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IntroductionActinobacillus pleuropneumoniae (A. pleuropneumoniae) is a member of Actinobacillus in family Pasteurellaceae. It is the causative agent of porcine pleuropneumonia, which has caused huge economic losses to pig industry over the world. Cysteine is a precursor of many important biomolecules and defense compounds in the cell. However, molecular mechanisms of cysteine transport in A. pleuropneumoniae are unclear.MethodsIn this study, gene-deleted mutants were generated and investigated, to reveal the roles of potential cysteine/cystine transport proteins FliY and YdjN of A. pleuropneumoniae.ResultsOur results indicated that the growth of A. pleuropneumoniae was not affected after fliY or ydjN single gene deletion, but absence of both FliY and YdjN decreased the growth ability significantly, when cultured in the chemically defined medium (CDM) supplemented with cysteine or cystine as the only sulfur source. A. pleuropneumoniae double deletion mutant ΔfliYΔydjN showed increased sensitivity to oxidative stress. Besides, trans-complementation of YdjN into ΔfliYΔydjN and wild type leads to increased biofilm formation in CDM. However, the virulence of ΔfliYΔydjN was not attenuated in mice or pigs.DiscussionThese findings suggest that A. pleuropneumoniae FliY and YdjN are involved in the cysteine/cystine acquisition, oxidative tolerance, and biofilm formation, but not contribute to the pathogenicity of A. pleuropneumoniae.

Summary Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe–S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe–S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW‐1. Comparative genomics of the order Thermotogales revealed that the feather‐degrading F. islandicum AW‐1 has a complete Suf‐like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S0). On the other hand, F. islandicum AW‐1 exhibited a significant retardation in the Suf system‐mediated keratin degradation in the presence of S0. Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS–SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf‐like machinery including the SufS–SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.

Currently the field of epilepsy lacks peripheral blood-based biomarkers that could predict the onset or progression of chronic seizures following an epileptogenic injury. Thiol/disulfide ratios have been shown to provide a sensitive means of assessing the systemic redox potential in tissue and plasma. In this study, we utilized a rapid, simple and reliable method for simultaneous determination of several thiol-containing amino acids in plasma using HPLC with electrochemical detection in kainic acid (KA) and pilocarpine rat models of epilepsy. In contrast to GSH and GSSG levels, the levels of cysteine (Cys) were decreased by 42% and 62% and cystine (Cyss) were increased by 46% and 23% in the plasma of KA- and pilocarpine-injected rats, respectively after 48 h. In chronically epileptic rats, plasma cysteine was decreased by 40.4% and 37.7%, and plasma GSSG increased by 33.8% and 35.0% following KA and pilocarpine, respectively. Treatment of rats with a catalytic antioxidant, 60 min after KA or pilocarpine significant attenuated the decrease of plasma Cys/Cyss ratios at the 48 h time point in both models. These observations suggest that the decreased cysteine and ratio of Cys/Cyss in plasma could potentially serve as redox biomarkers in temporal lobe epilepsy.

The chemotaxis system enables motile bacteria to search for an optimum level of environmental factors. Salmonella typhimurium senses the amino acid cysteine as an attractant and its oxidized dimeric form, cystine, as a repellent. We investigated the dose-response dependence of changes in chemotactic signaling activity upon exposure to cysteine and cystine of S. typhimurium LT2 using in vivo fluorescence resonance energy transfer (FRET) measurements. The dose-response curve of the attractant response to cysteine had a sigmoidal shape, typical for receptor-ligand interactions. However, in a knockout strain of the chemoreceptor genes tsr and tar, we detected a repellent response to cysteine solutions, scaling linearly with the logarithm of the cysteine concentration. Interestingly, the magnitude of the repellent response to cystine also showed linear dependence to the logarithm of the cystine concentration. This linear dependence was observed over more than four orders of magnitude, where detection started at nanomolar concentrations. Notably, low concentrations of another oxidized compound, benzoquinone, triggered similar responses. In contrast to S. typhimurium 14028, where no response to cystine was observed in a knockout strain of chemoreceptor genes mcpB and mcpC, here we showed that McpB/McpC-independent responses to cystine existed in the strain S. typhimurium LT2 even at nanomolar concentrations. Additionally, knocking out mcpB and mcpC did not affect the linear dose-response dependence, whereas enhanced responses were only observed to solutions that where not pH neutral (>100 μM cystine) in the case of McpC overexpression. We discuss that the linear dependence of the response on the logarithm of cystine concentrations could be a result of a McpB/C-independent redox-sensing pathway that exists in S. typhimurium LT2. We supported this hypothesis with experiments with defined cysteine/cystine mixed solutions, where a transition from repellent to attractant response occurred depending on the estimated redox potential.

