Efficient Adsorption and Extraction of Glutathione S-Transferases with Glutathione-Functionalized Graphene Oxide-Polyhedral Oligomeric Silsesquioxane Composite.

Glutathione S-transferases (GSTs) are important type-II detoxification enzymes that protect DNA and proteins from damage and are often used as protein tags for the expression of fusion proteins. In the present work, octa-aminopropyl caged polyhedral oligomeric silsesquioxane (OA-POSS) was prepared via acid-catalyzed hydrolysis of 3-aminopropyltriethoxysilane and polymerized on the surface of graphene oxide (GO) through an amidation reaction. Glutathione (GSH) was then modified to GO-POSS through a Michael addition reaction to obtain a GSH-functionalized GO-POSS composite (GPG). The structure and characteristics of the as-prepared GPG composite were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), thermogravity analysis, and surface charge analysis. The specific binding interactions between glutathione and GST gave GPG favorable adsorption selectivity towards GST, and other proteins did not affect GST adsorption. The adsorption behavior of GST on the GPG composite conformed to the Langmuir isotherm model, and the adsorption capacity of GST was high up to 364.94 mg g-1 under optimal conditions. The GPG-based solid-phase adsorption process was applied to the extraction of GST from a crude enzyme solution of pig liver, and high-purity GST was obtained via SDS-PAGE identification.

Diverse patterns of constitutive and inducible overexpression of detoxifying enzyme genes among resistant Aphis glycines populations.

Understanding the mechanisms of pyrethroid resistance is essential to the effective management of pesticide resistance in Aphis glycines Matsumura (Hemiptera: Aphididae). We mined putative detoxifying enzyme genes in the draft genome sequence of A. glycines for cytochrome oxidase P450 (CYP), glutathione-S-transferase (GST) and esterases (E4 and carboxylesterases-CES). Aphids from clonal populations resistant to pyrethroids from three sites in Minnesota, USA, were screened against a diagnostic LC99 concentration of either λ-cyhalothrin or bifenthrin and detoxifying enzyme genes expression in survivors was analyzed by qPCR. Their expression profiles were compared relative to a susceptible clonal population. We found 61 CYP (40 full-length), seven GST (all full-length), seven E4 (five full-length) and three CES (two full-length) genes, including 24 possible pseudogenes. The detoxifying enzymes had different expression profiles across resistant aphid populations, possibly reflecting differences in the genetic background and pyrethroid selection pressures as the number of constitutively overexpressed detoxifying enzyme genes was correlated with the level of resistance. Our findings will strengthen the understanding of the pyrethroid resistance mechanisms in A. glycines.

Effects of GST variants on the risk odds of hematological malignancy: A meta-analysis.

BACKGROUND: Whether glutathione S-transferases (GST) polymorphisms influence the risk odds of hematological malignancy remains controversial. Therefore, we performed this meta-analysis to better analyze correlations between GST polymorphisms and hematological malignancy. METHODS: Literature retrieve was conducted in PubMed, MEDLINE, and Embase. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. RESULTS: Sixty-two studies were enrolled for analyses. Significant associations with hematological malignancy were observed for GSTM1 (P < 0.0001, OR = 1.25, 95% CI, 1.14-1.38), GSTP1 (P = 0.002, OR = 1.20, 95% CI, 1.07-1.34), and GSTT1 (P < 0.0001, OR = 1.57, 95% CI, 1.39-1.76) polymorphisms in overall analyses. Further subgroup analyses by ethnicity revealed that GSTM1 and GSTT1 polymorphisms were both significantly correlated with hematological malignancy in Caucasians, East Asians, and West Asians, whereas GSTP1 polymorphism was only significantly correlated with hematological malignancy in Caucasians and West Asians. When we stratified data according to type of disease, positive results were found for all investigated polymorphisms in patients with certain types of acute leukemia. Moreover, GSTP1 polymorphism was also found to be significantly associated with chronic leukemia and lymphoma. CONCLUSIONS: Our findings indicated that GSTM1, GSTT1, and GSTP1 polymorphisms may serve as potential genetic biomarkers of hematological malignancy in certain ethnicities.

Effects of GST null genotypes on individual susceptibility to leukemia: A meta-analysis.

