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Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by difficulties in social interaction and communication, repetitive behaviors, and restricted interests. Unfortunately, the underlying molecular mechanism behind ASD remains unknown. It has been reported that oxidative and nitrosative stress are strongly linked to ASD. We have recently found that nitric oxide (NO•) and its products play an important role in this disorder. One of the key proteins associated with NO• is thioredoxin (Trx). We hypothesize that the Trx system is altered in the Shank3 KO mouse model of autism, which may lead to a decreased activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), resulting in oxidative stress, and thus, contributing to ASD-related phenotypes. To test this hypothesis, we conducted in vivo behavioral studies and used primary cortical neurons derived from the Shank3 KO mice and human SH-SY5Y cells with SHANK3 mutation. We showed significant changes in the levels and activity of Trx redox proteins in the Shank3 KO mice. A Trx1 inhibitor PX-12 decreased Trx1 and Nrf2 expression in wild-type mice, causing abnormal alterations in the levels of synaptic proteins and neurotransmission markers, and an elevation of nitrosative stress. Trx inhibition resulted in an ASD-like behavioral phenotype, similar to that of Shank3 KO mice. Taken together, our findings confirm the strong link between the Trx system and ASD pathology, including the increased oxidative/nitrosative stress, and synaptic and behavioral deficits. The results of this study may pave the way for identifying novel drug targets for ASD. [ABSTRACT FROM AUTHOR]

The nature of brain redox metabolism in health, aging, and disease remains to be fully established. Reversible oxidations, to disulfide bonds, of closely spaced (vicinal) protein thiols underlie the catalytic maintenance of redox homeostasis by redoxin enzymes, including thioredoxin peroxidases (peroxiredoxins), and have been implicated in redox buffering and regulation. We propose that non-peroxidase proteins containing vicinal thiols that are responsive to physiological redox perturbations may serve as intrinsic probes of brain redox metabolism. Using redox phenylarsine oxide (PAO)-affinity chromatography, we report that PAO-binding vicinal thiols on creatine kinase B and alpha-enolase from healthy rat brains were preferentially oxidized compared to other selected proteins, including neuron-specific (gamma) enolase, under conditions designed to trap in vivo protein thiol redox states. Moreover, measures of the extents of oxidations of vicinal thiols on total protein, and on creatine kinase B and alpha-enolase, showed that vicinal thiol-linked redox states were stable over the lifespan of rats and revealed a transient reductive shift in these redox couples following decapitation-induced global ischemia. Finally, formation of disulfide-linked complexes between peroxiredoxin-2 and brain proteins was demonstrated on redox blots, supporting a link between protein vicinal thiol redox states and the peroxidase activities of peroxiredoxins. The implications of these findings with respect to underappreciated aspects of brain redox metabolism in health, aging, and ischemia are discussed. [ABSTRACT FROM AUTHOR]

Ferroptosis, a unique type of non-apoptotic cell death resulting from iron-dependent lipid peroxidation, has a potential physiological function in tumor suppression, but its underlying mechanisms have not been fully elucidated. Here, we report that the long non-coding RNA (lncRNA) LncFASA increases the susceptibility of triple-negative breast cancer (TNBC) to ferroptosis. As a tumor suppressor, LncFASA drives the formation of droplets containing peroxiredoxin1 (PRDX1), a member of the peroxidase family, resulting in the accumulation of lipid peroxidation via the SLC7A11-GPX4 axis. Mechanistically, LncFASA directly binds to the Ahpc-TSA domain of PRDX1, inhibiting its peroxidase activity by driving liquid-liquid phase separation, which disrupts intracellular ROS homeostasis. Notably, high LncFASA expression indicates favorable overall survival in individuals with breast cancer, and LncFASA impairs the growth of breast xenograft tumors by modulating ferroptosis. Together, our findings illustrate the crucial role of this lncRNA in ferroptosis-mediated cancer development and provide new insights into therapeutic strategies for breast cancer. [ABSTRACT FROM AUTHOR]

This protocol describes a detailed fluorometric method for measuring peroxiredoxin (Prx) enzyme activity in vitro. Peroxide dissociation is the rate-limiting step in the Prx-controlled enzymatic reaction. To prevent interference by the catalase enzyme, we developed a peroxiredoxin assay that measures Prx activity using the substrate tert-Butyl hydroperoxide (t-BOOH). Prx enzyme activity is measured by incubating the enzymatic substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37 °C for 10 min in the presence of the desired volume of Prx enzyme. Next, the reagent N-(9-Acridinyl)maleimide (NAM) is used to stop the enzymatic reaction and form a fluorescent end product. Finally, Prx activity is measured by thiol fluorometry using a Box–Behnken design to optimize reaction conditions. This novel protocol was validated by evaluating Prx activity in matched samples against a reference assay. The correlation coefficient between our protocol and the reference assay was 0.9933, demonstrating its precision compared with existing methods. The NAM-Prx protocol instead uses t-BOOH as a substrate to measure Prx activity. Because catalase does not participate in the dissociation of t-BOOH, this approach does not require sodium azide. Furthermore, the method eliminates the need for concentrated acids to terminate the Prx enzymatic reaction since the NAM reagent can inhibit the enzymatic reaction regulated by the Prx enzyme. [ABSTRACT FROM AUTHOR]

Neuromyelitis optica spectrum disorders (NMOSD) are autoimmune disorders affecting central nervous system with immunoglobulin G positivity to aquaporin-4 and myelin oligodendrocyte glycoprotein. NMOSD are clinically featured by transverse myelitis and optic neuritis. It is also a demyelinating autoimmune disease which could affect the erythrocyte deformability and may contribute to the severity of the disease. To study the electrophoretogram profile of erythrocyte proteins and its comparative expression between control and neuromyelitis optica variants. The study involves 4 groups: control, Aqp-4- IgG, MOG-IgG and NMO with autoantibody negativity to MOG and AQP-4 with samples of 3 per each group. The electrophoretogram of erythrocyte proteins was done using SDS PAGE and comparative band intensity between control and NMOSD variants was observed. The study observed there was increase in band intensity in the region between 37 KDa and 50 KDa in Aqp-4 IgG seropositive group compared to control, whereas no differential expression was observed in MOG-IgG positive variant. The erythrocyte proteins in those regions could be G3PD, actin and dematin. The bands in the region corresponding to 25 KDa were stomatin or Aqp-4 which showed marginal decrease in band intensity in NMO variants compared to control and the same observation was made in the region of 20 KDa where the band possibly represented the redox protein, peroxiredoxin. The differential band expression pattern of erythrocyte proteins between control and NMOSD variants could help in identification of red blood cell proteins which aid in the diagnostics and prognostics of NMOSD. According to comparative electrophoretogram the differentially expressed proteins could be G3PD, actin, stomatin and peroxiredoxin. [ABSTRACT FROM AUTHOR]

