Effects of selenoprotein extracts from Cardamine hupingshanensis on growth, selenium metabolism, antioxidant capacity, immunity and intestinal health in largemouth bass Micropterus salmoides.

This study aimed to assess the impact of dietary selenoprotein extracts from Cardamine hupingshanensis (SePCH) on the growth, hematological parameters, selenium metabolism, immune responses, antioxidant capacities, inflammatory reactions and intestinal barrier functions in juvenile largemouth bass (Micropterus salmoides). The base diet was supplemented with four different concentrations of SePCH: 0.00, 0.30, 0.60 and 1.20 g/Kg (actual selenium contents: 0.37, 0.59, 0.84 and 1.30 mg/kg). These concentrations were used to formulate four isonitrogenous and isoenergetic diets for juvenile largemouth bass during a 60-day culture period. Adequate dietary SePCH (0.60 and 1.20 g/Kg) significantly increased weight gain and daily growth rate compared to the control groups (0.00 g/Kg). Furthermore, 0.60 and 1.20 g/Kg SePCH significantly enhanced amounts of white blood cells, red blood cells, platelets, lymphocytes and monocytes, and levels of hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin in the hemocytes. In addition, 0.60 and 1.20 g/Kg SePCH increased the mRNA expression levels of selenocysteine lyase, selenophosphate synthase 1, 15 kDa selenoprotein, selenoprotein T2, selenoprotein H, selenoprotein P and selenoprotein K in the fish liver and intestine compared to the controls. Adequate SePCH not only significantly elevated the activities of antioxidant enzymes (Total superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase), the levels of total antioxidant capacity and glutathione, while increased mRNA transcription levels of NF-E2-related factor 2, Cu/Zn-superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. However, adequate SePCH significantly decreased levels of malondialdehyde and H2O2 and the mRNA expression levels of kelch-like ECH-associated protein 1a and kelch-like ECH-associated protein 1b in the fish liver and intestine compared to the controls. Meanwhile, adequate SePCH markedly enhanced the levels of immune factors (alkaline phosphatase, acid phosphatase, lysozyme, complement component 3, complement component 4 and immunoglobulin M) and innate immune-related genes (lysozyme, hepcidin, liver-expressed antimicrobial peptide 2, complement component 3 and complement component 4) in the fish liver and intestine compared to the controls. Adequate SePCH reduced the levels of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin 8, interleukin 1ß and interferon γ), while increasing transforming growth factor ß1 levels at both transcriptional and protein levels in the liver and intestine. The mRNA expression levels of mitogen-activated protein kinase 13 (MAPK 13), MAPK14 and nuclear factor kappa B p65 were significantly reduced in the liver and intestine of fish fed with 0.60 and 1.20 g/Kg SePCH compared to the controls. Histological sections also demonstrated that 0.60 and 1.20 g/Kg SePCH significantly increased intestinal villus height and villus width compared to the controls. Furthermore, the mRNA expression levels of tight junction proteins (zonula occludens-1, zonula occludens-3, Claudin-1, Claudin-3, Claudin-5, Claudin-11, Claudin-23 and Claudin-34) and Mucin-17 were significantly upregulated in the intestinal epithelial cells of 0.60 and 1.20 g/Kg SePCH groups compared to the controls. In conclusion, these results found that 0.60 and 1.20 g/Kg dietary SePCH can not only improve growth, hematological parameters, selenium metabolism, antioxidant capacities, enhance immune responses and intestinal functions, but also alleviate inflammatory responses. This information can serve as a useful reference for formulating feeds for largemouth bass.

Glutathione Reductase Expression and Its Prognostic Significance in Colon Cancer.