Cysteine/cystine redox couple plays a vital role in maintaining the cellular redox state of living bio-systems. However, the dynamic changes of the intracellular cysteine/cystine pool mediated by cysteine/glutamate transporter (System X C −) in various cellular processes have remained incompletely understood. Here we established a series of strategies around fluorescent probe CP2 for effectively monitoring the intracellular cysteine level in living cells and in vivo zebrafish model, therefore for tracking dynamic changes of system X C −-mediated cysteine/cystine pool under ferroptosis. Living cell imaging and in vivo visualization both revealed that intracellular cysteine levels could be significantly reduced upon blocking system X C − activity and inducing ferroptotic cell death. This kind of blocking was potent against cisplatin-resistant A549R cancer cells, which might bring new potential for treating drug-resistant carcinoma through regulating system X C − and processes of ferroptosis. With further improvements in monitoring tools, understanding of system X C −-mediated cystine/cysteine pool and ferroptosis could certainly offer crucial information in more physiological or pathological processes. In this work, we established a series of strategies around fluorescent probe CP2 for effectively monitoring the intracellular cysteine level in living cells and zebrafish model, therefore for tracking dynamic changes of system X C −-mediated cysteine/cystine pool under ferroptosis. Living cell imaging and in vivo visualization both revealed that intracellular cysteine level could be significantly reduced upon treatment with system X C − inhibition and ferroptosis induction. This kind of blocking was potent against cisplatin-resistant A549R cancer cells, which might bring new potential for treating drug-resistant carcinoma through regulating system X C − and processes of ferroptosis. With monitoring tools further improved, further understanding of system X C −-mediated cystine/cysteine pool and ferroptosis could certainly offer crucial information in more physiological or pathological processes. Image 1 • New strategy for monitoring endogenous cysteine in cancer and dynamic changes of Cys/cystine pool in external stimulation. • Effectively monitoring intracellular cysteine in living cells and zebrafish thus tracking Cys/cystine changing. • The first report for imaging Cys/cystine pool dynamics during ferroptosis. • Revealing physiological or pathological role of the system XC--mediated Cys/cystine pool in ferroptosis> Providing possibility to circumvent drug resistance of cisplatin and identify inhibitors for ferroptosis-mediated therapy. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming is a hallmark of human cancer, and cancer-specific metabolism provides opportunities for cancer diagnosis, prognosis, and treatment. However, the underlying mechanisms by which metabolic pathways affect the initiation and progression of colorectal cancer (CRC) remain largely unknown. Here, we demonstrate that cysteine is highly enriched in colorectal tumors compared to adjacent non-tumor tissues, thereby promoting tumorigenesis of CRC. Synchronously importing both cysteine and cystine in colorectal cancer cells is necessary to maintain intracellular cysteine levels. Hypoxia-induced reactive oxygen species (ROS) and ER stress regulate the co-upregulation of genes encoding cystine transporters (SLC7A11, SLC3A2) and genes encoding cysteine transporters (SLC1A4, SLC1A5) through the transcription factor ATF4. Furthermore, the metabolic flux from cysteine to reduced glutathione (GSH), which is critical to support CRC growth, is increased due to overexpression of glutathione synthetase GSS in CRC. Depletion of cystine/cysteine by recombinant cyst(e)inase effectively inhibits the growth of colorectal tumors by inducing autophagy in colorectal cancer cells through mTOR-ULK signaling axis. This study demonstrates the underlying mechanisms of cysteine metabolism in tumorigenesis of CRC, and evaluates the potential of cysteine metabolism as a biomarker or a therapeutic target for CRC.