A novel fluorometric method is presented for accurately quantifying peroxiredoxin (Prx) enzyme activity in vitro. The rate-limiting step in the Prx-catalyzed reaction is the dissociation of peroxide. To avoid interference from catalase, we developed an assay using tert-butyl hydroperoxide (t-BOOH) as a substrate for specific Prx activity measurement. The assay involves incubating the enzyme substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37 °C for 10 min in a known volume of Prx enzyme. Following incubation, the reagent monobromobimane (mBB) is added to terminate the enzymatic reaction and produce a fluorescent product. Prx activity is subsequently determined by measuring thiol fluorescence, with reaction conditions optimized using a Bland-Altman plot. The efficacy of this novel protocol was rigorously validated by comparing Prx activity measurements from paired samples with those generated by a reference assay. A correlation coefficient of 0.995 was observed between the two methods, demonstrating superior precision and reliability compared to existing methods. The mBB-Prx protocol offers a significant safety advantage by using t-BOOH as a substrate for Prx activity measurement. As catalase does not catalyze t-BOOH dissociation, including sodium azide is unnecessary. Moreover, the method obviates the need for concentrated acids to terminate the Prx enzymatic reaction, as the mBB reagent efficiently inhibits Prx activity. This streamlined approach simplifies the assay and significantly improves its safety and usability, providing users with a reliable and convenient tool. The convenience of this method allows users to focus on their research without worrying about safety or complex procedures. [ABSTRACT FROM AUTHOR]

Neospora caninum, an apicomplexan parasite, is the etiological agent of neosporosis, a disease that leads to neurological symptoms in dogs and abortion in cattle. Vaccine or drug treatments for neosporosis remain to be determined. Therefore, it is of undeniable relevance to investigate new molecules involved in the parasite's successful survival within the host cell. The aim of this study was to characterize the N. caninum peroxiredoxin (NcPrx), an enzyme involved in the redox system of the parasite. The NcPrx amino acid sequence showed high identity and similarity compared to homologues representatives of Apicomplexa phylum. The recombinant NcPrx (rNcPrx) was cloned and expressed in Escherichia coli (BL21) with the predicted molecular weight (22 kDa), and the identity of monomer and dimer forms of rNcPrx was confirmed by mass spectrometry. Native and recombinant NcPrx were detected by ELISA and western blot, using the polyclonal anti-rNcPrx serum. Multiphoton analysis showed that NcPrx is localized in tachyzoite cytosol. H2O2 treatment increased the rNcPrx dimerization in vitro, and associated with the in silico data, we suggest that NcPrx belongs to typical 2-Cys Prx group (AhpC/Prx1 family). rNcPrx also increased the H2O2 clearance and protected plasmidial DNA under oxidative conditions. Finally, H2O2 increased the NcPrx dimerization in intracellular and extracellular tachyzoites suggesting that it is enrolled in H2O2 clearance and sensing in N. caninum. [ABSTRACT FROM AUTHOR]

NADPH provides the reducing power for decomposition of reactive oxygen species (ROS), making it an indispensable part during ROS defense. It remains uncertain, however, if living cells respond to the ROS challenge with an elevated intracellular NADPH level or a more complex NADPH-mediated manner. Herein, we employed a model fungus Aspergillus nidulans to probe this issue. A conditional expression of glucose-6-phosphate dehydrogenase (G6PD)-strain was constructed to manipulate intracellular NADPH levels. As expected, turning down the cellular NADPH concentration drastically lowered the ROS response of the strain; it was interesting to note that increasing NADPH levels also impaired fungal H2O2 resistance. Further analysis showed that excess NADPH promoted the assembly of the CCAAT-binding factor AnCF, which in turn suppressed NapA, a transcriptional activator of PrxA (the key NADPH-dependent ROS scavenger), leading to low antioxidant ability. In natural cell response to oxidative stress, we noticed that the intracellular NADPH level fluctuated "down then up" in the presence of H2O2. This might be the result of a co-action of the PrxA-dependent NADPH consumption and NADPH-dependent feedback of G6PD. The fluctuation of NADPH is well correlated to the formation of AnCF assembly and expression of NapA, thus modulating the ROS defense. Our research elucidated how A. nidulans precisely controls NADPH levels for ROS defense. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are cysteine-based peroxidases that play a central role in keeping the H2O2 at physiological levels. Eukaryotic cells express different Prxs isoforms, which differ in their subcellular locations and substrate specificities. Mitochondrial Prxs are synthesized in the cytosol as precursor proteins containing N-terminal cleavable presequences that act as mitochondrial targeting signals. Due to the fact that presequence controls the import of the vast majority of mitochondrial matrix proteins, the mitochondrial Prxs were initially predicted to be localized exclusively in the matrix. However, recent studies showed that mitochondrial Prxs are also targeted to the intermembrane space by mechanisms that remain poorly understood. While in yeast the IMP complex can translocate Prx1 to the intermembrane space, the maturation of yeast Prx1 and mammalian Prdx3 and Prdx5 in the matrix has been associated with sequential cleavages of the presequence by MPP and Oct1/MIP proteases. In this review, we describe the state of the art of the molecular mechanisms that control the mitochondrial import and maturation of Prxs of yeast and human cells. Once mitochondria are considered the major intracellular source of H2O2, understanding the mitochondrial Prx biogenesis pathways is essential to increase our knowledge about the H2O2-dependent cellular signaling, which is relevant to the pathophysiology of some human diseases. [ABSTRACT FROM AUTHOR]

2-Cys peroxiredoxins are abundant thiol proteins that react efficiently with a wide range of peroxides. Unlike other enzymes, their exceptionally high reactivity does not rely on cofactors. The mechanism of oxidation and reduction of peroxiredoxins places them in a good position to act as antioxidants as well as key players in redox signaling. Understanding of the intimate details of peroxiredoxin functioning is important for translational research. [ABSTRACT FROM AUTHOR]

Ischemia and reperfusion events, such as myocardial infarction (MI), are reported to induce remote organ damage severely compromising patient outcomes. Tissue survival and functional restoration relies on the activation of endogenous redox regulatory systems such as the oxidoreductases of the thioredoxin (Trx) family. Trxs and peroxiredoxins (Prxs) are essential for the redox regulation of protein thiol groups and for the reduction of hydrogen peroxide, respectively. Here, we determined whether experimental MI induces changes in Trxs and Prxs in the heart as well as in secondary organs. Levels and localization of Trx1, TrxR1, Trx2, Prx1, and Prx2 were analyzed in the femur, vertebrae, and kidneys of rats following MI or sham surgery. Trx1 levels were significantly increased in the heart (P = 0.0017) and femur (P < 0.0001) of MI animals. In the femur and lumbar vertebrae, Trx1 upregulation was detected in bone-lining cells, osteoblasts, megakaryocytes, and other hematopoietic cells. Serum levels of Trx1 increased significantly 2 days after MI compared to sham animals (P = 0.0085). Differential regulation of Trx1 in the bone was also detected by immunohistochemistry 1 month after MI. N-Acetyl-cysteine treatment over a period of 1 month induced a significant reduction of Trx1 levels in the bone of MI rats compared to sham and to MI vehicle. This study provides first evidence that MI induces remote organ upregulation of the redox protein Trx1 in the bone, as a response to ischemia–reperfusion injury in the heart. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx) refers to a family of thiol-dependent peroxidases that decompose hydrogen peroxide, lipid hydroperoxides, as well as peroxynitrite, and protect against oxidative and inflammatory stress. There are six mammalian Prx isozymes (Prx1–6), classified as typical 2-Cys, atypical 2-Cys, or 1-Cys Prxs based on the mechanism and the number of cysteine residues involved during catalysis. In addition to their well-established peroxide-scavenging activity, some Prxs also participate in the regulation of various cell signaling pathways. Extensive animal studies employing primarily gene knockout models provide substantial evidence supporting a critical protective role of Prxs in various disease processes involving oxidative and inflammatory stress. This review surveys recent research findings, published primarily in influential journals, on the involvement of various Prx isozymes in protecting against cardiovascular injury and related disorders, including diabetes, metabolic syndromes, and sepsis, whose pathophysiology all intimately involves oxidative stress and inflammation. [ABSTRACT FROM AUTHOR]

