and subsequent bioinformatics research revealed that ferroptosis and IDH1 play essential roles in foam cell production. We also confirmed that ox-LDL elevates macrophage ferroptosis and IDH1 protein levels considerably as compared with controls. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, reduced ox-LDL-induced elevated Fe2+ levels, lipid peroxidation (LPO) buildup, lactate dehydrogenase (LDH) buildup, glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4), ferritin heavy polypeptide 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11) protein downregulation. More crucially, inhibiting IDH1 reduced Fe2+ overload, lipid peroxidation, LDH, and glutathione depletion, and elevated GPX4, FTH1, and SLC7A11 protein expression, resulting in a reduction in ox-LDL-induced macrophage ferroptosis. IDH1 inhibition suppressed ox-LDL-induced macrophage damage and apoptosis while raising NRF2 protein levels. We have demonstrated that inhibiting IDH1 reduces ox-LDL-induced ferroptosis and foam cell formation in macrophages, implying that IDH1 may be an important molecule regulating foam cell formation and may be a promising molecular target for the treatment of atherosclerosis.
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Zhou, Xiaowen, and Yi Yao. "Unexpected Nephrotoxicity in Male Ablactated Rats Induced byCordyceps militaris: The Involvement of Oxidative Changes." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/786528.
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Recently, many nutraceutical products containing the powdered or extracted parts ofC. militarishave become available for health care. Due to the increased morbidity and mortality, poisonings associated with the use of herbs have raised the universal attention. Herein, we carried out the 28-day repeated toxicity test in male and female ablactated rats (three weeks old) givenC. militarispowder orally at 0 (control), 1, 2, and 3 g/kg per day. Noticeable increments of serum aspartate and alanine aminotransferase (ALT and AST) levels were observed for both sexes, suggestive of weak hepatic toxicity. Nephrotoxicity characterized by tubular epithelium degeneration and necrosis was observed at the high dose, and the male rats were more susceptible to renal toxicity than female rats. In addition, the genes and protein expressions of novel markers of kidney toxicity, such as kidney injury molecule-1 (KIM-1) were enlarged in the renal cortex and the urine. Moreover,C. militaristreatment significantly decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities. However, the ratio of glutathione oxidized form (GSSG)/glutathione reduced form (GSH) was increased byC. militaristreatment. We conclude that dietary contamination withC. militarismay have renal toxicity potentials, at least in part by causing oxidative damage to the kidney.
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Jha, Saurabh, John W. Calvert, Mark R. Duranski, Arun Ramachandran, and David J. Lefer. "Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling." American Journal of Physiology-Heart and Circulatory Physiology 295, no. 2 (August 2008): H801—H806. http://dx.doi.org/10.1152/ajpheart.00377.2008.
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Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with diverse physiological activity. The potential protective effects of H2S have not been evaluated in the liver. The purpose of the current study was to investigate if H2S could afford hepatoprotection in a murine model of hepatic ischemia-reperfusion (I/R) injury. Hepatic injury was achieved by subjecting mice to 60 min of ischemia followed by 5 h of reperfusion. H2S donor (IK1001) or vehicle were administered 5 min before reperfusion. H2S attenuated the elevation in serum alanine aminotransferase (ALT) by 68.6% and aspartate aminotransferase (AST) by 70.8% compared with vehicle group. H2S-mediated cytoprotection was associated with an improved balance between reduced glutathione (GSH) vs. oxidized glutathione (GSSG), an attenuated formation of lipid hydroperoxides, and an increased expression of thioredoxin-1 (Trx-1). Furthermore, H2S inhibited the progression of apoptosis after I/R injury by increasing the protein expression of heat shock protein (HSP-90) and Bcl-2. These results indicate that H2S protects the murine liver against I/R injury through an upregulation of intracellular antioxidant and antiapoptotic signaling pathways.
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Patel, Shreenal, Syeed Hussain, Richard Harris, Sunita Sardiwal, John M. Kelly, Shane R. Wilkinson, Paul C. Driscoll, and Snezana Djordjevic. "Structural insights into the catalytic mechanism of Trypanosoma cruzi GPXI (glutathione peroxidase-like enzyme I)." Biochemical Journal 425, no. 3 (January 15, 2010): 513–22. http://dx.doi.org/10.1042/bj20091167.
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Current drug therapies against Trypanosoma cruzi, the causative agent of Chagas disease, have limited effectiveness and are highly toxic. T. cruzi-specific metabolic pathways that utilize trypanothione for the reduction of peroxides are being explored as potential novel therapeutic targets. In the present study we solved the X-ray crystal structure of one of the T. cruzi enzymes involved in peroxide reduction, the glutathione peroxidase-like enzyme TcGPXI (T. cruzi glutathione peroxidase-like enzyme I). We also characterized the wild-type, C48G and C96G variants of TcGPXI by NMR spectroscopy and biochemical assays. Our results show that residues Cys48 and Cys96 are required for catalytic activity. In solution, the TcGPXI molecule readily forms a Cys48–Cys96 disulfide bridge and the polypeptide segment containing Cys96 lacks regular secondary structure. NMR spectra of the reduced TcGPXI are indicative of a protein that undergoes widespread conformational exchange on an intermediate time scale. Despite the absence of the disulfide bond, the active site mutant proteins acquired an oxidized-like conformation as judged from their NMR spectra. The protein that was used for crystallization was pre-oxidized by t-butyl hydroperoxide; however, the electron density maps clearly showed that the active site cysteine residues are in the reduced thiol form, indicative of X-ray-induced reduction. Our crystallographic and solution studies suggest a level of structural plasticity in TcGPXI consistent with the requirement of the atypical two-cysteine (2-Cys) peroxiredoxin-like mechanism implied by the behaviour of the Cys48 and Cys96 mutant proteins.
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Jo, Inseong, Nohra Park, In-Young Chung, You-Hee Cho, and Nam-Chul Ha. "Crystal structures of the disulfide reductase DsbM from Pseudomonas aeruginosa." Acta Crystallographica Section D Structural Biology 72, no. 10 (September 15, 2016): 1100–1109. http://dx.doi.org/10.1107/s2059798316013024.
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In bacteria, many Dsb-family proteins play diverse roles in the conversion between the oxidized and reduced states of cysteine residues of substrate proteins. Most Dsb enzymes catalyze disulfide formation in periplasmic or secreted substrate proteins. Recently, a DsbM protein has been found in a Gram-negative bacterium, and was characterized as a cytosolic Dsb member with the conserved CXXC motif on the basis of sequence homology to the Dsb-family proteins. The protein was implicated in the reduction of the cytoplasmic redox-sensor protein OxyR in Pseudomonas aeruginosa. Here, crystal structures of DsbM from P. aeruginosa are presented, revealing that it consists of a modified thioredoxin domain containing the CXXC motif and a lid domain surrounding the CXXC motif. In a glutathione-linked structure, a glutathione molecule is linked to the CXXC motif of DsbM and is bound in an elongated cavity region in the thioredoxin domain, which is also suited for substrate peptide binding. A striking structural similarity to a human glutathione S-transferase was found in the glutathione-binding pocket. Further, biochemical evidence is presented suggesting that DsbM is directly involved in the reduction of the disulfide of Cys199 and Cys208 in OxyR, resulting in the acceleration of OxyR reduction in the absence of reactive oxygen species stress. These findings may help to expand the understanding of the diverse roles of redox-related proteins that contain the CXXC motif.
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Gargallo, Pedro, Juan C. Colado, Alavaro Juesas, Amaya Hernando-Espinilla, Nuria Estañ-Capell, Lidia Monzó-Beltran, Paula García-Pérez, Omar Cauli, and Guillermo T. Sáez. "The Effect of Moderate- Versus High-Intensity Resistance Training on Systemic Redox State and DNA Damage in Healthy Older Women." Biological Research For Nursing 20, no. 2 (January 23, 2018): 205–17. http://dx.doi.org/10.1177/1099800417753877.
