Changes In Glutathione Metabolism Research Articles

Antifungal Activity and Mechanism of 4-Propylphenol Against Fusarium graminearum, Agent of Wheat Scab, and Its Potential Application.

Fusarium head blight (FHB), caused by Fusarium graminearum, is a predominant disease of wheat. Due to the lack of disease-resistant germplasm, chemical control is an important means to control wheat scab. Volatile substances produced in near-isogenic wheat lines were detected after inoculation with F. graminearum, and 4-propylphenol, which appears in FHB-resistant lines, was identified. In vitro and in vivo antifungal activity tests demonstrate that 4-propylphenol effectively inhibits the mycelial growth of F. graminearum. Metabolomics analysis showed changes in glutathione metabolism, indicating that 4-propylphenol triggered reactive oxygen species (ROS) stress. This was consistent with the increasing ROS levels in Fusarium cells treated with 4-propylphenol. Further results demonstrated that excessive accumulation of ROS induced DNA and cell membrane damage in the mycelium. Moreover, 4-propylphenol showed different degrees of inhibition against other soil-borne pathogens (fungi and oomycetes). These findings illustrated that 4-propylphenol has broad spectrum and high antifungal activity and should be considered for use as an ecological fungicide.

Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progredient neurodegenerative disease characterized by a degeneration of the first and second motor neurons. Elevated levels of reactive oxygen species (ROS) and decreased levels of glutathione, which are important defense mechanisms against ROS, have been reported in the central nervous system (CNS) of ALS patients and animal models. The aim of this study was to determine the cause of decreased glutathione levels in the CNS of the ALS model wobbler mouse. We analyzed changes in glutathione metabolism in the spinal cord, hippocampus, cerebellum, liver, and blood samples of the ALS model, wobbler mouse, using qPCR, Western Blot, HPLC, and fluorometric assays. Here, we show for the first time a decreased expression of enzymes involved in glutathione synthesis in the cervical spinal cord of wobbler mice. We provide evidence for a deficient glutathione metabolism, which is not restricted to the nervous system, but can be seen in various tissues of the wobbler mouse. This deficient system is most likely the reason for an inefficient antioxidative system and, thus, for elevated ROS levels.

Multiple Infusion Start Time Mass Spectrometry Imaging of Dynamic SIL-Glutathione Biosynthesis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization.

Due to the high association of glutathione metabolism perturbation with a variety of disease states, there is a dire need for analytical techniques to study glutathione kinetics. Additionally, the elucidation of microenvironmental effects on changes in glutathione metabolism would significantly improve our understanding of the role of glutathione in disease. We therefore present a study combining a multiple infusion start time protocol, stable isotope labeling technology, infrared matrix-assisted laser desorption electrospray ionization, and high-resolution accurate mass-mass spectrometry imaging to study spatial changes in glutathione kinetics across in sectioned mouse liver tissues. After injecting a mouse with the isotopologues [2-13C,15N]-glycine, [1,2-13C2]-glycine, and [1,2-13C2,15N]-glycine at three different time points, we were able to fully resolve and spatially map their metabolism into three isotopologues of glutathione and calculate their isotopic enrichment in glutathione. We created a tool in the open-source mass spectrometry imaging software MSiReader to accurately compute the percent isotope enrichment (PIE) of these labels in glutathione and visualize them in heat-maps of the tissue sections. In areas of high flux, we found that each label enriched an approximate median of 1.6%, 1.8%, and 1.5%, respectively, of the glutathione product pool measured in each voxel. This method may be adapted to study the heterogeneity of glutathione flux in diseased versus healthy tissues.