[Aims] To reveal the molecular mechanism of endophytic bacteria in Verticillium wilt-resistant cottons and deeply understand the interaction between soil and plants. [Methods] Active tracking screening was used for the isolation of antagonistic strain. Morphological and molecular analyses were conducted to determine its phenotypic and genotypic characteristics. MALDI-TOF/TOF MS was employed to identify the antifungal substance. Functional verification was performed by gene cloning, expression, knockout and complementation. [Results] Enterobacter sp. V1 was isolated from Verticillium-wilt-resistant cotton Xinhai 15, which showed significantly antagonistic abilities against cotton verticillium wilt. A peroxiredoxin named ProV1 with virulent against V. dahliae was obtained. The gene for the peroxiredoxin ProV1 was cloned, and the nucleotide sequence showed 85.5% similarity with peroxiredoxin C (WP 096152328) reported from E. hormaeche. The purified heterologously expressed ProV1 indicated strong inhibitory activities. Moreover, the suppression of V. dahliae also decreased in proV1 mutant strain compared with wild type strain. The inhibitory activities could be partially restored by a complementation of proV1 gene in the mutant strain. [Conclusion] The results of this study reveal that a novel peroxiredoxin from an endophytic bacterial strain Enterobacter sp. V1 can serve as a virulent factor against fungi V. dahliae, which helps understanding the interaction between endyphytic bacteria and plant pathogenic fungi, and improving the biological control of cotton Verticillium wilt. [ABSTRACT FROM AUTHOR]

Purpose: To study the presence and distribution of genes encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles.Methods: A class comparison study on existing granulosa cell transcriptome from primordial (n = 539 follicles) and primary (n = 261) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy was performed and interrogated.Results: In granulosa cells from primordial follicles, 30 genes were annotated ‘mitochondrial dysfunction’ including transcripts (PRDX5, TXN2) encoding enzymatic free radical scavengers peroxiredoxin 5 and thioredoxin 2. Several apoptosis regulation genes were noted (BCL2, CAS8, CAS9, AIFM1). In granulosa cells from primary follicles, mitochondrial dysfunction signalling pathway was annotated. High expression of transcripts encoding the free radical scavenger peroxiredoxin 3, as well as anti-apoptotic enzyme BCL2, was found. Interestingly, PARK7 encoding the deglycase (DJ-1) protein was expressed in granulosa cells from primary follicles. DJ-1 is implicated in oxidative defence and functions as a positive regulator of the androgen receptor and as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/serine-threonine protein kinase (AKT) signalling pathway suppressor PTEN.Conclusions: The results indicate extensive energy production and free radical scavenging in the granulosa cells of primordial follicles with potential implications for ovarian ageing, cigarette smoking, premature ovarian failure and polycystic ovarian syndrome. Furthermore, DJ-1 may be involved in androgen responsiveness and the regulation of follicle growth via PI3K/PTEN/AKT signalling pathway regulation in the granulosa cells of primary follicles. The involvement of mitochondrial free radical production in the age-related decline of competent oocytes is becoming apparent. [ABSTRACT FROM AUTHOR]

Preconditioning brain cultures with moderate concentrations of ethanol (EtOH) or trans-resveratrol (RES), key red wine constituents, can prevent amyloid-β (Aβ) neurotoxicity. Past studies have indicated that moderate EtOH activates synaptic N-methyl-D-aspartate receptors (NMDAR) that, in part, signal via protein kinase C (PKC) to increase protective antioxidant proteins such as peroxiredoxin-2 (Prx2). RES preconditioning also is reported to involve NMDAR and PKC. However, although moderate, the EtOH and RES concentrations used have been noticeably above circulating levels from two glasses of wine, a daily intake linked to reduced risk of cognitive decline among older social drinkers. Given their mechanistic parallels, we speculated that subprotective EtOH and RES concentrations in a combinatorial preconditioning paradigm might elicit synergistic neuroprotection. To examine this notion, rat cerebellar cultures were pretreated with 10 mM EtOH (circulating concentration after ~ 2 drinks), 5 μM RES, EtOH + RES combinatorially, or media alone (controls). After 3 days, media were removed, and fresh media aliquots containing Aβ25-35 (25 μM) were added. Assessing apoptosis 24 h later with Hoescht 33342, neurodegeneration did not differ from controls in cultures separately preconditioned with 10 mM EtOH or 5 μM RES. However, apoptosis was prevented in combinatorially preconditioned cultures. Also, immunoblotting revealed elevated Prx2 levels due to combinatorial pretreatment that correlated with subsequent neuroprotection, whereas Prx2 was unchanged in separately pretreated cultures. Although the protective mechanisms require clarification, synergistically upregulated NMDAR-PKC-Prx2 (and other antioxidant proteins) is a reasonable component. These findings imply that EtOH + RES antioxidant synergy could be involved in neurobenefits attributed to low-moderate wine consumption. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) and hydroxyl radicals (HO·) are generated through partial reduction of oxygen. The HO· are the most reactive and have a shorter half-life than H2O2, they are produced from comparatively stable H2O2 through Fenton reaction. Although controlling HO· is important and biologically advantageous for organisms, it may be difficult. Ticks are obligate hematophagous arthropods that need blood feeding for development. Ticks feed on vertebrate blood containing high levels of iron. Ticks also concentrate iron-containing host blood, leading to high levels of iron in ticks. Host-derived iron may react with oxygen in the tick body, resulting in high concentrations of H2O2. On the other hand, ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), to scavenge H2O2. Gene silencing of Prxs in ticks affects their blood feeding, oviposition, and H2O2 concentration. Therefore, Prxs could play important roles in ticks’ blood feeding and oviposition through the regulation of the H2O2 concentration. This review discusses the current knowledge of Prxs in hard ticks. Tick Prxs are also multifunctional molecules related to antioxidants and immunity like other organisms. In addition, tick Prxs play a role in regulating the host immune response for ticks’ survival in the host body. Tick Prx also can induce Th2 immune response in the host. Thus, this review would contribute to the further understanding of the tick’s antioxidant responses during blood feeding and the search for a candidate target for tick control. [ABSTRACT FROM AUTHOR]

