Passive immune-protection of small abalone against Vibrio alginolyticus infection by anti-Vibrio IgY-encapsulated feed

Small abalone (Haliotis diversicolor supertexta) is a high value-added shellfish. It however has been suffering Vibrio alginolyticus infections, which cause mass death of small abalone and thus great economic losses, particularly in artificial aquaculture. In this study, we attempted to treat small abalone with anti-Vibrio IgY to elicit a passive immunity directly against V. alginolyticus infections. Anti-Vibrio IgY was alginate encapsulated in egg powders as feed, which may avoid antibody inactivation in the gastrointestinal tract of small abalone. The feed was tested for the stability of anti-Vibrio IgY in a gastrointestinal mimic environment. The result showed anti-Vibrio IgY retained activity as high as 90% after 4 h exposure to pancreatic enzymes. Addition of 0, 5 or 10% anti-Vibrio IgY-encapsulated egg powders into a basal diet to form abalone diet formulae. Small abalones fed with the anti-Vibrio IgY formulae showed a relatively high respiratory burst activity than those without anti-Vibrio IgY treatments. The survival rates of small abalones fed with 5 or 10% anti-Vibrio IgY egg powders were in the range of 65–70% 14 days post-V. alginolyticus challenge (1 × 10⁶ c.f.u.), which was significantly higher than 0% of those fed without anti-Vibrio IgY. The anti-Vibrio IgY-encapsulated formulae were thus concluded to be an effective means to prevent small abalone from V. alginolyticus infection, and may be practical in use in abalone aquaculture.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

In vivo inhibition of selenium dependent glutathione peroxidase and superoxide dismutase in rats by diethyldithiocarbamate

Intraperitoneal injection of rats with diethyldithiocarbamate (1.2 g/kg body wt) led to maximum diminution of superoxide dismutase activity at 1 hr by 86 and 84% in liver and red blood cell respectively with a gradual return to the normal level at 48 hr after administration of injection. Significant inhibition of selenium-dependent glutathione peroxidase was also observed, which returned to normal at 48 hr after administration of injection. However, maximum decline in its activity was at 12 hr by 52 and 73% in liver and red blood cells respectively. No significant difference in tissue level of selenium-independent glutathione peroxidase was observed during time course study after diethyldithiocarbamate administration. It is possible that inhibition of superoxide dismutase by diethyldithiocarbamate leads to accumulation of superoxide anion which in turn inactivates selenium-dependent glutathione peroxidase by its reaction with selenium at the active site of the enzyme.     

The contribution of thioredoxin-2 reductase and glutathione peroxidase to H(2)O(2) detoxification of rat brain mitochondria

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H(2)O(2)) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60±20% and 20±15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Pretreatment with H(2) O(2) alleviates aluminum-induced oxidative stress in wheat seedlings

Hydrogen peroxide (H₂O₂) is a key reactive oxygen species (ROS) in signal transduction pathways leading to activation of plant defenses against biotic and abiotic stresses. In this study, we investigated the effects of H₂O₂ pretreatment on aluminum (Al) induced antioxidant responses in root tips of two wheat (Triticum aestivum L.) genotypes, Yangmai‐5 (Al‐sensitive) and Jian‐864 (Al‐tolerant). Al increased accumulation of H₂O₂ and O₂^?? leading to more predominant lipid peroxidation, programmed cell death and root elongation inhibition in Yangmai‐5 than in Jian‐864. However, H₂O₂ pretreatment alleviated Al‐induced deleterious effects in both genotypes. Under Al stress, H₂O₂ pretreatment increased the activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and monodehydroascorbate reductase, glutathione reductase and glutathione peroxidase as well as the levels of ascorbate and glutathione more significantly in Yangmai‐5 than in Jian‐864. Furthermore, H₂O₂ pretreatment also increased the total antioxidant capacity evaluated as the 2, 2‐diphenyl‐1‐picrylhydrazyl‐radical scavenging activity and the ferric reducing/antioxidant power more significantly in Yangmai‐5 than in Jian‐864. Therefore, we conclude that H₂O₂ pretreatment improves wheat Al acclimation during subsequent Al exposure by enhancing the antioxidant defense capacity, which prevents ROS accumulation, and that the enhancement is greater in the Al‐sensitive genotype than in the Al‐tolerant genotype.     

