The Role of the GSTF11 Gene in Resistance to Powdery Mildew Infection and Cold Stress

Oilseed rape (Brassica napus) is an economically important crop. In a temperate climate, powdery mildew Erysiphe crucifertaum can drastically reduce its yield. Nevertheless, cultivars resistant to this fungal disease have not yet been selected. Glutathione S-transferase GSTF11 is involved in glucosinolate (GSL) biosynthesis and response to stress, including fungal deceases. However, the impact of exogenous GSTF11 gene expression on resistance to powdery mildew has not yet been confirmed and requires further investigation. Transgenic B. napus was generated for this purpose. It demonstrated increased GST activity and a higher GSH:GSSG ratio under normal conditions. Powdery mildew Erysiphe crucifertaum caused 50% mortality in wild type (WT) plants. In most of transgenic plants, mycelium growth was inhibited. The infection contributed to higher GSTF11 expression and increased levels of glutathione (GSH) and oxidized glutathione (GSSG) in both transgenic and WT plants. In contrast, GSTF11 mRNA content, GST activity and GSSG level were lower only in WT plants. In transgenic plants, increased resistance to powdery mildew correlated with a lower GSH:GSSG ratio, indicating a higher content of neutralized toxic molecules. GSTF11 expression was also affected by cold stress, but not drought. At −1 °C, the expression level increased only in transgenic plants. Therefore, GSTF11 appears to be nonspecific and is able to protect plants under several types of stress. This gene could be used as a target in the production of stress tolerant cultivars.

Effect of Heavy Metals on the Activity of Catalase and Glutathione-S-Transferase in Nile Tilapia Fish (Oreochromis niloticus)

This study evaluated the response of antioxidant enzymes, such as Catalase (CAT) and Glutathione-S-transferase (GST) in freshwater Nile tilapia fish Oreochromis niloticus (O. niloticus) exposed to heavy metals (HMs) including copper (Cu), lead (Pb) and cadmium (Cd). Fish were expose to various concentrations of Cu2+, Pb2+ (0, 0.02, 0.05, 0.2 mg/l) and Cd2+ (0, 0.005, 0.01, 0.05 mg/l) for 15, 30, 45 and 60 days. The results indicated that enzyme activity was different according to the exposure time, concentration and type of heavy metals. CAT activity increased significantly beginning at day 45 of HMs exposure. After 60 days of exposure, CAT activity was steady with Cu and Pb but inhibited by Cd. While, GST induction was earlier observed from day 15 of HMs exposure. The increase of GST activity was found with the increase of exposure time in the treatment with Cu and Cd but not with Pb. Interestingly, GST activity was inhibited by Pb at longer exposure (45 and 60 days). Among tested metals, Cu showed the weaker effect on the activity of CAT and GST in comparison with Pb and Cd suggesting that these enzymes were less sensitive to Cu than other tested metals.

