Synthesis and application of novel probes for the detection of cysteine sulphenic acids

Cysteine is a thiol containing amino acid that readily undergoes oxidation by reactive oxygen species (ROS) to form sulphenic (R-SOH) sulphinic (RSO2H) and sulphonic (RSO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signalling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesise and use a chemical probe that selectively recognises the reversible oxidation state of cysteine sulphenic acid to understand more about oxidative signalling. The aim of this thesis was to investigate a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulphenic acids by fluorescence spectroscopy and confocal microscopy. N-[2-(Anthracen-2-ylamino)-2-oxoethyl]-3,5-dioxocyclohexanecarboxamide was synthesised in a multistep synthesis and characterised by nuclear magnetic resonance spectroscopy. The optimisation of conditions needed for sulphenic acid formation in a purified protein using human serum albumin (HSA) and the commercially available biotin tagged probe 3-(2,4-dioxocyclohexyl)propyl-5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate (DCP-Bio1) were identified. This approach was extended to detect sulphenic acids in Jurkat T cells and CD4+ T cells pre- and post-stimulus. Buthionine sulfoximine (BSO) was used to manipulate the endogenous antioxidant glutathione (GSH) in human CD4+ T cells. Then the surface protein thiol levels and sulphenic acid formation was examined. T cells were also activated by the lectin phytohaemagglutinin-L (PHA-L) and formation of sulphenic acid was investigated using SDS-PAGE, western blotting and confocal microscopy. Resting Jurkat cells have two prominent protein bands that have sulphenic acid modifications whereas resting CD4+ T cells have an additional band present. When cells were treated with BSO the number of bands increased whereas activation reduced the number of proteins that were modified. The identities of the protein bands containing sulphenic acids were explored by mass spectrometry. Cysteine oxidation was observed in redox, metabolic and cytoskeletal proteins. In summary, a novel fluorescent probe for detection of cysteine sulphenic acids has been synthesised alongside a model system that introduces cysteine sulphenic acid in primary T cells. This probe has potential application in the subcellular localisation of cysteine oxidation during T cell signalling.

The Nrf2-ARE activation protect neurones against oxidative stress

Oxidative stress has been implicated in the pathogenesis of many neurodegenerative diseases including Alzheimer’s disease. The transcription factor, Nrf2 (nuclear factor E2-related factor 2) that binds to the antioxidant responsive element (ARE) activates a battery of genes encoding enzymes and factors essential for neuronal survival. We have investigated the hypothesis that a downstream product of cyclooxygenase(COX-2), 15-deoxy-delta (12, 14)-prostagland in J2 (15d-PGJ2) has protective effects by activating the Nrf2 pathway during oxidative stress.Human neuroblastoma cells (SHSY5Y) were differentiated intoneuronal-like cells as described previously (Gimenez-Cassina et al.,2006). SHSY5Y cells were co-treated with 10 mM buthionine sulfoximine (BSO) 7 10 mM 15d-PGJ2. Cell viability was measured by MTT assay and cellular glutathione (GSH) levels were measured after treating cells for0.5-24 hours by GSH recycling assay. Cellular Nrf2 levels were determined by immunoblotting. IL-6 levels were measured by ELISA.15d-PGJ2 alone lowered GSH levels 30min after the treatment(12.870.64 nmol/mg protein) and returned to untreated control levels at 16hours (28.173.6 nmol/mg protein; Po0.01). Compared to intracellular GSH levels in untreated cells (27.871.8 nmol/mg protein) BSO treatment alone significantly decreased GSH (9.672.1 nmol/mg protein;Po0.001) but co-incubation with 15d-PGJ2 for 24 hours prevented the depletion elicited by BSO(21.372.7 nmol/mg protein). Compared to untreated cells BSO treatment decrease dIL-6 secretion (from 0.941.6ng/ml to 0.6971.3ng/ml) and total Nrf2 protein levels (by21%). Co-incubation with15d-PGJ2 for 24 hours with BSO did not change IL-6(0.6771.4ng/ml) or total Nrf2 level at any time point. This study suggests that neuronal toxicity resulting from glutathione depletion canbere stored by the induction of Nrf2-ARE pathway and the role of the Nrf2 signalling merits further investigation in neurodegenerative diseases.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human–hamster hybrid (AL) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5′,6′-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in AL cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration. Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector. Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage. Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP. We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite. Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.