High Glutathione and NAD(P)H Levels, and Increased Protein Folding Capacity Are Involved in Mediating Proteasome Inhibitor-Resistance in Multiple Myeloma

Background. Proteasome inhibitors (PI) are a backbone for the treatment of multiple myeloma (MM). However, most MM patients ultimately acquire PI-resistance and die from MM. The biology of PI resistance is poorly understood and established treatment options for PI-refractory MM are lacking. We have recently generated a next-generation in vitro model for PI-refractory MM, based on single MM cell-derived subclones with specific (50-100x) resistance to either bortezomib (AMO-BTZ) or carfilzomib (AMO-CFZ). These cells consistently show downregulation of IRE1/XBP1, a hallmark of BTZ-refractory primary MM cells, and do or do not carry the PSMB5 A310G mutation. Our recent quantitative proteomic analysis suggested that the PI-resistant phenotype of AMO-BTZ and AMO-CFZ is in particular characterized by massive metabolic changes that render MM cells largely independent from proteasome activity. To verify such metabolic adaptation as a hallmark of proteasome inhibitor resistance of MM, and to identify therapeutic targets, we performed whole metabolome profiling of proteasome inhibitor-sensitive AMO-1 cells in comparison to BTZ or CFZ-resistant clones from the same cell line.Methods. Metabolomic profiling was performed using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS); data were quantified and normalized by Metabolon, Inc. Metaboanalyst v3.0 software was used for Metabolite Set Enrichment Analysis (MSEA). Reactive oxygen species (ROS) formation was determined by flow cytometry after staining the cells with H2DCFDA and exposure to 0.08% H2O2. Protein folding was determined by MERO-GFP construct, viability was determined by MTS assay.Results. Based on MSEA, PI-adapted cells showed substantial metabolic changes in glutathione metabolism, protein biosynthesis, malate/aspartate shuttle, metabolism of purines, pyrimidines, amino acids and tricarboxylic acid (TCA). Relative to AMO-1, in both AMO-BTZ and AMO-CFZ, glutathione (l-γ-Glutamyl-l-cysteinyl-glycine; GSH and GSSG) was consistently identified as the metabolite with the most significant quantitative increase (fold change (FC) relative to AMO-1 for GSH in AMO-BTZ=52.92; in AMO-CFZ=56.79; FC for GSSG in AMO-BTZ=36.92; in AMO-CFZ=54.17), while many other metabolites in glycine/serine and cysteine/methionine pathways that are associated with glutathione synthesis were decreased. Consistently, PI-adapted cells showed accumulation of nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) (FC for NAD+ in AMO-BTZ=55.81; in AMO-CFZ=2.16; FC for NADH in AMO-BTZ=19.10; in AMO-CFZ=1.65), presumably to maintain balance between reduced and oxidized glutathione, together with changes in TCA and malate/aspartate shuttle fueling the NAD(P)H production and transport, respectively. PI-resistant cells in addition showed altered carbohydrate metabolism and a shift in availability of membrane lipids, from lysolipids to sphingomyelins, in comparison to non-adapted cells.The adaptive changes in these cells correspond well to the proteomic changes previously described, confirming that the metabolic adaptive changes of PI-refractory MM cells are in particular in the regulation of redox homeostasis and protein folding. Functional experiments confirmed that the PI-adapted cells show larger capacity to buffer ROS production and that protein re-folding after DTT challenge is more effective in PI-refractory MM, compared to PI-sensitive cells. Consequently, co-treatment of PI-adapted cells with respective proteasome inhibitors (bortezomib and carfilzomib) in the presence of the inhibitor of protein-disulfide isomerase (PDI), 16F16, re-sensitized cells to proteasome inhibition (FC of IC50 for bortezomib or carfilzomib alone vs bortezomib or carfilzomib + 16F16 in AMO-BTZ=1.26 or 1.88, respectively; in AMO-CFZ=1.25 or 2.59 respectively; in AMO-1=1.1 or 0.95, respectively.Conclusion. PI refractory MM cells show massive metabolic changes in particular in glutathione and NAD(P)H production. This is associated with increased protein folding capacity and likely results in decreased proteasome load. PI-refractory MM cells can be re-sensitized to PI treatment by reducing glutathione levels or by affecting protein folding. DisclosuresNo relevant conflicts of interest to declare.

Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction.

