Neurocognitive impairment in relation to glutathione S-transferase enzyme polymorphisms among medulloblastoma patients

Background. Medulloblastoma is a type of brain cancer that accounts for approximately 7-8% of all intracranial tumors and 20-30% of pediatric brain tumors. It is the most common type of malignant brain tumor in childhood. It was reported that majority of survivors with medulloblastoma have social problems, endocrine deficits, and neurological complications. Furthermore, all had significant deficits in neurocognitive functioning. Glutathione S-transferases belong to a family of isoenzymes that catalyze the glutathione conjugation of a variety of electrophilic compounds. ^ Objective. We aimed to determine whether the development of neurocognitive impairment is associated with GST polymorphisms among children and adolescents diagnosed with medulloblastoma (MB) after radiation therapy. ^ Methods. A pilot study composing of 16 children and adolescents diagnosed with MB at Texas Children's Cancer Center was conducted. The t-test was used to determine if the GST polymorphisms were related to neurocognitive impairment and logistic regression was performed to explore association between GST polymorphisms and gender, age at diagnosis, race/ethnicity, and risk group. ^ Results. An association was observed between GSTT1 polymorphism and cognitive impairment one year after radiation and GSTM1 polymorphism two years after radiation. It was observed that patients with GSTT1 null genotype have lower performance IQ (p=0.03) and full scale IQ (p=0.02) one year after radiation and patients with GSTM1 null genotype have lower verbal IQ (p=0.02) two years after radiation. Patients under age 8 have a statistically non-significant higher risk of having not null genotypes compared to those older than age 8 (OR= 7.5, 95%CI: 0.62-90.65 and OR= 2.63, 95%CI: 0.30-23.00 for GSTT1 and GSTM1 respectively). ^ Conclusion. There was a significant association between GSTT1 polymorphism and cognitive impairment one year after radiation and between GSTM1 polymorphism and cognitive impairment two years after radiation. Further large scale studies may be needed to confirm this finding and to examine the underlying mechanism of neurocognitive impairments after treatment of medulloblastoma patients.^

Signaling pathways in the transcriptional and post-translational regulation of the human glutathione S -transferase P1 gene

The human glutathione S-transferase P1 (GSTP1) protein is an endogenous inhibitor of c-jun N-terminal kinases (JNKs) and an important phase II detoxification enzyme. ^ Recent identification of a cAMP response element (CRE) in the 5 ′-region of the human GSTP1 gene and several putative phosphorylation sites for the Ser/Thr protein kinases, including, cAMP-dependent protein kinases (PKAs), protein kinases C (PKCs), and JNKs in the GSTP1 protein raised the possibility that signaling pathways may play an important role in the transcriptional and post-translational regulation of GSTP1 gene. This study examined (a) whether the signaling pathway mediated by CAMP, via the GSTP1 CRE, is involved in the transcriptional regulation of the GSTP1 gene, (b) whether signaling pathways mediated by the Ser/Thr protein kinases (PKAs, PKCs, and JNKs) induce post-translational modification, viz. phosphorylation of the GSTP1 protein, and (c) whether such phosphorylation of the GSTP1 protein alters its functions in metabolism and in JNK signaling. ^ The first major finding in this study is the establishment of the human GSTP1 gene as a novel CAMP responsive gene in which transcription is activated via an interaction between PKA activated CRE binding protein-1 (CREB-1) and the CRE in the 5′-regulatory region. ^ The second major finding in this study is the observation that the GSTP1 protein undergoes phosphorylation and functionally activated by second messenger-activated protein kinases, PKA and PKC, in tumor cells with activated signaling pathways. Following phosphorylation by PKA or PKC, the catalytic activity of the GSTP1 protein was significantly enhanced, as indicated by a decrease in its Km (2- to 3.6-fold) and an increase in Kcat/ Km (1.6- to 2.5-fold) for glutathione. Given the frequent over-expression of GSTP1 and the aberrant PKA/PKC signaling cascade observed in tumors, these findings suggest that phosphorylation of GSTP1 may contribute to the malignant progression and drug-resistant phenotype of these tumors. ^ The third major finding in this study is that the GSTP1 protein, an inhibitor of JNKs, undergoes significant phosphorylation in tumor cells with activated JNK signaling pathway and in those under oxidative stress. Following phosphorylation by JNK, the ability of GSTP1 to inhibit JNK downstream function, i.e. c-jun phosphorylation, was significantly enhanced, suggesting a feedback mechanism of regulation of JNK-mediated cellular signaling. (Abstract shortened by UMI.) ^

Gene expression and physiological changes of the Antarctic krill Euphausia superba under different hypoxia intensities

Antarctic krill (Euphausia superba) from South Georgia comprise one of the most northern and abundant krill stocks. South Georgia waters are undergoing rapid warming, as a result of climate change, which in turn could alter the oxygen concentration of the water. We investigated gene expression in Antarctic krill related to aerobic metabolism, antioxidant defence, and heat-shock response under severe (2.5% O2 saturation or 0.6 kPa) and threshold (20% O2 saturation or 4 kPa) hypoxia exposure compared to in situ levels (normoxic; 100% O2 saturation or 21 kPa). Biochemical metabolic and oxidative stress indicators complemented the genic expression analysis to detect in vivo signs of stress during the hypoxia treatments. Expression levels of the genes citrate synthase (CS), mitochondrial manganese superoxide dismutase (SODMn-m) and one heat-shock protein isoform (E) were higher in euphausiids incubated 6 h at 20% O2 saturation than in animals exposed to control (normoxic) conditions. All biochemical antioxidant defence parameters remained unchanged among treatments. Levels of lipid peroxidation were raised after 6 h of severe hypoxia. Overall, short-term exposure to hypoxia altered mitochondrial metabolic and antioxidant capacity, but did not induce anaerobic metabolism. Antarctic krill are swarming organisms and may experience short periods of hypoxia when present in dense swarms. A future, warmer Southern ocean, where oxygen saturation levels are decreased, may result in smaller, less dense swarms as they act to avoid greater levels of hypoxia.

