Protein Sulfhydryl Groups Research Articles

Cardioprotective effects of aqueous extract of Oxalis corniculata in experimental myocardial infarction

The present study evaluated the protective potential of aqueous extract of Oxalis corniculata (OCE) against isoproterenol (ISO) induced myocardial infarction in rats. Myocardial infarction in rats was induced by isoproterenol (200 mg/kg) at an interval of 24 h for 2 days. OCE was given to rats as pretreatment for 30 days orally using an intragastric tube. Isoproterenol caused a significant increase in the activity of cardiac injury marker enzymes like creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) and increased the concentration of serum lipids. OCE pretreatment significantly reduced the concentration of CPK, LDH, serum total cholesterol, LDL cholesterol and triglycerides. OCE also reduced the activity of lipogenic enzyme, glucose-6-phosphate dehydrogenase in ISO administered rats. Oxidative stress produced by isoproterenol was significantly lowered by the administration of OCE which was evident from increased activities of antioxidant enzymes (catalase and superoxide dismutase) and reduced concentration of lipid peroxidation products (TBARS and conjugated dienes). Concentration of vitamin C, protein sulfhydryl groups and reduced glutathione (GSH) was also high in OCE pretreated rats. Histopathology of heart of ISO administered rat pretreated with OCE showed normal myocardium with very little evidence of inflammatory infiltration. Results of our in vitro findings also confirmed that OCE exhibits significant antioxidant and radical scavenging activity against DPPH, superoxide and nitric oxide radicals. These findings provided evidence that O. corniculata was found to be protecting the myocardium against ischemic insult and the protective effect could attribute to its antioxidative and antihyperlipidemic activities.

Enhanced IL-12p40 production by phenylarsine oxide is mediated by cAMP response element in macrophages

Phenylarsine oxide (PAO), a membrane-permeable trivalent arsenical, is widely used as an inhibitor of protein tyrosine phosphatases. It reacts with vicinal sulfhydryl groups of proteins to form stable ring structures. Here we show the regulatory function of PAO in immune responses from macrophages. PAO significantly induced the secretion of interleukin (IL)-12p40 in lipopolysaccharide-stimulated macrophages. The mRNA expression and the gene promoter activity of IL-12p40 were enhanced by PAO. These results suggest that PAO may enhance IL-12p40 production at the transcriptional level. Furthermore, the effects of PAO on several signaling molecules regulating IL-12p40 expression were investigated. PAO attenuated the induced binding activity of cAMP response element (CRE), but not of NF-kappaB. Moreover, CRE promoter activity was dose-dependently inhibited by PAO and the increased secretion of IL-12p40 by PAO was reduced by forskolin, a cAMP activator. These results suggest that PAO enhances IL-12p40 production by inhibiting CRE activity.

Abstract 3785: Isothiocyanates vs. isoselenocyanates: Reactivity toward glutathione and cysteinyl residues in cell and cell-free systems