Relative contributions of heart mitochondria glutathione peroxidase and catalase to H(2)O(2) detoxification in in vivo conditions

This study was aimed at assessing the relative contributions to H(2)O(2) detoxification by glutathione peroxidase and catalase in the mitochondrial matrix of heart. For this purpose, mitoplasts from rat heart were used in order to minimize contamination with microperoxisomes, and the kinetic rate constants of both enzymatic activities were determined along with a simulation profile. Results show that the contribution of catalase to H(2)O(2) removal in heart mitochondria is not significant, even under strong oxidative conditions, such as those achieved in ischemia-reperfusion and involving extensive glutathione depletion and high H(2)O(2) concentrations. Conversely, maintenance of the steady state levels of H(2)O(2) in the heart mitochondrial matrix seems to be the domain of glutathione peroxidase. It is suggested that the physiological role of the low amounts of catalase found in heart mitochondria is related to its peroxidatic rather than catalatic activity.     

Response to oxidative stress caused by H(2)O(2) in Saccharomyces cerevisiae mutants deficient in trehalase genes

The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing. The most severe phenotype corresponded to the Delta nth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2). Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.     

The molecular mechanism of protecting cells against oxidative stress by 2-selenium-bridged beta-cyclodextrin with glutathione peroxidase activity

Ultraviolet B (UVB) induces apoptosis and lipid peroxidation of NIH3T3 cells by producing reactive oxygen species (ROS). Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in organism and it can scavenge ROS. 2-selenium-bridged beta-cyclodextrin (2-SeCD) is a GPX mimic generated in our lab. Its GPX activity is 7.4 U/mumol, which is 7.5 times as much as that of ebselen. In this paper, we have established a cell damage system using UVB radiation. Using this system, we have determined antioxidant effect of 2-SeCD by comparison of malondialdehyde (MDA) and H(2)O(2) contents in NIH3T3 cells before and after UVB radiation. Experimental results indicate that 2-SeCD can inhibit lipid peroxidation and protect the cells from the damage generated by UVB radiation. To evaluate the molecular mechanism of this protection, we determined the effect of 2-SeCD on the expression of p53 and Bcl-2 in NIH3T3 cells. The results showed that 2-SeCD inhibits the increase of p53 expression level and the decrease of expression of Bcl-2 induced by UVB radiation. Thus, we have concluded that protection of NIH3T3 cells against oxidative stress by 2-SeCD was carried out by regulation of the expression of Bcl-2 and p53.     

Pancreatic islet beta-cell and oxidative stress: the importance of glutathione peroxidase

Pancreatic beta-cell function continuously deteriorates in type 2 diabetes despite optimal treatment regimens, which has been attributed to hyperglycemia itself via formation of excess levels of reactive oxygen species (ROS). Glutathione peroxidase GPx), by virtue of its ability to catabolize both H(2)O(2) and lipid peroxides, is uniquely positioned to protect tissues from ROS. The level of this antioxidant in beta cells is extremely low and overexpression of GPx in islets provides enhanced protection against oxidative stress. This suggests that GPx mimetics may represent a valuable ancillary treatment that could add a novel layer of protection for the beta-cell.     

Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids

Patients with increased haemolytic haemoglobin (Hb) have 10-20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H(2)O(2) generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN(3), a peroxidase inhibitor, catalase, a H(2)O(2) decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H(2)O(2) production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN(3) and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.     