(Table 1) Antioxidant concentrations in liver of wild sea bird chicks from the Norwegian arctic region

The efficiency of antioxidant defenses and relationship with body burden of metal and organic contaminants has not been previously investigated in arctic seabirds, neither in chicks nor in adults. The objective of this study was to compare such defenses in chicks from three species, Black-legged kittiwake (Rissa tridactyla), Northern fulmar (Fulmarus glacialis), and Herring gull (Larus argentatus), and the relationship with tissue concentrations of essential metals such as selenium and iron and halogenated organic compounds, represented by polychlorinated biphenyl (PCB). The results showed significant species-specific differences in the antioxidant responses which also corresponded with metal and PCB levels in different ways. The capability to neutralize hydroxyl radicals (TOSC-HO°) and the activities of catalase and Se-dependent glutathione peroxidases (GPX) clearly increased in species with the higher levels of metals and PCBs, while the opposite trend was observed for Se-independent GPX, TOSC against peroxyl radicals (ROO°) and peroxynitrite (ONOOH). Less clear relationships were obtained for glutathione levels, GSH/GSSG ratio, glutathione reductase and superoxide dismutase. The results showed differences in antioxidant efficiency between the species, and some of these defenses exhibited dose-response-like relationships with measured levels of selenium, iron and XPCBs. PCBs, selenium and iron levels were positively related to the responses of antioxidants with potential to reduce HO°/H2O2 (Se-dependent GPX, CAT and TOSC against HO°). However, direct causal relationships between antioxidant responses and contaminant concentrations could not be shown on individual level. Varying levels of metals and contaminants due to different diet and age were probably the main explanations for the species differences in antioxidant defense.

Function of glutathione in Arabidopsis immunity and glucosinolate metabolism

Induced defense responses in plants usually involve biosynthesis of antimicrobial metabolites and their targeted secretion at the site of pathogen contact. Our recent study on the model plant Arabidopsis revealed a novel pathogen triggered metabolism pathway for glucosinolates, amino acid-derived thio-glucosides characteristic for crucifer plants that so far were mainly known as insect deterrents (Bednarek et al. 2009).

Impact of using new commercial glutathione enriched inactive dry yeast oenological preparations on the aroma and sensory properties of wines

The effect of the addition of a commercial enriched glutathione inactive dry yeast oenological preparation in the volatile and sensory properties of industrially manufactured rosé Grenache wines was evaluated during their shelf-life. In addition, triangle tests were performed at different times during wine aging (among 1 and 9 months) to determine the sensory differences between wines with and without glutathione inactive dry yeast preparations. Descriptive sensory analysis with a trained panel was carried out when sensory differences in the triangle test were noticed. In addition, consumer tests were performed in order to investigate consumers’ acceptability of wines. Results revealed significant sensory differences between control and glutathione inactive dry yeast wines after 9 months of aging. At that time, glutathione inactive dry yeast wines were more intense in fruity aromas (strawberry, banana) and less intense in yeast notes than control wine. The impact of the glutathione inactive dry yeast in the aroma might be the consequence of different effects that these preparations could induce in wine composition: modification of yeast byproducts during fermentation, release of volatile compounds from inactive dry yeast, interaction of wine volatile compounds with yeast macromolecules from inactive dry yeast and a possible antioxidant effect of the glutathione released by the inactive dry yeast preparation on some specific volatile compounds.

Persistent inhibition of cell respiration by nitric oxide: Crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1α and aggst1β) reported within are interrupted by introns. The gene aggst1α contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5′ exon encoding the N termini of the GST protein spliced to one of four distinct 3′ exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1α are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1β gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.

Overexpression of Glutathione Synthetase in Indian Mustard Enhances Cadmium Accumulation and Tolerance1

An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione. To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea). The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature-plant stages. Cd accumulation and tolerance were correlated with the gshII expression level. Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants. We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity.

Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.

Cyst(e)ine Is the Transport Metabolite of Assimilated Sulfur from Bundle-Sheath to Mesophyll Cells in Maize Leaves1

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, γ-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Location of a potential transport binding site in a sigma class glutathione transferase by x-ray crystallography.

The crystal structure of the sigma class glutathione transferase from squid digestive gland in complex with S-(3-iodobenzyl)glutathione reveals a third binding site for the glutathione conjugate besides the two in the active sites of the dimer. The additional binding site is near the crystallographic two-fold axis between the two alpha 4-turn-alpha 5 motifs. The principal binding interactions with the conjugate include specific electrostatic interactions between the peptide and the two subunits and a hydrophobic cavity found across the two-fold axis that accommodates the 3-iodobenzyl group. Thus, two identical, symmetry-related but mutually exclusive binding modes for the third conjugate are observed. The hydrophobic pocket is about 14 A from the hydroxyl group of Tyr-7 in the active site. This site is a potential transport binding site for hydrophobic molecules or their glutathione conjugates.