BACKGROUND: Whether glutathione S-transferases (GST) null genotypes influence individual susceptibility to leukemia remains controversial. Thus, we performed this meta-analysis to better analyze potential influences of GST null genotypes on individual susceptibility to leukemia. METHODS: Literature retrieve was conducted in PubMed, Web of Science and Embase. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. RESULTS: Totally fifty-one studies were enrolled for analyses. Significant associations with elevated individual susceptibility to leukemia were detected for GSTM1 (p < .0001, OR = 1.28, 95%CI 1.16-1.41), GSTP1 (p = .003, OR = 1.22, 95%CI 1.07-1.40) and GSTT1 (p < .0001, OR = 1.53, 95%CI 1.35-1.74) null genotypes in overall analyses. Further subgroup analyses by type of disease revealed that GSTM1, GSTP1 and GSTT1 null genotypes were all significantly associated with elevated individual susceptibility to acute lymphoblastic leukemia, GSTM1 and GSTT1 null genotypes were significantly associated with elevated individual susceptibility to acute myeloid leukemia, and GSTT1 null genotype was also significantly associated with elevated individual susceptibility to chronic leukemia. When we stratified data according to ethnicity of participants, positive results were found for all investigated variants in Caucasians and West Asians. Additionally, GSTM1 null genotype was also significantly correlated with elevated individual susceptibility to leukemia in East Asians. CONCLUSIONS: Our findings indicated that GSTM1, GSTT1 and GSTP1 null genotypes might serve as potential genetic biomarkers of leukemia in certain ethnicities.

Analysis of glutathione S-transferase allergen cross-reactivity in a North American population: Relevance for molecular diagnosis.

BACKGROUND: It is not clear whether cross-reactivity or cosensitization to glutathione S-transferases (GSTs) occurs in tropical and subtropical environments. In the United States, Bla g 5 is the most important GST allergen and lack of coexposure to GSTs from certain species allows a better assessment of cross-reactivity. OBJECTIVES: To examine the molecular structure of GST allergens from cockroach (Bla g 5), dust mites (Der p 8 and Blo t 8), and helminth (Asc s 13) for potential cross-reactive sites, and to assess the IgE cross-reactivity of sensitized patients from a temperate climate for these allergens for molecular diagnostic purposes. METHODS: Four crystal structures were determined. Sera from patients allergic to cockroach and mite were tested for IgE reactivity to these GSTs. A panel of 6 murine anti-Bla g 5 mAb was assessed for cross-reactivity with the other 3 GSTs using antibody binding assays. RESULTS: Comparisons of the allergen structures, formed by 2-domain monomers that dimerize, revealed few contiguous regions of similar exposed residues, rendering cross-reactivity unlikely. Accordingly, anti-Bla g 5 or anti-Der p 8 IgE from North American patients did not recognize Der p 8 or Bla g 5, respectively, and neither showed binding to Blo t 8 or Asc s 13. A weaker binding of anti-Bla g 5 IgE to Der p 8 versus Bla g 5 (∼ 100-fold) was observed by inhibition assays, similar to a weak recognition of Der p 8 by anti-Bla g 5 mAb. Patients from tropical Colombia had IgE to all 4 GSTs. CONCLUSIONS: The lack of significant IgE cross-reactivity among the 4 GSTs is in agreement with the low shared amino acid identity at the molecular surface. Each GST is needed for accurate molecular diagnosis in different geographic areas.

GLUTATHIONE S-TRANSFERASE Genes IN THE RICE LEAFFOLDER, Cnaphalocrocis medinalis (LEPIDOPTERA: PYRALIDAE): IDENTIFICATION AND EXPRESSION PROFILES.

In insects, glutathione S-transferases (GSTs) play critical roles in the detoxification of various insecticides, resulting in insecticide resistance. The rice leaffolder, Cnaphalocrocis medinalis, is an economically important pest of rice in Asia. GST genes have not been largely identified in this insect species. In the present study, by searching the transcriptome dataset, 25 candidate GST genes were identified in C. medinalis for the first time. Of these, 23 predicted GST proteins fell into five cytosolic classes (delta, epsilon, omega, sigma, and zeta), and two were assigned to the "unclassified" subgroup. Real-time quantitative PCR analysis showed that these GST genes were differentially expressed in various tissues, including the midgut, Malpighian tubules, and fat body of larvae, and the antenna, abdomen, and leg of adults, indicating diversified functions for these genes. Transcription levels of CmGSTd2, CmGSTe6, and CmGSTe7 increased significantly in larvae following exposure to chlorpyrifos, suggesting that these GST genes could be involved in the detoxification of this insecticide. The results of our study pave the way to a better understanding of the detoxification system of C. medinalis.

Expression profiles of selenium dependent glutathione peroxidase and glutathione S-transferase from Exopalaemon carinicauda in response to Vibrio anguillarum and WSSV challenge.