Summary: Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe–S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe–S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW‐1. Comparative genomics of the order Thermotogales revealed that the feather‐degrading F. islandicum AW‐1 has a complete Suf‐like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S0). On the other hand, F. islandicum AW‐1 exhibited a significant retardation in the Suf system‐mediated keratin degradation in the presence of S0. Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS–SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf‐like machinery including the SufS–SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions. [ABSTRACT FROM AUTHOR]

Sulfur is essential for biological processes such as amino acid biogenesis, iron-sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSCFliY cysteine/cystine import system. We found that the substrate- binding protein FliY binds L-cystine, L-cysteine, and D-cysteine with micromolar affinities. However, binding of the L- and D-enantiomers induced different conformational changes of FliY, where the L- enantiomer-substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by D-cysteine-bound FliY, and maximally by L-cysteine- or L-cystine-bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is prevalent in patients infected with HIV. The purpose of this study was to test the hypothesis that systemic oxidation correlates with loss of lung function in subjects with COPD, and that HIV infection can contribute to creating such an environment. Subjects were recruited at the University of Louisville in the following groups: HIV-infected (n = 36), COPD (n = 32), HIV and COPD (n = 28), and uninfected controls with normal lung function (n = 34). HIV infection was assessed by viral load and CD4 cell counts. Pulmonary function was determined by spirometry, and plasma was collected for measurement of cysteine (Cys), cystine (CySS), glutathione (GSH) and GSH disulfide (GSSG) by HPLC followed by estimation of redox potentials (E h) using the Nernst equation. Results showed that patients with COPD had more oxidized plasma E h Cys/CySS than patients with normal lung function, but plasma E h GSH/GSSG was unaltered. In addition, there was a correlation between the extent of plasma E h Cys/CySS oxidation and loss of lung function, and this correlation remained even after correcting for age, sex, race and body mass index. HIV infection per se was not associated with increased oxidation of plasma E h Cys/CySS, but plasma E h Cys/CySS was more oxidized in patients with lower CD4-positve T cell counts. In patients with both HIV infection and COPD, there was a significant correlation between CD4 cell counts and lung function. Thus, systemic oxidation correlated with decreased lung function in subjects with COPD and decreased CD4 counts in subjects infected with HIV. Thus, factors contributing to plasma E h Cys/CySS may represent novel mechanisms underlying the increased prevalence of COPD in people living with HIV. Image 1 • COPD is a major cause of morbidity and mortality in patients infected with HIV. • Oxidative stress has been implicated in the pathogenesis of both COPD and HIV. • Decreased lung function and CD4 counts paralleled oxidation of plasma cysteine/cystine redox potential (E h Cys/CySS). • Plasma E h Cys/CySS is a marker of oxidative stress that may be used to identify subjects at risk for lung function decline. [ABSTRACT FROM AUTHOR]

Human cell lines regulate the redox state ([E.sub.h]) of the cysteine/cystine (Cys/CySS) couple in culture medium to approximately -80 mV, a value similar to the average [E.sub.h] for Cys/CySS in human plasma. The mechanisms involved in regulation of extracellular [E.sub.h] of Cys/CySS are not known, but GSH is released from tissues at rates proportional to tissue GSH concentration, and this released GSH could react with CySS to contribute to maintenance of this balance. The present study was undertaken to determine whether depletion of cellular GSH alters regulation of extracellular Cys/CySS [E.sub.h]. Decrease of GSH in HT-29 cells by inhibiting synthesis with L-buthionine-[S,R]-sulfoximine showed no effect on the rate of reduction of extracellular CySS to achieve a stable [E.sub.h] for Cys/CySS in the culture medium. Limiting Cys and CySS in the culture medium also substantially decreased cellular GSH but resulted in no significant effect on extracellular Cys/CySS [E.sub.h]. Addition of CySS to these cells showed that extracellular Cys/CySS [E.sub.h] approached -80 mV at 4 h while cellular GSH and extracellular GSH/GSSG [E.sub.h] recovered more slowly. Together, these results show that HT-29 cells have the capacity to regulate the extracellular Cys/CySS [E.sub.h] by mechanisms that are independent of cellular GSH. The results suggest that transport systems for Cys and CySS and/or membranal oxidoreductases could be more important than cellular GSH in regulation of extracellular Cys/CySS [E.sub.h]. oxidative stress; thiol/disulfide redox control; amino acid deficiency; amino acid transport