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This study investigated effects of a 16-week progressive resistance training program (RTP) with elastic bands at two different intensities on systemic redox state, DNA damage, and physical function in healthy older women. Methods: Participants were randomly assigned to the high-intensity group (HIGH; n = 39), moderate-intensity group (MOD; n = 31), or control group (CG; n = 23). The exercise groups performed an RTP twice a week with three to four sets of 6 (HIGH) or 15 (MOD) repetitions of six overall body exercises at a perceived exertion rate of 8–9 on the OMNI-Resistance Exercise Scale for use with elastic bands. Thiol redox state was determined by reduced glutathione (GSH), oxidized glutathione (GSSG), and GSSG/GSH in blood mononuclear cells. Degree of DNA damage was assessed by presence of the oxidized DNA base molecule 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) in urine. Physical function monitoring was based on the arm curl, chair stand, up and go, and 6-min walk tests. Results: The HIGH group showed a significant increase in 8-OHdG (+71.07%, effect size [ES] = 1.12) and a significant decrease in GSH (−10.91, ES = −0.69), while the MOD group showed a significant decrease in 8-OHdG levels (−25.66%, ES = −0.69) with no changes in thiol redox state. GSH levels differed significantly between the HIGH and CG groups posttest. The exercise groups showed significant improvements in physical function with no differences between groups. Conclusion: RTP at a moderate rather than high intensity may be a better strategy to reduce DNA damage in healthy older women while also increasing independence.
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Annia, Rodríguez-Hernández, Enrique Romo-Arévalo, and Adela Rodríguez-Romero. "A Novel Substrate-Binding Site in the X-ray Structure of an Oxidized E. coli Glyceraldehyde 3-Phosphate Dehydrogenase Elucidated by Single-Wavelength Anomalous Dispersion." Crystals 9, no. 12 (November 26, 2019): 622. http://dx.doi.org/10.3390/cryst9120622.
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Escherichia coli (E. coli), one of the most frequently used host for the expression of recombinant proteins, is often affected by the toxic effect of the exogenous proteins that is required to express. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multi-functional protein that has been used as a control marker for basal function and it is known to undergo cysteine oxidation under different types of cellular stress. Here, we report the 3D structure of the endogenous GAPDH purified from stressed E. coli cells expressing a eukaryotic protein. The structure was solved at 1.64 Å using single-wavelength anomalous dispersion (SAD) phasing with a selenium-modified enzyme. Interestingly, each GAPDH monomer contains a molecule of glyceraldehyde-3 phosphate in a non-previously identified site. Furthermore, the catalytic Cys149 is covalently attached to a ~300 Da molecule, possibly glutathione. This modification alters the conformation of an adjacent alpha helix in the catalytic domain, right opposite to the NAD+ binding site. The conformation of the alpha helix is stabilized after soaking the crystals with NAD+. These results exemplify the effects that the overexpression of an exogenous protein has over the host proteins and sheds light on the structural changes that large oxidant molecules on the catalytic cysteine produce for the GAPDH enzyme.
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Murnan, Kevin, Serena Tommasini-Ghelfi, Lisa Hurley, Corey Dussold, Daniel Wahl, and Alexander Stegh. "EXTH-27. MOLECULAR CHARACTERIZATION AND PRECLINICAL DEVELOPMENT OF NOVEL SMALL MOLECULE INHIBITOR SPECIFIC FOR WILD-TYPE IDH1 FOR FERROPTOSIS INDUCTION IN GLIOBLASTOMA." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi169. http://dx.doi.org/10.1093/neuonc/noab196.666.
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Abstract Increased de novo synthesis, mobilization and uptake of fatty acids are required to provide sufficient lipids for membrane biogenesis in support of rapid tumor cell division and growth. In addition to their structural roles as components of the plasma membrane, fatty acid-derived lipids regulate ferroptotic cell death, a type of programmed cell death, when oxidized by iron-dependent lipoxygenase enzymes. De novo lipogenesis and the defense against oxidative lipid damage require large amounts of cytosolic NADPH. Our group has recently found that HGG up-regulate wild-type Isocitrate dehydrogenase 1 (referred to hereafter as ‘wt-IDH1high HGG’) to generate large quantities of cytosolic NADPH. RNAi-mediated knockdown of wt-IDH1, alone and in combination with radiation therapy (RT), slows the growth of patient-derived HGG xenografts, while overexpression of wt-IDH1 promotes intracranial HGG growth. Isotope tracer and liquid chromatography-based lipidomic studies indicated that wt-IDH1 supports the de novo biosynthesis of mono-unsaturated fatty acids (MUFAs) and promotes the incorporation of monounsaturated phospholipids into the plasma membrane, while displacing polyunsaturated fatty acid (PUFA) phospholipids. In addition, enhanced NADPH production in wt-IDH1high HGG increases glutathione (GSH) level, reduces reactive oxygen species (ROS), activates the phospholipid peroxidase glutathione peroxidase 4 (GPX4)-driven lipid repair pathway, and dampens the accumulation of PUFA-containing lipid peroxides, known executioners of ferroptosis. To pharmacologically target wt-IDH1,we have used and characterized wt-IDH1i-13, a first-in-class competitive α,β-unsaturated enone (AbbVie). wt-IDH1i-13 potently inhibits wt-IDH1 enzymatic activity, by covalently binding to the NADP+ binding pocket. Our data indicate that wt-IDH1i-13 promotes ferroptosis, which can be rescued by pre-treatment of cells with the peroxyl scavenger and ferroptosis inhibitor ferrostatin. wt-IDH1i-13 is brain-penetrant, and similar to genetic ablation, reduces progression and extends the survival of wt-IDH1high HGG bearing mice, alone and in combination with RT. These studies credential to wt-IDH1i-13 as a novel therapeutic modality for the treatment of wt-IDH1 gliomas.
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Ceder, Anna Sophia, Sofi E. Eriksson, Emarndeena Haji Cheteh, Vladimir J. N. Bykov, Lars Abrahmsen, and Klas G. Wiman. "Impact of combined MRP1 inhibition and mutant p53-targeting compound APR-246." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e14712-e14712. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e14712.
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e14712 Background: The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. Mutant p53 protein is often expressed at high levels and accompanied with gain-of-function activities that promote tumor development and resistance towards conventional treatment. APR-246 is a mutant p53-reactivating small molecule undergoing a Phase III clinical study in myelodysplastic syndrome (MDS), and several phase II studies. APR-246 is non-enzymatically converted to its active product methylene quinuclidinone (MQ) which binds to cysteine residues in p53. This stimulates proper folding of p53's DNA-binding core domain, leading to cell death. APR-246 also exhibits pro-oxidant activity as the electrophile MQ binds and inactivates important antioxidants such as glutathione and thioredoxin reductase, which both are essential for cellular defense against oxidative and electrophilic stress. Methods: All results are in vitro experiments by LC-MS as well as experiments in cultured cells, including 14C-APR-246/MQ detection, cell viability measurements, LC-MS analysis, enzyme recycling measurements and Western blotting. Results: We have utilized 14C-labelled APR-246 to investigate the effect of APR-246 and its active moiety MQ in tumor cell cultures upon inhibition of efflux transporter multidrug resistance protein 1 (MRP1) or cystine-glutamate transporter (xCT). Transient downregulation or small molecule inhibitors (MK-571, reversan, sulfasalazin) targeting MRP1 or xCT increased drug content and shifted intra- and extracellular thiol status. Missense mutant TP53-carrying cells exhibited higher sensitivity to APR-246 and combination therapies. MRP1 inhibition showed strong synergy with APR-246 and increased intracellular levels of MQ conjugated to glutathione (GS-MQ). We found that GS-MQ conjugate formation is reversible, and suggest that the intrinsic reversibility of MQ adduct formation is an important aspect of the mechanism of action of APR-246. This reversibility may also in part account for the benign safety profile reported from clinical studies with APR-246. Conclusions: Sensitivity to APR-246 is affected by MRP1 efflux activity and the redox status, reflected in ratios of cysteine/cystine and reduced/oxidized glutathione (GSH/GSSG).
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Badmus, Olufunto O., and Lawrence A. Olatunji. "Dexamethasone causes defective glucose-6-phosphate dehydrogenase dependent antioxidant barrier through endoglin in pregnant and nonpregnant rats." Canadian Journal of Physiology and Pharmacology 98, no. 10 (October 2020): 667–77. http://dx.doi.org/10.1139/cjpp-2018-0351.