Calcineurin expression and activity is regulated by the intracellular redox status and under hypertension in human neutrophils

Calcineurin (protein phosphatase 2B) (CN) comprises a family of serine/threonine phosphatases that play a pivotal role in signal transduction cascades in a variety of cells, including neutrophils. Angiotensin II (Ang II) increases both activity and de novo synthesis of CN in human neutrophils. This study focuses on the role that intracellular redox status plays in the induction of CN activity by Ang II. Both de novo synthesis of CN and activity increase promoted by Ang II were downregulated when cells were treated with l-buthionine-(S,R)-sulfoximine, an inhibitor of synthesis of the antioxidant glutathione. We have also investigated the effect of pyrrolidine dithiocarbamate and phenazine methosulfate, which are antioxidant and oxidant compounds, respectively, and concluded that the intracellular redox status of neutrophils is highly critical for Ang II-induced increase of CN expression and activity. Results obtained in neutrophils from hypertensive patients were very similar to those obtained in these cells on treatment with Ang II. We have also addressed the possible functional implication of CN activation in the development of hypertension. Present findings indicate that downregulation of hemoxygenase-1 expression in neutrophils from hypertensive subjects is likely mediated by CN, which acts by hindering translocation to the nucleus of the transcription factor NRF2. These data support and extend our previous results and those from other authors on modulation of CN expression and activity levels by the intracellular redox status.

Chloroplastic glutamine synthetase from normal and water stressed safflower (Carthamus tinctorius L.) leaves

The chloroplastic isoform of glutamine synthetase (GS(2), EC 6.3.1.2) from normal and water stressed safflower (Carthamus tinctorius L. cv.A-300) leaves has been purified to apparent electrophoretic homogeneity by a procedure involving anion-exchange, hydrophobic and size-exclusion chromatography followed by electroelution of the protein from preparative polyacrylamide gels. The observed molecular weight of the native protein varied from 305-330 kDa depending on the sizing column employed. The native protein is composed of 44 kDa subunits. Under conditions of saturating ammonium and at ATP levels of 0.1-10 mM, double-reciprocal plots with respect to glutamate are biphasic and concave downward at high concentrations of the varied substrate for normal enzyme but are linear for enzyme from water-stressed plants. Under subsaturating ATP levels, K-Glu is over 18-fold lower for enzyme from stressed leaves. The K-m, (ATP) varies with Mg2+ levels in the assay mixture. Double-reciprocal plots of initial velocity with respect to ATP at changing fixed levels of NH4+ are linear for normal enzyme but are curved upwards for enzyme from stressed leaves. Initial velocity data of 1/v vs. 1/ammonium for the enzyme from both the sources are non-linear (curved upwards) when ATP is saturating. At subsaturating ATP levels, the data are linear for normal enzyme but are still non-linear for the enzyme from stressed leaves. The results obtained suggest positively cooperative binding of NH4+ A V-max(/2) value of 3.6 mM for Mg2+ was obtained at 5 mM ATP. The isoelectric point of the native protein from normal and stressed leaves was determined to be, respectively, 5.6 and 6.1. The mixed competitive and competitive inhibitors, methionine sulfoximine and ADP and K-i values of 0.086 mM (0.017 for the enzyme from stressed leaves) and 2.15 mM (1.70 for the enzyme from stressed leaves), respectively. Enzyme from stressed leaves is not inhibited by 5 mM proline. The observed kinetic constants of GS(2) from normal and water stressed safflower seedlings are discussed in relation to the known water-stress tolerance of this crop plant.

Cross-Hetero-Dehydrogenative Coupling Reaction of Phosphites: A Catalytic Metal-Free Phosphorylation of Amines and Alcohols

Phosphorylation of amines, alcohols, and sulfoximines are accomplished using molecular iodine as a catalyst and H2O2 as the sole oxidant under mild reaction conditions. This method provides an easy route for synthesizing a variety of phosphoramidates, phosphorus triesters and sulfoximine-derived phosphoramidates which are of biological importance.

谷氨酰胺合成酶在固氮鱼腥藻固氮调节中的作用

在MSX(methlonine sulfoximine,谷氨酰胺合成酶的不可逆抑制剂)存在下,固氮鱼腥藻(Anabaena azotica)所分泌的氨量和谷氨酰胺合成酶(GS)活力有较好的负相关性,证明谷氨酰胺合成酶-谷氨酸合成酶(GS-GOGAT)是固氮鱼腥藻氨同化的重要途径。在蛋白质合成受到氯霉素拟制时,NH_4~+对固氮酶的失活是迅速的,同时GS活力有较大下降,表明NH_4~+的调控酶的失活或降解。在氮固定条件下,固氮酶活力半衰期小于4小时,GS活力半衰期大于10小时,则GS并不是固氮酶的正调节因子