Maintaining a balanced redox state within cells is crucial for the sustenance of life. The process involves continuous cytosolic disulfide reduction reactions to restore oxidized proteins to their reduced thiol forms. There are two main cellular antioxidant pathways-the thioredoxin (Trx) and glutathione (GSH)/glutaredoxin (Grx) systems. In the GSH/Grx system, glutathione reductase (GR; GSR) catalyses the reduction of GSH disulfide (GSSG) to its sulfhydryl form (GSH), which can then further reduce oxidized Grxs. GR is an essential enzyme that helps in maintaining the supply of reduced glutathione-GSH, which is a significant reducing thiol found in most cells and known for its antioxidant properties. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of GR protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of GR and tumour histological grade, depth of invasion, regional lymph node involvement, staging, and PCNA immunohistochemical expression. It was found that 95% of patients with stage I had low levels of GR expression, whereas 89% of patients with stage III had high levels of immunohistochemical expression. A high level of expression was also detected in the patients with stage II of the disease, where almost 63% were characterized by a high expression of GR. The Western blot method revealed that the highest level of expression was found in the LS 174T cell line, which corresponds to stage II. The results of our study indicate that the immunohistochemical expression of GR may act as an independent prognostic factor associated with colon adenocarcinoma patients' prognosis.

Redox perturbations in yeast cells lacking glutathione reductase.

Cellular redox homeostasis has a major effect on cell functions and its maintenance is supported by glutathione and protein thiols which serve as redox buffers in cells. The regulation of the glutathione biosynthetic pathway is a focus of a lot of scientific research. However, still little is known about how complex cellular networks influence glutathione homeostasis. In this work was used an experimental system based on an S. cerevisiae yeast mutant with a lack of the glutathione reductase enzyme and allyl alcohol as a precursor of acrolein inside the cell to determine the cellular processes influencing glutathione homeostasis. The absence of Glr1p slows down the growth rate of the cell population, especially in the presence of allyl alcohol, but does not lead to complete inhibition of the cell's reproductive capacity. It also amends the GSH/GSSG ratio and the share of NADPH and NADP+ in the total NADP(H) pool. The obtained results show that potential pathways involved in the maintenance of redox homeostasis are based from one side on de novo synthesis of GSH as indicated by increased activity of γ-GCS and increased expression of GSH1 gene in the Δglr1 mutant, from the other hand, on increased the level of NADPH. This is because the lower ratio of GSH/GSSG can be counterbalanced with the NADPH/NADP+ alternative system. The higher level of NADPH can be used by the thioredoxin system and other enzymes requiring NADPH to reduce cytosolic GSSG and maintain glutathione redox potential.

Overexpression of tomato glutathione reductase (SlGR) in transgenic tobacco enhances salt tolerance involving the S-nitrosylation of GR.

S-nitrosylation, a post-translational modification (PTM) dependent on nitric oxide, is essential for plant development and environmental responsiveness. However, the function of S-nitrosylation of glutathione reductase (GR) in tomato (SlGR) under NaCl stress is yet uncertain. In this study, sodium nitroprusside (SNP), an exogenous NO donor, alleviated the growth inhibition of tomato under NaCl treatment, particularly at 100 µM. Following NaCl treatment, the transcripts, enzyme activity, and S-nitrosylated level of GR were increased. In vitro, the SlGR protein was able to be S-nitrosylated by S-nitrosoglutathione (GSNO), significantly increasing the activity of GR. SlGR overexpression transgenic tobacco plants exhibited enhanced germination rate, fresh weight, and increased root length in comparison to wild-type (WT) seedlings. The accumulation of reactive oxygen species (ROS) was lower, whereas the expression and activities of GR, ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT); the ratio of ascorbic acid/dehydroascorbic acid (AsA/DHA), reduced glutathione/oxidized glutathione (GSH/GSSG), total soluble sugar and proline contents; and the expression of stress-related genes were higher in SlGR overexpression transgenic plants in comparison to the WT plants following NaCl treatment. The accumulation of NO and S-nitrosylated levels of GR in transgenic plants was higher in comparison to WT plants following NaCl treatment. These results indicated that S-nitrosylation of GR played a significant role in salt tolerance by regulating the oxidative state.