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Glucocorticoid therapy has been associated with adverse cardiometabolic effects during pregnancy. Inflammation-mediated cardiac dysfunction, an independent risk factor for morbidity and mortality, has been linked to defective glucose-6-phosphate dehydrogenase (G6PD) dependent antioxidant defenses and increased endoglin expression. We therefore sought to investigate the effects of dexamethasone (DEX) on cardiac endoglin and G6PD-dependent antioxidant defense. Twenty-four rats were randomly assigned to nonpregnant (PRE(–)), DEX-exposed nonpregnant (PRE(–) + DEX), pregnant (PRE(+)), and DEX-exposed pregnant (PRE(+) + DEX) rats, respectively (n = 6 per group). PRE(–) and PRE(+) rats received vehicle (per oral (po)), while PRE(–) + DEX and PRE(+) + DEX groups were administered DEX (0.2 mg/kg po) between gestational days 14 and 19, respectively. Results showed that DEX caused increased cardiac pro-inflammatory markers (adenosine deaminase (ADA) activity, endoglin, vascular cell adhesion molecule-1 (VCAM-1), tissue injury markers (LDH, GGT, AST, ALT, and ALP), metabolic disturbances (elevated fasting plasma glucose, free fatty acid (FFA), lactate, cardiac FFA, and lactate) and depressed G6PD-dependent antioxidant defenses (G6PD activity, reduced glutathione/oxidized glutathione ratio, and nitric oxide) in pregnant and nonpregnant rats. The present study demonstrates that DEX led to increased cardiac endoglin and VCAM-1 that is accompanied by defective G6PD-dependent antioxidant defenses but not cardiac lipid accumulation in both pregnant and nonpregnant rats.
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Dey, Prasanta, Amit Kundu, Ha Eun Lee, Babli Kar, Vineet Vishal, Suvakanta Dash, In Su Kim, Tejendra Bhakta, and Hyung Sik Kim. "Molineria recurvata Ameliorates Streptozotocin-Induced Diabetic Nephropathy through Antioxidant and Anti-Inflammatory Pathways." Molecules 27, no. 15 (August 5, 2022): 4985. http://dx.doi.org/10.3390/molecules27154985.
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Molineria recurvata (MR) has been traditionally used to manage diabetes mellitus in India. However, the molecular mechanism of MR on the diabetic-induced nephropathy has not been clearly investigated. Thus, this study investigates the protective effects of the MR extract on nephropathy in streptozotocin (STZ)-induced diabetic rats. Diabetes was instigated by a single intraperitoneal injection of STZ (45 mg/kg) in male Sprague-Dawley rats. Once the diabetes was successfully induced, the MR extract (200 mg/kg/day) or metformin (200 mg/kg/day) was orally administered for 14 days. Renal function, morphology changes and levels of inflammatory cytokines were measured. Blood glucose concentrations were considerably reduced in STZ-induced diabetic rats following treatment with the MR extract. The administration of the MR extract substantially restored the abnormal quantity of the oxidative DNA damage marker 8-hydroxy-2′-deoxy-guanosine (8-OHdG), malondialdehyde, glutathione, oxidized glutathione, superoxide dismutase, catalase, interleukin (IL)-1β, IL-6, IL-10, and transforming growth factor-β (TGF-β). The urinary excretion of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), selenium binding protein 1 (SBP1), and pyruvate kinase M2 (PKM2) was significantly reduced in diabetes rats after administration of the MR extracts. In the kidneys of STZ-induced diabetic rats, the MR extracts markedly downregulated the expression of fibronectin, collagen-1, and α-smooth muscle actin (α-SMA). In particular, the MR extracts markedly increased the level of SIRT1 and SIRT3 and reduced claudin-1 in the kidney. These results suggest that the MR extracts exhibits therapeutic activity in contrast to renal injury in STZ-induced diabetic rats through repressing inflammation and oxidative stress.
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Ventimiglia, Leslie, and Bulent Mutus. "The Physiological Implications of S-Nitrosoglutathione Reductase (GSNOR) Activity Mediating NO Signalling in Plant Root Structures." Antioxidants 9, no. 12 (November 30, 2020): 1206. http://dx.doi.org/10.3390/antiox9121206.
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Nitrogen remains an important macronutrient in plant root growth due to its application in amino acid production, in addition to its more elusive role in cellular signalling through nitric oxide (NO). NO is widely accepted as an important signalling oxidative radical across all organisms, leading to its study in a wide range of biological pathways. Along with its more stable NO donor, S-nitrosoglutathione (GSNO), formed by NO non-enzymatically in the presence of glutathione (GSH), NO is a redox-active molecule capable of mediating target protein cysteine thiols through the post translational modification, S-nitrosation. S-nitrosoglutathione reductase (GSNOR) thereby acts as a mediator to pathways regulated by NO due to its activity in the irreversible reduction of GSNO to oxidized glutathione (GSSG) and ammonia. GSNOR is thought to be pleiotropic and often acts by mediating the cellular environment in response to stress conditions. Under optimal conditions its activity leads to growth by transcriptional upregulation of the nitrate transporter, NRT2.1, and through its interaction with phytohormones like auxin and strigolactones associated with root development. However, in response to highly nitrosative and oxidative conditions its activity is often downregulated, possibly through an S-nitrosation site on GSNOR at cys271, Though GSNOR knockout mutated plants often display a stunted growth phenotype in all structures, they also tend to exhibit a pre-induced protective effect against oxidative stressors, as well as an improved immune response associated with NO accumulation in roots.
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Taniguchi, Misako, Nobuko Mori, Chizuru Iramina, and Akira Yasutake. "Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver." Scientific World Journal 2016 (2016): 1–7. http://dx.doi.org/10.1155/2016/6382467.
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Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level,γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule.
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Bavunoglu, Isil, Habibe Genc, Dildar Konukoglu, Hayriye Cicekci, Volkan Sozer, Remise Gelisgen, and Hafize Uzun. "Oxidative stress parameters and inflammatory and immune mediators as markers of the severity of sepsis." Journal of Infection in Developing Countries 10, no. 10 (October 31, 2016): 1045–52. http://dx.doi.org/10.3855/jidc.7585.
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Introduction: Sepsis is a complex inflammatory syndrome with diverse etiology and wide spectrum of severity. The aim of this study was to investigate whether inflammatory mediators, in comparison with oxidative parameters, are associated with severity of sepsis. Methodology: Plasma neopterin, adenosine deaminase (ADA), vascular cell adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNF-α), as inflammatory mediators, and serum nitric oxide (NOx), nitrotyrosine (NT), oxidized LDL (oxLDL) levels, serum paraoxonase 1 (PON1) activity, and erythrocyte glutathione (GSH) levels as oxidative stress parameters of 12 patients with mild sepsis, 25 patients with severe sepsis, and 20 healthy control subjects were evaluated. NOx, GSH levels and PON1 activity were determined by colorimetric methods, whereas neopterin, VCAM, ICAM, IL-1, IL-6, TNF-α, NT, and oxLDL levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: All parameters in mild and severe sepsis were significantly different from those of healthy subjects, except ADA activities. Patients with severe sepsis exhibited higher IL-6, TNF-α, NT, and oxLDL levels than patients with mild sepsis. GSH (98%, 98%), oxLDL (98%, 98%), VCAM-1 (99%, 99%), and ICAM-1 (99%, 99%) have much more sensitivitiy and specificity in sepsis. Conclusions: Our results suggest that the oxidative stress and inflammatory response in patients with sepsis were increased and that serum IL-6, TNF-α, NT, and oxLDL levels were correlated with the severity of sepsis. Therefore, increases in these parameters may contribute to the dysfunction or failure of one or more organs, or even death, in sepsis.
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Si, Meiru, Lei Zhang, Muhammad Tausif Chaudhry, Wei Ding, Yixiang Xu, Can Chen, Ali Akbar, Xihui Shen, and Shuang-Jiang Liu. "Corynebacterium glutamicum Methionine Sulfoxide Reductase A Uses both Mycoredoxin and Thioredoxin for Regeneration and Oxidative Stress Resistance." Applied and Environmental Microbiology 81, no. 8 (February 13, 2015): 2781–96. http://dx.doi.org/10.1128/aem.04221-14.
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ABSTRACTOxidation of methionine leads to the formation of theSandRdiastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown inActinomycetes. Here, we report that aCorynebacterium glutamicummethionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of themsrAgene inC. glutamicumresulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of anS-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditionsin vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.
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Cate, Shelby A., Tahsin Ozpolat, Junmei Chen, Colette Norby, Barbara A. Konkle, Jose A. Lopez, and Xiaoyun Fu. "Quantitative Analysis of Small Molecular Weight Thiols and Disulfides in Blood from a Sickle Cell Disease Patient Infused with N-Acetyl-L-Cysteine." Blood 124, no. 21 (December 6, 2014): 2662. http://dx.doi.org/10.1182/blood.v124.21.2662.2662.