Few data are available on plasma redox responses to sulfur amino acid (SAA) loads. In this study, we had 2 aims: to determine whether the SAA content of a meal affected postprandial plasma cysteine (Cys), cystine (CySS), or redox potential ([E.sub.h]CySS) in humans and whether SAA intake level (adequate or inadequate) in the days preceding the meal challenge affected these postprandial levels. Eight healthy individuals aged 18-36 y were equilibrated for 3 d to adequate SAA, fed chemically defined meals without SAA for 5 d (inadequate SAA) and then fed isoenergetic, isonitrogenous meals with adequate SAA for 5 d. On the first and last days with the chemically defined meals, a morning meal containing 60% of the daily food intake was given, and plasma Cys, CySS, and [E.sub.h]CySS were determined over an 8-h postprandial time course. Following equilibration to adequate intake, provision of the meal with SAA resulted in increased plasma Cys and CySS concentrations and more reduced plasma [E.sub.h]CySS compared with the postprandial values following the same meal without SAA. Equilibration to inadequate SAA intake for the days preceding the meal challenge did not affect this response. The magnitude of the difference in postprandial plasma [E.sub.h]CySS (10 mV) due to meal content of SAA was comparable to those which alter physiologic signaling and/or are associated with disease risk. Consequently, the SAA content of meals could affect physiologic signaling and associated disease mechanisms in the postprandial period by changes in Cys, CySS, or [E.sub.h]CySS. doi: 10.3945/jn.109.116764

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

Previous research shows that stimulation of proliferation of colon carcinoma (Caco-2) cells by a more reduced extracellular cysteine/cystine (Cys/CySS) redox state occurs with no apparent effect on intracellular glutathione and that this stimulation is lost on addition of epidermal growth factor. The purpose of the present study was to determine whether a more reduced extracellular Cys/CySS redox state activates the mitogenic p44/p42 mitogen-activated protein kinase (MAPK) pathway and whether this is signaled through the epidermal growth factor receptor (EGFR). Caco-2 cells were exposed to a range of physiological extracellular redox conditions from--150 to 0 mV. In the absence of added growth factors, the most reduced (-150 mV) redox state induced an 80% increase in EGFR phosphorylation, and this was followed by a marked increase in phosphorylation of p44/p42 MAPK. Inhibitors of EGFR (AG1478) and p44/p42 MAPK (U0126) phosphorylation blocked redox-dependent p44/p42 phosphorylation, indicating that signaling occurred by EGFR. These effects were inhibited by pretreatment with a nonpermeant alkylating agent, showing that signaling involved thiols accessible to the extracellular space. The EGFR ligand TGF-[alpha] was increased in culture medium at more reduced redox states. Redox-dependent phosphorylation of EGFR was completely prevented by a metalloproteinase inhibitor (GM6001), and an antibody to TGF-[alpha] partially inhibited the phosphorylation of p44/p42 MAPK by redox. Thus the data show that a redox-dependent activation of metalloproteinase can stimulate the mitogenic p44/p42 MAPK pathway by a TGF-[alpha]-dependent mechanism. Because Cys availability and Cys/CySS redox are dependent on nutrition, disease, and environmental exposures, the results suggest that cell proliferation could be influenced physiologically by Cys-dependent redox effects on growth factor signaling pathways. oxidation reduction; intestine; transforming growth factor-[alpha]; glutathione

Oxidant stress has been implicated in the pathogenesis of chronic lung disorders like idiopathic pulmonary fibrosis. However, mechanisms that link oxidant stress to fibrogenesis remain partially elucidated. Emerging data suggest an important role for the extracellular thiol/disulfide redox environment. The cysteine (Cys)/cystine (CySS) redox couple represents the predominant low-molecular-weight thiol/disulfide pool found in plasma and is sensitive to aging, smoking, and other host factors. We hypothesized that an oxidized extracellular Cys/CySS redox potential ([E.sub.h] Cys/CySS) affects lung fibroblasts by inducing intracellular signals that stimulate proliferation and matrix expression. We tested this hypothesis in primary murine lung fibroblasts and found that an oxidized [E.sub.h] Cys/CySS (-46 mV) stimulated lung fibroblast proliferation. Furthermore, it stimulated their expression of fibronectin, a matrix glycoprotein highly expressed in fibrotic lung diseases and implicated in lung injury. This stimulatory effect was dependent on protein kinase C activation. Oxidant stress also increased the phosphorylation of cAMP response element binding protein, a transcription factor known for its ability to stimulate fibronectin expression, and increased the expression of mRNAs and proteins coding for the transcription factors nuclear factor (NF)-[kappa]B and mothers against decapentaplegic homolog 3. Fibroblasts cultured in normal (-80 mV) or reduced (-131 mV) [E.sub.h] Cys/CySS showed less induction. Furthermore, fibronectin expression in response to an oxidized [E.sub.h] Cys/CySS was associated with expression of transforming growth factor-[beta]1 (TGF-[beta]1) and was inhibited by an anti-TGF-[beta]1 antibody and SB-431542, a TGF-[beta]1 receptor inhibitor. These studies suggest that extracellular oxidant stress activates redox-sensitive pathways that stimulate lung fibroblast proliferation and matrix expression through upregulation of TGF-[beta]1. redox potential; oxidant stress; fibroblasts; fibrosis; fibronectin; proliferation; redox signaling