Molecular cloning of glutathione reductase from Oryza sativa, demonstrating its peroxisomal localization and upregulation by abiotic stresses.

Abiotic stress is a major constraint on crop productivity and in the agricultural field, multiple abiotic stresses act synchronously leading to substantial damage to plants. A common after-effect of abiotic stress-induced damage in plants is an increased concentration of reactive oxygen species (ROS) leading to oxidative damage. Glutathione reductase (GR) plays a significant role in curtailing ROS. Apart from the GR enzyme, the peroxisome as an organelle also plays a significant role in ROS homeostasis. Here, we report a peroxisome localized GR, whose expression was found to be upregulated by various abiotic stresses. The in silico analysis also revealed that the peroxisomal localization of GR could be a common phenomenon in angiosperms, suggesting that it could be a suitable candidate against abiotic stress combinations.

Functional expression, localization, and biochemical characterization of thioredoxin glutathione reductase from air-breathing magur catfish, Clarias magur.

The glutathione (GSH) and thioredoxin (Trx) systems regulate cellular redox homeostasis and maintain antioxidant defense in most eukaryotes. We earlier reported the absence of gene coding for the glutathione reductase (GR) enzyme of the GSH system in the facultative air-breathing catfish, Clarias magur. Here, we identified three thioredoxin reductase (TrxR) genes, one of which was later confirmed as a thioredoxin glutathione reductase (TGR). We then characterized the novel recombinant TGR enzyme of C. magur (CmTGR). The tissue-specific expression of the txnrd genes and the tissue-specific activity of the TrxR enzyme were analyzed. The recombinant CmTGR is a dimer of ~133 kDa. The protein showed TrxR activity with 5,5'-diothiobis (2-nitrobenzoic acid) reduction assay with a Km of 304.40 µM and GR activity with a Km of 58.91 µM. Phylogenetic analysis showed that the CmTGR was related to the TrxRs of fishes and distantly related to the TGRs of platyhelminth parasites. The structural analysis revealed the conserved glutaredoxin active site and FAD- and NADPH-binding sites. To our knowledge, this is the first report of the presence of a TGR in any fish. This unusual presence of TGR in C. magur is crucial as it helps maintain redox homeostasis under environmental stressors-induced oxidative stress.

Induced defense responses in cultivated and wild chickpea genotypes against Helicoverpa armigera infestation.

Five desi (GL 12,021, GL 29,095, GL 29,078, H11 22 and CSJ 515) and three wild (GLW 22, GLW 58 and GLW 187) chickpea cultivars showed induced defense response against Helicoverpa armigera infestation as a result of enhanced activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione reductase, polyphenol oxidase, phenylalanine ammonia lyase, tyrosine ammonia lyase in leaves, pod walls and seeds. Catalase activity increased in leaves of GL 12,021, H11 22, GL 29,095, CSJ 515, GLW 22, and GL 29,078 after infestation compared to resistant check; catalase and peroxidase activities in GL 29,095 and GL 29,078; ascorbate peroxidase and glutathione reductase activities in leaves of GLW 58. The increased activity of superoxide dismutase in pod wall of H1122; catalase in pod wall of 29,078, GL 29,095 and GL 22; ascorbate peroxidase and glutathione reductase in pod wall of GLW 58; phenylalanine ammonia lyase and tyrosine ammonia lyase in pod wall of GLW 187, H11 22, GL 20,978, GLW 22 and GLW 58 after infestation as compared to resistant check might be responsible for mitigating infestation induced oxidative stress. MDA content decreased in leaves, pod wall and seeds of GLW 187 and GL 12,021 after infestation. Lower percent pod damage (9.58-12.44%) in GL 12,021, GLW 187, GL 29,095, H11 22, GL 29,078, GLW 22 and GLW 58 as compared to resistant (16.18%) and susceptible (21.50) checks might be attributed to differential induced defense mechanism in them. The identified desi and wild genotypes might be used in breeding program to develop cultivars with improved resistance to herbivore.