Abstract Isothiocyanates (ITCs) are naturally occurring compounds found in cruciferous vegetables. Both naturally occurring phenylalkyl ITCs (BITC, PEITC) and longer chain synthetic ITCs (PBITC, PHITC) have been studied extensively and are shown to inhibit carcinogenesis due to inhibition of CYP450 enzymes, as well as induction of Phase II detoxification enzymes. These actions are thought to be mediated by the reactivity of the ITC functional group (N=C=S) with sulfhydryl groups of cell thiols, mainly glutathione and proteins (cysteinyl residues). Our lab has modified both the naturally occurring and synthetic phenyl alkyl ITCs by isosterically replacing sulfur with selenium to make isoselenocyanates (ISCs): ISC1, ISC2, ISC4, and ISC6. We have found these compounds to be more potent in cancer cell viability assays and in xenograft mouse model at inhibiting tumor growth than corresponding ITCs. Our hypothesis is the better potency of ISC compounds may be due to their modified reactivity rates toward cell thiols. To test this, A549 lung cancer cells were treated with our panel of ITC compounds or corresponding ISC compounds from 500 nM to 15 µM for 1 hour. GSH depletion appeared to be dose dependent for all compounds except for hexyl chain compounds PHITC and ISC6. For the remaining compounds, ISC compounds depleted GSH in cells to a greater extent than corresponding ITCs. 15 µM of ISC1, ISC2, and ISC4 depleted GSH levels to 58%, 53%, and 63% of control, respectively, whereas BITC, PEITC, and PBITC only depleted GSH to 69%, 86%, and 86%, respectively. These results may be related to redox cycling capabilities which measures the reactivity of compounds with GSH and O2 to form superoxide. In a chemiluminesence assay measuring redox cycling, ISC compounds showed a much greater ability to redox cycle compared to corresponding ITCs. The redox cycling capabilities were as follows: ISC2≫PEITC>ISC1=ISC4=ISC6>PHITC>PBITC>BITC. In a cell free system, overall binding of ITC and ISC compounds were analyzed for GSH using the Ellman assay. In this cell free system both ITCs and ISCs showed a trend of increasing binding with decreasing chain length: BITC>PEITC>PBITC>PHITC and ISC1>ISC2>ISC4>ISC6. However, corresponding ITC and ISC compounds bound similarly to GSH. Similar experiment using tubulin protein exhibited the same trend of reactivity with ITCs, as with GSH. However, among ISC compounds, ISC2 showed the greatest binding followed by ISC1=ISC4>ISC6. These results suggest that cell environment is playing a role in the reactivity of these compounds with glutathione and proteins. In conclusion, ISC compounds depleted GSH in cells to a greater extent than corresponding ITC compounds and were also better able to redox cycle. Future studies will analyze the exact rate of reactivity of ISC compounds toward GSH, identifying protein targets of these compounds, and binding preferences of different proteins. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3785.

Detection of the Sulfhydryl Groups in Proteins with Slow Hydrogen Exchange Rates and Determination of Their Proton/Deuteron Fractionation Factors Using the Deuterium-Induced Effects on the 13Cβ NMR Signals

A method for identifying cysteine (Cys) residues with sulfhydryl (SH) groups exhibiting slow hydrogen exchange rates has been developed for proteins in aqueous media. The method utilizes the isotope shifts of the C(beta) chemical shifts induced by the deuteration of the SH groups. The 18.2 kDa E. coli peptidyl prolyl cis-trans isomerase b (EPPIb), which was selectively labeled with [3-(13)C;3,3-(2)H(2)]Cys, showed much narrower line widths for the (13)C(beta) NMR signals, as compared to those of the proteins labeled with either [3-(13)C]Cys or (3R)-[3-(13)C;3-(2)H]Cys. The (13)C(beta) signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-(13)C;3,3-(2)H(2)]Cys, split into four signals in H(2)O/D(2)O (1:1) at 40 degrees C and pH 7.5, indicating that the exchange rates of the side-chain SH's and the backbone amides are too slow to average the chemical shift differences of the (13)C(beta) signals, due to the two- and three-bond isotope shifts. By virtue of the well-separated signals, the proton/deuteron fractional factors for both the SH and amide groups of the two Cys residues in EPPIb could be directly determined, as approximately 0.4-0.5 for [SD]/[SH] and 0.9-1.0 for [ND]/[NH], by the relative intensities of the NMR signals for the isotopomers. The proton NOE's of the two slowly exchanging SH's were clearly identified in the NOESY spectra and were useful for the determining the local structure of EPPIb around the Cys residues.

Protein S-nitrosylation and cardioprotection.

Nitric oxide (NO) plays an important role in the regulation of cardiovascular function. In addition to the classic NO activation of the cGMP-dependent pathway, NO can also regulate cell function through protein S-nitrosylation, a redox dependent, thiol-based, reversible posttranslational protein modification that involves attachment of an NO moiety to a nucleophilic protein sulfhydryl group. There are emerging data suggesting that S-nitrosylation of proteins plays an important role in cardioprotection. Protein S-nitrosylation not only leads to changes in protein structure and function but also prevents these thiol(s) from further irreversible oxidative/nitrosative modification. A better understanding of the mechanism regulating protein S-nitrosylation and its role in cardioprotection will provide us new therapeutic opportunities and targets for interventions in cardiovascular diseases.