Proteomic Analysis of Glutathione Transferases from Lucilia Cuprina

<p>The glutathione transferases are a family of multifunctional enzymes involved in detoxification of xenobiotic and endogenous electrophilic compounds. Interest in insect GSTs has primarily focused on their role in insecticide resistance. The sheep blowfly, Lucilia cuprina is a major economic problem for the sheep meat and wool industries in Australasia and hence this thesis has attempted the study of the Lucilia cuprina GST family, using proteomics, with a view to eventually determining their role in insecticide resistance. Combinations of different affinity matrices (glutathione-Sepharose matrix (GSH) followed by dinitrophenyl-glutathione-Sepharose matrix (DNP-GSH)) and two-dimensional electrophoresis has successfully isolated members from major four insect GST classes: Sigma, Delta, Epsilon and Omega. Drosophila melanogaster has been used as a model insect throughout as a basis for comparison. To characterise Lucilia GSTs, the whole metazoan fragmentation database was used for sequence alignment with Lucilia peptides. This approach is broad and speculative but predicts a possible classification of the GSTs based on % similarity and % identity. This method of characterisation yielded match scores that provided a basis for classification, which must at present be regarded as tentative and in need of confirmation. In D. melanogaster and L. cuprina, GSH affinity-purified extracts showed the presence of only Sigma and Delta GSTs. In D. melanogaster, the DNP-GSH affinity-purified GSTs showed mostly the presence of Epsilon and Omega GSTs whereas in L. cuprina no Omega GSTs were detected. In both species, the migration pattern of Delta GST on 2D PAGE gel indicated possible post-translational modification. The results from analysis of LC-MS/MS data by the software PEAKS suggested deamidation at asparagine and glutamine residues in a limited number of the matched peptides of Delta GST. GST activity was present in all developmental stages of L. cuprina. The number of isoenzymes and their extent of expression vary as the insect develops. Delta GSTs were present in all developmental stages. The Sigma GST started expressing from the larval stage and was abundantly present in adult stage. The DNP-GSH affinity matrix purified GSTs which have been tentatively classified as Mu-like GSTs were present in egg, larvae and pupae but totally absent in adult stage. The GST families were characterised by proteomics in the main body sections of L. cuprina. Higher GST activity towards 1-chloro-2, 4-dinitrobenzene (CDNB) was found in the thorax (65.2 %) followed by the abdomen (19.6%) and the head (15.2%). The cytosolic GSTs of a resistant strain (PY81) of L. cuprina had significantly higher (2.26- and 2.6- fold) activity than the susceptible strains (NSW and CSIRO) towards CDNB and 2, 3-dichloro, 4-nitrobenzene (DCNB) respectively. The proteomic analysis of DNP-GSH purified extract from susceptible and resistant strains showed quantitatively higher expression of GSTs on 2D PAGE gel of the PY81 strain. The in vitro interaction of purified GSTs and model insecticides studied by high performance liquid chromatography revealed that Delta and DNP-GSH affinity-purified GSTs catalyse the conjugation of the insecticides to reduced glutathione but Sigma GST had almost no activity.</p>

Proteomic Analysis of Glutathione Transferases from Lucilia Cuprina

<p>The glutathione transferases are a family of multifunctional enzymes involved in detoxification of xenobiotic and endogenous electrophilic compounds. Interest in insect GSTs has primarily focused on their role in insecticide resistance. The sheep blowfly, Lucilia cuprina is a major economic problem for the sheep meat and wool industries in Australasia and hence this thesis has attempted the study of the Lucilia cuprina GST family, using proteomics, with a view to eventually determining their role in insecticide resistance. Combinations of different affinity matrices (glutathione-Sepharose matrix (GSH) followed by dinitrophenyl-glutathione-Sepharose matrix (DNP-GSH)) and two-dimensional electrophoresis has successfully isolated members from major four insect GST classes: Sigma, Delta, Epsilon and Omega. Drosophila melanogaster has been used as a model insect throughout as a basis for comparison. To characterise Lucilia GSTs, the whole metazoan fragmentation database was used for sequence alignment with Lucilia peptides. This approach is broad and speculative but predicts a possible classification of the GSTs based on % similarity and % identity. This method of characterisation yielded match scores that provided a basis for classification, which must at present be regarded as tentative and in need of confirmation. In D. melanogaster and L. cuprina, GSH affinity-purified extracts showed the presence of only Sigma and Delta GSTs. In D. melanogaster, the DNP-GSH affinity-purified GSTs showed mostly the presence of Epsilon and Omega GSTs whereas in L. cuprina no Omega GSTs were detected. In both species, the migration pattern of Delta GST on 2D PAGE gel indicated possible post-translational modification. The results from analysis of LC-MS/MS data by the software PEAKS suggested deamidation at asparagine and glutamine residues in a limited number of the matched peptides of Delta GST. GST activity was present in all developmental stages of L. cuprina. The number of isoenzymes and their extent of expression vary as the insect develops. Delta GSTs were present in all developmental stages. The Sigma GST started expressing from the larval stage and was abundantly present in adult stage. The DNP-GSH affinity matrix purified GSTs which have been tentatively classified as Mu-like GSTs were present in egg, larvae and pupae but totally absent in adult stage. The GST families were characterised by proteomics in the main body sections of L. cuprina. Higher GST activity towards 1-chloro-2, 4-dinitrobenzene (CDNB) was found in the thorax (65.2 %) followed by the abdomen (19.6%) and the head (15.2%). The cytosolic GSTs of a resistant strain (PY81) of L. cuprina had significantly higher (2.26- and 2.6- fold) activity than the susceptible strains (NSW and CSIRO) towards CDNB and 2, 3-dichloro, 4-nitrobenzene (DCNB) respectively. The proteomic analysis of DNP-GSH purified extract from susceptible and resistant strains showed quantitatively higher expression of GSTs on 2D PAGE gel of the PY81 strain. The in vitro interaction of purified GSTs and model insecticides studied by high performance liquid chromatography revealed that Delta and DNP-GSH affinity-purified GSTs catalyse the conjugation of the insecticides to reduced glutathione but Sigma GST had almost no activity.</p>