Full textAbstract:
Abstract N-acetyl-L-cysteine (NAC) is an FDA approved drug used to treat acetaminophen overdose or as a mucolytic agent in respiratory disorders. The commonly accepted mechanism of action is that NAC undergoes deacetylation to cysteine, which is then used to synthesize glutathione (GSH), a major intracellular antioxidant. Like other thiol-containing compounds, NAC can also act as a reducing agent to break protein disulfide bonds or as a scavenger of reactive oxygen species. Due to its antioxidant properties, NAC has been proposed as a potential treatment for many diseases associated with oxidative stress, including sickle cell disease (SCD), neurological disorders, infectious diseases, and cancers. Though NAC has been widely studied, a full understanding of the mechanism by which NAC is effective in vivo has been limited by challenges in accurately quantifying NAC and its metabolites. As part of a clinical trial of NAC therapy in SCD, we have developed a liquid chromatography-mass spectrometry (LC-MS) based assay to quantify small molecule free thiols and disulfides using isotopically labeled internal standards. We applied this method to quantify small molecular thiols and disulfides in whole blood, red blood cells, and plasma from a SCD patient before (pre) and at 1, 8, 24 and 72 hr time points of intravenous administration of NAC at a dose of 300 mg/kg (a bolus infusion of 150 mg/kg for 1 hour followed by 150 mg/kg given over the next 7 hr). The cysteine concentration in whole blood increased to 286 μM at 1 hr from 97 μM at baseline, indicating that NAC is indeed rapidly metabolized (deacetylated) to cysteine. Interestingly, although cysteine concentration in RBCs increased over 4 fold at 1 hr and remained high compared to baseline, the highest concentration of total GSH in blood was observed at 24 hr (743 μM compared to 494 μM at baseline). Intracellular availability of cysteine is known as a rate-limiting step for GSH synthesis, and the delayed accumulation of GSH may suggest that NAC is involved in the extracellular deficit of reducing equivalents before it serves as a substrate in GSH synthesis. To explore this possibility, we quantitated NAC and its oxidation products, homo- and mixed disulfides. We found that total NAC concentration reached 1.58 mM in whole blood at 1 hr, but 44% of NAC was oxidized to N-acetyl-cystine (NAC-ss) or formed mixed disulfides with GSH (GS-ss-NAC) and Cys (Cys-ss-NAC), whereas the NAC used for infusion contained less than 0.5% in the oxidized form (NAC-ss). Concurrent with the formation of NAC disulfides, the levels of oxidized GSH (GSSG, GS-ss-Cys) and cysteine (cystine) were significantly decreased. These observations suggest that NAC administration of SCD not only increases GSH levels by raising the cysteine concentration, but also directly functions as an antioxidant to reduce oxidative stress. SCD patients are known to have low levels of GSH and frequently experience oxidative stress. NAC treatment is likely to address both issues. We plan to analyze the effects of NAC on blood small molecule thiol concentrations in several more SCD patients. Disclosures No relevant conflicts of interest to declare.
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Homma, Takujiro, Sho Kobayashi, and Junichi Fujii. "Methionine Deprivation Reveals the Pivotal Roles of Cell Cycle Progression in Ferroptosis That Is Induced by Cysteine Starvation." Cells 11, no. 10 (May 10, 2022): 1603. http://dx.doi.org/10.3390/cells11101603.
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Ferroptosis, a type of iron-dependent necrotic cell death, is triggered by the accumulation of excessive lipid peroxides in cells. Glutathione (GSH), a tripeptide redox molecule that contains a cysteine (Cys) unit in the center, plays a pivotal role in protection against ferroptosis. When the transsulfuration pathway is activated, the sulfur atom of methionine (Met) is utilized to generate Cys, which can then suppress Cys-starvation-induced ferroptosis. In the current study, we cultured HeLa cells in Met- and/or cystine (an oxidized Cys dimer)- deprived medium and investigated the roles of Met in ferroptosis execution. The results indicate that, in the absence of cystine or Met, ferroptosis or cell cycle arrest, respectively, occurred. Contrary to our expectations, however, the simultaneous deprivation of both Met and cystine failed to induce ferroptosis, although the intracellular levels of Cys and GSH were maintained at low levels. Supplementation with S-adenosylmethionine (SAM), a methyl group donor that is produced during the metabolism of Met, caused the cell cycle progression to resume and lipid peroxidation and the subsequent induction of ferroptosis was also restored under conditions of Met/cystine double deprivation. DNA methylation appeared to be involved in the resumption in the SAM-mediated cell cycle because its downstream metabolite S-adenosylhomocysteine failed to cause either cell cycle progression or ferroptosis to be induced. Taken together, our results suggest that elevated lipid peroxidation products that are produced during cell cycle progression are involved in the execution of ferroptosis under conditions of Cys starvation.
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Faruqi, R. M., E. J. Poptic, T. R. Faruqi, C. De La Motte, and P. E. DiCorleto. "Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression." American Journal of Physiology-Heart and Circulatory Physiology 273, no. 2 (August 1, 1997): H817—H826. http://dx.doi.org/10.1152/ajpheart.1997.273.2.h817.
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We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.
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Ozpolat, Hasan Tahsin, Junmei Chen, Xiaoyun Fu, Shelby A. Cate, Jennie Le, Minhua Ling, Colette Norby, Dominic W. Chung, Barbara A. Konkle, and Jose A. Lopez. "A Pilot Study of High-Dose N-Acetylcysteine Infusion in Patients with Sickle Cell Disease." Blood 128, no. 22 (December 2, 2016): 1299. http://dx.doi.org/10.1182/blood.v128.22.1299.1299.
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Abstract We have completed a clinical trial evaluating the safety of N-acetylcysteine (NAC) infusion in adult patients with sickle cell disease (SCD) at disease baseline (NCT01800526). This trial was inspired by the hypothesis that von Willebrand factor (VWF) has an important role in the pathophysiology of vaso-occlusion and hemolysis in SCD and that NAC can reduce signs and symptoms of the disease by reducing the activity of VWF. A secondary hypothesis was that NAC could also act by reversing or preventing some of the oxidative changes associated with SCD. VWF is stored in the Weibel-Palade bodies of endothelial cells and α-granules of platelets and released from endothelial cells upon activation and mediates the attachment of platelets to the vessel wall, and secondarily, of erythrocytes and leukocytes. We previously showed that VWF levels and adhesive activity in SCD plasma were elevated and that total active VWF correlated with the extent of hemolysis (determined by plasma LDH levels), suggesting that VWF participates in the pathophysiology of SCD (Chen et al, Blood. 2011; 117:3680‐3). In another study, we showed that NAC was able to reduce VWF multimer size and platelet-binding activity ex vivo in human plasma and in vivo in mice (Chen et al, JCI, 2011, 121:522). In this clinical trial, we enrolled 5 subjects at disease baseline, 4 with SCD and 1 with sickle trait. All subjects received two high-dose NAC infusions: initially 150 mg/kg and 4 weeks later 300 mg/kg. NAC infusion was administered in subjects 1 and 2 as a bolus infusion of half of the dose in the 1sthour and the remaining half in 7 hr. Subjects 3-5 received NAC as constant infusion for 8 hr. Blood was collected immediately before infusion, during infusion (at 1 and 4 hr), immediately after infusion (8 hr), and 24 and 72 hr after infusion. We examined blood for red blood cell (RBC) fragments, dense cells, platelet activation status, platelet-monocyte complexes and the concentrations of reduced and oxidized glutathione. We also examined plasma for VWF concentration and multimer distribution, ADAMTS13 antigen and activity, and evaluated plasma redox status by measuring the concentrations of NAC and cysteine in their reduced, oxidized, and mixed disulfide forms, as well as the concentration of protein-bound cysteine. The small molecule thiols were measured by mass spectrometry. Results:1) All subjects tolerated NAC infusion well, except that subjects 1 and 2 experienced pruritus during the bolus infusion at the 300 mg/kg dose. We therefore changed the protocol to deliver the drug over 8 hr by continuous infusion for subjects 3 through 5. 2) During NAC infusion, the percentage of dense cells and concentration of RBC fragments decreased rapidly (average decrease: low dose 44%; high dose 31%) and the change persisted up to 72 hr. 3) Platelet activation also decreased, as determined by reduced P-selectin expression (low dose, 7.7 ± 0.7% to 5.0 ± 1.9%; high dose, 17.8 ± 0.07% to 7.3% ± 0.4), PAC-1 binding (evaluates activated αΙΙbβ3), and formation of platelet-monocyte complexes (monocytes with attached platelets: low dose, 52 ± 16% to 27 ± 12%; high dose, 77 ± 12% to 50 ± 17%). 4) The highest molecular weight VWF multimers transiently disappeared during the NAC infusion and recovered by 24 hr. There were no appreciable changes in VWF antigen or ADAMTS13 antigen or activity with NAC infusion. 5) With NAC infusion, the concentrations of Cys and NAC increased in plasma, both total and reduced forms, with a concomitant decrease in protein-bound Cys in the one patient studied (free thiol Cys increased 23 fold with the high dose infusion at 8 hr; protein-bound Cys decreased 87%). Similarly, the concentrations of reduced and oxidized glutathione increased in whole blood. Summary: NAC infusion in SCD patients at disease baseline appears safe and is well tolerated. NAC infusion decreases RBC fragments, dense cells, and the size of large VWF multimers, and inhibits platelet activation and formation of platelet-monocyte complexes. In addition, NAC infusion increases the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in SCD. Our data suggest that NAC may be of benefit for SCD patients during vaso-occlusive crisis through a variety of mechanisms, which include improvements in red cell parameters, decreased platelet adhesion, reduced adhesive activity of VWF, and protection against oxidative damage. Disclosures No relevant conflicts of interest to declare.
