Omics for Investigating Chitosan as an Antifungal and Gene Modulator

Chitosan is a biopolymer with a wide range of applications. The use of chitosan in clinical medicine to control infections by fungal pathogens such as Candida spp. is one of its most promising applications in view of the reduced number of antifungals available. Chitosan increases intracellular oxidative stress, then permeabilizes the plasma membrane of sensitive filamentous fungus Neurospora crassa and yeast. Transcriptomics reveals plasma membrane homeostasis and oxidative metabolism genes as key players in the response of fungi to chitosan. A lipase and a monosaccharide transporter, both inner plasma membrane proteins, and a glutathione transferase are main chitosan targets in N. crassa. Biocontrol fungi such as Pochonia chlamydosporia have a low content of polyunsaturated free fatty acids in their plasma membranes and are resistant to chitosan. Genome sequencing of P. chlamydosporia reveals a wide gene machinery to degrade and assimilate chitosan. Chitosan increases P. chlamydosporia sporulation and enhances parasitism of plant parasitic nematodes by the fungus. Omics studies allow understanding the mode of action of chitosan and help its development as an antifungal and gene modulator.

Rearing effect of biofloc on antioxidant and antimicrobial transcriptional response in Litopenaeus stylirostris shrimp facing an experimental sub-lethal hydrogen peroxide stress

This study compares the antioxidant and antimicrobial transcriptional expression of blue shrimps reared according to two different systems, BioFloc Technology (BFT) and Clear sea Water (CW) and their differential responses when facing an experimental sublethal hydrogen peroxide stress. After 30 days of rearing, juvenile shrimps were exposed to H2O2 stress at a concentration of 30 ppm during 6 hours. The oxidative stress caused by H2O2 was examined in the digestive glands of the shrimp, in which antioxidant enzyme (AOE) and antimicrobial peptide (AMP) gene expression were analysed by quantitative real-time PCR. Results showed that rearing conditions did not affect the expression of genes encoding AOEs or AMPs. However, H2O2 stress induced a differential response in expression between shrimps from the two rearing treatments (BFT and CW). Comparative analysis of the expression profiles indicates that catalase transcripts were significantly upregulated by H2O2 stress for BFT shrimps while no change was observed for CW shrimps. In contrast, H2O2 caused down-regulation of superoxide dismutase and glutathione transferase transcripts and of the three AMP transcripts studied (penaeidin 2 and 3, and crustin) for CW shrimps, while no effect was observed on BFT shrimp transcript levels. These results suggested that BFT shrimps maintained antioxidant and AMP responses after stress and therefore can effectively protect their cells against oxidative stress, while CW shrimp immune competence seems to decrease after stress.

Investigation of glutathione S-transferase zeta and the development of sporadic breast cancer

Background Certain genes from the glutathione S-transferase superfamily have been associated with several cancer types. It was the objective of this study to determine whether alleles of the glutathione S-transferase zeta 1 (GSTZ1) gene are associated with the development of sporadic breast cancer. Methods DNA samples obtained from a Caucasian population affected by breast cancer and a control population, matched for age and ethnicity, were genotyped for a polymorphism of the GSTZ1 gene. After PCR, alleles were identified by restriction enzyme digestion and results analysed by chi-square and CLUMP analysis. Results Chi-squared analysis gave a χ2 value of 4.77 (three degrees of freedom) with P = 0.19, and CLUMP analysis gave a T1 value of 9.02 with P = 0.45 for genotype frequencies and a T1 value of 4.77 with P = 0.19 for allele frequencies. Conclusion Statistical analysis indicates that there is no association of the GSTZ1 variant and hence the gene does not appear to play a significant role in the development of sporadic breast cancer.

Polymorphisms of glutathione S-transferase genes (GSTM1, GSTP1 and GSTT1) and breast cancer susceptibility

The glutathione S-transferase (GST) family of enzymes function in the body to detoxify carcinogenic compounds. Several genes that code for these enzymes are polymorphic, with particular genotypes previously shown to confer an increased cancer risk. In this study, we investigated the role of three GST genes (GSTM1, GSTP1 and GSTT1) in the development of sporadic breast cancer. Genotypes were determined in 129 breast cancer affected and 129 age and sex matched control individuals. Results did not support an involvement of these specific GST gene polymorphisms, either independently or in combination, in susceptibility to sporadic breast cancer in the tested Australian Caucasian population.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

The acute effects of microcystin LR on the transcription of nine glutathione S-transferase genes in common carp Cyprinus carpio L.