The metabolism of cysteine and other related compounds was investigated. This involved the utilization of an isolated perfused hindquarter of rats in order to evaluate the ability of its skeletal muscle for metabolizing cysteine, cystine, the disulfide of cysteine and the major plasma form of cyst(e)ine. Results demonstrate the very limited capacity of rat skeletal muscles for cysteine metabolism.

A unique PED waveform is applied for detection of the sulfur-containing compounds of interest following their separation on a chromatographic column having both cation-exchange and reversed-phase properties. The mobile phase is 0.10 M HClO4/0.15 M NaClO4/5% MeCN, which results in virtually no PED response for carbohydrates and non-sulfur-containing amino acids. The PED waveform utilizes current integration throughout the period of a fast cyclic potential scan to eliminate the large background current normally observed for the conventional three-step PED waveform as a consequence of formation of surface oxide, which is required by the detection mechanism. Limits of detection for the compounds of interest are at the picomolar level.

The chemotaxis system enables motile bacteria to search for an optimum level of environmental factors. Salmonella typhimurium senses the amino acid cysteine as an attractant and its oxidized dimeric form, cystine, as a repellent. We investigated the dose-response dependence of changes in chemotactic signaling activity upon exposure to cysteine and cystine of S. typhimurium LT2 using in vivo fluorescence resonance energy transfer (FRET) measurements. The dose-response curve of the attractant response to cysteine had a sigmoidal shape, typical for receptor-ligand interactions. However, in a knockout strain of the chemoreceptor genes tsr and tar, we detected a repellent response to cysteine solutions, scaling linearly with the logarithm of the cysteine concentration. Interestingly, the magnitude of the repellent response to cystine also showed linear dependence to the logarithm of the cystine concentration. This linear dependence was observed over more than four orders of magnitude, where detection started at nanomolar concentrations. Notably, low concentrations of another oxidized compound, benzoquinone, triggered similar responses. In contrast to S. typhimurium 14028, where no response to cystine was observed in a knockout strain of chemoreceptor genes mcpB and mcpC, here we showed that McpB / McpC-independent responses to cystine existed in the strain S. typhimurium LT2 even at nanomolar concentrations. Additionally, knocking out mcpB and mcpC did not affect the linear dose-response dependence, whereas enhanced responses were only observed to solutions that where not pH neutral (>100 μM cystine) in the case of McpC overexpression. We discuss that the linear dependence of the response on the logarithm of cystine concentrations could be a result of a McpB / C-independent redox-sensing pathway that exists in S. typhimurium LT2. We supported this hypothesis with experiments with defined cysteine / cystine mixed solutions, where a transition from repellent to attractant response occurred depending on the estimated redox potential. [ABSTRACT FROM AUTHOR]