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Xin, Yufeng, Yaxin Wang, Honglin Zhang, Yu Wu, Yongzhen Xia, Huanjie Li, and Xiaohua Qu. "Cupriavidus pinatubonensis JMP134 Alleviates Sulfane Sulfur Toxicity after the Loss of Sulfane Dehydrogenase through Oxidation by Persulfide Dioxygenase and Hydrogen Sulfide Release." Metabolites 13, no. 2 (February 2, 2023): 218. http://dx.doi.org/10.3390/metabo13020218.
Full textAbstract:
An incomplete Sox system lacking sulfane dehydrogenase SoxCD may produce and accumulate sulfane sulfur when oxidizing thiosulfate. However, how bacteria alleviate the pressure of sulfane sulfur accumulation remains largely unclear. In this study, we focused on the bacterium Cupriavidus pinatubonensis JMP134, which contains a complete Sox system. When soxCD was deleted, this bacterium temporarily produced sulfane sulfur when oxidizing thiosulfate. Persulfide dioxygenase (PDO) in concert with glutathione oxidizes sulfane sulfur to sulfite. Sulfite can spontaneously react with extra persulfide glutathione (GSSH) to produce thiosulfate, which can feed into the incomplete Sox system again and be oxidized to sulfate. Furthermore, the deletion strain lacking PDO and SoxCD produced volatile H2S gas when oxidizing thiosulfate. By comparing the oxidized glutathione (GSSG) between the wild-type and deletion strains, we speculated that H2S is generated during the interaction between sulfane sulfur and the glutathione/oxidized glutathione (GSH/GSSG) redox couple, which may reduce the oxidative stress caused by the accumulation of sulfane sulfur in bacteria. Thus, PDO and H2S release play a critical role in alleviating sulfane sulfur toxicity after the loss of soxCD in C. pinatubonensis JMP134.
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Vatolin, Sergei, and Jaroslaw P. Maciejewski. "Novel Small Molecule Stimulants of Hematopoietic Stem Cells and Their Mode of Action." Blood 132, Supplement 1 (November 29, 2018): 1302. http://dx.doi.org/10.1182/blood-2018-99-114838.
Full textAbstract:
Abstract Agents that can improve the function and/or numbers of hematopoietic progenitor and stem cells (HSC) are of great importance for the treatment of bone marrow failures of different etiology. However, except for the hematopoietic growth factors, which lead to significant depletion HSC via simultaneous differentiation and some anabolic steroids, the list of capable agents that can improve the function or numbers of HSC is very short. However, such drugs would have a tremendous range of application from ex vivo expansion, to bone marrow regeneration in aplastic anemia (AA) post chemotherapy, HSC transplantation or to improve the function of normal HSC in aging. While performing a multidrug screen for the agents that could simultaneously decrease senescence and overcome proliferative block in pre-senescent cells, we have identified two compounds violuric acid (VA) and 1-naphthoquinone-2-monoxime (N2N1). Both compounds considerably extended the replicative life span (RLS) of normal cells. We applied these drugs to stromal/mesenchymal cells obtained from healthy bone marrows, primary human normal dermal fibroblasts, progeroid primary cells derived from the patients diagnosed with Werner or Bloom syndromes and small panel of cancer cell lines (SKM-1, K562, KG-1, THP-1). Both compounds, in dose dependent manner prolonged the RLS of replicatively pre-aged cells. On an average, 10-15 additional population doublings (PD) were achieved after addition of N2N1 at 1μM. VA treatment has added 8-10 extra population doublings. If compared with untreated controls that can propagate up to 45-50 PD, the treatment with VA or N2N1 adds from 16 to 30% increase in replicative life span. To compare, the effect of rapamycin (1nM) on human fibroblasts showed the RLS increase ranged from 5 to 10%. Treatment with both VA and N2N1 results in restoration of cell cycle progression, decreased activity of SAβG, down-regulation of p16, p21 and γH2A.X and, up-regulation of lamin B1 protein. Treatment with both compounds resulted in maintenance of normal telomere length. In term of HSC these agents in vivo increased the performance of HSC in competitive repopulation assay. Bone marrow cells were isolated from mice (CD45.1) treated with the vehicle or experimental drugs for three weeks. After that, they were mixed with the equal number of competitor bone marrow cells CD45.2 and injected into irradiated CD45.2 host animals. In three weeks, we observed a substantial domination of CD45.1 cells over CD45.2 in experimental groups, while control (vehicle) group exhibited equal representation of both genotypes. In vitro, treatment with VA or N2N1 contributed to prolonged availability of HSC in serial replating CFU assays in methylcellulose and long-term culture initiating cell (LTC-IC) assays. Addition of VA or N2N1 to the short-term cultures (7-14 days) of normal bone marrow cells in a medium containing a cocktail of growth factors (Il6, IL3, FLT3L, TPO, SCF) resulted in maintenance and growth of HSC or progenitors. Gated on lymphocyte sub-population, treated with N2N1 or VA samples revealed ~0.3%±0.02 or 0.2% ±0.02 of CD34+, CD45+ cells correspondingly, while control samples had 0.08% of these cells (the result of three independent experiments). Most importantly, we observed colonies formation, after application of these drugs to the bone marrow isolated from the patients diagnosed with severe AA. Further studies also indicated that these agents do not promote growth of leukemic cell. Analysis of mechanism of action showed that VA and N1N2 function as redox co-factors in oxidations of NAD(P)H. VA transfers electrons non-enzymaticly from NAD(P)H to oxidized glutathione or peroxides. N2N1 is a redox co-factor for the NAD(P)H dehydrogenase (quinone) 1 (NQO1) and together they move electrons from NAD(P)H to cytochrome c or CoQ10. As such, we presented here a comprehensive prove that pharmacologic manipulation of redox balance controlled by glutathione or NQO1 activity via redox catalysts can ameliorate the detrimental consequences of HSC loss during normal aging by interfering with direct ROS mediated signaling or attenuating collateral ROS mediated damages. Figure. Figure. Disclosures Maciejewski: Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.
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Cobbold, Christian, Miriam Windsor, James Parsley, Ben Baldwin, and Thomas Wileman. "Reduced redox potential of the cytosol is important for African swine fever virus capsid assembly and maturation." Journal of General Virology 88, no. 1 (January 1, 2007): 77–85. http://dx.doi.org/10.1099/vir.0.82257-0.
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Assembly of African swine fever virus (ASFV) involves the transfer of the major capsid protein, p73, from the cytosol onto the cytoplasmic face of endoplasmic reticulum-derived membranes. During this process, the folding of p73 is dependent upon transient association with a specific viral chaperone, CAP80. The cell cytoplasm maintains high concentrations of reduced glutathione, leading to a reducing environment. Here, the effects of redox environment on the assembly of ASFV have been studied. Diamide, which oxidizes the cell cytosol, slowed the folding of p73 and prevented release from CAP80 and subsequent binding of p73 to membranes. Similarly, cell oxidation slowed the assembly of p73 molecules already bound to membranes into virus capsid precursors. Interestingly, addition of oxidized glutathione to newly assembled virus capsid precursors in vitro led to disassembly; however, virus particles released from cells were resistant to oxidized glutathione. These data show that assembly of ASFV requires the reducing environment that prevails in the cytosol, but as the virus matures, it becomes resistant to oxidation, possibly indicating preparation for release from the cell.
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Xu, Xiuling, Katharina von Loehneysen, Deborah Noack, Andrew Vu, and Jeff S. Friedman. "A Novel Approach for In Vivo Measurement of Red Cell Redox Status." Blood 116, no. 21 (November 19, 2010): 2036. http://dx.doi.org/10.1182/blood.v116.21.2036.2036.
