Mitogen-Activated Protein Kinase Cascade and Reactive Oxygen Species Metabolism are Involved in Acibenzolar-S-Methyl-Induced Disease Resistance in Apples.

Apple fruits were subjected to dipping treatment to explore the effects of acibenzolar-S-methyl (ASM) and the mitogen-activated protein kinase (MAPK) inhibitor PD98059 on lesion growth in fruits inoculated with Penicillium expansum. We investigated the roles of the MAPK cascade and reactive oxygen species metabolism in disease resistance in apples. ASM treatment inhibited lesion growth; suppressed catalase (CAT) activity; increased H2O2 content; reduced glutathione and ascorbic acid contents; and increased glutathione reductase, ascorbate peroxidase, peroxidase, superoxide dismutase, and NADPH oxidase activities. Moreover, ASM upregulated MdSOD, MdPOD, MdGR, MdAPX, MdMAPK4, MdMAPK2, and MdMAPKK1 expressions and downregulated MdCAT and MdMAPK3 expressions. PD98059 + ASM treatment increased CAT activity and MdCAT and MdMAPK3 expressions; inhibited MdSOD, MdPOD, MdGR, MdAPX, MdMAPK4, MdMAPK2, and MdMAPKK1 expressions; reduced superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase activities; and reduced glutathione content in apples. These findings indicate that ASM induces disease resistance in apples by regulating the expressions of key genes involved in reactive oxygen species metabolism and the MAPK cascade.

Analgesic and Anti-Inflammatory Activities of Rosa taiwanensis Nakai in Mice.

In this study, we evaluated the analgesic and anti-inflammatory activities of a 70% ethanol extract from Rosa taiwanensis Nakai (RTEtOH). The analgesic effect was determined using acetic acid-induced writhing response and formalin test. The anti-inflammatory activity was evaluated by λ-carrageenan-induced paw edema in mice. The anti-inflammatory mechanism of RTEtOH was examined by measuring the levels of cyclooxygenase-2 (COX-2), nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and malondialdehyde (MDA) in the paw edema tissue and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GRd) in the liver tissue. The betulinic acid and oleanolic acid contents of RTEtOH were assayed by HPLC. The results showed that RTEtOH decreased the acetic acid-induced writhing responses (1.0 g/kg) and the late phase of the formalin-induced licking time (0.5 and 1.0 g/kg). In the anti-inflammatory models, RTEtOH (0.5 and 1.0 g/kg) reduced the paw edema at 3, 4, and 5 h after λ-carrageenan administration. Moreover, the anti-inflammatory mechanisms might be due to the decreased levels of COX-2, TNF-α, IL-1ß, and IL-6, as well as the inhibition of NO and MDA levels through increasing the activities of SOD, GPx, and GRd. The contents of two active compounds, betulinic acid and oleanolic acid, were quantitatively determined. This study demonstrated the analgesic and anti-inflammatory activities of RTEtOH and provided evidence to support its therapeutic use in inflammatory diseases.

Immunoglobulin and cytokine changes induced following immunization with a DNA vaccine encoding Toxoplasma gondii selenium-dependent glutathione reductase protein.

Toxoplasma gondii Glutathione Reductase (TgGR) plays important role during the survival of the parasite. In this investigation, immunological changes and protection efficiency of this protein delivered as a DNA vaccine (pTgGR) have been evaluated. Mice were immunized with pTgGR, followed by challenge with virulent T. gondii RH strain, 2 weeks after the booster immunization. Compared to the control groups pVAX1, PBS and Blank groups, the results showed that pTgGR stimulated specific humoral response defined by significant titers of total IgG, subclasses IgG1 and IgG2a, classes IgA and IgM, but not IgE. Analysis of IFN-γ, IL-4, IL-17 and TGF-ß1 cytokines after immunization and compared with the control groups showed significant increments in pTgGR group. Additionally, T lymphocytes subpopulation CD4(+) T was positively recruited with significant percentage detected, while subset CD8(+) appeared not to be involved in response to this antigen. Vaccinated mice showed a significantly longer survival time, 15 days, in contrast with control groups which died within 8-10 days after challenge. These results demonstrated that TgGR could induce significant humoral and cell mediated responses leading to a considerable level of resistance against toxoplasmosis infection.

A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits.