Depletion of Tip60/Kat5 affects the hepatic antioxidant system in mice.

Tat-interactive protein 60 kDa (TIP60, also known as lysine acetyltransferase 5 [KAT5]) is a member of the MYST protein family with histone acetyltransferase activity. Recent studies have reported that TIP60 has multiple functions in many signal transduction mechanisms, especially p53-mediated apoptosis. Although the activation of apoptosis signaling pathways requires the presence of cellular reactive oxygen species (ROS) at a certain level, an imbalance between the production and consumption of ROS in cells results in oxidative stress (OS). In this study, we investigated for the first time how the absence of the Tip60 gene in the liver affects gene expression, enzyme activity, and protein expression of the hepatic antioxidant members localized in the cytoplasm, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). First, we successfully generated liver-specific Tip60 knockout mice (mutants) using Cre/LoxP recombination. The reduced glutathione level and nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, a marker of OS, increased significantly in the Tip60 mutant liver. Gene expression, activity, and protein expression of the enzymatic antioxidant system, including SOD, CAT, GR, GPx, and GST were investigated in mutants and control groups. Despite a significant correlation between the gene, enzyme activity, and protein content for CAT and GR, this was not true for SOD and GPx. The overall results suggest that TIP60 acts on the hepatic antioxidant system both at the gene and protein levels, but the actual effect of the deletion of Tip60 is observed at the protein level, especially for SOD and GPx.

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra.

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Elucidating the genetic architecture controlling antioxidant status and ionic balance in barley under salt stress.

KEY MESSAGE: Association genetic analysis empowered us to identify candidate genes underlying natural variation of morpho-physiological, antioxidants, and grain yield-related traits in barley. Novel intriguing genomic regions were identified and dissected. Salinity stress is one of the abiotic stresses that influence the morpho-physiological, antioxidants, and yield-related traits in crop plants. The plants of a core set of 138 diverse barley accessions were analyzed after exposure to salt stress under field conditions during the reproductive phase. A genome-wide association scan (GWAS) was then conducted using 19,276 single nucleotide polymorphisms (SNPs) to uncover the genetic basis of morpho-physiological and grain-related traits. A wide range of responses to salt stress by the accessions was explored in the current study. GWAS detected 263 significantly associated SNPs with the antioxidants, K+/Na+ content ratio, and agronomic traits. Five genomic regions harbored interesting putative candidate genes within LD ± 1.2 Mbp. Choromosome 2H harbored many candidate genes associated with the antioxidants ascorbic acid (AsA) and glutathione (GSH), such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), under salt stress. Markedly, an A:C SNP at 153,773,211 bp on chromosome 7H is located inside the gene HORVU.MOREX.r3.7HG0676830 (153,772,300-153,774,057 bp) that was annotated as L-gulonolactone oxidase, regulating the natural variation of SOD_S and APX_S. The allelic variation at this SNP reveals a negative selection of accessions carrying the C allele, predominantly found in six-rowed spring landraces originating from Far-, Near-East, and central Asia carrying photoperiod sensitive alleles having lower activity of enzymatic antioxidants. The SNP-trait associations detected in the current study constitute a benchmark for developing molecular selection tools for antioxidant compound selection in barley.

Mitochondrial DNA insertions into nuclear DNA affecting chromosome segregation: Insights for a novel mechanism of immunosenescence in mice.

Mitochondrial DNA sequences were found inserted in the nuclear genome of mouse peritoneal T lymphocytes that increased progressively with aging. These insertions were preferentially located at the pericentromeric heterochromatin. In the same individuals, binucleated T-cells with micronuclei showed a significantly increased frequency associated with age. Most of them were positive for centromere sequences, reflecting the loss of chromatids or whole chromosomes. The proliferative capacity of T lymphocytes decreased with age as well as the glutathione reductase activity, whereas the oxidized glutathione and malondialdehyde concentrations exhibited a significant increase. These results may point to a common process that provides insights for a new approach to understanding immunosenescence. We propose a novel mechanism in which mitochondrial fragments, originated by the increased oxidative stress status during aging, accumulate inside the nuclear genome of T lymphocytes in a time-dependent way. The primary entrance of mitochondrial fragments at the pericentromeric regions may compromise chromosome segregation, causing genetic loss that leads to micronuclei formation, rendering aneuploid cells with reduced proliferation capacity, one of the hallmark of immunosenescence. Future experiments deciphering the mechanistic basis of this phenomenon are needed.