Does carbonyl stress cause increased blood viscosity during storage?

The structural and biochemical changes to erythrocytes during storage, called 'storage lesion', are important factors that contribute to decreases in the efficacy and safety of blood transfusions. However, the biochemical mechanisms are only partly understood. Fresh whole blood in citrate phosphate dextrose anticoagulant was preserved in storage bags at 1-4 degrees C for over 20 days. Aliquots of stored blood were withdrawn for analysis at the 1st, 5th, 10th, 15th and 20th day of storage. Whole blood viscosity, plasma reactive carbonyl species, erythrocyte membrane protein carbonylation, protein (tryptophan) fluorescence and the contents of thiols were quantified concurrently. There were significant increases in blood viscosity and plasma reactive carbonyl species level during storage. There were significant time-dependent increases in membrane protein carbonylation, increases in protein (tryptophan) fluorescence and a decrease in the content of thiols. Reactive carbonyl species, by attacking the amino and/or sulfhydryl groups of erythrocyte membrane proteins, induce a series of structural alterations in erythrocytes. These lead to an increase in blood viscosity and reduce the efficacy and safety of storage and transfusion.

Diet supplementation with acai ( Euterpe oleracea Mart.) pulp improves biomarkers of oxidative stress and the serum lipid profile in rats

Objective We investigated the antioxidant potential and hypocholesterolemic effects of acai ( Euterpe oleracea Mart.) pulp ingestion in rats fed a standard or hypercholesterolemic diet. Methods Female Fischer rats were fed a standard AIN-93 M diet (control) or a hypercholesterolemic diet that contained 25% soy oil and 1% cholesterol. The test diet was supplemented with 2% acai pulp (dry wt/wt) for control (group CA) and hypercholesterolemic rats (group HA) for 6 wk. At the end of the experimental period, rats were sacrificed and the blood and livers were collected. To evaluate the effect of acai consumption, levels of protein carbonyl and sulfhydryl groups, superoxide dismutase and paraoxonase activities, and lipid profiles of the sera were measured. Results Animals that were fed the hypercholesterolemic diet presented increased levels of total and non–high-density lipoprotein cholesterol and decreased levels of high-density lipoprotein cholesterol. Supplementing the diet of this group with acai caused a hypocholesterolemic effect by reducing total and non–high-density lipoprotein cholesterol. Serum levels of carbonyl proteins and total, free, and protein sulfhydryl groups were reduced by acai ingestion in animals receiving the standard or hypercholesterolemic diet. Acai supplementation induced a significant reduction in superoxide dismutase activity only in the hypercholesterolemic rats, indicating an association between diet and acai treatment. Also, acai supplementation increased paraoxonase activity in the CA and HA groups. Conclusion These results suggest that the consumption of acai improves antioxidant status and has a hypocholesterolemic effect in an animal model of dietary-induced hypercholesterolemia.

Acrylamide catalytically inhibits topoisomerase II in V79 cells

The vinyl monomer acrylamide is characterized by the presence of an α,β-unsaturated carbonyl group that makes it reactive towards thiol, hydroxyl or amino groups and towards the nucleophilic centers in DNA. The ability of acrylamide to chemically modify protein thiols has prompted us to consider topoisomerase II as one possible target of acrylamide, since agents targeting protein sulfhydryl groups act as either catalytic inhibitors or poisons of topoisomerase II. Nuclear extracts from V79 Chinese hamster cells incubated with acrylamide reduced topoisomerase II activity as inferred by an inability to convert kinetoplast DNA to the decatenated form. Nuclear extracts incubated with acrylamide pre-incubated with DTT converted kinetoplast DNA to the decatenated form, suggesting that acrylamide influences topoisomerase II activity through reaction with sulfhydryl groups on the enzyme. Furthermore, acrylamide did not induce the pBR322 DNA cleavage, as assessed by cleavage assay; thus, it cannot be regarded as a poison of topoisomerase II. As a catalytic inhibitor, acrylamide antagonizes the effect of etoposide, a topoisomerase II poison, as determined by clonogenic assay in V79 cells. This antagonism is confirmed by band depletion assay, from which it can be inferred that acrylamide reduces the level of catalytically active cellular topoisomerase II available for the action of etoposide.