Environmental stressors inducing oxidative stress such as ionizing radiation may influence cognitive function and neuronal plasticity. Recent studies have shown that transgenic mice deficient of DNA glycosylases display unexpected cognitive deficiencies related to changes in gene expression in the hippocampus. The main objectives of the present study were to determine learning and memory performance in C57BL/6NTac 8-oxoguanine DNA glycosylase 1 (Ogg1)+/− (heterozygote) and Ogg1+/+ (wild type, WT) mice, to study whether a single acute X-ray challenge (0.5 Gy, dose rate 0.457 Gy/min) influenced the cognitive performance in the Barnes maze, and if such differences were related to changes in gene expression levels in the hippocampus. We found that the Ogg1+/− mice exhibited poorer early-phase learning performance compared to the WT mice. Surprisingly, X-ray exposure of the Ogg1+/− animals improved their early-phase learning performance. No persistent effects on memory in the late-phase (6 weeks after irradiation) were observed. Our results further suggest that expression of 3 (Adrb1, Il1b, Prdx6) out of in total 35 genes investigated in the Ogg1+/− hippocampus is correlated to spatial learning in the Barnes maze. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to evaluate peroxiredoxin I (Prx I) participation in the cellular antioxidant response to low-dose X-rays through the analysis of its expression in buccal mucosa cells from patients of different ages following panoramic dental radiography.Materials and methods: Of the 50 patients included in this study, oral mucosa cells from six adults were collected for the immunofluorescence cytological analysis. The other 44 patients, 11 patients aged below 20 years; 22 patients aged between 20 and 50 years; and 11 patients aged above 50 years, were submitted to panoramic dental radiography, and oral mucosa cells were collected for the gene expression analysis before and 1 hour after exposure.Results: The results demonstrated Prx I expression in the cytoplasm of oral mucosa cells either before or after radiation exposure. The quantitative analysis showed that in oral mucosa cells from patients aged below 50 years the mRNA levels of PRDX1 were significantly increased after radiation exposure. On the other hand, the cells from patients aged above 50 years presented significantly lower PRDX1 transcript levels after radiation exposition.Conclusions: Panoramic radiography leads to increased Prx I expression in buccal mucosa cells, probably as an adaptive response to eliminate X-ray-induced ROS, except in cells from elderly people.Clinical relevance: Even low doses of radiation employed for dental purposes are capable to provoke stress to cells, which was demonstrated via the induction of the antioxidant gene PRDX1. In elderly patients, such mechanism was demonstrated to be impaired. [ABSTRACT FROM AUTHOR]

Ticks require blood feeding on vertebrate animals throughout their life cycle, and also concentrate the iron-containing blood, resulting in a high concentration of hydrogen peroxide (H2O2). High concentrations of H2O2 are harmful to organisms, due to their serious damage of macromolecules. Ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), that scavenge H2O2. Prxs may have important roles in regulating the H2O2 concentration in ticks during blood feeding and oviposition. Moreover, Prxs are considered potential vaccine candidates in other parasites, such as Leishmania and Fasciola. In the present study, the efficacy of a tick Prx (HlPrx2) as a vaccine candidate antigen was evaluated. First, recombinant HlPrx2 (rHlPrx2) was expressed in Escherichia coli, and then, its purity and endotoxin levels were confirmed prior to administration. The rHlPrx2 proteins were of high purity with acceptably low endotoxin levels. Second, the ability of rHlPrx2 administration to stimulate mouse immunity was evaluated. The rHlPrx2 protein, with or without an adjuvant, could stimulate immunity in mice, especially the IgG1 of Th2 immune response. Using Western blot analysis, we also observed whether rHlPrx2-immunized mice sera could recognize native HlPrx2 protein in crude tick midgut proteins. Western blot analysis demonstrated that rHlPrx2-administrated mouse sera could detect the native HlPrx2. Finally, the effects of rHlPrx2 immunization in mice were studied using nymphal ticks. Although the challenged ticks were not affected by rHlPrx2 immunization, rHlPrx2 still might be considered as a vaccine candidate against ticks because of its high immunogenicity. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (HO) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under HO and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of HO, superoxide (O ) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress. [ABSTRACT FROM AUTHOR]

Bismuth drugs have been used clinically to treat infections from Helicobacter pylori, a pathogen that is strongly related to gastrointestinal diseases even stomach cancer. Despite extensive studies, the mechanisms of action of bismuth drugs are not fully understood. Alkyl hydroperoxide reductase subunit C (AhpC) is the most abundant 2-cysteine peroxiredoxin, crucial for H. pylori survival in the host by defense of oxidative stress. Herein we show that a Bi(III) antiulcer drug (CBS) binds to the highly conserved cysteine residues (Cys49 and Cys169) with a dissociation constant ( K ) of Bi(III) to AhpC of 3.0 (±1.0) × 10 M. Significantly the interaction of CBS with AhpC disrupts the peroxiredoxin and chaperone activities of the enzyme both in vitro and in bacterial cells, leading to attenuated bacterial survival. Moreover, using a home-made fluorescent probe, we demonstrate that Bi(III) also perturbs AhpC relocation between the cytoplasm and membrane region in decomposing the exogenous ROS. Our study suggests that disruption of redox homeostasis by bismuth drugs via interaction with key enzymes such as AhpC contributes to their antimicrobial activity. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (PRX) defines a family that provides antioxidant defense in different cell types by removing reactive oxygen species (ROS) through conserved active cysteines, with the support of other types of antioxidants such as thioredoxin, glutaredoxin, and glutathione peroxidase. By regulation of intracellular ROS levels, the mammalian PRXs influence a variety of reproductive processes including gamete maturation, fertilization, and embryo development. Experimental mice lacking PRXs developed normally, but some showed accelerated decrease in fertility with aging, suggesting that deficiency of PRXs did not have lethal consequences for reproduction. The aim of this review is to summarize the role of mammalian PRXs in the reproductive performance. [ABSTRACT FROM AUTHOR]

The minimal genome of the mollicute Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia, encodes a limited repertoire of antioxidant enzymes that include a single and atypical peroxiredoxin (MhPrx), whose evolution and function were studied here. MhPrx has only one catalytic cysteine, in contrast with some of its possible ancestors (2-Cys peroxiredoxins), which have two. Although it is more similar to 2-Cys orthologs, MhPrx can still function with a single peroxidatic cysteine (Cys), using non-thiolic electron donors to reduce it. Therefore, MhPrx could be a representative of a possible group of 2-Cys peroxiredoxins, which have lost the resolving cysteine (Cys) residue without losing their catalytic properties. To further investigate MhPrx evolution, we performed a comprehensive phylogenetic analysis in the context of several bacterial families, including Prxs belonging to Tpx and AhpE families, shedding light on the evolutionary history of Mycoplasmataceae Prxs and giving support to the hypothesis of a relatively recent loss of the Cys within this family. Moreover, mutational analyses provided insights into MhPrx function with one, two, or without catalytic cysteines. While removal of the MhPrx putative Cys caused complete activity loss, confirming its catalytic role, the introduction of a second cysteine in a site correspondent to that of the Cys of a 2-Cys orthologue, as in the MhPrx supposed ancestral form, was compatible with enzyme activity. Overall, our phylogenetic and mutational studies support that MhPrx recently diverged from a 2-Cys Prx ancestor and pave the way for future studies addressing structural, functional, and evolutive aspects of peroxiredoxin subfamilies in Mollicutes and other bacteria. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) constitute a group of thiol-specific antioxidant enzymes which are present in bacteria, yeasts, and in plant and animal cells. Although Prxs are mainly localized in the cytosol, they are also present in mitochondria, chloroplasts, and nuclei, but there is no evidence of the existence of Prxs in plant peroxisomes. Using soluble fractions (matrices) of peroxisomes purified from leaves of pea ( Pisum sativum L.) plants, the immunological analysis with affinity-purified IgG against yeast Prx1 revealed the presence of an immunoreactive band of about 50 kDa. The apparent molecular mass of the peroxisomal Prx was not sensitive to oxidizing and reducing conditions what could be a mechanism of protection against the oxidative environment existing in peroxisomes. Postembedment, EM immunocytochemical analysis with affinity-purified IgG against yeast Prx1 antibodies, confirmed that this protein was present in the peroxisomal matrix, mitochondria, and chloroplasts. In pea plants grown under oxidative stress conditions, the protein level of peroxisomal Prx was differentially modulated, being slightly induced by growth of plants with 50 µM CdCl, but being significantly reduced by treatment with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The presence in the matrix of peroxisomes of a protein immunorelated to Prx of about 50 kDa, which is in the range of molecular mass of the dimeric form of other Prxs, opens new questions on the molecular properties of Prxs, but also on their function in the metabolism of reactive oxygen and nitrogen species (ROS/RNS) in these plant cell organelles, where they could be involved in the regulation of hydrogen peroxide and/or peroxynitrite. [ABSTRACT FROM AUTHOR]