Radiation therapy for head and neck cancer causes damage to the surrounding salivary glands, resulting in salivary gland hypofunction and xerostomia. Current treatments do not provide lasting restoration of salivary gland function following radiation; therefore, a new mechanistic understanding of the radiation-induced damage response is necessary for identifying therapeutic targets. The purpose of the present study was to investigate the metabolic phenotype of radiation-induced damage in parotid salivary glands by integrating transcriptomic and metabolomic data. Integrated data were then analyzed to identify significant gene-metabolite interactions. Mice received a single 5 Gy dose of targeted head and neck radiation. Parotid tissue samples were collected 5 days following treatment for RNA sequencing and metabolomics analysis. Altered metabolites and transcripts significantly converged on a specific region in the metabolic reaction network. Both integrative pathway enrichment using rank-based statistics and network analysis highlighted significantly coordinated changes in glutathione metabolism, energy metabolism (TCA cycle and thermogenesis), peroxisomal lipid metabolism, and bile acid production with radiation. Integrated changes observed in energy metabolism suggest that radiation induces a mitochondrial dysfunction phenotype. These findings validated previous pathways involved in the radiation-damage response, such as altered energy metabolism, and identified robust signatures in salivary glands, such as reduced glutathione metabolism, that may be driving salivary gland dysfunction.

N-Myc-induced metabolic rewiring creates novel therapeutic vulnerabilities in neuroblastoma

N-Myc is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of Myc family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of Myc proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of N-Myc overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of N-Myc induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of N-Myc. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of N-Myc overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered N-Myc induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.

Different source of commercial vegetable oils may regulate metabolic, inflammatory and redox status in healthy rats

• The vegetable oils intake induces changes in metabolic and inflammatory parameters. • Coconut oil induces glycometabolic and hepatic alterations in healthy rats. • Linseed oil reduces serum TAG and IL-6. • Sunflower oil increases superoxide dismutase and catalase activity and reduces total glutathione, IL-6 e TNF. • Sunflower oil increases hepatic lipogenesis and reduces serum lipids. Our goal was to carry out a comparative study to evaluate the metabolic and inflammatory effects and the redox status of commercial vegetable oils supplementation [linseed (LO), coconut (VCO), and sunflower (SO)] in metabolically healthy rats. The results found in this study showed that the LO group decreased the HOMA-IR and hepatic cholesterol, and increased the serum levels of IL-6. Supplementation with VCO increased glucose and HOMA-IR, cholesterol concentration and serum triacylglycerol (TAG). In this group, there was also an increase in TBARS. In the SO group there was a decrease in serum concentrations of cholesterol and TAG and an increase in hepatic concentration of these lipids. In addition, in the SO group there was a decrease in hepatic and serum concentrations of IL-6 and hepatic levels of TNF, as well as a decrease in the GSH/GSSG ratio, suggesting changes in glutathione metabolism and inflammatory mediators.

Metabolic Profiling of Human Gastric Cancer Cells Treated With Salazosulfapyridine.

Purpose:The adhesion molecule cluster of differentiation 44v9 interacts with and stabilizes the cystine/glutamate exchanger protein, which functions as a transporter of cystine. Stabilized cystine/glutamate exchanger increases extracellular cystine uptake and enhances glutathione production. Augmented levels of reduced glutathione mitigate reactive oxygen species and protect cancer cells from apoptosis. Salazosulfapyridine blocks cystine/glutamate exchanger activity and mitigates the supply of cystine to increase intracellular ROS production, thereby increasing cell susceptibility to apoptosis. This enhances the effect of anticancer drugs such as cisplatin. Currently, salazosulfapyridine is being developed as a promising anticancer agent. In the present study, we elucidated the molecular mechanism associated with salazosulfapyridine by investigating the salazosulfapyridine-induced changes in glutathione metabolism in cultured gastric cancer cell lines.Methods:The effect of salazosulfapyridine treatment on glutathione metabolism was investigated in 4 gastric cancer (AGS, MKN1, MKN45, and MKN74) and 2 colorectal cancer (HCT15 and HCT116) cell lines using metabolomic analyses. In addition, the effect of inhibition of the reduced form of nicotinamide adenine dinucleotide phosphate by 2-deoxyglucose on glutathione metabolism was evaluated.Results:Under hypoxia, enhanced glycolysis resulted in lactate accumulation with an associated reduction in nicotinamide adenine dinucleotide phosphate. Salazosulfapyridine treatment decreased the cysteine content and inhibited the formation of glutathione. Combined treatment with salazosulfapyridine and 2-deoxyglucose significantly inhibited cell proliferation. 2-Deoxyglucose, an inhibitor of glycolysis, depleted nicotinamide adenine dinucleotide phosphate required for the formation of glutathione.Conclusions:Our results indicate that in cancer cells having a predominant glycolytic pathway, metabolomic analyses under hypoxic conditions enable the profiling of global metabolism. In addition, inhibiting the supply of nicotinamide adenine dinucleotide phosphate by blocking glycolysis is a potential treatment strategy for cancer, in addition to cystine blockade by salazosulfapyridine.