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Abstract Abstract 2036 Maintenance of a reducing redox balance is a critical physiologic function of red cell metabolic machinery. Perturbation of this balance, whether inherited or acquired, is found in a variety of red cell pathologies. Methods for evaluation of red cell redox status include direct approaches such as determining glutathione (GSH, GSSG) levels, and indirect approaches such as measuring fluorescence of oxidation sensitive dyes. Here we describe an alternative method for evaluation of red cell redox status that can be used in vivo and in real-time assays. Engineered variants of GFP possessing two solvent accessible cysteine residues function as molecular redox sensors with distinct fluorescence characteristics. Excitation spectrum shifts upon the oxidation of cysteine residues forming a disulfide. A higher ratio of fluorescence when comparing excitation at 405nm versus 488nm indicates rising levels of oxidized GFP and a shift in cellular redox status. To validate redox GFPs in erythroid cells, we first performed in vitro assays with MEL cells over-expressing several related GFP sensors (ro-GFPs), selecting the brightest molecule (roGFP2) for further study. The sensor function of roGFP2 in MEL cells was verified by stimulation with exogenous oxidant (1mM H202) or reductant (10 mM DTT) as shown in the figure below. In order to create a physiologic in vivo model for study of red cell redox status, transgenic mice expressing roGFP2 specifically in the erythroid lineage were generated. roGFP2 expressing red cells demonstrate the expected shift in fluorescence upon exposure to H202 or t-butyl peroxide in a short-term assay. In vivo, we have measured red cell lifespan (using biotin-labeling) in roGFP2 transgenic animals to follow redox status of red cells as a function of cell age. Expression of roGFP2 has no effect on red cell survival. Interestingly, when comparing old red cells (age > 50days) with younger cells (age < 50days), a shift in GFP fluorescence ratio indicating that a higher fraction of the sensor is oxidized in the aged cells was observed. This observation is consistent with the hypothesis that metabolic changes, in particular a decline in ability to reduce oxidative damage, contribute to red cell senescence. We are generating several murine strains with defined red cell defects also expressing roGFP2 in order to assess the role of changes in intra-erythrocyte redox status in a range of pathologic conditions. In vitro and in vivo assays using roGFP2 transgenic cells/mice are in process to determine the potential utility of this system as a screen for hematoxicity of drugs and other compounds. Figure 1 Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Figure 1. Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Disclosures: No relevant conflicts of interest to declare.
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Rubino, Federico Maria. "The Redox Potential of the β-93-Cysteine Thiol Group in Human Hemoglobin Estimated from In Vitro Oxidant Challenge Experiments." Molecules 26, no. 9 (April 26, 2021): 2528. http://dx.doi.org/10.3390/molecules26092528.
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Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive β-93-Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E0 of the β-93-Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E0 of −121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione–glutathione disulfide and of glutathionyl–hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.
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Ke, Yi-Yuan, Yuan-Tay Shyu, and Sz-Jie Wu. "Evaluating the Anti-Inflammatory and Antioxidant Effects of Broccoli Treated with High Hydrostatic Pressure in Cell Models." Foods 10, no. 1 (January 15, 2021): 167. http://dx.doi.org/10.3390/foods10010167.
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Isothiocyanates (ITCs) are important functional components of cruciferous vegetables. The principal isothiocyanate molecule in broccoli is sulforaphane (SFN), followed by erucin (ERN). They are sensitive to changes in temperature, especially high temperature environments where they are prone to degradation. The present study investigates the effects of high hydrostatic pressure on isothiocyanate content, myrosinase activity, and other functional components of broccoli, and evaluates its anti-inflammatory and antioxidant effects. Broccoli samples were treated with different pressures and for varying treatment times; 15 min at 400 MPa generated the highest amounts of isothiocyanates. The content of flavonoids and vitamin C were not affected by the high-pressure processing strategy, whereas total phenolic content (TPC) exhibited an increasing tendency with increasing pressure, indicating that high-pressure processing effectively prevents the loss of the heat-sensitive components and enhances the nutritional content. The activity of myrosinase (MYR) increased after high-pressure processing, indicating that the increase in isothiocyanate content is related to the stimulation of myrosinase activity by high-pressure processing. In other key enzymes, the ascorbate peroxidase (APX) activity was unaffected by high pressure, whereas peroxidase (POD) and polyphenol oxidase (PPO) activity exhibited a 1.54-fold increase after high-pressure processing, indicating that high pressures can effectively destroy oxidases and maintain food quality. With regards to efficacy evaluation, NO production was inhibited and the expression levels of inducible nitric oxide synthase (iNOS) and Cyclooxygenase-2 (COX-2) were decreased in broccoli treated with high pressures, whereas the cell viability remained unaffected. The efficacy was more significant when the concentration of SFN was 60 mg·mL−1. In addition, at 10 mg·mL−1 SFN, the reduced/oxidized glutathione (GSH/GSSG) ratio in inflammatory macrophages increased from 5.99 to 9.41. In conclusion, high-pressure processing can increase the isothiocyanate content in broccoli, and has anti-inflammatory and anti-oxidant effects in cell-based evaluation strategies, providing a potential treatment strategy for raw materials or additives used in healthy foods.
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Zimring, James C., Nicole H. Smith, Sean R. Stowell, Richard O. Francis, Eldad A. Hod, Jeanne E. Hendrickson, Larry J. Dumont, John Roback, and Steven L. Spitalnik. "A Genetic Basis for Donor Variation in Generation of Prostaglandins and Leukotrienes in Stored RBCs Using a Mouse Model." Blood 120, no. 21 (November 16, 2012): 844. http://dx.doi.org/10.1182/blood.v120.21.844.844.
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Abstract Abstract 844 Background: RBC transfusion has clear efficacy in treating various types of anemia. However, in recent decades, there is a renewed focus on the potential for negative clinical sequelae from transfusing stored RBCs. Whether or not older, stored RBC units are associated with adverse outcomes remains controversial. However, there are clear cellular and biochemical data that RBC units accumulate particles and molecules over time with known toxicity when administered to animals and/or humans. Among the most potent of these are prostaglandins and leukotrienes (jointly known as eicosanoids), which are potent mediators of inflammation and vascular pathology. Indeed, arachidonic acid (AA), and 5-, 12-, and 15-hydroxyeicsotetranoic acid (HETE) accumulate during storage of human RBCs and are biologically active in priming neutrophils (Silliman et al., Transfusion 2011 51(12):2549-54). It is well known that there is substantial donor-to-donor variation in how well RBCs store from the standpoint of post-transfusion RBC recovery; however, it is unclear whether the accumulation of eicosanoids varies substantially among donors. If differences exist, then it may be useful to screen RBC units prior to transfusion into patients with illnesses likely to be affected by eicosanoid exposure. Using a well characterized mouse model of RBC storage, and different strains of donor mice, we tested the hypothesis that there are genetic determinants affecting eicosanoid levels in stored RBCs. Methods: RBCs from C57BL/6 (B6) and FVB mice were collected in CPDA-1, filter leukoreduced, and stored under conditions previously shown to model human RBC storage. Samples collected on days 0, 5, 9, and 14 were analyzed by small molecule mass spectrometry. The study was repeated 3 times and combined data were analyzed. Results: AA accumulated over storage time in both B6 and FVB RBC units to a similar level. In contrast, although essentially no accumulation of eicosanoids was observed in B6 RBC units, substantial time-dependent increases (compared to day of collection) were observed in FVB RBC units for 5-HETE (4-fold) and 15-HETE (12-fold). In addition, a greater than 10-fold increase was observed for prostaglandin E2 in FVB RBC units with no detectable prostaglandin E2 in B6 RBC units [ findings were consistent in all 3 experiments and all differences had p values of <.05]. As oxidized phospholipids are better substrates for phospholipases that generate AA, we hypothesized that oxidative damage would be increased in FVB RBCs. Additional metabolomic analysis demonstrated that B6 RBCs had decreased levels of oxidative damage compared to FVB RBCs, including substantially lower levels of 9,10-epoxystearate, an oxidized membrane lipid. Consumption of glucose and production of lactate were similar in both strains; however, B6 RBCs had increased total glutathione (GSH), oxidized GSH, and cysteine-GSH disulfides. No difference in flux through the pentose-phosphate shunt was observed (including similar NADPH levels); this suggests that the increased GSH in C57BL/6 RBCs was due to de novo synthesis and not to regeneration by GSH reductase. C57BL/6 RBCs also had increased levels of natural anti-oxidants, including ergothioneine and alpha-tocopherol. [All the above differences achieved a p value of <.05]. Conclusion: The current findings take advantage of a mouse model of RBC storage. Given that two inbred, genetically distinct strains demonstrate significant differences in the rate and magnitude of eicosanoid generation, these findings strongly support both the existence of donor variability in eicosanoid accumulation during RBC storage and a genetic basis thereof (in the context of a mouse system). In addition to providing a tractable platform to dissect the genetics of donor-specific RBC storage biology, these findings suggest the hypothesis that similar differences in eicosanoid generation occur with human RBC storage. Subsequent analysis of human specimens will need to be performed to test whether these findings extend to stored human RBC units. Disclosures: No relevant conflicts of interest to declare.