The glutathione S-transferases play important roles in the detoxification of microcystin. In this experiment, nine glutathione S-transferase genes including cytosolic GSTs (rho, mu, theta, alpha and pi), mitochondrial GST (kappa) and microsomal GSTs (mGST1, mGST2 and mGST3) were cloned from common carp Cyprinus carpio. The mRNA abundance of each carp GST isoform in liver was analyzed by real time PCR. The relative changes after stimulation with microcystin LR were also analyzed: increased levels of transcription of GST alpha, rho and mGST3 isoforms were detected at 6 h post stimulation; the transcription of mu, theta and mGST2 isoforms were relatively stable; and all the GST isoforms except GST kappa and rho recovered to original levels compared with controls at 72 h. It is suggested that MC-LR showed different effects on the transcription of nine carp GST isoforms. (c) 2006 Elsevier B.V. All rights reserved.

PLLA-PCys co-electrospun fibers for capture and elution of glutathione S-transferase

The copolymer poly(L-lactic acid)-b-poly(L-cysteine) (PLA-b-PCys) was co-electrospun with PLGA into ultrafine fibers. The reduced glutathione (GSH) was conjugated to the fiber surfaces via disulfide bonds. The glutathione S-transferase (GST) was captured onto the GSH fibers via specific substrate-enzyme interaction between the bound GSH and GST. The captured GST was eluted with free GSH aqueous solution and lyophilized to get pure GST powders. The results show that the GSH moieties on the fiber surface retain the bioactivity of the free GSH and thus they can bind specifically with GST and the GST in solution is captured onto the fiber surface. In addition, the bound GSH is not as active as free GSH so that the captured GST can be eluted off from the fiber by free GSH aqueous solution. Based on this principle, GST itself or its fused proteins can be separated and purified very easily. The preliminary purification efficiency is 6.5 mg center dot(g(PCys))(-1). Further improvements are undertaken.

Determination of the molecular weight distribution of non-enzymatic browning products formed by roasting of glucose and glycine and studies on their effects on NADPH-cytochrome c-reductase and glutathione-S-transferase in Caco-2 cells

After thermal treatment of a mixture of glucose and glycine for 2 h at 125 degreesC, about 60% of the starting material was converted into nonsoluble, black pigments, whereas 40% of the mixture was still water-soluble. Dialysis of the latter fraction revealed 30.4% of low molecular weight compounds (LMWs; MW <10 000 De) and 10.0% high-molecular weight products [HMWs; MW greater than or equal to 10000 Dal. The water-soluble Maillard reaction products (MRPs) were separated by gel permeation chromatography and ultrafiltration, revealing that 60% of the water-soluble products of the total carbohydrate/amino acid mixture had MWs <1 000 Da and consisted mainly of non-coloured reaction products. MRPs with MWs between 1000 and 30000 Da were Found in comparatively low yields (about 1.3%). In contrast, about 31.1% of the MRPs exhibited MWs > 30000 Da, amongst which 14.5% showed MWs > 100000 Da, thus indicating an oligomerisation of LMWs to melanoidins under roasting conditions. To investigate the physiological effects of these MRPs, xenobiotic enzyme activities were analysed in intestinal Caco-2 cells. For Phase-I NADPH-cytochrome c-reductase, the activity in the presence of the LMW and HMW fraction was decreased by 13% and 22%: respectively. Phase-II glutathione-S-transferase activity decreased by 15% and 18%, respectively, after incubation with the LMW and the HMW fractions. Considering the different yields, 30% and 10%, respectively, of the LMW and the HMW fractions, the total amount of the LMW fraction present in the glucose-glycine mixture is more active in modulating three enzyme activities than that of the HMW fraction.

Novel glutathione disulfide transferase function of CC-glutaredoxins involved in disease resistance and flower development

