Functional characterization of two variant human GSTO 1-1s (Ala140Asp and Thr217Asn)

Glutathione-S-transferase class Omega (GSTO 1-1) belongs to a new subfamily of GSTs, which is identical with human monomethylarsonic acid (MMA(V)) reductase, the rate limiting enzyme for biotransformation of inorganic arsenic, environmental carcinogen. Recombinant GSTO 1-1 variants (Ala140Asp and Thr217Asn) were functionally characterized using representative substrates. No significant difference was observed in GST activity towards 1-chloro-2,4-dinitrobenzene, whereas thioltransferase activity was decreased to 75% (Ala140Asp) and 40% (Thr217Asn) of the wild-type GSTO 1-1. For MMA(V) reductase activity, the Ala140Asp variant exhibited similar kinetics to wild type, while the Thr217Asn variant had lower V(max) (56%) and K(m) (64%) values than the wild-type enzyme. The different activities of the enzyme variants may influence both the intracellular thiol status and arsenic biotransformation. This can help explain the variation between individuals in their susceptibility to oxidative stress and inorganic arsenic.     

Reduced expression of GSTM2 and increased oxidative stress in spontaneously hypertensive rat

Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. The spontaneously hypertensive rat (SHR) is a well-characterized experimental model for essential hypertension. By comparative proteomics, we previously identified glutathione S-transferase, mu 2 (GSTM2), a protein involved in detoxification of reactive oxygen species, which had a significant reduction in left ventricles of 16-week-old SHR compared with WKY rats. In parallel, Western blotting and RT-PCR showed a similar reduction of GSTM2 in left ventricles and aortas of 4-, 8-, and 16-week-old SHR, which is before the onset of hypertension. This suggests that differential expression is not attributable to long-term changes in blood pressure. Meanwhile, the activities of GSTM2 were significantly decreased in different ages old SHR. Conversely, there was an enhanced generation of superoxide anion and activation of NADPH oxidase in SHR, which was accompanied by an increase in the protein expression of p47phox, a subunit of NADPH oxidase. These data suggest that it maybe a reduction in antioxidant defenses, evident by a reduced expression and activity of GSTM2, in the left ventricles and aortas of SHR that leads to increased levels of superoxide anion and activation of NADPH oxidase.     

Relationship between glutathione S-transferase, catalase, oxygen consumption, lipid peroxidation and oxidative stress in eggs and larvae of Boophilus microplus (Acarina: Ixodidae)

Glutathione S-transferases (GSTs) are enzymes that act in excretion of physiologic and xenobiotic substances, protecting cells against chemical toxicity and stress. In this work, we characterized the enzymatic activity of GST in eggs and larvae of cattle tick Boophilus microplus, on different days after oviposition and eclosion. The results showed that the GST activity varied depending on the time elapsed after oviposition and eclosion. Molecules involved in mechanism of protection from oxidative stress are correlated with the increase in GST activity. The oxygen consumption kinetics showed a positive correlation with the increase in GST activity during embryogenesis. A high content of thiobarbituric acid reactive substances were observed in egg and larva extracts, indicating that ticks face high oxidative stress during embryogenesis and aging. In eggs and larvae, GST activity can be correlated to kinetic parameters of oxidative stress such as catalase and glutathione. In addition, GST activity showed strong positive correlation with lipid peroxidation, an indication that it plays a role in oxidant defences in eggs.     

A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses

A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO₃ stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO₃), drought (PEG) and metals (CuCl_2, ZnCl₂, CdCl₂). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g⁻¹ dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu²⁺, Zn²⁺ and Cd²⁺ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl₂, ZnCl₂ or CdCl₂, respectively, compared with control after 6 h culture.     

Nitric oxide alleviates arsenic toxicity by reducing oxidative damage in the roots of Oryza sativa (rice)

Nitric oxide (NO) is a bioactive gaseous, multifunctional molecule playing a central role and mediating a variety of physiological processes and responses to biotic and abiotic stresses including heavy metals. The present study investigated whether NO applied exogenously as sodium nitroprusside (SNP) has any protective role against arsenic (As) toxicity in Oryza sativa (rice). Treatment with 50 μM SNP (a NO donor) significantly ameliorated the As-induced (25 or 50 μM) decrease in root and coleoptile length of rice. Further, As-induced oxidative stress measured in terms of malondialdehyde (MDA), superoxide ion (), root oxidizability and H₂O₂ content was lesser upon supplementation of NO. It indicated a reactive oxygen species (ROS) scavenging activity of NO. NO addition reversed (only partially) the As-induced increase in activities of antioxidant enzymes – superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase. The study concludes that exogenous NO provides resistance to rice against As-toxicity and has an ameliorating effect against As-induced stress.     