Non-targeted metabolomic analysis on multidrug resistance hepatocellular carcinoma cell and reversal effect of annonaceous acetogenins

Multidrug-resistance (MDR) has been shown to play a critical role in the development of many diseases. In this study, we used metabolomic method to evaluate the MDR in hepatocellular carcinoma, and investigate regulatory of annonaceous acetogenins on MDR of hepatocellular carcinoma. Multivariate statistical analysis revealed that the MDR of SMMC 7721 together with changes in glutathione metabolism, arginine and proline metabolism, sphingolipid metabolism. Annonaceous acetogenins impact these metabolism pathways. Metabolic pathway analysis coupled with stoichiometry analysis can be an effective tool to understand MDR mechanism and to potentially find new MDR reversal agents.

High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome

The optimization of bioprocesses for biopharmaceutical manufacturing by Chinese hamster ovary (CHO) cells can be a challenging endeavor and, today, heavily relies on empirical methods treating the bioreactor process and the cells as black boxes. Multi-omics approaches have the potential to reveal otherwise unknown characteristics of these systems and identify culture parameters to more rationally optimize the cultivation process. Here, the authors have applied both metabolomic and proteomic profiling to a perfusion process, using CHO cells for antibody production, to explore how cell biology and reactor environment change as the cell density reaches ≥200 × 106 cells mL-1 . The extracellular metabolic composition obtained in perfusion mode shows a markedly more stable profile in comparison to fed-batch, despite a far larger range of viable cell densities in perfusion. This stable profile is confirmed in the extracellular proteosome. Furthermore, the proteomics data shows an increase of structural proteins as cell density increases, which could be due to a higher shear stress and explain the decrease in cell diameter at very high cell densities. Both proteomic and metabolic results shows signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The authors suggest the methodology presented herein to be a powerful tool for optimizing processes of recombinant protein production.

The protective effect of metformin on hypertriglyceridemia-induced dicarbonyl stress in serum and tissues