Assisted reproductive techniques expose gametes to excessive concentrations of reactive oxygen species. The present study aimed to evaluate the effects of oxidative stress on apoptosis in goat oocytes and embryonic development. The results demonstrated that the addition of 100 µM hydrogen peroxide (H2O2) into media produces an oxidative environment during oocyte maturation. The number of cumulus cells positive for terminal deoxynucleotidyl transferase UTP nick end labeling, and the activity of caspase 3 in mature oocytes were increased, compared with the control group (P<0.05). In addition, the expression levels of mitochondrial regulators, including peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) and nuclear respiratory factor-1 (NRF-1) were increased in the oxidative oocytes, compared with those in the control group (P<0.05). The ratio of the proapoptotic gene, B cell lymphoma (Bcl-2)-associated X pr otein (BAX), to the anti-apoptotic gene, BCL-2, was higher in the H2O2 group, compared with the control group (P<0.05). To overcome oxidative stress in oocytes and embryos cultured in vitro, 200 µM cysteine and 200 µM cystine were added to the media, thereby increasing the concentration of intracellular glutathione (GSH) and assisting in maintaining the redox state of the cells. In conclusion, cysteine and cystine reduced the oxygen tension caused by H2O2, thereby providing a novel strategy for optimizing in vitro embryonic development systems. [ABSTRACT FROM AUTHOR]

Background/Aims: Oxidative stress has been considered a nontraditional risk factor for cardiovascular disease in the chronic kidney disease (CKD) population, possibly triggered by uremic toxicity. Methods: A chromatographic method with coulometric detection was adapted to directly and simultaneously determine cysteine (Cys) and cystine (Cyss) in plasma samples. Healthy subjects and CKD subjects in different stages were analyzed. The free Cys and free Cyss levels in their plasma were determined, and the reduction potential [Eh(Cyss/2Cys)] was calculated with the Nernst equation. Results: Healthy plasma presented Eh(Cyss/2Cys) of -123 ± 7 mV. Plasma Eh(Cyss/2Cys) correlated significantly with creatinine levels (p < 0.0001, r = 0.62). Conclusion: Plasma Eh(Cyss/2Cys) correlated with increased levels of plasma creatinine, supporting the view that uremia triggers oxidative stress. In addition, it may be used as a quantitative oxidative stress biomarker in uremic conditions. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The selective detection of cysteine and cystine amino acids over other standard amino acids was possible with the naked eye using silver nanoparticles (AgNPs) in a simple procedure at room temperature. The change in color and the aggregation of NPs were studied using UV–vis spectroscopy and transmission electron microscopy, respectively. It was observed that when the derivative and substructures of cysteine were employed as analytes the detection was possible only when there is free SH or S–S group present in the analytes. The method was extended to a dipeptide ‘cys-gly’ as a model peptide where the detection was successful due to the presence of SH moiety. [Copyright &y& Elsevier]

Few data are available on plasma redox responses to sulfur amino acid (SAA) loads. In this study, we had 2 aims: to determine whether the SAA content of a meal affected postprandial plasma cysteine (Cys), cystine (CySS), or redox potential (EhCySS) in humans and whether SAA intake level (adequate or inadequate) in the days preceding the meal challenge affected these postprandial levels. Eight healthy individuals aged 18-36 y were equilibrated for 3 d to adequate SAA, fed chemically defined meals without SAA for 5 d (inadequate SM) and then fed isoenergetic, isonitrogenous meals with adequate SAA for 5 d. On the first and last days with the chemically defined meals, a morning meal containing 60% of the daily food intake was given, and plasma Cys, CySS, and ECySS were determined over an 8-h postprandial time course. Following equilibration to adequate intake, provision of the meal with SAA resulted in increased plasma Cys and CySS concentrations and more reduced plasma EhCySS compared with the postprandial values following the same meal without SM. Equilibration to inadequate SAA intake for the days preceding the meal challenge did not affect this response. The magnitude of the difference in postprandial plasma EhCySS (10 mV) due to meal content of SAA was comparable to those which alter physiologic signaling and/or are associated with disease risk. Consequently, the SAA content of meals could affect physiologic signaling and associated disease mechanisms in the postprandial period by changes in Cys, CySS, or EhCySS. [ABSTRACT FROM AUTHOR]