RNA-Seq揭示Foc4在外源氧化胁迫(H_2O_2)下的基因表达及细胞代谢变化

尖孢镰刀菌古巴专化型4号小种(Fusarium oxysporum f. sp. cubense race 4,Foc4)是香蕉枯萎病的强致病性病原菌。Foc4在侵染香蕉植株早期必须面对寄主的活性氧迸发。【目的】了解Foc4应对外源氧化胁迫的分子机制。【方法】利用Illumina 2500 RNA-Seq测序平台分析了经外源氧化胁迫(H_2O_2)处理的Foc4与对照在转录组水平的基因表达差异。【结果】在外源氧化胁迫条件下,Foc4的生长受到抑制。转录组测序获得了超过2千万条clean reads。进一步的差异基因表达分析以差异倍数FC (fold change)≥2且FDA值≤0.001为选择标准,发现496个基因表达上调,298个基因表达下调。GO功能富集分析显示,429个基因比对到GO功能分析数据库,在这些差异表达基因中,许多与代谢过程、生物调节、细胞过程和刺激应答有关。KEGG通路富集分析显示,有141个表达差异显著基因比对到KEGG中的50条代谢途径。其中,主要是各类氨基酸代谢途径、脂肪酸代谢途径。同时也包括与抗氧化胁迫直接相关的代谢途径,包括DNA的损伤修复、类胡萝卜素的生物合成、过氧化物酶体、谷胱甘肽代谢等。【结论】这些结果暗示,为了在强氧化胁迫环境下生存,Foc4细胞从包括直接应对氧化胁迫的信号调控途径在内的物质代谢和能量代谢均发生改变以应对环境变化的胁迫。     

Association of glutathione S-transferase gene polymorphisms (GSTM1 and GSTT1) of vitiligo in Korean population

Vitiligo is an acquired pigmentary disorder of the skin involving melanocyte dysfunction. It has been reported that melanocyte impairment could be related to increased oxidative stress. The glutathione S-transferases (GSTs) are group of polymorphic enzymes that are important in protection against oxidative stress. To find the relationship between GSTM1 and GSTT1 polymorphisms with vitiligo susceptibility, GSTM1 and GSTT1 (homozygous deletion vs. non-deleted) polymorphisms between vitiligo patients (n=310) and healthy controls (n=549) were analyzed. We observed significant association in null alleles of the GSTM1 (P<0.001, OR=2.048, 95% CI=1.529-2.743). GSTM1 null type was also statistically different between two vitiligo subtypes and controls (Focal P<0.001, OR=2.224, 95% CI=1.499-3.298; Generalized P=0.001, OR=1.974, 95% CI=1.342-2.904). However, no significant association in GSTT1 (P=0.869, OR=1.024, 95% CI=0.775-1.353) was observed with vitiligo. In combined analysis of GSTM1 and GSTT1, both null type and GSTM1/GSTT1 (null/present) group showed significant differences between controls and vitiligo patients. These results suggest that GSTM1 null type might be associated with vitiligo susceptibility in Korean population.     

Reduction of oxidative stress and liver injury following silymarin and praziquantel treatment in mice with Mesocestoides vogae (Cestoda) infection

Oxidative stress is a common mechanism contributing to hepatic damage and fibrogenesis in a variety of liver disorders. The liver is the target organ for many parasitic infections, hence there is a great demand for the development of novel treatment strategies. In the present study conducted on mice infected with larval stage of Mesocestoides vogae, we investigated effects of therapy with praziquantel (PZQ) alone and in combination with silymarin on liver GSH content, lipid peroxidation and larval reduction. Proliferation of liver cells by means of BrdU incorporation into DNA and production of superoxide anions by peritoneal adherent cells was measured to assess the antioxidant activity of silymarin. Drug administration was carried on from day 15 post infection (p.i.) for ten consecutive days and examination was performed during 20 days of follow-up the therapy. Larval M. vogae infection caused liver damage and triggered extensive oxidative stress, resulting in the abolishment of GSH redox balance and ROS-induced lipid peroxidation. PZQ administration caused short-term decline of GSH levels in healthy mice. Low GSH levels in infected mice were elevated gradually in response to the drug, but respiratory burst in cells was not reduced. Silymarin in combination with PZQ showed strong direct antioxidant capacity and stimulated the larvicidal effect of praziquantel. Treatment with PZQ and silymarin downregulated the generation of superoxide anions, prevented lipid peroxidation, stimulated GSH synthesis and proliferation of hepatocytes in infected livers. These findings demonstrated that silymarin can markedly decrease the liver injury and its co-administration with PZQ potentiate effect of therapy, probably due to the down-regulation of fibrogenesis.     