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Tomás-Simó, Patricia, Luis D’Marco, María Romero-Parra, Mari Carmen Tormos-Muñoz, Guillermo Sáez, Isidro Torregrosa, Nuria Estañ-Capell, Alfonso Miguel, José Luis Gorriz, and María Jesús Puchades. "Oxidative Stress in Non-Dialysis-Dependent Chronic Kidney Disease Patients." International Journal of Environmental Research and Public Health 18, no. 15 (July 23, 2021): 7806. http://dx.doi.org/10.3390/ijerph18157806.
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Background: Cardiovascular complications are the leading cause of morbidity and mortality at any stage of chronic kidney disease (CKD). Moreover, the high rate of cardiovascular mortality observed in these patients is associated with an accelerated atherosclerosis process that likely starts at the early stages of CKD. Thus, traditional and non-traditional or uremic-related factors represent a link between CKD and cardiovascular risk. Among non-conventional risk factors, particular focus has been placed on anaemia, mineral and bone disorders, inflammation, malnutrition and oxidative stress and, in this regard, connections have been reported between oxidative stress and cardiovascular disease in dialysis patients. Methods: We evaluated the oxidation process in different molecular lines (proteins, lipids and genetic material) in 155 non-dialysis patients at different stages of CKD and 45 healthy controls. To assess oxidative stress status, we analyzed oxidized glutathione (GSSG), reduced glutathione (GSH) and the oxidized/reduced glutathione ratio (GSSG/GSH) and other oxidation indicators, including malondialdehyde (MDA) and 8-oxo-2’-deoxyguanosine (8-oxo-dG). Results: An active grade of oxidative stress was found from the early stages of CKD onwards, which affected all of the molecular lines studied. We observed a heightened oxidative state (indicated by a higher level of oxidized molecules together with decreased levels of antioxidant molecules) as kidney function declined. Furthermore, oxidative stress-related alterations were significantly greater in CKD patients than in the control group. Conclusions: CKD patients exhibit significantly higher oxidative stress than healthy individuals, and these alterations intensify as eGFR declines, showing significant differences between CKD stages. Thus, future research is warranted to provide clearer results in this area.
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Mandal, Subhrangshu, Moidu Jameela Rameez, Sumit Chatterjee, Jagannath Sarkar, Prosenjit Pyne, Sabyasachi Bhattacharya, Rahul Shaw, and Wriddhiman Ghosh. "Molecular mechanism of sulfur chemolithotrophy in the betaproteobacterium Pusillimonas ginsengisoli SBSA." Microbiology 166, no. 4 (April 1, 2020): 386–97. http://dx.doi.org/10.1099/mic.0.000890.
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Chemolithotrophic sulfur oxidation represents a significant part of the biogeochemical cycling of this element. Due to its long evolutionary history, this ancient metabolism is well known for its extensive mechanistic and phylogenetic diversification across a diverse taxonomic spectrum. Here we carried out whole-genome sequencing and analysis of a new betaproteobacterial isolate, Pusillimonas ginsengisoli SBSA, which is found to oxidize thiosulfate via the formation of tetrathionate as an intermediate. The 4.7 Mb SBSA genome was found to encompass a soxCDYZAXOB operon, plus single thiosulfate dehydrogenase (tsdA) and sulfite : acceptor oxidoreductase (sorAB) genes. Recombination-based knockout of tsdA revealed that the entire thiosulfate is first converted to tetrathionate by the activity of thiosulfate dehydrogenase (TsdA) and the Sox pathway is not functional in this bacterium despite the presence of all necessary sox genes. The ∆soxYZ and ∆soxXA knockout mutants exhibited a wild-type-like phenotype for thiosulfate/tetrathionate oxidation, whereas ∆soxB, ∆soxCD and soxO::KanR mutants only oxidized thiosulfate up to tetrathionate intermediate and had complete impairment in tetrathionate oxidation. The substrate-dependent O2 consumption rate of whole cells and the sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, indicated that glutathione plays a key role in SBSA tetrathionate oxidation. The present findings collectively indicate that the potential glutathione : tetrathionate coupling in P. ginsengisoli involves a novel enzymatic component, which is different from the dual-functional thiol dehydrotransferase (ThdT), while subsequent oxidation of the sulfur intermediates produced (e.g. glutathione : sulfodisulfane molecules) may proceed via the iterative action of soxBCD .
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Rossi, Claudio, Alessandro Donati, Sergio Ulgiati, and Maria Rosaria Sansoni. "Dynamic behaviour of oxidized glutathione in solution investigated by nuclear magnetic resonance." Canadian Journal of Chemistry 71, no. 4 (April 1, 1993): 506–11. http://dx.doi.org/10.1139/v93-071.
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In view of the important biological functions of oxidized glutathione (GSSG), we investigated the correlation between dynamic and conformational properties and biological activity. Anisotropic molecular motion characterizes different GSSG molecular districts. This information, obtained by NMR carbon relaxation investigations, suggests that the peptide does not independently assume any stable structure in solution. Analysis of the effects of the addition of a stable spin label to the solution confirmed the absence of conformation of GSSG in D2O. Moreover, the paramagnetic effects observed on proton and carbon nuclei of oxidized glutathione suggest that the dipolar term is the main source of paramagnetic relaxation.
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Chebib, Soraya, and Wilfried Schwab. "Microscale Thermophoresis Reveals Oxidized Glutathione as High-Affinity Ligand of Mal d 1." Foods 10, no. 11 (November 11, 2021): 2771. http://dx.doi.org/10.3390/foods10112771.
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Pathogenesis-related (PR)-10 proteins, due to their particular secondary structure, can bind various ligands which could be important for their biological function. Accordingly, the PR-10 protein Mal d 1, the major apple allergen, probably also binds molecules in the hydrophobic cavity of its secondary structure, but it has not yet been investigated in this respect. In this study, various natural products found in apples such as flavonoids, glutathione (GSH), and glutathione disulfide (GSSG) were investigated as possible ligands of Mal d 1 using microscale thermophoresis. Dissociation constants of 16.39 µM, 29.51 µM, 35.79 µM, and 0.157 µM were determined for catechin, quercetin-3-O-rhamnoside, GSH, and GSSG, respectively. Molecular docking was performed to better understand the underlying binding mechanism and revealed hydrophobic interactions that stabilize the ligands within the pocket while hydrophilic interactions determine the binding of both GSH derivatives. The binding of these ligands could be important for the allergenicity of the PR-10 protein and provide further insights into its physiological role.
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Tomin, Tamara, Matthias Schittmayer, and Ruth Birner-Gruenberger. "Addressing Glutathione Redox Status in Clinical Samples by Two-Step Alkylation with N-ethylmaleimide Isotopologues." Metabolites 10, no. 2 (February 16, 2020): 71. http://dx.doi.org/10.3390/metabo10020071.
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Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) glutathione to oxidized (bound) glutathione. We aimed to facilitate this ratio determination in order to aid its incorporation as a routine clinical parameter. To this end, we developed a simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from reduced and oxidized glutathione (after its chemical reduction) for mass spectrometric analysis. A third isotopologue can be used as isotopic standard for simultaneous absolute quantification. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origins can only impact method sensitivity but not quantification accuracy. Robustness, simplified data analysis, cost effectiveness by one common standard, and highly improved mass spectrometric sensitivity by conversion of oxidized glutathione to an alkylated glutathione isotopologue are the main advantages of our approach. We present a method fully optimized for blood, plasma, serum, cell, and tissue samples. In addition, we propose production of N-ethylmaleimide customized blood collection tubes to even further facilitate the analysis in a clinical setting.
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Østergaard, Henrik, Christine Tachibana, and Jakob R. Winther. "Monitoring disulfide bond formation in the eukaryotic cytosol." Journal of Cell Biology 166, no. 3 (July 26, 2004): 337–45. http://dx.doi.org/10.1083/jcb.200402120.
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Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein–based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be −289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.