Glutaredoxins are oxidoreductases capable of reducing protein disulfide bridges and glutathione mixed disulfides through the process of deglutathionylation and glutathionylation. Lately, redox-mediated modifications of functional cysteine residues of TGA1 and TGA8 transcription factors have been postulated. Namely, GRX480 and ROXY1 glutaredoxins have been previously shown to interact with TGA proteins and have been suggested to regulate redox state of these proteins. TGA1, together with TGA2, is involved in systemic acquired resistance (SAR) establishment in the plant Arabidopsis thaliana through PR1 (Pathogenesis related 1) gene activation. They both form an enhanceosome complex with the NPR1 protein (non-expressor of pathogenesis related gene 1) which leads to PR1 transcription. Although TGA1 is capable of activating PR1 transcription, the ability of the TGA1 NPR1 enhanceosome complex to assembly is based on the redox status of TGA1. We identified GRX480 as a glutathionylating enzyme that catalyzes the TGA1 glutathione disulfide transferase reaction with a Km of around 20μM GSSG (oxidized glutathione). Out of four cysteine residues found within TGA1, C172 and C266 were found to be glutathionylated by this enzyme. We also confirmed TGA1 glutathionylation in vivo and showed that this modification takes place while TGA1 is associated with the PR1 promoter enzymatically via GRX480. Furthermore, we show that glutathionylation via GRX480 abolishes TGA1's interaction with NPR1 and consequently prevents the TGA1-NPR1 transcription activation of PR1. When glutathionylated, TGA1 is recruited to the PR1 promoter and acts as a repressor. Therefore, glutathionylation is a mechanism that prevents TGA1 NPR1 interaction, allowing TGA1 to function as a repressor of PR1 transcription. Surprisingly, GRX480 was not able to deglutathionylate proteins demonstrating the irreversible nature of the reaction. Moreover, we demonstrate that other members of CC-class glutaredoxins, namely ROXY1 and ROXY2, can also catalyze protein glutathionylation. The TGA8 protein was previously shown to interact with NPR1 analogs, BOP1 and BOP2 proteins. However, unlike the case of TGA1 NPR1 interaction, here we demonstrate that TGA8-BOP1 interaction is not redox regulated and that TGA8 glutathionylation by ROXY1 and ROXY2 enzymes does not abolish this interaction in vitro. However, TGA8 glutathionylation results in TGA8 oligomer disassembly into smaller complexes and monomers. Our results suggest that CC-Grxs are unable to reduce mixed disulfides, instead they efficiently catalyze the opposite reaction which distinguishes them from traditional glutaredoxins. Therefore, they should not be classified as glutaredoxins but as protein glutathione disulfide transferases.

Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system

Epidemiological studies have shown that ingestion of isoflavone-rich soy products is associated with a reduced risk for the development of breast cancer. In the present study, we investigated the hypothesis that genistein modulates the expression of glutathione S-transferases (GSTs) in human breast cells, thus conferring protection towards genotoxic carcinogens which are GST substrates. Our approach was to use human mammary cell lines MCF-10A and MCF-7 as models for non-neoplastic and neoplastic epithelial breast cells, respectively. MCF-10A cells expressed hGSTA1/2, hGSTA4-4, hGSTM1-1 and hGSTP1-1 proteins, but not hGSTM2-2. In contrast, MCF-7 cells only marginally expressed hGSTA1/2, hGSTA4-4 and hGSTM1-1. Concordant to the protein expression, the hGSTA4 and hGSTP1 mRNA expression was higher in the non-neoplastic cell line. Exposure to genistein significantly increased hGSTP1 mRNA (2.3-fold), hGSTP1-1 protein levels (3.1-fold), GST catalytic activity (4.7-fold) and intracellular glutathione concentrations (1.4-fold) in MCF-10A cells, whereas no effects were observed on GST expression or glutathione concentrations in MCF-7 cells. Preincubation of MCF-10A cells with genistein decreased the extent of DNA damage by 4-hydroxy-2-nonenal (150 mu M) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (50 mu M), compounds readily detoxified by hGSTA4-4 and hGSTP1-1. In conclusion, genistein pretreatment protects non-neoplastic mammary cells from certain carcinogens that are detoxified by GSTs, suggesting that dietary-mediated induction of GSTs may be a mechanism contributing to prevention against genotoxic injury in the aetiology of breast cancer.

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole and ivermectin in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.

The effect of sixteen medicinal plants used in the Brazilian pharmacopoeia on the expression and activity of glutathione S-transferase in hepatocytes and leukemia cells

Glutathione S-transferase (GST) is a family of enzymes involved in the detoxification of electrophilic compounds. Different classes of GST are expressed in various organs, such as liver, lungs, stomach and others. Expression of GST can be modulated by diet components and plant-derived compounds. The importance of controlling GST expression is twofold: increasing levels of GST are beneficial to prevent deleterious effects of toxic and carcinogenic compounds, while inhibition of GST in tumor cells may help overcoming tumor resistance to chemotherapy. A screening of 16 plants used in the Brazilian pharmacopoeia tested their effects on GST expression in hepatocytes and Jurkat (leukemia) T-cells. The methanol extracts of five plants inhibited GST expression in hepatocytes. Three plants significantly inhibited and four others induced GST expression in Jurkat cells. Among these, the extracts of Bauhinia forficata Link. (Leguminosae) and Cecropia pachystachya Trec. (Urticaceae) inhibited GST expression at relatively low concentrations. With the exception of B. forficata, all plants were cytotoxic when administered to Jurkat cells at high doses (1 mg/mL) and some extracts were considerably cytotoxic even at lower concentrations.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.