Antioxidant systems are fundamental components of host-parasite interactions, and often play a key role in parasite survival. Here, we report the cloning, heterologous expression, and characterization of a thioredoxin glutathione reductase (TGR) from Fasciola hepatica. The deduced polypeptide sequence of the cloned open reading frame (ORF) confirmed the experimental N-terminus previously determined for a native F. hepatica TGR showing thioredoxin reductase (TR) activity. The sequence revealed the presence of a fusion between a glutaredoxin (Grx) and a TR domain, similar to that previously reported in Schistosoma mansoni and Echinococcus granulosus. The F. hepatica TGR sequence included an additional redox active center (ACUG; U being selenocysteine) located at the C-terminus. The addition of a recombinant selenocysteine insertion sequence (SECIS) element in the Escherichia coli expression vector, or the substitution of the native selenocysteine by a cysteine, indicated the relevance of this unusual amino acid residue for the activity of F. hepatica TGR. Rabbit vaccination with recombinant F. hepatica TGR reduced the worm burden by 96.7% following experimental infection, further supporting the relevance of TGR as a promising target for anti Fasciola treatments.

[Clinical and pathogenetic significance of antibodies to antioxidative enzymes in patients with Raynaud's syndrome].

Patients presenting with systemic scleroderma were found to have antibodies to antioxidative enzymes the levels of which increased with activity of the disease. Taking into account the important role of immune disorders in the development of systemic sclerosis, it can be conjectured that antienzyme antibodies may cause dysregulation of enzymatic systems. Serum antibodies were detected by the original modification of indirect enzyme immunoassay using immobilized antigenic forms of enzymes. All patients with high antibody titers presented with class II-IV Raynaud"s syndrome (RS). Pathogenetic mechanisms of RS are complicated and poorly known, it is supposed to be a multifactor pathology associated with disturbances in neural, vascular, mediator, and immune systems. Reperfusion and free oxygen radicals may also contribute to the development of ischemia in RS. The antioxidative system is known to involve a number of enzymes that neutralize pathogenic effects of free oxygen species. Disturbance of equilibrium between aggressive and protective factors under pathological conditions leads to further aggravation of tissue lesions. The presence of antienzyme antibodies in the blood of patients with primary RS may be regarded as an unfavourable prognostic factor preceding a systemic disease of connective tissue.

Differences in associations between markers of antioxidative defense and asthma are sex specific.

BACKGROUND: Lungs are exposed to high levels of oxygen, air pollutants, and smoke, all of which stimulate the production of reactive oxygen species (ROS). In addition, inflammatory cells produce ROS, and thus there may be increased demand for antioxidants, including antioxidant enzymes, in inflammatory lung diseases such as asthma. Sex-specific differences have been noted for asthma, which in postpubertal subjects is predominantly found in females. These sex-specific differences may be associated with differences on the molecular level as well. OBJECTIVE: The aim of this cross-sectional study was to examine associations between markers of antioxidative defense and asthma, and to investigate whether these associations were different between women and men. METHODS: Based on the European Community Respiratory Health Survey protocol, subjects were enrolled in a study of asthma risk factors. The multicenter study was conducted in 5 west Danish counties between 2003 and 2006, and the subjects were recruited as a case-enriched random sample of 10,000 Danish inhabitants aged 20 to 44 years selected by their civil registration number. Participants were identified by positive answers to asthma questions on a screening questionnaire, random sampling, or both. Serum selenium concentrations and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase [GPX], glutathione reductase [GR], and glucose-6-phosphate dehydrogenase [G6PD]) in erythrocytes were measured. Asthma was defined as either current asthma symptoms with bronchial hyperresponsiveness (BHR) or a continuous asthma score based on 8 questions. RESULTS: A total of 1191 mostly white women and men (mean [SD] age, 34.0 [7.1] and 35.1 [7.1] years, respectively) were enrolled in the study. Current asthma symptoms were present in 29.9% (200/670) of women and 22.5% (117/521) of men, with women reporting more positive answers (51.1% vs 40.9%, respectively; P < 0.01) to asthma questions. Serum selenium concentrations were measured in 1151 subjects (640 women, 511 men), and antioxidant enzyme activities were measured in 295 subjects (161 women, 134 men). Women had higher enzyme activities of most antioxidant enzymes (GPX, P = 0.006; GR, P < 0.001; and G6PD, P = 0.009) than did men. Although the serum selenium concentration was inversely associated with asthma in both sexes, there was a female preponderance, with 3.5% lower serum selenium in subjects with current asthma symptoms with BHR (n = 77) compared with controls (n = 287). GR activity was associated with asthma in men, with 5.7% higher enzyme activity in subjects with current asthma symptoms with BHR (n = 14) compared with controls (n = 77). However, a significant interaction with gender was observed for analyses of GR (P = 0.02), but not for analyses of selenium. CONCLUSIONS: In this study of asthma risk factors, women had higher levels of enzyme activities than did men in a randomly selected Danish population, and sex-specific differences were found in the associations between markers of antioxidative defense and asthma.