Cysteine-based peroxidases, known as peroxiredoxins (Prx) or thioredoxin peroxidases (TPx), are important antioxidant enzymes that prevent oxidative damage caused by reactive oxygen species (ROS). In this study, we identified a novel mitochondrial 2-Cys Prx, BbTPx-2, from a bovine Babesia parasite, B. bovis. BbTPx-2 complementary DNA (cDNA) encodes a polypeptide of 254 amino acid residues. This protein has a mitochondrial targeting peptide at the N-terminus and two conserved cysteine residues of the typical 2-Cys Prx. By using a thiol mixed-function oxidation assay, the antioxidant activity of recombinant BbTPx-2 was revealed, and its antioxidant activity was comparable to that of a cytosolic 2-Cys Prx from B. bovis, BbTPx-1. Notably, we confirmed that BbTPx-2 was expressed in the mitochondrion of B. bovis merozoites. Taken together, the results suggest that the mitochondrial BbTPx-2 is an antioxidative enzyme for scavenging ROS in B. bovis. [ABSTRACT FROM AUTHOR]

α-Tocopheryl phosphate (αTP) is a phosphorylated form of α-tocopherol. Since it is phosphorylated in the hydroxyl group that is essential for the antioxidant property of α-tocopherol, we hypothesized that αTP would modulate the antioxidant system, rather than being an antioxidant agent per se. α-TP demonstrated antioxidant activity in vitro against iron-induced oxidative stress in a mitochondria-enriched fraction preparation treated with 30 or 100 µM α-TP. However, this effect was not observed ex vivo with mitochondrial-enriched fraction from mice treated with an intracerebroventricular injection of 0.1 or 1 nmol/site of αTP. Two days after treatment (1 nmol/site αTP), peroxiredoxin 2 (Prx2) and glutathione reductase (GR) expression and GR activity were decreased in cerebral cortex and hippocampus. Glutathione content, glutathione peroxidase, and thioredoxin reductase activities were not affected by αTP. In conclusion, the persistent decrease in GR and Prx2 protein content is the first report of an in vivo effect of αTP on protein expression in the mouse brain, potentially associated to a novel and biologically relevant function of this naturally occurring compound. [ABSTRACT FROM AUTHOR]

Therapeutic benefits offered by common chemotherapy drugs, such as oxaliplatin, are limited due to the development of resistance, which contributes to treatment failure and metastasis. The epithelial-mesenchymal transition (EMT) is a key event contributing to the development of resistance to chemotherapeutics. Although the relationship between oxaliplatin and chemotherapy resistance has been described for decades, the molecular mechanisms have remained elusive. The aim of the present study was to investigate the underlying mechanisms of oxaliplatin-mediated metastasis. Here, we identify reactive oxygen species (ROS) as mediators that promote the oxaliplatin-induced EMT. Following oxaliplatin treatment, the messenger RNA (mRNA) levels of most peroxiredoxin family genes, except for peroxiredoxin 1 (prdx1) gene, were constant or even decreased, resulting in ROS abundance. And the antioxidant guardian Nrf2 was unconspicuously raised both transcriptionally and translationally with oxaliplatin treatment as compared to those induced by topotecan treatment, which has been proved with no induced metastasis. In addition, the study evaluated high levels of ROS leading to EMT via activation of the known oncogenes Akt and Snail. Using the Akt inhibitor LY294002 or knocking down Snail expression via RNA interference (RNAi) reversed the effects of oxaliplatin on the EMT and metastasis. Our studies establish a role for the ROS-Akt-Snail axis as a mechanism by which chemotherapeutics induce EMT and cancer metastasis. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are peroxidases that reduce hydrogen peroxide (HO) and various alkyl hydroperoxides and act as reductants. A full-length cDNA encoding for a Prx polypeptide was isolated and cloned from grape ( Vitis vinifera L. cv. Askari) berries. The cDNA was 773 nucleotides in length with a deduced amino acid of 162 residues, possessing one conserved cysteine, which belongs to the type II Prx C. The calculated molecular mass and the predicted isoelectric point of the deduced polypeptide are 17.26 kD and 5.15, respectively. The deduced protein sequence showed a high similarity to PrxII C from other plants, in particular from cotton ( Gossypium hirsutum), poplar ( Populus trichocarpa), and Citrus sp. The in silico analysis of the promoter region of grape Prx demonstrated the presence of a number of potential cis-acting elements to respond to environmental signals, suggesting that VvPrxII C may respond to a variety of environmental signals, including dehydration, heat, heavy metals, light, pathogens, wounding, and plant hormones. The grape Prx gene was also analyzed for its expressional response to abiotic stress, oxidative stress, and antioxidants application. The results revealed a highly induced response to abiotic stress conditions due to the presence of different putative regulatory elements in its promoter. [ABSTRACT FROM AUTHOR]

The activity of typical 2-cys peroxiredoxin (Prxs) can be regulated by hyperoxidation with a consequent loss of redox activity. Here we developed a simple assay to monitor the level of hyperoxidation of different typical 2-cys prxs simultaneously. This assay only requires standard equipment and can compare different samples on the same gel. It requires much less time than conventional 2D gels and gives more information than Western blotting with an antibody specific for hyperoxidized peroxiredoxin. This method could also be used to monitor protein modification with a charge difference such as phosphorylation. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (HO) produces hydroxyl radicals that directly attack a variety of biomolecules and cause severe cellular dysfunction. An extremely thermophilic bacterium, Thermus thermophilus HB8, possesses at least three enzymes that can scavenge HO: manganese-containing catalase (TTHA0122, MnCAT), a possible peroxiredoxin homologue (TTHA1300), and a possible heme peroxidase (HPX) homologue (TTHA1714). To investigate the roles of these proteins, we attempted to disrupt each of these genes in T. thermophilus HB8. Although we were able to completely disrupt ttha1300, we were unable to completely delete ttha0122 and ttha1714 because of polyploidy. Quantitative real-time PCR showed that, compared to the wild type, 31 % of ttha0122 and 11 % of ttha1714 remained in the ∆ ttha0122 and ∆ ttha1714 disruption mutants, respectively. Mutants with reduced levels of ttha0122 or ttha1714 exhibited a significant increase in spontaneous mutation frequency. ∆ ttha1714 grew slower than the wild type under normal conditions. ∆ ttha0122 grew very poorly after exposure to HO. Moreover, ∆ ttha0122 did not show HO-scavenging activity, whereas ∆ ttha1300 and ∆ ttha1714 scavenged HO, a property similar to that exhibited by the wild type. MnCAT purified from T. thermophilus HB8 cells scavenged HO in vitro. The recombinant form of the possible HPX homologue, reconstituted with hemin, showed peroxidase activity with HO as an oxidant substrate. Based on these results, we propose that not only MnCAT but also the possible HPX homologue is involved in protecting the cell from oxidative stress in T. thermophilus. [ABSTRACT FROM AUTHOR]