Identification, analysis of deleterious SNPs of the human GSR gene and their effects on the structure and functions of associated proteins and other diseases.

Hereditary glutathione reductase deficiency, caused by mutations of the GSR gene, is an autosomal recessive disorder characterized by decreased glutathione disulfide (GSSG) reduction activity and increased thermal instability. This study implemented computational analysis to screen the most likely mutation that might be associated with hereditary glutathione reductase deficiency and other diseases. Using ten online computational tools, the study revealed four nsSNPs among the 17 nsSNPs identified as most deleterious and disease associated. Structural analyses and evolutionary confirmation study of native and mutant GSR proteins using the HOPE project and ConSruf. HOPE revealed more flexibility in the native GSR structure than in the mutant structure. The mutation in GSR might be responsible for changes in the structural conformation and function of the GSR protein and might also play a significant role in inducing hereditary glutathione reductase deficiency. LD and haplotype studies of the gene revealed that the identified variations rs2978663 and rs8190955 may be responsible for obstructive heart defects (OHDs) and hereditary anemia, respectively. These interethnic differences in the frequencies of SNPs and haplotypes might help explain the unpredictability that has been reported in association studies and can contribute to predicting the pharmacokinetics and pharmacodynamics of drugs that make use of GSR.

The role of Acanthamoeba castellanii (T4 genotype) antioxidant enzymes in parasite survival under H2O2-induced oxidative stress.

Acanthamoeba castellanii (A. castellanii) is an important opportunistic parasite. Induction of oxidative stress by the host immune system is one of the most important defense strategies against parasites. Hence, parasites partly deal with oxidative stress by different mechanisms. Identifying resistance mechanisms of A. castellanii parasites against oxidative stress is important to achieve a new therapeutic approach. Thus, this study aimed to understand the resistance mechanisms of A. castellanii, against oxidative stress. Trophozoites of A. castellanii were treated with different concentrations of H2O2. The half maximal inhibitory concentration (IC50) of H2O2 was determined using the MTT assay. The induction of oxidative stress was confirmed by flow cytometer. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined. The gene expression levels of CAT and SOD were measured by qRT-PCR. Furthermore, 3-amino-1:2:4-triazole (3-AT) and potassium cyanide (KCN) were used as specific inhibitors of CAT and SOD, respectively. Cell cycle assay and the apoptosis were evaluated by flow cytometer. The activities of SOD, CAT, GR, and GPx, showed an increase in oxidative stress. The cell cycle analysis revealed that most of the cellular population was in G0 and G1 phases. The apoptosis increased in oxidative stress conditions. Moreover, the apoptosis significantly increased after the specific inhibition of CAT and SOD under oxidative stress. The gene expression levels of CAT and SOD significantly increased under oxidative stress. A. castellanii can resist the host immune system through various mechanisms, including evoking its antioxidant enzymes. Therefore, by reducing or inhibiting the activity of the parasite's antioxidant enzymes such as SOD and CAT, it is possible to cope with A. castellanii.

ADH2/GSNOR1 is a key player in limiting genotoxic damage mediated by formaldehyde and UV-B in Arabidopsis.