[Interrelationship between antibodies to enzymes of antioxidant system and cardiac involvement in patients with systemic scleroderma].

In blood of patients with systemic scleroderma we detected antibodies to the antioxidant system enzymes. Level of specific immunoglobulins rose with increase of the disease activity. Antioxidant system of the body is represented by a number of enzymes called to negate pathogenic effect of active forms of oxygen. In pathological states balance between factors of aggression and defense is disturbed. This leads to even more deep damage of tissues. Taking into consideration important role of immunological shifts in development of atherosclerosis one can suggest that autoantibodies to enzymes represent one of mechanisms of derangement of the work of enzymatic systems. Significantly higher levels of antibodies were detected in patients with symptoms of involvement of the cardiovascular system. We measured blood serum antibodies according to elaborated by us method of indirect immuno enzyme analysis with the use of immobilized antigenic forms of enzymes.

Effect of Prototheca zopfii on neutrophil function from bovine milk.

This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H(2)O(2)) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative "California Mastitis Test" (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H(2)O(2) production was measured using the phenol red method. Catalase activity was measured following H(2)O(2) reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey's test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H(2)O(2) production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.

Macrophages in the development of protective immunity against experimental Brugia malayi infection.

The present report compares the macrophage function in rodent hosts susceptible and resistant to the human lymphatic filariid Brugia malayi. Macrophages from both mastomys (resistant) and gerbil (susceptible) infected intraperitoneally (i.p.) with the infective larvae (L3) of B. malayi were isolated from peritoneal lavage at different time-intervals and formation rate of NO, H2O2, O2-, TNF-alpha, glutathione peroxidase and reductase was assayed. NO release was found to be significantly increased in resistant mastomys as compared to gerbils and the release was markedly suppressed by i.p. administration of the NOS inhibitor aminoguanidine (AG). The AG-treated mastomys also demonstrated significantly greater establishment of larvae which correlated well with suppressed formation of NO. Nitric oxide synergizes with superoxide to form peroxynitrite radical (potent oxidant), which is known to be more toxic per se than NO. Results indicate the possible involvement of peroxynitrite in the rapid killing of larvae in the peritoneal cavity of mastomys. In contrast, the production of H2O2 was found to be enhanced in both species indicating that B. malayi L3 could withstand the toxic effects of H2O2. The higher level of glutathione peroxidase and reductase, as observed in mastomys compared with the gerbil after larval introduction, possibly protects the cell against the injurious effect of H2O2. The TNF-alpha level remained virtually unchanged in both the hosts, suggesting an insignificant role for this cytokine in parasite establishment.

Glutathione reductase is expressed at high levels in pancreatic islet cells.

Reactive oxygen species are, at least partly, involved in the diabetogenic agent-induced dysfunction of pancreatic beta-cells because the expression of antioxidative and redox proteins is low. We examined the levels of antioxidant/redox proteins, peroxiredoxins-1, -4, and -6 and glutathione reductase (GR), by immunohistochemistry and found that the expression of GR was very high in pancreatic islet cells compared to exocrine cells. When diabetes was induced by an intravenous injection of streptozotocin, the pre-administration of 1,3-bis[2-chloroethyl]-1-nitrosourea, an irreversible inhibitor of GR, made islet cells more vulnerable to streptozotocin. These data point to a pivotal role of the glutathione redox system in pancreatic islet cells against diabetogenic stress.

Immunosuppression and oxidative stress induced by acute and chronic exposure to cocaine in rat.