Hydrogen sulfide increases calcium-activated potassium (BK) channel activity of rat pituitary tumor cells

Hydrogen sulfide (H(2)S) is the third gasotransmitter found to be produced endogenously in living cells to exert physiological functions. Large conductance (maxi) calcium-activated potassium channels (BK), which play an important role in the regulation of electrical activity in many cells, are targets of gasotransmitters. We examined the modulating action of H(2)S on BK channels from rat GH(3) pituitary tumor cells using patch clamp techniques. Application of sodium hydrogen sulfide as H(2)S donor to the bath solution in whole cell experiments caused an increase of calcium-activated potassium outward currents. In single channel recordings, H(2)S increased BK channel activity in a concentration-dependent manner. Hydrogen sulfide induced a reversible increase in channel open probability in a voltage-dependent, but calcium independent manner. The reducing agent, dithiothreitol, prevented the increase of open probability by H(2)S, whereas, the oxidizing agent thimerosal increased channel open probability in the presence of H(2)S. Our data show that H(2)S augments BK channel activity, and this effect can be linked to its reducing action on sulfhydryl groups of the channel protein.

Characterization of S‐thiolation on secreted proteins fromE. coliby mass spectrometry

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.

Structural modification of soy protein by 13-hydroperoxyoctadecadienoic acid

To reveal the role of primary products of lipid peroxidation during soy protein oxidation process, oxidative modification of soy protein by 13-hydroperoxyoctadecadienoic acid (13-HPODE) generated by lipoxygenase-catalyzed oxidation of linoleic acid was investigated in this article. Incubation of soy protein with increasing concentration of 13-HPODE resulted in generation of protein carbonyl derivatives and loss of protein sulfhydryl groups. Circular dichroism spectra indicated that exposure of soy protein to 13-HPODE led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity gradually decreased, accompanied by loss and burial of some tryptophan residues. The results of surface hydrophobicity and tryptophan fluorescence implied that aggregation was induced by oxidation. Size exclusion chromatogram indicated that the extent of aggregation was increased in a 13-HPODE dose-dependent manner. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that non-disulfide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to 13-HPODE than glycinin.

Analysis of the Structure and Function of YfcG from Escherichia coli Reveals an Efficient and Unique Disulfide Bond Reductase

YfcG is one of eight glutathione (GSH) transferase homologues encoded in the Escherichia coli genome. The protein exhibits low or no GSH transferase activity toward a panel of electrophilic substrates. In contrast, it has a very robust disulfide-bond reductase activity toward 2-hydroxyethyldisulfide on par with mammalian and bacterial glutaredoxins. The structure of YfcG at 2.3 A-resolution from crystals grown in the presence of GSH reveals a molecule of glutathione disulfide in the active site. The crystallographic results and the lack of functional cysteine residues in the active site of YfcG suggests that the reductase activity is unique in that no sulfhydryl groups in the YfcG protein are covalently involved in the redox chemistry.

Carboplatin-induced alteration of the thiol homeostasis in the isolated perfused rat kidney

Carboplatin elicits minor side effects with respect to its first generation analogue cisplatin. Nevertheless, a dose-dependent nephrotoxicity of the drug has been reported to occur both in patients and in rats and a possible pathogenic role have been attributed to oxidative stress. We have studied the effect of carboplatin administration on the thiol/disulfide balance, on other biomarkers of oxidative stress and on antioxidant enzymes in the isolated perfused rat kidney. A 5–500 μM carboplatin dose range did not alter renal function but significantly decreased levels of cysteine, glutathione and exposed protein sulfhydryl groups. Only a minimal increment in disulfides was observed, whereas malonyldialdehyde and protein carbonyls did not increase significantly. Among the antioxidant enzymes studied, only thioltransferase was inhibited by the treatment. Our data suggest that a minimal oxidative stress is present under our experimental conditions, thus indicating that platinum-based drugs do not produce significant amount of reactive oxygen species.