s / Atherosclerosis 241 (2015) e32ee71 e58 EAS-0142. THE PROTECTIVE EFFECT OF METFORMIN ON HYPERTRIGLYCERIDEMIAINDUCED DICARBONYL STRESS IN SERUM AND TISSUES V. Skop, H. Malinska, J. Trnovska, L. Kazdova. Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Prague Czech Republic, Prague 4, Czech Republic Biogenesis of reactive dicarbonyls leads to the production of advanced glycation end products, increases oxidative stress, and play a key role in the development of diabetic vascular complications. Metformin is the most widely prescribed glucose-lowering agent; however, its effect on dicarbonyls metabolism in tissues is not clarified. In our study we analyzed levels of individual reactive dicarbonyls e methylglyoxal (MG), glyoxal (GL) and 3-deoxyglucosone (3-DG) in relation to lipid disorders and after metformin administration (300 mg/kg b.wt.) in non-obese hereditary hypertriglyceridemic (HHTg) rats. The concentration of dicarbonyls were determined by derivatisation with 1,2-diamino-4,5-dimethoxy-benzene and HPLC-method with fluorescence detection. Compared with control Wistar rats, HHTg rats exhibited markedly increased serum level of GL (+89%) and MG (+179%), increased levels of GL (+189%), MG (+73%) and 3-DG (+114%) in myocardium and increased level of GL (+59%) and MG (+57%) in kidney cortex. Metformin treatment significantly reduced level of reactive dicarbonyls in the myocardium (Gl: -31%; MG: -15%; 3-DG: -49%) and reduced level of MG (-49%) in serum. However, dicarbonyls concentrations in the kidney cortex were not affected by metformin. Changes in dicarbonyls concentrations were accompanied by changes in glutathione metabolism. Metformin prevents negative effect of hypertriglyceridemia on GSH/GSSG ratio and total glutathione content in myocardium, but neither hypertriglyceridemia nor metformin affects glutathione metabolism in kidney cortex. Results indicate that hypertriglyceridemia is associated with increased dicarbonyl stress in serum and tissues. Beneficial effect of metformin therapy on reactive dicarbonyls in heart could contribute to its cardioprotective effects. Supported by the grant GACR P303/13-04420S EAS-0202. MILK INTAKE IS NOT CAUSALLY ASSOCIATED WITH RISK OF ISCHEMIC HEART DISEASE OR MYOCARDIAL INFARCTION e A MENDELIAN RANDOMIZATION STUDY IN 98,529 DANISH INDIVIDUALS H.K.M. Bergholdt , B.G. Nordestgaard , A. Varbo , C. Ellervik . Dept. of Clinical Biochemistry, Naestved Hospital, Naestved, Denmark; Dept. of Clinical Biochemistry, Copenhagen University Hospital, Herlev, Denmark; 3 Faculty of Health and Medical Sciences, University of Copenhagen,

Effectiveness of Methylcobalamin and Folinic Acid Treatment on Adaptive Behavior in Children with Autistic Disorder Is Related to Glutathione Redox Status

Treatments targeting metabolic abnormalities in children with autism are limited. Previously we reported that a nutritional treatment significantly improved glutathione metabolism in children with autistic disorder. In this study we evaluated changes in adaptive behaviors in this cohort and determined whether such changes are related to changes in glutathione metabolism. Thirty-seven children diagnosed with autistic disorder and abnormal glutathione and methylation metabolism were treated with twice weekly 75 µg/Kg methylcobalamin and twice daily 400 µg folinic acid for 3 months in an open-label fashion. The Vineland Adaptive Behavior Scale (VABS) and glutathione redox metabolites were measured at baseline and at the end of the treatment period. Over the treatment period, all VABS subscales significantly improved with an average effect size of 0.59, and an average improvement in skills of 7.7 months. A greater improvement in glutathione redox status was associated with a greater improvement in expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills. Age, gender, and history of regression did not influence treatment response. The significant behavioral improvements observed and the relationship between these improvements to glutathione redox status suggest that nutritional interventions targeting redox metabolism may benefit some children with autism.

CE/LC‐MS multiplatform for broad metabolomic analysis of dietary polyphenols effect on colon cancer cells proliferation

In this study, an analytical multiplatform is presented to carry out a broad metabolomic study on the anti-proliferative effect of dietary polyphenols on human colon cancer cells. CE, RP/UPLC, and HILIC/UPLC all coupled to TOF MS were combined to achieve a global metabolomic examination of the effect of dietary polyphenols on HT29 colon cancer cells. By the use of a nontargeted metabolomic approach, metabolites showing significant different expression after the polyphenols treatment were identified in colon cancer cells. It was demonstrated that this multianalytical platform provided extensive metabolic information and coverage due to its complementary nature. Differences observed in metabolic profiles from CE-TOF MS, RP/UPLC-TOF MS, and HILIC/UPLC-TOF MS can be mainly assigned to their different separation mechanisms without discarding the influence of the different tools used for data processing. Changes in glutathione metabolism with an enhanced reduced glutathione/oxidized glutathione (GSH/GSSG) ratio were detected in polyphenols-treated cells. Moreover, significant alterations in polyamines content with important implications in cancer proliferation were observed after the treatment with polyphenols. These results from metabolomics can explain the chemopreventive effect of the tested dietary polyphenols on colon cancer and may be of importance for future prevention and/or treatment of this disease.