The objective of the present study was to verify if immunosuppression caused by cocaine (CO) can be mediated, at least in part, by increased formation of oxidative metabolites and reactive oxygen species (ROS) in rat. Pharmacokinetics of cocaine and its metabolites, cell-mediated immune function and cytokines production, biomarkers of cell redox state maintenance and lipidic peroxidation, and variations of activity in the enzymatic systems involved in cell antioxidant defence were measured in spleen of Wistar rats acutely and chronically treated with cocaine.C(max), AUC, and t(1/2) of norcocaine (NC) significantly increased after chronic exposure to cocaine while kinetic parameters of benzoylecgonine (BE) significantly decreased. A decrease in cultured T-lymphocytes proliferation and natural killer (NK) cell activity, a high increase of immunosuppressive cytokines and a switch from Th1-type cytokines to Th2-type cytokines together with an unbalance toward anti-inflammatory cytokines recovered within 4 h after acute treatment while subsisted for 14 days after chronic treatment. A significant increase in ascorbic acid (AA), reduced glutathione and glutathione reductase (GR) with a simultaneous decrease in oxidized glutathione were observed in the first hours after acute administration. Conversely, the increase in oxidized glutathione and malondialdehyde (MDA) production and the simultaneous depletion of reduced glutathione and ascorbic acid persisted at least 24 h after chronic cocaine treatment as well as the increase in the activities of glutathione reductase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). The results suggest that chronic cocaine administration affects cellular enzyme and non-enzyme-mediated antioxidant defence systems and promotes immunotoxicity in rat. Cocaine N-oxidative metabolism may be an indirect contributor, via oxidative stress.

Glutathione reductase directly mediates the stimulation of yeast glucose-6-phosphate dehydrogenase by GSSG.

Yeast glucose-6-phosphate dehydrogenase was inhibited by low NADPH concentrations in cell-free extracts, and de-inhibited by GSSG; extensive dialysis of the crude extract did not diminish the GSSG effect. Immunoprecipitation of glutathione reductase abolished the de-inhibition of glucose-6-phosphate dehydrogenase by GSSG. Purified glucose-6-phosphate dehydrogenase was inhibited by NADPH but not de-inhibited by GSSG, and upon addition of pure glutathione reductase GSSG completely de-inhibited the glucose-6-phosphate dehydrogenase.

Purification and immunological studies of glutathione reductase from rat liver. Evidence for an antigenic determinant at the nucleotide-binding domain of the enzyme.

Glutathione reductase has been purified to homogeneity by a method which is an improvement of an earlier procedure (Carlberg, I. and Mannervik, B. (1975) J. Biol. Chem. 250, 5475-5480). The new steps in the purification scheme include affinity chromatography on 2',5' ADP-Sepharose 4B. Antibodies to glutathione reductase from rat liver were raised in rabbits and used for analysis of the enzyme by quantitative 'rocket' immunoelectrophoresis. Glutathione reductase from human erythrocytes, porcine erythrocytes, and calf-liver gave precipitin lines showing partial identity with the rat liver enzyme in Ouchterlony double diffusion experiments. Enzyme from spinach, yeast (Saccharomyces cerevisiae), and the photosynthetic bacterium Rhodospirillum rubrum did not give precipitates with the antibodies to the enzyme from rat liver. Titration of glutathione reductase from the different sources with antibodies confirmed the cross-reactivity of the mammalian enzymes; the human enzyme giving the strongest heterologous reaction. No reaction was observed with the enzyme from spinach, yeast, and Rhodospirillum rubrum. NADPH, NADP+, and 2',5' ADP were found to inhibit the interaction between antibodies and glutathione reductase from rat liver and human erythrocytes. NADH, glutathione, or glutathione disulfide did not protect the enzyme from reacting with the antibodies. It is concluded that glutathione reductase has an antigenic binding site for the antibodies at the pyridine nucleotide-binding site of the enzyme molecule.

Glutathione reductase deficiency in a kindred with hereditary spherocytosis.

A kindred with hereditary spherocytosis was found to have erythrocyte glutathione reductase deficiency. The glutathione reductase was evaluated enzymatically and immunologically. The enzyme activity could not be restored either by flavin adenine dinucleotide (FAD) in vitro, or by oral administration of FAD. Kinetic and electrophoretic studies, heat stability tests, and pH curves revealed no evidence of a varient enzyme. The results of electroimmunodiffusion and neutralization tests using an antiserum indicated that the deficiency is caused by a diminished production of enzyme protein. Hereditary spherocytosis and glutathione reductase deficiency traits segregated independently. The amount of reduced glutathione in the erythrocyte was normal, and the deficiency state without hereditary spherocytosis was asymptomatic. All deficient members in this kindred were heterozygous and the residual enzyme activity in the heterozygote might be sufficient to maintain the reduced glutathione level to prevent oxidative stress.