The PsbQ-like protein, termed CyanoQ, found in the cyanobacterium Synechocystis sp. PCC 6803 is thought to bind to the lumenal surface of photosystem II (PSII), helping to shield the MnCaO oxygen-evolving cluster. CyanoQ is, however, absent from the crystal structures of PSII isolated from thermophilic cyanobacteria raising the possibility that the association of CyanoQ with PSII might not be a conserved feature. Here, we show that CyanoQ (encoded by tll2057) is indeed expressed in the thermophilic cyanobacterium Thermosynechococcus elongatus and provide evidence in support of its assignment as a lipoprotein. Using an immunochemical approach, we show that CyanoQ co-purifies with PSII and is actually present in highly pure PSII samples used to generate PSII crystals. The absence of CyanoQ in the final crystal structure is possibly due to detachment of CyanoQ during crystallisation or its presence in sub-stoichiometric amounts. In contrast, the PsbP homologue, CyanoP, is severely depleted in isolated PSII complexes. We have also determined the crystal structure of CyanoQ from T. elongatus to a resolution of 1.6 Å. It lacks bound metal ions and contains a four-helix up-down bundle similar to the ones found in Synechocystis CyanoQ and spinach PsbQ. However, the N-terminal region and extensive lysine patch that are thought to be important for binding of PsbQ to PSII are not conserved in T. elongatus CyanoQ. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prdxs) comprise a large family of cysteine-based peroxidases that are involved in a wide variety of physiological processes. At the single-cell level co-expression of Prdxs has not been studied. We performed gene expression profiling in mouse olfactory neurons and found that individual cells express distinct prdxs in multiple combinations. In the case of conventional interpretation of RT-PCR data, differential expression patterns indicate that each olfactory neuron selects specific set of prdxs to express. However, a new concept of stochastic dynamics of gene expression may provide an alternative interpretation of our data. Here we suggest that the lack of synchronization in transcriptional states of individual genes may give rise to temporal shifts in gene expression pattern, thus constituting a plausible source of variability detected at the single-cell level. If so, the olfactory neuron is likely to transcribe genes for each of the six Prdxs yet with entirely different transcriptional phases. Based on the discontinuous mode of gene expression, the composition of Prdx transcripts within a cell may be variable, and therefore, the outcome of gene expression analysis is likely to depend on the moment of cell harvesting. This assumption easily reconciles the apparent discrepancy between transcriptional profiles derived from single cells and from population of cells. [ABSTRACT FROM AUTHOR]

Objective: Although peroxiredoxin 3 (PRX3) was reported to be overexpressed in liver cancer, the precise function of PRX3 in the development and/or progression of liver cancer remained to be obscure. The present study was conducted to investigate the response of PRX3 to oxidative stress in hepatocellular carcinoma (HCC) cells. Methods: After successful knockdown of PRX3 expression by small interfering RNA, we treated HCC cell lines Hep-3b and Hep-G2 with gradient concentrations of HO and detected cell proliferation, apoptosis, and the level of reactive oxygen species (ROS) in the cells. Results: After low-dose (5-20 μmol/l) HO treatment, the ROS level was significantly higher in PRX3-knockdown Hep-3b cells than in controls. In addition, PRX3 down-regulation resulted in decreased proliferation, increased apoptosis, and increased caspase 3 activity of Hep-3b cells. We did not notice significant difference between PrxIII knockdown and control Hep-G2 cells in ROS level, cell viability or apoptosis. Conclusion: Our results suggest that PRX3 is an indispensable ROS scavenger that protects tumor cells against oxidative damage and subsequent apoptosis, which provides a clue that PRX3 may be involved in the chemotherapeutic resistance of liver cancer. The underlying mechanism for PRX3 function needs further investigation. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) play important roles in antioxidant defense and redox signaling pathways. A Prx isozyme cDNA (TcPrx2, 745 bp, EF552425) was cloned from Taiwanofungus camphorata and its recombinant protein was overexpressed. The purified protein was shown to exist predominantly as a dimer by sodium dodecyl sulfate-polyacrylamide gel electrolysis in the absence of a reducing agent. The protein in its dimeric form showed no detectable Prx activity. However, the protein showed increased Prx activity with increasing dithiothreitol concentration which correlates with dissociation of the dimer into monomer. The TcPrx2 contains two Cys residues. The Cys located in the conserved active site is the putative active peroxidatic Cys. The role of Cys was investigated by site-directed mutagenesis. The C31S mutant (C → S) exists predominantly as a monomer with noticeable Prx activity. The Prx activity of the mutant was higher than that of the corresponding wild-type protein by nearly twofold at 12 μg/mL. The substrate preference of the mutant was HO > cumene peroxide > t-butyl peroxide. The Michaelis constant ( K) value for HO of the mutant was 0.11 mM. The mutant enzyme was active under a broad pH range from 6 to 10. The results suggest a role of Cys in dimerization of the TcPrx2, a role which, at least in part, may be involved in determining the activity of Prx. The C residue does not function as a resolving Cys and therefore the TcPrx2 must follow the reaction mechanism of 1-Cys Prx. This TcPrx2 represents a new isoform of Prx family. [ABSTRACT FROM AUTHOR]

Patients with end-stage renal disease are exposed to increased oxidative stress and impairment of antioxidant mechanisms. We focused on dialysis renal cell carcinoma (RCC), including epithelial hyperplasia in acquired cystic disease of the kidney (ACDK). We attempted to obtain insight into the carcinogenesis and tumor progression in terms of cellular defense mechanisms associated with oxidative stress by investigating the expression of antioxidant proteins by immunohistochemistry. We evaluated retrospectively 43 cases of dialysis RCC and, as a control group, 49 cases of sporadic RCC. Peroxiredoxin (Prx) 1, 3, 4, 5, and 6 expression in dialysis RCC was positively correlated with the duration of dialysis. In epithelial hyperplasia, in 17 cases of acquired cystic disease of the kidney, Prxs and thioredoxin were highly expressed. Moreover, in dialysis RCC, Prx 3, 4, and 5 immunoreactivity and nuclear expression of Y-box-binding protein-1 were higher than in sporadic RCC. In dialysis RCC, Prx 3, 4, and 5 immunoreactivity positively correlated with the Fuhrman nuclear grade. These data suggest that oxidative stress during dialysis enhances antioxidant activity, with an inhibiting effect on carcinogenesis. Once cancer has developed, antioxidant activity might have a stimulating effect on the progression of dialysis RCC. [ABSTRACT FROM AUTHOR]

There is no doubt as to the important role that free radicals and reactive oxygen species play in the cell. Disturbances in intracellular redox proteins are often accompanied by common pathologies, including diabetes, myocardial infarction, neurodegeneration, bronchopulmonary diseases, cancer, etc. Numerous antioxidant enzymes are related to various redox biology systems, the thiol oxidoreductase superfamily playing a key role. The superfamily includes thioredoxin, glutaredoxin, peroxiredoxin, protein disulfide isomerase, and glutathione peroxidase families and a number of other proteins. Apart from their antioxidant function, thiol oxidoreductases are capable of recycling hydroperoxyde to produce specific disulfide bonds within and between proteins, which significantly expands their functional range. In view of this, it is a topical problem of redox biology to characterize the superfamily members biochemically and to study their functional mechanisms. [ABSTRACT FROM AUTHOR]