Glutathione S-Transferase and Clusterin, New Players in the Ischemic Preconditioning Renal Protection in a Murine Model of Ischemia and Reperfusion

BACKGROUND/AIMS: Renal ischemia and reperfusion injury (IRI) involves oxidative stress, disruption of microvasculature due to endothelial cell damage, loss of epithelial cell polarity secondary to cytoskeletal alterations, inflammation, and the subsequent transition into a mesenchymal phenotype. Ischemic preconditioning (IPC) has been proposed as a therapeutic strategy to avoid/ameliorate the IRI. Since previous results showed that IPC could have differential effects in kidney cortex vs. kidney medulla, in the present study we analyzed the effectiveness and molecular mechanisms implicated in IPC in both kidney regions. METHODS: We evaluated 3 experimental groups of BALB/c male mice: control (sham surgery); renal ischemia (30 min) by bilateral occlusion of the renal pedicle and reperfusion (48 hours) (I/R); and renal IPC (two cycles of 5 min of ischemia and 5 min of reperfusion) applied just before I/R. Acute kidney injury was evaluated by glomerular filtration rate (GFR), Neutrophil Gelatinase-Associated Lipocalin (NGAL) blood level, and histologic analysis. Oxidative stress was studied measurement the Glutathione S-Transferase (GST) activity, GSH/GSSG ratio, and lipoperoxidation levels. Inflammatory mediators (IL-1β, IL-6, Foxp3, and IL-10) were quantified by qRT-PCR. The endothelial (PECAM-1), epithelial (AQP-1), mesenchymal (Vimentin, Fascin, and Hsp47), iNOS, clusterin, and Hsp27 expression were evaluated (qRT-PCR and/or Western blot). RESULTS: The IPC protocol prevented the decrease of GFR, reduced the plasma NGAL, and ameliorated morphological damage in the kidney cortex after I/R. The IPC also prevented the downregulation of GST activity, lipoperoxidation and ameliorated the oxidized glutathione. In addition, IPC prevented the upregulation of vimentin, fascin, and Hsp47, which was associated with the prevention of the downregulation of AQP1 after I/R. The protective effect of IPC was associated with the upregulation of Hsp27, Foxp3, and IL-10 expression in the renal cortex. However, the upregulation of iNOS, IL-1β, IL-6, and clusterin by I/R were not modified by IPC. CONCLUSION: IPC conferred better protection in the kidney cortex as compared to the kidney medulla. The protective effect of IPC was associated with amelioration of oxidative stress, tubular damage, and the induction of markers of Treg lymphocytes activity in the cortical region. Further studies are needed to evaluate if lower tubular cell stress/damage after I/R may explain the preferential induction of Treg response in the kidney cortex induced by IPC.

