Regulation of superoxide dismutase isoforms in resistant and susceptible strawberry cultivars subjected to leaf spot disease

Abstract

Macroscopic symptoms were observed in two strawberry cultivars, with the degree of symptom intensity varying depending on the susceptibility of the cultivars, i.e. resistant or susceptible. The symptoms presented as red spots and were observed 30 d following leaf tissue inoculation with the Mycosphaerella fragariae pathogen. A comparison of the superoxide dismutase isoform profiles obtained by gel electrophoresis in all samples extracted from both resistant and susceptible cultivars indicated one constant sharp band, identified as Mn[sbnd]SOD with a molecular mass of 19 kDa. The intensity of this band was higher in all samples derived from the resistant cultivar than in those from the susceptible cultivar. Another superoxide dismutase (SOD) isoform, identified as CuZn[sbnd]SOD with a molecular mass of 16 kDa, was detected in all soluble proteins derived from the resistant cultivar. This isoform was not observed in the susceptible cultivar; however, following an incremental increase in the amount of loaded protein, it was illuminated as a faint band in a sample collected 3 d after inoculation, indicating insufficient production of the CuZn[sbnd]SOD isoform in the susceptible cultivar during an oxidative burst induced by the M. fragaria pathogen. Several bands were also characterized in both cultivars containing Fe and Mn as their co-factors (Fe, Mn[sbnd]SOD). Unlike in the resistant cultivar, where the activity of Fe, Mn[sbnd]SOD isoforms gradually and regularly increased and reached its highest level on the third day after inoculation, the activity of the isoforms changed irregularly over 20 days of study in the susceptible cultivar.     

Time-related alterations of superoxide radical levels in diverse organs of bile duct-ligated rats

The time-related alterations of superoxide radical measured in vivo by employing an ultrasensitive fluorescent assay in the liver, intestine, kidney and brain of rats with experimentally induced obstructive jaundice was investigated. Eighteen rats were randomly divided into Group A, rats subjected to sham operation, and Group B, rats subjected to bile duct ligation (BDL). Three rats from each group were subsequently killed at different time points post-operatively (1, 5 and 10 days). As compared to sham-operated, BDL rats showed a gradual increase with time of superoxide radical in the intestine, liver, kidney and brain: for animals sacrificed on the 1^st, 5^th and 10^th day the increase was 45%, 50% and 96% in the liver, 76%, 81% and 118% in the intestine, 64%, 71% and 110% in the kidney and 76%, 95% and 142% in the brain, respectively. This study provides direct evidence of an early appearance of oxidative stress in diverse organs, implying a uniform systemic response to biliary obstruction and emphasizing the need of early bile flow restoration.     

Studies on the mechanism of toxicity of the mycotoxin, sporidesmin. I. Generation of superoxide radical by sporidesmin

Sporidesmin (SDMS2), the mycotoxin responsible for 'facial eczema' in ruminants, contains a disulphide group which appears to be intimately involved in its toxic action. The reduced (dithiol) form of sporidesmin has been shown readily to undergo autoxidation in vitro in a reaction which generates superoxide radical (O2-). The autoxidation reaction, which takes place over a wide pH range, is strongly catalysed by trace amounts of copper, although the reaction was inhibited at high concentrations of this metal. Inhibition of the autooxidation of reduced sporidesmin (SDM(SH)2) was also observed in the presence of nickel, cobalt and manganese. Superoxide radical is also generated from SDMS2 itself in a cyclic reduction/autoxidation reaction with glutathione and other thiols; in view of the known toxicity of superoxide and its derivatives, it is suggested that oxygen-free-radicals may be involved in the initiation of the deleterious effects of the mycotoxin.     

Identification of superoxide dismutase isoenzymes in tobacco pollen

We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetra-zolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).     

Triarylmethyl-based biradical as a superoxide probe

Abstract

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM–TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7?±?0.2)?×?10³ M^?1 s^?1) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.     

Ionol (BHT) produces superoxide anion

In aqueous medium etiolated wheat seedlings release superoxide anion ( ). Interaction of a synthetic antioxidant, butylated hydroxytoluene (BHT, ionol), with oxygen in the aqueous medium is accompanied by formation. This suggests that under certain conditions BHT behaves as a prooxidant. A natural antioxidant, superoxide dismutase (SOD), and also a wound healing preparation, emulsified denatured placenta (EDP), do not exhibit the prooxidant properties. In contrast to BHT, they reduce production by the etiolated wheat seedling system.     