We studied the expression of peroxiredoxin genes ( PRDX1, PRDX2, PRDX3, and PRDX6) in human erythroleukemia K652, human breast carcinoma MCF-7, and human ovarian carcinoma SKOV-3 cells during cisplatin resistance development. It was found that drug resistance formation was accompanied by a significant increase in the expression of PRDX1, PRDX2, PRDX3, PRDX6 genes in all cancer cell strains, which confirms the important contribution of redox-dependent mechanisms into the development of cisplatin resistance of cancer cells. [ABSTRACT FROM AUTHOR]

Single bouts of exercise induce an acute state of oxidative stress. It is largely unknown what this means in the context of diseases which are associated with increased oxidative stress, e.g., type 2 diabetes mellitus (T2DM). Free radicals can destroy the structure of erythrocytes and reduce their deformability. Antioxidative peroxiredoxins are highly abundant in erythrocytes. Therefore, we immunohistochemically examined whether the free radical-induced erythrocyte lipid-peroxidation measured by 8-iso-prostaglandin-F2α (8-Iso-PGF) as well as the erythrocyte contents of overoxidized peroxiredoxins (PRDX-SO) differ between overweight/obese T2DM men ( n = 15, years = 59 ± 10 (mean ± SD)) and overweight/obese non-diabetic control subjects ( n = 12, years = 53 ± 4) during acute exercise (WHO-step test). We further studied whether physical training affects the oxidative stress response to acute exercise. Seven men belonging to the diabetic group took part in a moderate intensity cycling endurance training. Erythrocyte 8-Iso-PGF significantly increased during acute exercise and decreased in the 30-min recovery phase in untrained diabetic and non-diabetic men ( P ≤ 0.05). Increases/decreases in 8-Iso-PGF in relation to exercise/recovery time were similar in both groups. A significant exercise-induced increase in the contents of erythrocyte PRDX-SO was only observed in T2DM men ( P ≤ 0.05). PRDX-SO contents were not reduced during recovery. Following physical training, the magnitude of exercise-induced increases in 8-Iso-PGF (relative to exercise time) was significantly lower in the erythrocytes of T2DM men ( P ≤ 0.05), whereas increases in PRDX-SO were significantly higher ( P ≤ 0.05). Exercise-induced erythrocyte lipid-peroxidation is similar in untrained overweight/obese T2DM patients and overweight/obese control subjects, while antioxidative mechanisms differ. Physical training might improve oxidative stress in T2DM men's erythrocytes during acute exercise. [ABSTRACT FROM AUTHOR]

Purpose: Peroxiredoxins, which reduced intracellular peroxides as a noval kind of antioxidant protein, were extensively expressed in various types of cancers and were thought as a biomarker of cancer cells. In this work, we performed genotyping analyses for tag SNP of Prdx 1, 2 and 6, and then evaluated the association with susceptibility and clinic stage of esophageal squamous cell carcinoma (ESCC) in a case-control study. Methods: The protein level of these Prdx isoforms in ESCC cancer samples was evaluated by Western blot. Then 356 ESCC cancer cases and 315 controls were genotyped by SNPshot assay. Differences in frequencies of the genotypes of the SNPs variant between the cases and controls were evaluated by using the chi-square test. Results: Our result of Western blot confirmed the aberrant expression of Prdx 1, 2 and 6 in ESCC samples, which was coincident with other studies. After genotyping by SNPshot assay, the result showed that the allele and genotype frequencies did not differ between the patients and controls. And no association between the polymorphism and the progression of ESCC including tumor grade and stage was found. Conclusions: Our data suggested that polymorphisms of Prdx 1, 2 and 6 were not associated with esophageal cancer. [ABSTRACT FROM AUTHOR]

Zearalenone is a mycotoxin mainly produced by Fusarium mold, which has been associated with hyperestrogenism and other reproductive disorders in pigs, sheep, and other farm animals. Since zearalenone engendered economic losses to farm animal production, its detoxification in contaminated grains or by-products would be advantageous. In this study, enzymes from the Acinetobacter sp. SM04 extracellular extracts of liquid cultures were isolated by Sephadex G-100 column, and an active fraction capable of efficiently degrading zearalenone was obtained. Zearalenone could be oxidized into smaller estrogenic products by the active fraction, and two intermediate products, ZEN-1([M-H] at m/z 489) and ZEN-2([M-H] at m/z 405), were found. Zearalenone degradation activity of the active fraction was significantly inhibited by low oxygen gas content, protease, SDS, and EDTA treatment. Further, enzymes in the active fraction were analysed by SDS-PAGE and MALDI-TOF/TOF/MS, and three proteins were found that matched the database for Acinetobacter genus with great homology. They were identified as peroxiredoxin, a possible cytochrome and a putative fimbrial protein precursor. [ABSTRACT FROM AUTHOR]

Peroxiredoxin 6 (Prx6) belongs to the family of thiol-dependent peroxidases that catalyze the reduction of hydrogen peroxide, organic peroxides, and peroxynitrite. Peroxiredoxins (Prxs) are increasingly recognized as a multi-functional proteins involved in various cellular processes. Accordingly, individual Prxs have been found to interact with multiple partners. Although the list of Prxs-binding proteins is rapidly growing, interactions reported so far show very limited overlap with each other. Our earlier studies indicated that Prx6 is a major cytosolic protein of rat olfactory epithelium and an important component of antioxidant defense system in this tissue. Here we used a combination of biochemical methods including pull-down assay, chemical cross-linking with a tri-functional cross-linker sulfo-SBED, co-immunoprecipitation, and mass spectrometry-based detection to conduct a random search for the Prx6-binding partners in water-soluble extracts from rat olfactory epithelium. Our findings showed that recombinant Prx6 formed complexes with peroxiredoxin 1, cytoskeletal proteins actin and tubulin, metabolic protein glyceraldehyde-3-phosphate dehydrogenase, heat shock proteins 70 and 90, and native Prx6. A common property of the identified proteins is the presence of conserved redox-sensitive Cys residue in their molecules. Novel interactions of rat Prx6 were validated by several independent methods, thus eliminating technical false-positives. However, the conventional methodological approaches to capture reproducibly real physical interactions have intrinsic limitations in distinguishing between randomly and functionally associated proteins, since not all of the naturally occurring protein complexes necessarily bear functional significance. We hypothesize that protein-protein interactions of Prx6 in vivo might induce conformational changes in its molecule, thus increasing the accessibility of the active-site Cys to general cellular reducing agents, such as thioredoxin or glutathione. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs), a group of antioxidant enzymes, are an important component of the oxidative defense system and have been demonstrated to function as peroxidases, sensors of HO-mediated signaling and/or chaperones. In this study, a cDNA library was constructed from a halotolerant alga, Dunaliella viridis, and was used in a functional complementation screen for antioxidative genes in an oxidative sensitive yeast mutant. Two Prx genes, DvPrx1 and DvPrx2, were obtained from this screen. These two genes were classified as type II Prx and 2-Cys Prx based on amino acid sequence and phylogenetic analysis. When over-expressed in yeast cells, both Prx genes were able to confer better oxidative tolerance and decrease the level of reactive oxygen species (ROS). Subcellular localization experiments in tobacco cells revealed that both DvPrx1 and DvPrx2 were localized in the cytosol. The transcription of DvPrx1 and DvPrx2 can be induced by hypersalinity shock, but is not obviously affected by treatment with high levels of oxidant. Our results shed light on the function and regulation of Prx genes from Dunaliella and their potential roles in salt tolerance. [ABSTRACT FROM AUTHOR]