Structural modification of soy protein by the lipid peroxidation product malondialdehyde

AbstractBACKGROUND: Protein oxidation results in covalent modification of structure and deterioration of functional properties of target protein. Oxidation extent of soy protein was affected by the content and type of lipid peroxidation (LPO) products in defatted soybean flours during storage and processing. Malondialdehyde (MDA) was selected as a secondary byproduct of LPO to investigate the effects of oxidative modification of LPO‐derived reactive aldehyde on soy protein structure.RESULTS: MDA reacted with ε‐amino and sulfhydryl groups of soy protein, and resulted in an increase in protein carbonyl groups but a decrease in sulfhydryl/disulfide, free amines and lysine. The decrease in solubility, surface hydrophobicity and intrinsic fluorescence indirectly indicated that MDA induced soy protein aggregation, and results of high‐performance size‐exclusion chromatography directly showed that gradual aggregation of soy protein was induced by increasing concentration of MDA. Results of electrophoresis indicated that MDA caused soy protein aggregation, and non‐disulfide covalent bonds were involved in aggregate formation.CONCLUSION: The results showed that sensitivity of soy protein was related to MDA concentration. Soy protein gradually aggregated with increase of MDA concentration; β‐conglycinin was more sensitive to MDA modification than glycinin. Copyright © 2009 Society of Chemical Industry

Hypovolaemy and oxidative stress in burned patients

Adequate treatment of the developing hypovolemia caused by marked oedema formation plays a key role in the care of burned patients. Oxidative stress may be an underlying cause of the excessive oedema formation. The aim of the study was to asses how can different resuscitation schemata be adapted better to the requirement of each individual patient. Effect of the use of different schemata on organ-function and developing oxidative stress and correlation between the extent of burn and oxidative stress has also been studied. Patients admitted to our level one intensive ward were involved in the study. Inclusion criterion was the start of our fluid resuscitation schema at least 3 hours after injury. Hemodynamic measurements were performed by transpulmonary thermodilution technique. Blood samples were taken on admission and on the 5 consecutive days thereafter. White blood cell count of the patients, the percentage of granulocytes, lymphocytes and monocytes, the levels of malondialdehyde, protein sulfhydryl groups in plasma, reduced glutathione, catalase, and superoxide dismutase enzyme activities, and phorbol myristate acetate induced free radical generating capacity were assessed. Significantly more fluid was administered than predicted by the Parkland formula (p < 0.05). Patients in whom fluid resuscitation was guided by invasive hemodynamic parameters ScvO 2 was significantly higher (p < 0.05), multi organ dysfunction scores were lower (p < 0.05) than in the hourly urine output group. Patients whom fluid resuscitation was guided by invasive hemodynamic parameters relative number of the granulocytes and maximal rate of reactive oxygen species production (p < 0.05) were significantly higher (p < 0.05), the lag phases were significantly shorter (p < 0.05). With the use of correction factors the Parkland formula can be more precise, still, fluid resuscitation guided by invasive hemodynamic measurements can provide the best results. The type of fluid resuscitation schema influences mainly the pro-oxidant system.

The effect of dihydroquercetin on active and passive ion transport systems in plant vacuolar membrane

The effect of dihydroquercetin (DHQ) on proton pumps of the vacuolar membrane (H(+)-ATPase and H(+)-pyrophosphatase), slow vacuolar (SV) channel, lipid peroxidation, and stability of isolated vacuoles was studied. The results of experiments showed that DHQ affected active and passive transport systems of the vacuolar membrane. The mechanism of action of DHQ may be based on its combined effect on the sulfhydryl groups of proteins and the lipid component of the membrane. The strong stabilizing effect of DHQ on the membranes of isolated vacuoles may be associated not only with its antioxidant properties but also with changes in the membrane permeability affecting the ion channels.