Activity of biochemical biomarkers in grasshoppers Abracris flavolineata (De Geer, 1773) (Orthoptera: Acrididae: Ommatolampidinae)

Biochemical biomarkers are commonly used in environmental monitoring programs due to their sensitivity to certain pollutants. From this perspective, their responses can be used as indicators of environmental quality. The present study aimed to determine the activity of the catalase (CAT) and glutathione-S-transferase (GST) enzymes in grasshoppers Abracris flavolineata (De Geer, 1773) from two forest remnant areas in Serra da Jiboia (BA) and compare them between males and females. The specimens were collected at two sites in Serra da Jiboia (Bahia, Brasil), named ‘Baixa de Areia’ and ‘Baixa Grande’. The animals were actively collected in the morning using a sweep net and a 2.5 h sampling effort. In total, 160 individuals were collected, with 80 individuals from each sampling site, 50 males and 30 females. After identification, an incision was made in the lateral region of the abdomen to remove the midgut, which was used to extract the CAT and GST enzymes. The results obtained demonstrated that CAT and GST activity did not vary significantly between sampling areas. However, with regard to sex, enzyme activity was significantly higher in males (p<0.005) in both locations. This is a pioneer study on the responses of CAT and GST activity in grasshoppers in Brazil.

The activity of markers of oxidative and nitrosative stresses in blood plasma of Parkinson’s disease patients at the early stages

The aim of the study is to investigate activity of markers of oxidative and nitrosative stresses in blood plasma of patients in the I–II stages of Parkinson’s disease (PD) and to determine correlations between their concentrations and severity of non-motor PD symptoms. Materials and methods. 67 patients at I–II PD stages and 20 healthy controls took part in the research. Cognitive functions were examined due to the Montreal Cognitive Assessment test – MoCA test. For the severity of psycho-emotional disorders evaluation the following scales and questionnaires were used: Night Sleep Assessment Questionnaire by A. M. Vein, Zung test for anxiety, apathy Starkstein scale, Boston stress-resistance test, Beck Depression Inventory (BDI-II). We performed ELISA test for determination of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities and 3-nitrotyrosine (3-NT) level in blood plasma of participants (Elabscience® kit). Results. The middle age of PD patients and healthy controls was 64.35 ± 1.22 and 66.40 ± 0.70 years, respectively. GPx activity in plasma of patients at І–ІІ PD stages was significantly lower than in healthy controls (P < 0.001) and was higher at the I stage compared to the II PD stage (P = 0.003). Also GPx activity in PD patients with normal cognition was higher than in PD patients with cognitive impairment (P = 0.042). The GST activity in plasma of PD patients with anxiety was significantly lower (P = 0.002) compared to those without anxiety, and 3-NT blood plasma level in PD patients with moderate anxiety was higher than in those without one (P = 0.029). Conclusions. The activity of antioxidant GPx was significantly lower in PD patients at early stages compared to healthy controls, and in PD patients in the II stage of the disease compared to the I stage, and it was significantly lower in PD patients with cognitive impairment. PD patients with moderate anxiety had lower 3-NT levels and GST activity in blood plasma.

Long-term Exposure to Phenanthrene-Induced Gene Expressions and Enzyme Activities of Cyprinus Carpio Below the Safe Concentration