路易斯碱催化还原胺化反应研究

胺及其衍生物是很多重要生物活性分子的结构单元，是合成天然产物和手性药物的重要中间体。 直接还原胺化由于其合成步骤简单而成为制备二级胺和三级胺的简便方法。为了发展一种较为简便的直接还原胺化反应，我们把研究的重点放在开发一种简便实用的有机小分子催化方法上。由文献调研可知，现已报道的直接还原胺化方法大多是催化醛或酮与一级胺或者脂肪二级胺的直接还原胺化，而醛或酮与芳香二级胺的直接还原胺化却尚无报道。在本文中，我们发现用简单的四甲基乙二胺(TEMED)在室温下以二氯甲烷为溶剂即可催化三氯氢硅对酮和芳香二级胺之间的直接还原胺化反应，并取得了高达92%的收率。该反应条件温和，底物普适性广，各种类型的酮均可以与芳香二级胺进行直接还原胺化，并且得到比较满意的收率。 同时，我们从手性Sulfoximine出发，设计和合成了一系列的Sulfoximine类新衍生物，并将其应用于间接还原胺化反应中。遗憾的是我们并没有得到预期的不对称催化效果。 Amines and their derivatives are basic structural motifs in natural products and pharmaceuticals and highly versatile building blocks for various organic substrates. Direct reductive amination (DRA) is a convenient method for the preparation of secondary and tertiary amines owing to its operational simplicity. In an effort to develop a simple and convenient procedure for direct reductive amination reaction, we focused our study on search for a mild and efficient organocatalytic system. In the literature, there are many reports concerning DRA between aldehydes or ketones and either primary amines or secondary aliphatic amines. But there are no reports concerning DRA between aldehydes or ketones and secondary aromatic amines. In this study, we have developed a highly practical method for the synthesis of tertiary amines by the direct reductive amination of ketones and secondary aromatic amines with tetramethylethylenediamine (TEMED) as the catalyst using HSiCl3 as the reducing agent in dichloromethane (affording up to 92% yield). This method can be carried out under mild conditions and is compatible with many functional groups. A variety of ketones were efficiently aminated with secondary aromatic amines to afford the corresponding amines in good to excellent yields. Starting from chiral sulfoximine, we designed and synthesized a series of new sulfoximine derivatives and tested their efficiencies as asymmetric organocatalysts for the reduction of imines, which, unfortunately, only exhibited low catalytic activity and enantioselectivity.

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions

Hyperglycaemia-induced oxidative stress may play a key role in the pathogenesis of diabetic vascular disease. The purpose of the present study was to determine the effects of glucose on levels of glutathione (a major intracellular antioxidant), the expression of gamma-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione de novo synthesis) and DNA damage in human vascular smooth muscle cells in vitro. High glucose conditions and buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, reduced intracellular glutathione levels in vascular smooth muscle cells. This reduction was accompanied by a decrease in the mRNA expression of both subunits of gamma-glutamylcysteine synthetase as well as an increase in DNA damage. In high glucose conditions incubation of the vascular smooth muscle cells with alpha-lipoic acid and L-cystine restored glutathione levels. We suggest that the decrease in GSH levels seen in high glucose conditions is mediated by the availability of cysteine (rate-limiting substrate in de novo glutathione synthesis) and the gene expression of the gamma- glutamylcysteine synthetase enzyme. Glutathione depletion is associated with an increase in DNA damage, which can be reduced when glutathione levels are restored.

The role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells. I

Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s-1 to 144 s-1, and for lethal lesion precursors from 533 s-1 to 165 s-1. This is generally consistent with the role of GSH in the repair-fixation model of radiation damage at the critical DNA lesions. However, the reduction in chemical repair rate was not proportional to the severe thiol depletion (down to almost-equal-to 1% for GSH) and a residual repair capacity remained (almost-equal-to 30%). This was found not to be due to compartmentalization of residual GSH in the nucleus, as the repair rate for dsb precursors in isolated nuclei, washed virtually free of GSH, was identical to that found in GSH-depleted cells (144 s-1), also the OER remained substantially above unity. This suggests that other reducing agents may have a role to play in the chemical repair of oxygen-dependent damage. One possible candidate is the significant level of protein sulphydryls present in isolated nuclei.

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO center dot) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage. (c) 2007 Elsevier B.V. All rights reserved.

Flux control of sulphate assimilation in Arabidopsis thaliana : adenosine 5′-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied l-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5′-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing l-cysteine to the nutrient solution increased internal cysteine, γ-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm l-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm l-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of l-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm l-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using 35SO42– in the presence of 0.5 mm l-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.