Aging Impairs Skeletal Muscle Mitochondrial Bioenergetic Function

This study investigated the influence of age on the functional status of mitochondria isolated from skeletal muscle of C57BL/6 mice aged 3 and 18 months. We hypothesized that skeletal muscle mitochondria isolated from aged animals will exhibit a decreased respiratory function. Mitochondrial respiratory functional measures (ie, State 3 and 4 respiration, respiratory control ratio and number of nanomoles of ADP phosphorylated by nanomoles of O(2) consumed per mitochondrion) and biochemical markers of oxidative damage (aconitase activity, protein carbonyl derivatives, sulfhydryl groups, and malondialdehyde) were measured in isolated mitochondrial suspensions. Along with traditional tests of mitochondrial function, an in vitro repetitive ADP-stimulation test was used to evaluate the mitochondrial capacity to reestablish the homeostatic balance between successive ADP stimulations. The number of mitochondria per mitochondrial suspension, calculated by transmission electron microscopy, was used to normalize functional and biochemical data. Our results confirm the existence of an age-associated decline in mitochondrial function of mixed skeletal muscle, which is significantly correlated with higher levels of mitochondrial oxidative damage.

Expressional Control of a Cadmium-transporting P1B-type ATPase by a Metal Sensing Degradation Signal

Cadmium is a highly toxic environmental contaminant implicated in various diseases. Our previous data demonstrated that Pca1, a P1B-type ATPase, plays a critical role in cadmium resistance in yeast S. cerevisiae by extruding intracellular cadmium. This illustrates the first cadmium-specific efflux pump in eukaryotes. In response to cadmium, yeast cells rapidly enhance expression of Pca1 by a post-transcriptional mechanism. To gain mechanistic insights into the cadmium-dependent control of Pca1 expression, we have characterized the pathway for Pca1 turnover and the mechanism of cadmium sensing that leads to up-regulation of Pca1. Pca1 is a short-lived protein (t1/2 < 5 min) and is subject to ubiquitination when cells are growing in media lacking cadmium. Distinct from many plasma membrane transporters targeted to the vacuole for degradation via endocytosis, cells defective in this pathway did not stabilize Pca1. Rather, Pca1 turnover was dependent on the proteasome. These data suggest that, in the absence of cadmium, Pca1 is targeted for degradation before reaching the plasma membrane. Mapping of the N terminus of Pca1 identified a metal-responding degradation signal encompassing amino acids 250-350. Fusion of this domain to a stable protein demonstrated that it functions autonomously in a metal-responsive manner. Cadmium sensing by cysteine residues within this domain circumvents ubiquitination and degradation of Pca1. These data reveal a new mechanism for substrate-mediated control of P1B-type ATPase expression. Cells have likely evolved this mode of regulation for a rapid and specific cellular response to cadmium.

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.

Phenotypic Analysis of Arabidopsis Mutants: Heavy Metal Stress

INTRODUCTIONArsenic (As) is a major environmental contaminant, arising from both industrial and agricultural use, and is one of the metals given top priority for remediation by the U.S. Department of Energy on the basis of its toxicity and widespread contamination. However, many of the molecular and genetic mechanisms contributing to arsenic toxicity and detoxification remain unknown in plants. The toxicity of arsenic depends on the arsenic oxidation state. The main arsenic oxidation states are As(III) and As(V), both of which are highly toxic to plants and animals. In plants, As(III) disrupts the sulfhydryl groups of proteins and induces the production of hydrogen peroxide, which leads to membrane damage, whereas As(V) uncouples phosphorylation in mitochondria and at high levels can inhibit seed germination. As described in this protocol, resistance or sensitivity to arsenic can be easily scored by measuring germination and root bending in exposed seedlings.