The experimental sulfur K-edge X-ray absorption near-edge structure (XANES) spectra of the amino acids cysteine, homocysteine, penicillamine, methionine, including the oxidation products methionine sulfoxide and the disulfide cystine, have been analyzed by transition potential DFT calculations. The absolute energies and intensities of the main pre-edge sulfur 1s electron transitions have been computed to determine the character of the receiving unoccupied molecular orbitals (MO), and to investigate the influence of external interactions, especially by introducing water molecules hydrogen-bonded to the ionic species present in different pH ranges. When the thiol group deprotonates for cysteine, homocysteine and penicillamine and also for the cysteine residue in glutathione the energy of the main transition, to an MO with antibonding σ*(S-H) character, reduces by ∼1.1 eV and the receiving MO obtains σ*(S-C) character. The changes in transition energy due to hydrogen-bonding were in most cases found to be relatively small, although the transition intensities could vary significantly due to the changes induced in the molecular charge distribution, thereby affecting the shapes of the spectral features. For the cysteine and penicillamine zwitterions deconvolution of the experimental spectra allowed the microscopic acid dissociation constants to be extracted separately for the thiol and the protonated amine groups, pKa S = 8.5 ± 0.1 and 8.2 ± 0.1, and pKa N = 8.9 ± 0.1 and 8.8 ± 0.1, respectively, with the thiol group in both cases being the more acidic. Coordination of cysteine to nickel(II) or mercury(II) introduced a new low energy transition involving metal ion orbitals in the receiving LUMO. The small experimentally observed energy differences between the similar main absorption features of the cysteine and methionine zwitterions, 0.2-0.3 eV in comparable surrounding, as well as a minor difference in their intensities, are reflected in the calculated transitions. The S K-edge XANES spectrum of the disulfide cystine displays a characteristic double peak with the lower energy transition (2469.9 eV) into the antibonding σ*(S-S) MO. The second peak, at 2471.5 eV in aqueous solution, contains several transitions into MOs with σ*(S-C) character involving also charge transfer to the water molecules hydrating the protonated amine groups (NH3 +) of cystine. For solid cystine without hydrogen bonding the experimental energy difference between the two peaks is 0.2 eV larger, while no such increase occurs for the oxidized disulfide of glutathione, with a similar -S-S- bond between its cysteine residues as in cystine, because the amine groups are engaged in peptide bonds. This study shows that externally induced changes in the intramolecular bonding, e.g., by coordination, conformation geometry or hydrogen-bonding, can significantly influence the S K-edge spectra, and emphasizes the importance of a similar chemical surrounding when choosing the model compounds for standard spectra of sulfur functional groups, used to deconvolute composite experimental spectra. [ABSTRACT FROM AUTHOR]

The experimental sulfur K-edge X-ray absorption near-edge structure (XANES) spectra of the amino acids cysteine, homocysteine, penicillamine, methionine, including the oxidation products methionine sulfoxide and the disulfide cystine, have been analyzed by transition potential DFT calculations. The absolute energies and intensities of the main pre-edge sulfur 1s electron transitions have been computed to determine the character of the receiving unoccupied molecular orbitals (MO), and to investigate the influence of external interactions, especially by introducing water molecules hydrogen-bonded to the ionic species present in different pH ranges. When the thiol group deprotonates for cysteine, homocysteine and penicillamine and also for the cysteine residue in glutathione the energy of the main transition, to an MO with antibonding σ*(S–H) character, reduces by ∼1.1 eV and the receiving MO obtains σ*(S–C) character. The changes in transition energy due to hydrogen-bonding were in most cases found to be relatively small, although the transition intensities could vary significantly due to the changes induced in the molecular charge distribution, thereby affecting the shapes of the spectral features. For the cysteine and penicillamine zwitterions deconvolution of the experimental spectra allowed the microscopic acid dissociation constants to be extracted separately for the thiol and the protonated amine groups, pKaS= 8.5 ± 0.1 and 8.2 ± 0.1, and pKaN= 8.9 ± 0.1 and 8.8 ± 0.1, respectively, with the thiol group in both cases being the more acidic. Coordination of cysteine to nickel(II) or mercury(II) introduced a new low energy transition involving metal ion orbitals in the receiving LUMO. The small experimentally observed energy differences between the similar main absorption features of the cysteine and methionine zwitterions, 0.2–0.3 eV in comparable surrounding, as well as a minor difference in their intensities, are reflected in the calculated transitions. The S K-edge XANES spectrum of the disulfide cystine displays a characteristic double peak with the lower energy transition (2469.9 eV) into the antibonding σ*(S–S) MO. The second peak, at 2471.5 eV in aqueous solution, contains several transitions into MOs with σ*(S–C) character involving also charge transfer to the water molecules hydrating the protonated amine groups (NH3+) of cystine. For solid cystine without hydrogen bonding the experimental energy difference between the two peaks is 0.2 eV larger, while no such increase occurs for the oxidized disulfide of glutathione, with a similar –S–S– bond between its cysteine residues as in cystine, because the amine groups are engaged in peptide bonds. This study shows that externally induced changes in the intramolecular bonding, e.g., by coordination, conformation geometry or hydrogen-bonding, can significantly influence the S K-edge spectra, and emphasizes the importance of a similar chemical surrounding when choosing the model compounds for standard spectra of sulfur functional groups, used to deconvolute composite experimental spectra. [ABSTRACT FROM AUTHOR]