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Krezel, A., and W. Bal. "Coordination chemistry of glutathione." Acta Biochimica Polonica 46, no. 3 (September 30, 1999): 567–80. http://dx.doi.org/10.18388/abp.1999_4129.
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The metal ion coordination abilities of reduced and oxidized glutathione are reviewed. Reduced glutathione (GSH) is a very versatile ligand, forming stable complexes with both hard and soft metal ions. Several general binding modes of GSH are described. Soft metal ions coordinate exclusively or primarily through thiol sulfur. Hard ones prefer the amino acid-like moiety of the glutamic acid residue. Several transition metal ions can additionally coordinate to the peptide nitrogen of the gamma-Glu-Cys bond. Oxidized glutathione lacks the thiol function. Nevertheless, it proves to be a surprisingly efficient ligand for a range of metal ions, coordinating them primarily through the donors of the glutamic acid residue.
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Asanuma, Masato, and Ikuko Miyazaki. "Glutathione and Related Molecules in Parkinsonism." International Journal of Molecular Sciences 22, no. 16 (August 13, 2021): 8689. http://dx.doi.org/10.3390/ijms22168689.
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Glutathione (GSH) is the most abundant intrinsic antioxidant in the central nervous system, and its substrate cysteine readily becomes the oxidized dimeric cystine. Since neurons lack a cystine transport system, neuronal GSH synthesis depends on cystine uptake via the cystine/glutamate exchange transporter (xCT), GSH synthesis, and release in/from surrounding astrocytes. Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a detoxifying master transcription factor, is expressed mainly in astrocytes and activates the gene expression of various phase II drug-metabolizing enzymes or antioxidants including GSH-related molecules and metallothionein by binding to the antioxidant response element (ARE) of these genes. Accumulating evidence has shown the involvement of dysfunction of antioxidative molecules including GSH and its related molecules in the pathogenesis of Parkinson’s disease (PD) or parkinsonian models. Furthermore, we found several agents targeting GSH synthesis in the astrocytes that protect nigrostriatal dopaminergic neuronal loss in PD models. In this article, the neuroprotective effects of supplementation and enhancement of GSH and its related molecules in PD pathology are reviewed, along with introducing new experimental findings, especially targeting of the xCT-GSH synthetic system and Nrf2–ARE pathway in astrocytes.
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Harwood, D. Tim, Anthony J. Kettle, and Christine C. Winterbourn. "Production of glutathione sulfonamide and dehydroglutathione from GSH by myeloperoxidase-derived oxidants and detection using a novel LC–MS/MS method." Biochemical Journal 399, no. 1 (September 13, 2006): 161–68. http://dx.doi.org/10.1042/bj20060978.
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GSH is rapidly oxidized by HOCl (hypochlorous acid), which is produced physiologically by the neutrophil enzyme myeloperoxidase. It is converted into, mainly, oxidized glutathione. Glutathione sulfonamide is an additional product that is proposed to be covalently bonded between the cysteinyl thiol and amino group of the γ-glutamyl residue of GSH. We have developed a sensitive liquid chromatography–tandem MS assay for the detection and quantification of glutathione sulfonamide as well as GSH and GSSG. The assay was used to determine whether glutathione sulfonamide is a major product of the reaction between GSH and HOCl, and whether it is formed by other two-electron oxidants. At sub-stoichiometric ratios of HOCl to GSH, glutathione sulfonamide accounted for up to 32% of the GSH that was oxidized. It was also formed when HOCl was generated by myeloperoxidase and its yield increased with the flux of oxidant. Of the other oxidants tested, only hypobromous acid and peroxynitrite produced substantial amounts of glutathione sulfonamide, but much less than with HOCl. Chloramines were able to generate detectable levels only when at a stoichiometric excess over GSH. We conclude that glutathione sulfonamide is sufficiently selective for HOCl to be useful as a biomarker for myeloperoxidase activity in biological systems. We have also identified a novel oxidation product of GSH with a molecular weight two mass units less than GSH, which we have consequently named dehydroglutathione. Dehydroglutathione represented a few percent of the total products and was formed with all of the oxidants except H2O2.
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Chan, Alvin C. "Partners in defense, vitamin E and vitamin C." Canadian Journal of Physiology and Pharmacology 71, no. 9 (September 1, 1993): 725–31. http://dx.doi.org/10.1139/y93-109.
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In addition to the enzymic mechanism of free-radical removal, essential nutrients that can scavenge free radicals, such as vitamins E and C, constitute a strong line of defense in retarding free radical induced cellular damage. Distinct pathways for the repair of oxidized vitamin E in human cells have been recently identified. Within 0.5 min after the addition of arachidonic acid to a human platelet homogenate, over half of the platelet vitamin E and added arachidonate were metabolized by platelet cyclooxygenase and lipoxygenase pathways. After adding nordihydroguaiaretic acid, a lipoxygenase inhibitor and a strong reductant, over 60% of the oxidized vitamin E was regenerated. To test other physiological, water-soluble reductants that may help regenerate vitamin E, eicosatetraynoic acid, a lipoxygenase inhibitor that is not an antioxidant, was used. In this system, both ascorbate and glutathione provided significant vitamin E regeneration. Kinetic analysis and studies of vitamin E regeneration in a protein-denaturing system revealed that ascorbate regenerates vitamin E by a nonenzymic mechanism, whereas glutathione regenerates vitamin E enzymatically. These studies suggest that significant interaction occurs between water- and lipid-soluble molecules at the membrane–cytosol interface and that vitamin C may function in vivo to repair the membrane-bound oxidized vitamin E.Key words: vitamin C, vitamin E, vitamin E cycle, glutathione, human platelets.
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Stio, M., T. Iantomasi, F. Favilli, P. Marraccini, B. Lunghi, M. T. Vincenzini, and C. Treves. "Glutathione metabolism in heart and liver of the aging rat." Biochemistry and Cell Biology 72, no. 1-2 (January 1, 1994): 58–61. http://dx.doi.org/10.1139/o94-010.
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A comprehensive study on glutathione metabolism in rat heart and liver as a function of age was performed. In the heart, reduced glutathione, total glutathione, and the glutathione redox index showed a decrease during aging, while oxidized glutathione levels increased in 5-month-old rats with respect to the young animals and remained quite constant in 14- and 27-month-old rats. In the liver, the highest levels of reduced glutathione were found in the 2-month-old rats, while oxidized glutathione reached a peak at 5 months. Glutathione-associated enzymes showed age-related changes. Glutathione peroxidase, unaffected by aging in the heart, decreased in the liver of the 27-month-old rats. In the heart and the liver, the highest values of glutathione S-transferase were found at 5 months and 27 months, respectively. Glucose-6-phosphate dehydrogenase followed a similar trend in both heart and liver. Glutathione reductase also showed the same behaviour in heart and in liver, increasing in old rats with respect to the other age groups. A decrease in γ-glutamylcysteine synthetase was found in the heart and liver of 27-month-old rats in comparison with the 2-month-old ones. In conclusion, a decreased antioxidant capability has been demonstrated in both heart and liver of old rats.Key words: glutathione metabolism, age, rat heart, rat liver.
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Yeh, H. I., C. H. Hsieh, L. Y. Wang, S. P. Tsai, H. Y. Hsu, and M. F. Tam. "Mass spectrometric analysis of rat liver cytosolic glutathione S-transferases: modifications are limited to N-terminal processing." Biochemical Journal 308, no. 1 (May 15, 1995): 69–75. http://dx.doi.org/10.1042/bj3080069.
Full textAbstract:
Cytosolic glutathione S-transferases (GSTs) from rat livers were purified using an S-hexylglutathione affinity column. The GST subunits were resolved by reverse-phase HPLC and their molecular masses were determined by electrospray mass spectrometry. The major hepatic GSTs detected were subunits 1, 1′, 2, 3 and 4, with molecular mass of 25,520, 25,473, 25,188, 25,782 and 25,571 Da respectively. Subunits 6, 7 and 10 are minor components, with molecular mass of 25,551, 23,308 and 25,211 Da respectively. Alternatively, the hepatic GSTs were purified using a glutathione affinity column. Subunits 1, 1′, 2, 8 and 10 were eluted from this column with GSSG, the oxidized form of glutathione. Subunit 8 has a molecular mass of 25,553 Da. The remaining proteins on the glutathione affinity column were removed with glutathione and S-hexylglutathione. Subunits 2, 3, 4 and 6 could be detected in the eluate. We could not detect any significant difference in molecular mass between GSTs isolated from male and female rat livers. Cytosolic GSTs were isolated from livers of buthionine sulphoximine-treated female rats for MS analysis. The molecular masses obtained were identical to those determined for the controls.