Maintenance of genome stability is an essential requirement for all living organisms. Formaldehyde and UV-B irradiation cause DNA damage and affect genome stability, growth and development, but the interplay between these two genotoxic factors is poorly understood in plants. We show that Arabidopsis adh2/gsnor1 mutant, which lacks alcohol dehydrogenase 2/S-nitrosoglutathione reductase 1 (ADH2/GSNOR1), are hypersensitive to low fluence UV-B irradiation or UV-B irradiation-mimetic chemicals. Although the ADH2/GSNOR1 enzyme can act on different substrates, notably on S-hydroxymethylglutathione (HMG) and S-nitrosoglutathione (GSNO), our study provides several lines of evidence that the sensitivity of gsnor1 to UV-B is caused mainly by UV-B-induced formaldehyde accumulation rather than other factors such as alteration of the GSNO concentration. Our results demonstrate an interplay between formaldehyde and UV-B that exacerbates genome instability, leading to severe DNA damage and impaired growth and development in Arabidopsis, and show that ADH2/GSNOR1 is a key player in combating these effects.

Exploration of glutathione reductase for abiotic stress response in bread wheat (Triticum aestivum L.).

KEY MESSAGE: A total of seven glutathione reductase (GR) genes were identified in Triticum aestivum, which were used for comparative structural characterization, phylogenetic analysis and expression profiling with the GR genes of other cereal plants. The modulated gene expression and enzyme activity revealed the role of GRs in abiotic stress response in T. aestivum. Glutathione reductase (GR) is an enzymatic antioxidant that converts oxidized glutathione (GSSG) into reduced glutathione (GSH) through the ascorbate-glutathione cycle. In this study, a total of seven GR genes forming two homeologous groups were identified in the allohexaploid genome of bread wheat (Triticum aestivum). Besides, we identified three GR genes in each Aegilops tauschii, Brachypodium distachyon, Triticum urartu and Sorghum bicolor, which were used for comparative characterization. Phylogenetic analysis revealed the clustering of GR proteins into two groups; class I and class II, which were predicted to be localized in cytoplasm and chloroplast, respectively. The exon-intron and conserved motif patterns were almost conserved in each group, in which a maximum of 10 and 17 exons were present in chloroplastic and cytoplasmic GRs, respectively. The protein structure analysis confirmed the occurrence of conserved pyridine nucleotide disulfide oxidoreductase (Pyr_redox) and pyridine nucleotide disulfide oxidoreductase dimerization (Pyr_redox_dim) domains in each GR. The active site of GR proteins consisted of two conserved cysteine residues separated by four amino acid residues. Promoter analysis revealed the occurrence of growth and stress-related cis-active elements. Tissue-specific expression profiling suggested the involvement of GRs in both vegetative and reproductive tissue development in various plants. The differential expression of TaGR genes and enhanced GR enzyme activity suggested their roles under drought, heat, salt and arsenic stress. Interaction of GRs with other proteins and chemical compounds of the ascorbate-glutathione cycle revealed their coordinated functioning. The current study will provide a foundation for the validation of the precise role of each GR gene in future studies.

Short-term effects of cadmium on leaf growth and nutrient transport in rice plants.

Consumption of rice grains contaminated with high concentrations of cadmium (Cd) can cause serious long-term health problems. Moreover, even low Cd concentrations present in the soil can result in the abatement of plant performance, leading to lower grain yield. Studies examining the molecular basis of plant defense against Cd-induced oxidative stress could pave the way in creating superior rice varieties that display an optimal antioxidative defense system to cope with Cd toxicity. In this study, we showed that after one day of Cd exposure, hydroponically grown rice plants exhibited adverse shoot biomass and leaf growth effects. Cadmium accumulates especially in the roots and the leaf meristematic region, leading to a disturbance of manganese homeostasis in both the roots and leaves. The leaf growth zone showed an increased amount of lipid peroxidation indicating that Cd exposure disturbed the oxidative balance. We propose that an increased expression of genes related to the glutathione metabolism such as glutathione synthetase 2, glutathione reductase and phytochelatin synthase 2, rather than genes encoding for antioxidant enzymes, is important in combating early Cd toxicity within the leaves of rice plants. Furthermore, the upregulation of two RESPIRATORY BURST OXIDASE HOMOLOG genes together with a Cd concentration-dependent increase of abscisic acid might cause stomatal closure or cell wall modification, potentially leading to the observed leaf growth reduction. Whereas abscisic acid was also elevated at long term exposure, a decrease of the growth hormone auxin might further contribute to growth inhibition and concomitantly, an increase in salicylic acid might stimulate the activity of antioxidative enzymes after a longer period of Cd exposure. In conclusion, a clear interplay between phytohormones and the oxidative challenge affect plant growth and acclimation during exposure to Cd stress.