In vivo protection of synaptosomes from oxidative stress mediated by Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride by the glutathione mimetic tricyclodecan-9-yl-xanthogenate

D609 (tricyclodecan-9-yl-xanthogenate) is a phosphatidylcholine-specific phospholipase C inhibitor that also has been reported to protect rodents against oxidative damage caused by lethal doses of ionizing radiation. We previously showed that D609 mimics glutathione. D609 has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies from our laboratory have also shown that D609 reduces the Alzheimer amyloid beta-peptide (1-42)-induced oxidative stress and cytotoxicity in neuronal cell culture. The present study was undertaken to test the hypothesis that D609 would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes isolated from gerbils, previously injected intraperitoneally (ip) with D609, were treated with the oxidants Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH), which produce free radicals. Synaptosomes isolated from the gerbils ip injected with D609 and treated with Fe2+/H2O2 or AAPH showed significant reduction in reactive oxygen species, levels of protein carbonyl, protein-bound hydroxynonenal (a lipid peroxidation product), and 3-nitrotyrosine (another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to oxidative stress in synaptosomes isolated from gerbils that were injected with saline, but treated with Fe2+/H2O2 or AAPH. These results are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders.     

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H(2)O(2) challenge in Scylla paramamosain

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135?bp full-length cDNA contains a 9?bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562?bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec(40), or U(40)) residue which is encoded by an opal codon, (127)TGA(129), and forms an active site with residues Q(74) and W(142). Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 ((64)LAFPCNQF(71)), an active site motif ((152)WNFEKF(157)), a potential N-glycosylation site ((76)NTT(78)), and two residues (R(90) and R(168)) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p?相似文献   

Inhibition of selenium dependent glutathione peroxidase and superoxide dismutase in rats by diethyldithiocarbamate: effect of pre-administration of alpha-tocopherol

Rats pre-administered with alpha-tocopherol (10 mgs/day) for 7 days afforded a significant protection at the tissue level against the lowering of superoxide dismutase and glutathione peroxidase, especially the selenium-dependent glutathione peroxidase. The protective action of alpha-tocopherol in the diethyldithiocarbamate treated rats may be attributed to its antioxidant/free radical scavenging action. It is concluded that selenium-dependent glutathione peroxidase and alpha-tocopherol act in a complementary fashion to block free radical formation.     

Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus)

Three novel glutathione S-transferase (GSTs) cDNAs were cloned from a disk abalone (Haliotis dicus discus) cDNA library. Multiple alignment and phylogenetic analysis of three GSTs revealed that their closest relationship is with insect sigma GSTs. Recombinant GSTs were over-expressed in Escherichia coli as soluble fusion proteins. HdGSTS1 and HdGSTS2 were active towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid, whereas HdGSTS3 appeared to be a non-enzymatic GST. Two active GSTs had similar optimum conditions for enzymatic reaction at pH 8.0 and temperature of approximately 30 degrees C. Molecular modeling analysis of three GSTs implicates their diverse active sites as being responsible for their different enzymatic features. Three sigma GSTs had significantly different expression patterns and levels of expression in abalone tissues, indicating their different functions. After 48 h-exposure to three model marine pollutants, only HdGSTS1 exhibited a proper inducibility, exhibiting its good biomarker potential for organic contaminants in marine environment. In contrast, the other two sigma GSTs revealed a minor role in the response of pollutants exposure.     

The molecular mechanism of protecting cells against oxidative stress by 2-selenium-bridged β-cyclodextrin with glutathione peroxidase activity

Ultraviolet B (UVB) induces apoptosis and lipid peroxidation of NIH3T3 cells by producing reactive oxygen species (ROS). Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in organism and it can scavenge ROS. 2-selenium-bridged β-cyclodextrin (2-SeCD) is a GPX mimic generated in our lab. Its GPX activity is 7.4 U/μmol, which is 7.5 times as much as that of ebselen. In this paper, we have established a cell damage system using UVB radiation. Using this system, we have determined antioxidant effect of 2-SeCD by comparison of malondialdehyde (MDA) and H₂O₂ contents in NIH3T3 cells before and after UVB radiation. Experimental results indicate that 2-SeCD can inhibit lipid peroxidation and protect the cells from the damage generated by UVB radiation. To evaluate the molecular mechanism of this protection, we determined the effect of 2-SeCD on the expression of p53 and Bcl-2 in NIH3T3 cells. The results showed that 2-SeCD inhibits the increase of p53 expression level and the decrease of expression of Bcl-2 induced by UVB radiation. Thus, we have concluded that protection of NIH3T3 cells against oxidative stress by 2-SeCD was carried out by regulation of the expression of Bcl-2 and p53.