Biochemical disorders associated with antiproliferative effect of dehydroepiandrosterone in hepatoma cells as revealed by LC-based metabolomics

DHEA is known to have chemopreventive and antiproliferative activities, and was initially thought to be mediated by inhibition of G6PD. Our previous study has shown that DHEA may act through interference with energy metabolism. To study the effect of pharmacological dose of DHEA on cellular metabolism, and to further delineate the mechanism underlying its antiproliferative effect, we applied a metabolomic approach to globally profile the changes in metabolites in SK-Hep1 cells underexpressing G6PD (Sk-Gi) and control cells (Sk-Sc) after DHEA treatment. RRLC-TOF-MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. DHEA induced changes in glutathione metabolism, lipid metabolism, s-adenosylmethionine (SAM) metabolism, as well as lysine metabolism. Elevation in level of glutathione disulfide, together with a concomitant decrease in level of reduced glutathione, was indicative of increased oxidative stress. Depletion of carnitine and its acyl derivatives reflected decline in fatty acid catabolism. These changes were associated with mitochondrial malfunction and reduction in cellular ATP content. Cardiolipin (CL) and phosphatidylcholine (PC) levels decreased significantly, suggesting that alterations in lipid composition are causally related to decline in mitochondrial function after DHEA treatment. The decline in cellular SAM content was accompanied by decreased expression of methionine adenosyltransferase genes MAT2A and MAT2B. SAM supplementation partially rescued cells from DHEA-induced growth stagnation. Our findings suggest that DHEA causes perturbation of multiple pathways in cellular metabolism. Decreased SAM production, and cardiolipin depletion and the resulting mitochondrial dysfunction underlie the antiproliferative effect of DHEA.

Evidence for light wavelength-specific photoelectrophysiological signaling and memory of excess light episodes in Arabidopsis.

Although light is essential for photosynthesis, excess light can damage the photosynthetic apparatus and deregulate other cellular processes. Thus, protective integrated regulatory responses that can dissipate excess of absorbed light energy and simultaneously optimize photosynthesis and other cellular processes under variable light conditions can prove highly adaptive. Here, we show that the local and systemic responses to an excess light episode are associated with photoelectrophysiological signaling (PEPS) as well as with changes in nonphotochemical quenching and reactive oxygen species levels. During an excess light incident, PEPS is induced by quantum redox changes in photosystem II and in its proximity and/or by changes in glutathione metabolism in chloroplasts. PEPS is transduced, at least in part, by bundle sheath cells and is light wavelength specific. PEPS systemic propagation speed and action potential are dependent on ASCORBATE PEROXIDASE2 function. Excess light episodes are physiologically memorized in leaves, and the cellular light memory effect is specific for an excess of blue (450 nm) and red (650 nm) light of similar energy. It is concluded that plants possess a complex and dynamic light training and memory system that involves quantum redox, reactive oxygen species, hormonal, and PEPS signaling and is used to optimize light acclimation and immune defenses.

Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and sysemic lupus erythematosus

Nitric oxide (NO) has been shown to regulate T cell functions under physiological conditions, but overproduction of NO may contribute to T lymphocyte dysfunction. NO-dependent tissue injury has been implicated in a variety of rheumatic diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Several studies reported increased endogenous NO synthesis in both SLE and RA, and recent evidence suggests that NO contributes to T cell dysfunction in both autoimmune diseases. The depletion of intracellular glutathione may be a key factor predisposing patients with SLE to mitochondrial dysfunction, characterized by mitochondrial hyperpolarization, ATP depletion and predisposition to death by necrosis. Thus, changes in glutathione metabolism may influence the effect of increased NO production in the pathogenesis of autoimmunity.