Phenanthrene (PHE) is a typical food chain biomagnified compound which endangers human health and is generally accumulated from marine lives. Previous PHE-stressed Carp acute toxicity test showed that the safe concentration of PHE to carp was 1.12 mg/L. In this study, the carp was long-term exposed to PHE below safe concentration up to 25 days. The gene expression levels and cytochrome P450 (CYP1A/EROD (7-Ethoxylesorufin O-deethylase)) and glutathione S-transferase (GST) activities were determined in the carp liver and brain tissues. The results showed that both the CYP1A mRNA expression and EROD activity in the liver were continuously stimulated after induction with the increase in exposure time and exposure concentration. However, with the increase of PHE concentration, GST mRNA expression in the liver was firstly induced and then inhibited and the induction was significant in the treatment with 0.1 mg/L PHE in the 15th day (almost 2-fold). In the brain, after the 15th day, GST mRNA expression was suppressed, but GST activity was induced. Correlation analysis results showed that the CYP1A mRNA expression was significantly correlated with the activity of EROD in both tissues (liver, r = 0.602, P < 0.01; brain, r = 0.508, P < 0.01), but the correlation between GST mRNA expression and GST activity was poor (liver, r = 0.385, P < 0.01; brain, r = 0.293, P < 0.01). This experiment revealed the self-regulation mechanism of carp exposed to lower than safe concentrations of PHE for a long time, indicating the toxicological risk of PHE in the ecosystem.

Resistance Mechanism to Metsulfuron-Methyl in Polypogon fugax

Polypogon fugax is a common winter weed in China and other Asia countries. We have previously found a P. fugax biotype (R) resistant to acetyl co-enzyme A carboxylase (ACCase) herbicides also cannot be effectively controlled by some acetolactate synthase (ALS) herbicides. This study evaluated the level of resistance to four ALS herbicides (metsulfuron-methyl, chlorsulfuron, monosulfuron, pyribambenz isopropyl) in the R biotype and the associated resistance mechanism. The R biotype exhibited moderate level of resistance to metsulfuron-methyl (6.0-fold) compared with the sensitive biotype (S). Sequence analysis of ALS gene revealed that two ALS genes existed in P. fugax. However, no substitution associated with ALS resistance mechanism were found in ALS genes between the S and R biotypes. The activity of ALS enzyme isolated from the R biotype was inherently higher and less sensitive to metsulfuron-methyl than the S biotype. Glutathione S-transferases (GST) activity was also less sensitive to metsulfuron-methyl in the R than as the S biotypes. Malathion, a cytochrome P450 (CYP) monooxygenase inhibitor, had much greater synergistic effect with metsulfuron-methyl on the R than as the S plants, reducing the ED50 value (herbicide dose to inhibit growth by 50%) of metsulfuron-methyl by 23- and 6-fold, respectively, suggesting that CYP mediated enhanced metabolism might contribute to the resistance to ALS herbicides. These results suggest that metsulfuron-methyl resistance in the R biotype was associated with the up-regulated ALS enzymatic activity and the GST and CYP-mediated enhanced herbicide metabolism.

Characterization of glutathione S-transferase enzymes in Dictyostelium discoideum suggests a functional role for the GSTA2 isozyme in cell proliferation and development

In this report, we extend our previous characterization of Dictyostelium discoideum glutathione S-transferase (DdGST) enzymes that are expressed in the eukaryotic model organism. Transcript profiling of gstA1-gstA5 (alpha class) genes in vegetative, log phase cells identified gstA2 and gstA3 with highest expression (6–7.5-fold, respectively) when compared to other gstA transcripts. Marked reductions in all gstA transcripts occurred under starvation conditions, with gstA2 and gstA3 exhibiting the largest decreases (-96% and -86.6%, respectively). When compared to their pre-starvation levels, there was also a 60 percent reduction in total GST activity. Glutathione (GSH) pull-down assay and mass spectroscopy detected three isozymes (DdGSTA1, DdGSTA2 and DdGSTA3) that were predominantly expressed in vegetative cells. Biochemical and kinetic comparisons between rDdGSTA2 and rDdGSTA3 shows higher activity of rDdGSTA2 to the CDNB (1-chloro-2,4-dinitrobenzene) substrate. RNAi-mediated knockdown of endogenous DdGSTA2 caused a 60 percent reduction in proliferation, delayed development, and altered morphogenesis of fruiting bodies, whereas overexpression of rDdGSTA2 enzyme had no effect. These findings corroborate previous studies that implicate a role for phase II GST enzymes in cell proliferation, homeostasis, and development in eukaryotic cells.