A selenium-dependent glutathione peroxidase cDNA was obtained from the ridgetail white prawn Exopalaemon carinicauda (EcGPx) by rapid amplification of cDNA ends (RACE) methods. The full-length cDNA of EcGPx was 946 bp, which contains a 5'-untranslated region (UTR) of 105 bp, 3'-UTR of 280 bp with a poly (A) tail, an open reading frame (ORF) of 561 bp, encoding a 186 amino-acid polypeptide with the predicted molecular weight of 21.35 kDa and estimated isoelectric point of 7.65. It involves a putative selenocysteine (U39) residue which is encoded by an opal codon, (220)TGA(222), and forms an active site with residues Q73 and W141. Sequence analysis revealed that a GPx signature motif 2 ((63)LAFPCNQF(70)), an extra active site motif ((151)WNFEKF(156)), two putative N-glycosylation site ((75)NNT(77) and (107)NGS(109)), and two arginine residues (R89 and R167) were observed in the EcGPx sequence. Comparison of amino acid sequences showed that white shrimp GPx is more closely related to GPx1 and GPx2 subgroups. Quantitative real-time RT-PCR analysis indicated that two glutathione antioxidant enzymes of E. carinicauda, glutathione peroxidase (designated EcGPx) and glutathione S-transferase (designated EcGST) were widely expressed in all the tested tissues, but showed different expression patterns. After Vibrio anguillarum and WSSV challenge, EcGPx and EcGST transcripts both in hemocytes and hepatopancreas increased in the first 6 h and 3 h, respectively. The results suggested that EcGPx and EcGST might be associated with the immune defenses to V. anguillarum and WSSV in E. carinicauda.

Quizalofop-p-ethyl resistance in Polypogon fugax involves glutathione S-transferases.

BACKGROUND: Asia minor bluegrass (Polypogon fugax) is one of the main weeds invading Chinese canola fields. The P. fugax resistant population SC-R, which survived quizalofop-p-ethyl at the field-recommended rate (67.5 g ha-1 ), was collected from a canola field in Qingsheng County in China. The present study aimed to (1) characterize the SC-R resistance pattern to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides, and (2) investigate the mechanism of quizalofop-p-ethyl resistance in this population. RESULTS: Dose-response studies showed that resistance to quizalofop-p-ethyl and haloxyfop occurred in the SC-R population. Four transcripts/genes encoding the plastidic ACCase carboxyl-transferase domain were isolated from the P. fugax plants. No mutations in the four ACCase genes were detected in the SC-R population compared to the SC-S population. Pre-treatment with the known glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBDCl), reversed resistance to quizalofop-p-ethyl and partially reversed resistance to haloxyfop-R-methyl in the resistant population (SC-R). However, the cytochrome P450 inhibitor malathion did not reverse the resistance. There was no difference in basal GST activity (using CDNB as a substrate), but there was higher inducible GST activity in SC-R relative to SC-S. Two GST genes, GST2c and GSTL3, were constitutively overexpressed in the resistant SC-R population. CONCLUSION: This study confirmed that resistance to quizalofop-p-ethyl in the resistant P. fugax population is likely nontarget-site based involving GST, and this resistance mechanism also partially confers haloxyfop-R-methyl resistance. © 2020 Society of Chemical Industry.

Influence of Multidrug Resistance-Associated Proteins on the Excretion of the ABCC1 Imaging Probe 6-Bromo-7-[11C]Methylpurine in Mice.

PURPOSE: Multidrug resistance-associated proteins (MRPs) mediate the hepatobiliary and renal excretion of many drugs and drug conjugates. The positron emission tomography (PET) tracer 6-bromo-7-[11C]methylpurine is rapidly converted in tissues by glutathione-S-transferases into its glutathione conjugate, and has been used to measure the activity of Abcc1 in the brain and the lungs of mice. Aim of this work was to investigate if the activity of MRPs in excretory organs can be measured with 6-bromo-7-[11C]methylpurine. PROCEDURES: We performed PET scans with 6-bromo-7-[11C]methylpurine in groups of wild-type, Abcc4(-/-) and Abcc1(-/-) mice, with and without pre-treatment with the prototypical MRP inhibitor MK571. RESULTS: 6-Bromo-7-[11C]methylpurine-derived radioactivity predominantly underwent renal excretion. In blood, MK571 treatment led to a significant increase in the AUC and a decrease in the elimination rate constant of radioactivity (kelimination,blood). In the kidneys, there were significant decreases in the rate constant for radioactivity uptake from the blood (kuptake,kidney), kelimination,kidney, and the rate constant for tubular secretion of radioactivity (kurine). Experiments in Abcc4(-/-) mice indicated that Abcc4 contributed to renal excretion of 6-bromo-7-[11C]methylpurine-derived radioactivity. CONCLUSIONS: Our data suggest that 6-bromo-7-[11C]methylpurine may be useful to assess the activity of MRPs in the kidneys as well as in other organs (brain, lungs), although further work is needed to identify the MRP subtypes involved in the disposition of 6-bromo-7-[11C]methylpurine-derived radioactivity.