Enhancement Glutathione Peroxidase Activity and α2-Macroglobulin Gene Expression of Macrobrachium Rosenbergii Fed with Aqueous Morinda Citrifolia Leaves Extract-Supplemented Diet

Morinda citrifolia, known commercially as noni is often used for enhance immunity, these plant rich of phenolic compound with antioxidant properties. In the present study, Macrobrachium rosenbergii were fed with diets containing of aqueous M. citrifolia leaves extract (AMLE) at 0.6, 4 and 6 g kg-1. Glutathione peroxidase (GPx) activity was conducted to measure immune parameter, these parameter was evaluated before and after 7, 21, 35, 49 and 63 days of feeding trial. Immune gene expression of α2-macroglubulin (α2-M) was evaluated in this study. The results showed that after 63 days of feeding treatment, significantly increased in GPx activity. Gene expressions of α2-macroglubulin was significantly upregulated. These results recommend that administration of AMLE can be used as an immunostimulant and regulated immune response and immune gene expression in M. rosenbergii

Insulin-like Growth Factor 1 and Transforming Growth Factor-β Stimulate Cystine/Glutamate Exchange Activity in Dental Pulp Cells

Introduction The growth factors insulin-like growth factor (IGF-1) and transforming growth factor-β (TGF-β) are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line (Henry Schein Inc, New York, NY). Because the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-β were through the enhancement of an endogenous antioxidant mechanism. Methods We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-β, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-). Results We found that the toxicity of Durafill VS and Flow Line was attenuated by the addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis, we found that IGF-1 and TGF-β were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Because levels of cellular cystine are the limiting factor in the production of glutathione, we tested the effects of IGF-1 and TGF-β on cystine uptake. Both growth factors stimulated system xc–mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line. Conclusions The results suggest that IGF-1 and TGF-β are protective through the stimulation of system xc–mediated cystine uptake, leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells

Expression of Heterologous OsDHAR Gene Improves Glutathione (GSH)-Dependent Antioxidant System and Maintenance of Cellular Redox Status in Synechococcus elongatus PCC 7942.

An excess of reactive oxygen species (ROS) can cause severe oxidative damage to cellular components in photosynthetic cells. Antioxidant systems, such as the glutathione (GSH) pools, regulate redox status in cells to guard against such damage. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of oxidized ascorbate (dehydroascorbate) and contains a redox active site and glutathione binding-site. The DHAR gene is important in biological and abiotic stress responses involving reduction of the oxidative damage caused by ROS. In this study, transgenic Synechococcus elongatus PCC 7942 (TA) was constructed by cloning the Oryza sativa L. japonica DHAR (OsDHAR) gene controlled by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter (Ptrc) into the cyanobacterium to study the functional activities of OsDHAR under oxidative stress caused by hydrogen peroxide exposure. OsDHAR expression increased the growth of S. elongatus PCC 7942 under oxidative stress by reducing the levels of hydroperoxides and malondialdehyde (MDA) and mitigating the loss of chlorophyll. DHAR and glutathione S-transferase activity were higher than in the wild-type S. elongatus PCC 7942 (WT). Additionally, overexpression of OsDHAR in S. elongatus PCC 7942 greatly increased the glutathione (GSH)/glutathione disulfide (GSSG) ratio in the presence or absence of hydrogen peroxide. These results strongly suggest that DHAR attenuates deleterious oxidative effects via the glutathione (GSH)-dependent antioxidant system in cyanobacterial cells. The expression of heterologous OsDHAR in S. elongatus PCC 7942 protected cells from oxidative damage through a GSH-dependent antioxidant system via GSH-dependent reactions at the redox active site and GSH binding site residues during oxidative stress

Adaptive response to oxidative stress in the filamentous fungus Aspergillus niger B1-D

In the present study, we used a recombinant filamentous fungus strain, Aspergillus niger B1-D, as a model system, and investigated the antioxidant defences in this organism. Our findings indicate that pretreatment with low concentrations of H2O2 completely prevents killing by this oxidant at high concentrations. It shows that A. niger adapts to exposure to H2O2 by reducing growth and inducing a number of antioxidant enzyme activities, including superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, of which the induction of catalase is the most pronounced. Moreover the decline of these antioxidant enzymes activities after H2O2 detoxification, coincides with recommencement of growth. Results from monitoring the extracellular H2O2 concentration clearly indicate a very rapid detoxification rate for H2O2 in adapted A. niger cultures. A mathematical model predicts only very low concentrations of intracellular H2O2 accumulating in such cultures. Our results also show that glutathione plays a role in the oxidative defence against H2O2 in A. niger. On addition of H2O2, the intracellular pool of glutathione increases while the redox state of glutathione becomes more oxidized

Glutathione-Mediated Neuroprotection Against Methylmercury Neurotoxicity in Cortical Culture is Dependent on MRP1

Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5 μM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30 min and a late increase at 6 h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3 μM) caused an increase in system xc− mediated 14C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system xc−. Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux

Glutathione accelerates sodium channel inactivation in excised rat axonal membrane patches