Functioning of the Antioxidant Defense System in Rotenone-Induced Parkinson's Disease.

A comprehensive study of the functioning of antioxidant system in rats with rotenone-induced parkinsonism was conducted. The development of pathology led to inhibition of the majority of the studied antioxidant enzymes in the brain and blood serum of animals, which can be associated with decompensation of oxidative stress under conditions of prolonged mitochondrial dysfunction. These changes apparently make an important contribution into neuronal degeneration in the cerebral cortex and striatum and motor disorders in experimental animals.

Micro RNAs in Regulation of Cellular Redox Homeostasis.

In living cells Reactive Oxygen Species (ROS) participate in intra- and inter-cellular signaling and all cells contain specific systems that guard redox homeostasis. These systems contain both enzymes which may produce ROS such as NADPH-dependent and other oxidases or nitric oxide synthases, and ROS-neutralizing enzymes such as catalase, peroxiredoxins, thioredoxins, thioredoxin reductases, glutathione reductases, and many others. Most of the genes coding for these enzymes contain sequences targeted by micro RNAs (miRNAs), which are components of RNA-induced silencing complexes and play important roles in inhibiting translation of their targeted messenger RNAs (mRNAs). In this review we describe miRNAs that directly target and can influence enzymes responsible for scavenging of ROS and their possible role in cellular redox homeostasis. Regulation of antioxidant enzymes aims to adjust cells to survive in unstable oxidative environments; however, sometimes seemingly paradoxical phenomena appear where oxidative stress induces an increase in the levels of miRNAs which target genes which are supposed to neutralize ROS and therefore would be expected to decrease antioxidant levels. Here we show examples of such cellular behaviors and discuss the possible roles of miRNAs in redox regulatory circuits and further cell responses to stress.

Glutathione reductase: A cytoplasmic antioxidant enzyme and a potential target for phenothiazinium dyes in Neospora caninum.

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 µM, 11 ± 2 µM, 0.7 ± 0.1 µM, and 0.9 ± 0.2 µM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.

Genome-wide identification of ascorbate-glutathione cycle gene families in soybean (Glycine max) reveals gene duplication events and specificity of gene members linked to development and stress conditions.

Ascorbate-glutathione (AsA-GSH) cycle plays an important role in tuning beneficial ROS accumulation for intracellular signals and imparts plant tolerance to oxidative stress by detoxifying excess of ROS. Here, we present genome-wide identification of AsA-GSH cycle genes (APX, MDHAR, DHAR, and GR) in several leguminous species and expression analyses in G. max during stress, germination and tissue development. Our data revealed 24 genes in Glycine genus against the maximum of 15 in other leguminous species, which was due to 9 pars of duplicated genes mostly originated from sub/neofunctionalization. Cytosolic APX and MDHAR genes were highly expressed in different tissues and physiological conditions. Germination induced genes encoding AsA-GSH proteins from different cell compartments, whereas vegetative phase (leaves) stimulated predominantly genes related to chloroplast/mitochondria proteins. Moreover, cytosolic APX-1, 2, MDHAR-1a, 1b and GR genes were the primary genes linked to senescence and biotic stresses, while stAPX-a, b and GR (from organelles) were the most abiotic stress related genes. Biotic and abiotic stress tolerant genotypes generally showed increased MDHAR, DHAR and/or GR mRNA levels compared to susceptible genotypes. Overall, these data clarified evolutionary events in leguminous plants and point to the functional specificity of duplicated genes of the AsA-GSH cycle in G. max.