How I became a biochemist

How I became a biochemist

Knee replacement surgery as a human clinical model of the effects of ischaemia/reperfusion upon skeletal muscle

The temporal pattern of metabolic alterations in muscle tissue during total ischaemia and reperfusion are not well-characterized in humans with respect to glutathione, amino acids and energy-rich compounds. In the present study, knee replacement surgery was used as a clinical model to elucidate this pattern of metabolic alterations. Patients (n=15) undergoing elective knee replacement surgery employing tourniquet ischaemia were studied. Muscle biopsies were taken from the quadriceps femoris muscle on the operated side preoperatively, at maximal ischaemia and after 24 h of reperfusion. The biopsies were analysed for glutathione, amino acids and energy-rich compounds. In addition the patients were randomized to receive either glucose or a mannitol infusion in the 24 h following tourniquet ischaemia. During ischaemia, muscle lactate increased by 400% (P<0.05) and phosphocreatine decreased by 70% (P<0.05). During the subsequent 24 h of reperfusion, muscle-reduced glutathione and total glutathione decreased by 27% and 22% (P<0.05) respectively. The muscle amino acid pattern changed during ischaemia with an increase in alanine by 65% (P<0.001) and a decrease in glutamate by 29% (P<0.001). During the reperfusion part of the study, no differences attributable to the infusion of mannitol or glucose were observed. During tourniquet ischaemia and subsequent reperfusion, changes in glutathione metabolism developed, indicating oxidative stress. Knee replacement surgery as a clinical model was useful during the ischaemia period, whereas the reperfusion period was dominated by the general changes seen postoperatively.

Mitochondrial dysfunction, peroxidation damage and changes in glutathione metabolism in PARK6

Oxidative stress and protein aggregation are biochemical hallmarks of Parkinson's disease (PD), a frequent sporadic late-onset degenerative disorder particularly of dopaminergic neurons in the substantia nigra, resulting in impaired spontaneous movement. PARK6 is a rare autosomal-recessively inherited disorder, mimicking the clinical picture of PD with earlier onset and slower progression. Genetic data demonstrated PARK6 to be caused by mutations in the protein PINK1, which is localized to mitochondria and has a serine-threonine kinase domain. To study the effect of PINK1 mutations on oxidative stress, we used primary fibroblasts and immortalized lymphoblasts from three patients homozygous for G309D-PINK1. Oxidative stress was evident from increases in lipid peroxidation and in antioxidant defenses by mitochondrial superoxide dismutase and glutathione. Elevated levels of glutathione reductase and glutathione-S-transferase were also observed. As a putative cause of oxidation, a mild decrease in complex I activity and a trend to superoxide elevation were detectable. These data indicate that PINK1 function is critical to prevent oxidative damage and that peripheral cells may be useful for studies of progression and therapy of PARK6.

Improved development of microspore‐derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism

Glutathione has been shown to play an important role during embryo development in both plant and animal systems. The effects of altered glutathione metabolism during microspore‐derived embryos (MDEs) of Brassica napus were investigated following exogenous application of reduced glutathione (GSH), its oxidized form (GSSG) and buthionine sulfoximine (BSO), an inhibitor of glutathione de novo synthesis. Applications of BSO which lowered the cellular glutathione redox status, i.e. GSH/(GSH + GSSG), enhanced significantly the quality of the embryos and their ability to convert into viable plants. Histological analyses revealed that inclusions of BSO in the culture medium altered the pattern of storage product accumulation in the embryos and improved the architecture of the shoot apical meristems (SAMs). Compared with their control counterparts which showed severe signs of SAM deterioration, such as the formation of intercellular spaces and differentiation of the meristematic cells, BSO‐treated embryos had well‐organized SAMs. The improved SAM organization observed in the presence of BSO also correlated with the proper localization pattern of WUSCHEL, a SAM molecular marker gene which was miss‐expressed in control embryos. The beneficial effects of BSO on embryo development and conversion were ascribed to the increasing levels of ABA. The concentration of this growth regulator in BSO‐treated embryos was always higher than that of control embryos during the second half of the maturation period. Furthermore, many structural alterations induced by BSO could be reproduced in embryos cultured in the presence of ABA. Taken together, these results suggest that a lowering of the glutathione redox status during embryo development may represent a metabolic switch needed for increasing the endogenous levels of ABA, which is required for successful completion of the developmental program.