The effects of glutathione were studied on the gating behaviour of sodium channels in membrane patches of rat axons. Depolarizing pulses from –120 to –40 mV elicited sodium currents of up to 500 pA, indicating the simultaneous activation of up to 250 sodium channels. Inactivation of these channels in the excised, inside-out configuration was fitted by two time constants ( h1=0.81 ms; h2= 5.03 ms) and open time histograms at 0 mV revealed a biexponential distribution of channel openings ( short=0.28 ms; long=3.68 ms). Both, the slow time constant of inactivation and the long lasting single channel openings disappeared after addition of the reducing agent glutathione (2–5 mM) to the bathing solution. Sodium channels of excised patches with glutathione present on the cytoplasmatic face of the membrane had inactivation kinetics similar to channels recorded in the cell-attached configuration. These observations indicate that redox processes may contribute to the gating of axonal sodium channels

Understanding cisplatin resistance using cellular models

Many mechanisms of cisplatin resistance have been proposed from studies of cellular models of resistance including changes in cellular drug accumulation, detoxification of the drug, inhibition of apoptosis and repair of the DNA adducts. A series of resistant models were developed from CCRF-CEM leukaemia cells with increasing doses of cisplatin from 100 ng/ml. This produced increasing resistance up to 7-fold with a treatment dose of 1.6 μg/ml. Cisplatin resistance in these cells correlated with increases in the antioxidant glutathione, yet treatment with buthionine sulphoximine, an inhibitor of glutathione synthesis, had no effect on resistance, suggesting that the increase in glutathione was not directly involved in cisplatin resistance. Two models were developed from H69 SCLC cells, H69-CP and H69CIS200 using 100 ng/ml or 200 ng/ml cisplatin respectively. Both cell models were 2-4 fold resistant to cisplatin, and have decreased expression of p21 which may increase the cell’s ability to progress through the cell cycle in the presence of DNA damage. Both the H69-CP and H69CIS200 cells showed no decrease in cellular cisplatin accumulation. However, the H69-CP cells have increased levels of cellular glutathione and are cross resistant to radiation whereas the H69CIS200 cells have neither of these changes. This suggests that increases in glutathione may contribute to cross-resistance to other drugs and radiation, but not directly to cisplatin resistance. There are multiple resistance mechanisms induced by cisplatin treatment, even in the same cell type. How then should cisplatin-resistant cancers be treated? Cisplatin-resistant cell lines are often more sensitive to another chemotherapeutic drug paclitaxel (H69CIS200), or are able to be sensitised to cisplatin with paclitaxel pre-treatment (H69-CP). The understanding of this sensitisation by paclitaxel using cell models of cisplatin resistance will lead to improvements in the clinical treatment of cisplatin resistant tumours

A reverse chemical ecology approach to explore wood natural durability

The natural durability of wood species, defined as their inherent resistance to wood‐destroying agents, is a complex phenomenon depending on many biotic and abiotic factors. Besides the presence of recalcitrant polymers, the presence of compounds with antimicrobial properties is known to be important to explain wood durability. Based on the advancement in our understanding of fungal detoxification systems, a reverse chemical ecology approach was proposed to explore wood natural durability using fungal glutathione transferases. A set of six glutathione transferases from the white‐rot Trametes versicolor were used as targets to test wood extracts from seventeen French Guiana neotropical species. Fluorescent thermal shift assays quantified interactions between fungal glutathione transferases and these extracts. From these data, a model combining this approach and wood density significantly predicts the wood natural durability of the species tested previously using long‐term soil bed tests. Overall, our findings confirm that detoxification systems could be used to explore the chemical environment encountered by wood‐decaying fungi and also wood natural durability

Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg(257)/Lys(260), and unmasking of acid-base catalysis

Glyoxalase II (GloII) is a ubiquitous thioester hydrolase catalyzing the last step of the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Here, we present a detailed structure-function analysis of cGloII from the malaria parasite Plasmodium falciparum. The activity of the enzyme was salt-sensitive and pH-log k(cat) and pH-log k(cat)/K-m profiles revealed acid-base catalysis. An acidic pK(a)(app) value of approximately 6 probably reflects hydroxide formation at the metal center. The glutathione-binding site was analyzed by site-directed mutagenesis. Substitution of residue Arg(154) caused a 2.5-fold increase of K-m(app), whereas replacements of Arg(257) or Lys(260) were far more detrimental. Although the glutathione-binding site and the catalytic center are separated, six of six single mutations at the substrate-binding site decreased the k(cat)(app) value. Furthermore, product inhibition studies support a Theorell-Chance Bi Bi mechanism with glutathione as the second product. We conclude that the substrate is predominantly bound via ionic interactions with the conserved residues Arg(257) and Lys(260), and that correct substrate binding is a pH-and salt-dependent rate-limiting step for catalysis. The presented mechanistic model is presumably also valid for GloII from many other organisms. Our study could be valuable for drug development strategies and enhances the understanding of the chemistry of binuclear metallohydrolases