Oxidant stress has been implicated in the pathogenesis of chronic lung disorders like idiopathic pulmonary fibrosis. However, mechanisms that link oxidant stress to fibrogenesis remain partially elucidated. Emerging data suggest an important role for the extracellular thiol/disulfide redox environment. The cysteine (Cys)/cystine (CySS) redox couple represents the predominant low-molecular-weight thiol/disulfide pool found in plasma and is sensitive to aging, smoking, and other host factors. We hypothesized that an oxidized extracellular Cys/CySS redox potential (Eh Cys/CySS) affects lung fibroblasts by inducing intracellular signals that stimulate proliferation and matrix expression. We tested this hypothesis in primary murine lung fibroblasts and found that an oxidized Eh Cys/CySS (–46 mV) stimulated lung fibroblast proliferation. Furthermore, it stimulated their expression of fibronectin, a matrix glycoprotein highly expressed in fibrotic lung diseases and implicated in lung injury. This stimulatory effect was dependent on protein kinase C activation. Oxidant stress also increased the phosphorylation of cAMP response element binding protein, a transcription factor known for its ability to stimulate fibronectin expression, and increased the expression of mRNAs and proteins coding for the transcription factors nuclear factor (NF)-κB and mothers against decapentaplegic homolog 3. Fibroblasts cultured in normal (–80 mV) or reduced (–131 mV) Eh Cys/CySS showed less induction. Furthermore, fibronectin expression in response to an oxidized Eh Cys/CySS was associated with expression of transforming growth factor-β1 (TGF-β1) and was inhibited by an anti-TGF-β1 antibody and SB-43 1542, a TGF-β1 receptor inhibitor. These studies suggest that extracellular oxidant stress activates redox-sensitive pathways that stimulate lung fibroblast proliferation and matrix expression through upregulation of TGF43 1. [ABSTRACT FROM AUTHOR]

Objective: The prevalence and costs associated with treating pressure ulcers (PU) are at high levels. Frequently, PUs heal slowly or not at all, which may be due to the patient's catabolic state which may include protein energy malnutrition. The objective of this open label clinical trial was to improve healing rates by providing patients with a patented, high-quality protein containing all essential amino acids to ensure positive nitrogen balance. An additional benefit of this protein is the delivery of bioavailable cysteine (cystine) to promote glutathione (GSH) synthesis which supports immune function and heightens antioxidant defences. Methods: Patients with category II, III and IV PUs were fed 20g BID whey protein dietary supplement for 16–120 days, without change in ongoing 'best practice' PU management and their progress recorded. Results: A total of 10 patients were recruited, with an average age of 77 years. Most had shown no improvement in healing for ≥2 months before treatment and usually had other complications including chronic obstructive pulmonary disease (COPD), diabetes and various cardiovascular diseases. There were a total of 23 PUs, with some patients having more than one. Of these, 44% (n=10) showed complete resolution 83% (n=19) had better than 75% resolution over the observation period. Healing rates ranged from 16.9–0.2cm2/month (healed PUs) and 60.0–1.6cm2/month for resolving PUs. Conclusion: By providing the necessary amino acids to rebuild tissues and bioactive cysteine (cystine) to promote synthesis of intracellular GSH and positive nitrogen balance, improvement in PUs healing was achieved. [ABSTRACT FROM AUTHOR]

Presents an overview of a study which showed that steady-state redox potential of the cysteine/cystine couple in cells is independent of the redox state of Glutathione(GSH)/GSSG and is sufficiently oxidized relative to the GSH/GSSG redox couple for the cysteine/cystine couple to function as an oxidant in redox switching. Redox state of cysteine/cystine in proliferating HT29 cells; Culture of HT29 cells in cysteine-free medium; Impact of glutathione synthesis with buthionine sulfoximine on glutathione concentration.