The techniques of homology cloning and anchored PCR were used to clone the peroxiredoxin (Prx) gene from black tiger shrimp ( Penaeus monodon). The full length cDNA of black tiger shrimp Prx (PmPrx) contained a 5′ untranslated region (UTR) of 51 bp, an ORF (open reading frame) of 582 bp encoding a polypeptide of 193 amino acids with an estimated molecular mass of 22.15 kDa and a 3′ UTR of 948 bp. Sequence comparison showed that PmPrx shared higher identities with Prx IVs than that with other isoforms of Prx, indicating PmPrx was a member of the Prx IV family. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of PmPrx in different tissues and the temporal expression of PmPrx in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of PmPrx was detected in the tissues of hepatopancreas, gonad and heart. The expression of PmPrx in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that PmPrx was a constitutive and inducible expressed protein and could be induced by LPS. [ABSTRACT FROM AUTHOR]

The antioxidant protein, adhesin thiol peroxidase ( HpTpx or HP0390), plays an important role in enabling Helicobacter pylori to survive gastric oxidative stress. The bacterium colonizes the host stomach and produces gastric cancer. However, little information is available about the biochemical characteristics of HpTpx. We expressed recombinant HpTpx in Escherichia coli, purified to homogeneity, and characterized it. The results showed that HpTpx existed in a monomeric hydrodynamic form and the enzyme fully retained its peroxidase and antioxidant activities. The catalytic reaction of the enzyme was similar to an atypical 2-cysteine peroxiredoxin (Prx). The conformation of the enzyme was observed in the presence and absence of dithiothreitol (DTT); similar to other known thiol peroxidases, conformational change was observed in HpTpx by the addition of DTT. [ABSTRACT FROM AUTHOR]

The genome of Synechococcus elongatus PCC 7942 encodes six peroxiredoxins (Prx). Single genes are present each for a 1-Cys Prx and a 2-Cys Prx, while four genes code for PrxQ-like proteins ( prxQ-A1, - A2, - A3 and B). Their transcript accumulation varies with growth conditions in a gene-specific manner (Stork et al. in J Exp Bot 56:3193–3206, ). To address their functional properties, members of the prx gene family were produced as recombinant proteins and analysed for their peroxide detoxification capacity and quaternary structure by size exclusion chromatography. Independent of the reduction state, the 2-Cys Prx separated as oligomer, the 1-Cys Prx as dimer and the PrxQ-A1 as monomer. PrxQ-A2 was inactive in our assays, 1-Cys Prx activity was unaffected by addition of TrxA, while all others were stimulated to a variable extent by addition of E. coli thioredoxin. Sensitivity towards cumene hydroperoxide treatment of E. coli BL21 cells expressing the cyanobacterial PrxQ-A1 to A3 proteins was greatly reduced, while expression of the other Prx had no effect. The study shows differentiation of Prx functions in S. elongatus PCC 7942 which is discussed in relation to potential roles in site- and stress-specific defence. [ABSTRACT FROM AUTHOR]

The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCPlO. The bcp + mRNA level in the pBCPlO-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCPIO exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels. [ABSTRACT FROM AUTHOR]

Among the key antioxidant enzymes, thioredoxin and glutaredoxin systems play an important role in cell defense against oxidative stress and maintenance of redox homeostasis owing to the regulation of thiol—disulfide exchange. The thioredoxin isoforms Trx1 (cytoplasmic form) and Trx2 (mitochondrial form) can reduce inter- and intramolecular disulfide bonds in proteins, in particular, in oxidized peroxiredoxins, which disrupt organic hydroperoxides, H2O2, and peroxynitrite. NADPH-dependent thioredoxin reductase, which reduces a broad range of substrates including oxidized form of thioredoxin, can also directly reduce lipid hydroperoxides, H2O2, and dehydroascorbic and lipoic acids. Glutaredoxin, whose major isoforms in mammals are Grx1, Grx2, and Grx5, as well as thioredoxin, catalyzes S-glutathionylation and deglutathionylation of proteins to protect SH-groups from oxidation and restore functionally active thiols. However, in contrast to thioredoxin, glutaredoxin reduces GSH-mixed disulfides and catalyzes the reaction not only via a dithiol mechanism but also via monothiol reduction. In addition to the role in cellular antioxidant defense, all of the reviewed redox proteins (thioredoxin, thioredoxin reductase, peroxiredoxin, and glutaredoxin) have a number of significant functions required for cell viability: they regulate transcription factor activities, play the role of growth factors, serve as enzyme cofactors, take part in regulation of cell cycle, and are involved in antiapoptotic mechanisms. [ABSTRACT FROM AUTHOR]

Frozen tumor tissues from a patient who showed rapid progression to anaplastic oligodendroglioma after near total resection of oligodendroglioma were used to examine differential expression of proteins to gain better understanding of the pathogenesis of malignant transformation. We have determined their protein profiles using a 2D gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry approach. Among 23 differentially expressed spots, overexpression of peroxiredoxin 6 and underexpression of rho GDP dissociation inhibitor alpha were confirmed to be valid after western blot and immunocytochemical analysis of oligodendroglioma tissue. Abnormal expression of peroxiredoxin 6 and rho GDP dissociation inhibitor alpha may be associated with malignant transformation in oligodendroglioma and these proteins might be candidates of molecular predictive factors. [ABSTRACT FROM AUTHOR]

Recent reports showing successful inhibition of cancer and leukemia cell growth using histone deacetylase inhibitor (HDACi) compounds have highlighted the potential use of HDACi as anti-cancer agents. However, high incidence of toxicity and low stability in vivo were observed with hydroxamic acid-based HDACi such as suberoylanilide hydroxamic acid (SAHA), thus limiting its clinical applicability. In this study, we found that a novel non-hydroxamate HDACi NCH-51 could inhibit the cell growth of a variety of lymphoid malignant cells through apoptosis induction, more effectively than SAHA. Activation of caspase-3, -8 and -9, but not -7 was detected after the treatment with NCH-51. Gene expression profiles showed that NCH-51 and SAHA similarly upregulated p21 and downregulated anti-apoptotic molecules including survivin, bcl-w and c-FLIP. Proteome analysis using two-dimensional electrophoresis revealed that NCH-51 upregulated anti-oxidant molecules including peroxiredoxin 1 and 2 and glutathione S-transferase at the protein level. Interestingly, NCH-51 induced reactive oxygen species (ROS) after 8 h whereas SAHA continuously declined ROS. Pretreatment with an antioxidant, N-acetyl-L-cysteine, abolished the cytotoxicity of NCH-51. These findings suggest that NCH-51 exhibits cytotoxicity by sustaining ROS at the higher level greater than SAHA. This study indicates the therapeutic efficacy of NCH-51 and novel insights for anti-HDAC therapy.Leukemia (2007) 21, 2344–2353; doi:10.1038/sj.leu.2404902; published online 9 August 2007 [ABSTRACT FROM AUTHOR]