2-Benzoxazolinone (BOA) induced oxidative stress, lipid peroxidation and changes in some antioxidant enzyme activities in mung bean (Phaseolus aureus)

2-Benzoxazolinone (BOA), a well-known allelochemical with strong phytotoxicity, is a potential herbicidal candidate. The aim of the present study was to determine whether phytotoxicity of BOA is due to induction of oxidative stress caused by generation of reactive oxygen species (ROS) and the changes in levels of antioxidant enzymes induced in response to BOA. Effect of BOA was studied on electrolyte leakage, lipid peroxidation (LP), hydrogen peroxide (H(2)O(2)) generation, proline (PRO) accumulation, and activities of antioxidant enzymes-superoxide dismutase (SOD, 1.15.1.1), ascorbate peroxidase (APX, 1.11.1.11), guaiacol peroxidase (GPX, 1.11.1.7), catalase (CAT, 1.11.1.6) and glutathione reductase (GR, 1.6.4.2) in Phaseolus aureus (mung bean). BOA significantly enhanced malondialdehyde (MDA) content, a product of LP, in both leaves and roots of mung bean. The amount of H(2)O(2), a product of oxidative stress, and endogenous PRO increased many-fold in response to BOA. Accumulation of PRO, MDA and H(2)O(2) indicates the cellular damage in the target tissue caused by ROS generated by BOA. In response to BOA, there was a significant increase in the activities of scavenging enzymes SOD, APX, GPX, CAT, and GR in root and leaf tissue of mung bean. At 5 mM BOA, GR activity in roots showed a nearly 22-fold increase over that in control. The present study concludes that BOA induces oxidative stress in mung bean through generation of ROS and upregulation of activities of various scavenging enzymes.     

Genome-wide analysis of the stress associated protein (SAP) gene family containing A20/AN1 zinc-finger(s) in rice and their phylogenetic relationship with Arabidopsis

Proteins with the A20/AN1 zinc-finger domain are present in all eukaryotes and are well characterized in animals, but little is known about their function in plants. Earlier, we have identified an A20/AN1 zinc-finger containing stress associated protein 1 gene (SAP1) in rice and validated its function in abiotic stress tolerance. In this study, genome-wide survey of genes encoding proteins possessing A20/AN1 zinc-finger, named SAP gene family, has been carried out in rice and Arabidopsis. The genomic distribution and gene architecture as well as domain structure and phylogenetic relationship of encoded proteins numbering 18 and 14 in rice and Arabidopsis, respectively, have been studied. Expression analysis of the rice SAP family was done to investigate their response under abiotic stress conditions. All the genes were inducible by one or the other abiotic stresses indicating that the OsSAP gene family is an important component of stress response in rice. Manipulation of their expression and identification of their superior alleles should help confer stress tolerance in target crops.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A long-term study on interactions between the Adh and αGpdh allozyme polymorphisms and the chromosomal inversion In(2L)t in a seminatural population of D. melanogaster

The Adh and αGpdh allozyme loci (both located on the second chromosome) showed considerable fluctuations in allele frequencies in a seminatural population of Drosophila melanogaster during 1972–97. Both long-term and short-term fluctuations were observed. The short-term fluctuations occurred within almost all years and comparison of allele frequencies between winters and summers showed significantly higher Adh^S (P < 0.001) and αGpdh^F (P < 0.01) allele frequencies in summers. Frequencies of these alleles were significantly positively correlated with environmental temperature, suggesting the adaptive significance of these allozyme polymorphisms. Frequency changes of the Odh locus (located on the third chromosome) showed no seasonal pattern and were not correlated with environmental temperature. Almost all short-term and long-term increases in Adh^S frequency were accompanied by a corresponding decrease in αGpdh^S frequency (r = –0.82, P < 0.001) and vice versa. Further analysis showed that gametic disequilibria between the Adh and αGpdh loci, which frequently occurred, were due to the presence of inversion In(2L)t located on the same chromosome arm and In(2L)t frequencies were positively correlated with environmental temperature. Gametic disequilibria between Adh and Odh and between Odh and αGpdh were hardly observed. Because In(2L)t is exclusively associated with the Adh^S/αGpdh^F allele combination, the observed correlated response in Adh/αGpdh allele frequencies is (at least partly) explained by hitchhiking effects with In(2L)t. This means that the adaptive value of the allozyme polymorphisms has been overestimated by ignoring In(2L)t polymorphism. Fluctuations in Adh allele frequencies are fully explained by selection on In(2L)t polymorphism, whereas we have shown that αGpdh frequency fluctuations are only partly explained by chromosomal hitchhiking, indicating the presence of selective differences among αGpdh genotypes in relation with temperature and independent of In(2L)t. Frequency fluctuations of αGpdh and In(2L)t are consistent with their latitudinal distributions, assuming that temperature is the main environmental factor varying with latitude that causes directly or indirectly these frequency distributions. However, the results of the tropical greenhouse population show no correlation of Adh (independent of In(2L)t) and Odh allele frequencies with environmental temperature, which may indicate that the latitudinal distribution in allele frequencies for these loci is not the result of selection on the F/S polymorphism in a direct way.