Regulation of superoxide flashes by transient and steady mitochondrial calcium elevations

The mitochondria play essential roles in both intracellular calcium and reactive oxygen species signaling.As a newly discovered universal and fundamental mitochondrial phenomenon,superoxide flashes reflect transient bursts of superoxide production in the matrix of single mitochondria.Whether and how the superoxide flash activity is regulated by mitochondrial calcium remain largely unknown.Here we demonstrate that elevating mitochondrial calcium either by the calcium ionophore ionomycin or by increasing the bathing calcium in permeabilized HeLa cells increases superoxide flash incidence,and inhibition of the mitochondrial calcium uniporter activity abolishes the flash response.Quantitatively,the superoxide flash incidence is correlated to the steady-state mitochondrial calcium elevation with 1.7-fold increase per 1.0?F/F0 of Rhod-2 signal.In contrast,large mitochondrial calcium transients(e.g.,peak△F/F0~2.8,duration~2 min)in the absence of steady-state elevations failed to alter the flash activity.These results indicate that physiological levels of sustained,but not transient,mitochondrial calcium elevation acts as a potent regulator of superoxide flashes,but its mechanism of action likely involves a multi-step,slow-onset process.     

A new perspective on the role of CuZn superoxide dismutase (SOD1)

The CuZn superoxide dismutase (SOD1), a member of a group of isoenzymes involved in the scavenger of superoxide anions, is a dimeric carbohydrate free protein, mainly localized in the cytosol. The reactive oxygen species (ROS) are involved in many pathophysiological events correlated with mutagenesis, cancer, degenerative processes and aging. In the first part of this mini-review the well known role of SOD1 and ROS are briefly summarized. Following, a potential novel biological action that SOD1 could exert is described, based on the recent researches demonstrating the secretion of this enzyme in many cellular lines. Moreover, the role of impaired mutant SOD1 secretion, associated with cytoplasmic toxic inclusion, which occurs in familial amyotrophic lateral sclerosis (ALS), is summarized. In addition, a depolarization-dependent release of SOD1 in pituitary GH3 cells and in rat synaptosomes through a calcium and SNARE-dependent mechanism is reported.     

Subcutaneous administration of bovine superoxide dismutase protects lungs from radiation-induced lung injury

Abstract

Background. The objective of the present study was to determine whether single administration of the antioxidant enzyme bovine superoxide dismutase (bSOD) after radiation therapy (RT) mitigates development of pulmonary toxicity in rats. Methods. Female F344 rats (n = 60) were divided among six experimental groups: (1) RT, single dose of 21 Gy to the right hemithorax; (2) RT + 5 mg/kg bSOD; (3) RT + 15 mg/kg bSOD; (4) No RT; (5) sham RT + 5 mg/kg bSOD; and (6) sham RT + 15 mg/kg bSOD. A single subcutaneous injection of bSOD (5 or 15 mg/kg) was administered 24 h post-radiation. The effects of bSOD on radiation-induced lung injury were assessed by measurement of body weight, breathing frequency, and histopathological changes. Immunohistochemistry was used to evaluate oxidative stress (8-OHdG⁺, NOX4⁺, nitrotyrosine⁺, and 4HNE⁺ cells), macrophage activation (ED1⁺), and expression of profibrotic transforming growth factor-β or TGF-β in irradiated tissue. Results. Radiation led to an increase in all the evaluated parameters. Treatment with 15 mg/kg bSOD significantly decreased levels of all the evaluated parameters including tissue damage and breathing frequency starting 6 weeks post-radiation. Animals treated with 5 mg/kg bSOD trended toward a suppression of radiation-induced lung damage but did not reach statistical significance. Conclusions. The single application of bSOD (15 mg/kg) ameliorates radiation-induced lung injury through suppression of reactive oxygen species/reactive nitrogen species or ROS/RNS-dependent tissue damage.     

Attenuation of oxidative stress by Allylpyrocatechol in synovial cellular infiltrate of patients with Rheumatoid Arthritis

Abstract

Free radicals are involved in the pathogenesis of Rheumatoid arthritis, a systemic autoimmune disorder characterized by unchecked synovial inflammation. Allylpyrocatechol, a phytoconstituent of Piper betle leaves, has potent anti-inflammatory activity and this study evaluated its anti-oxidant effect on the synovial infiltrate of patients with Rheumatoid arthritis. The ex vivo effect of allylpyrocatechol upon generation of reactive oxygen species in neutrophils, macrophages and lymphocytes was measured by flow cytometry using dichlorodihydrofluorescein diacetate, wherein it significantly decreased basal levels as also scavenged phorbol myristate acetate generated reactive oxygen species. Furthermore, its effect on generation of superoxide and hydroxyl radicals produced within infiltrated neutrophils was measured by cytochrome c and deoxyribose assay, respectively. Allylpyrocatechol significantly scavenged superoxide and hydroxyl radicals in infiltrated neutrophils. The effect of allylpyrocatechol on nitric oxide was measured in macrophages using 4,5-diaminofluorescein diacetate by flow cytometry wherein it decreased production of nitric oxide in infiltrated macrophages, which correlated with its in vitro nitric oxide scavenging activity. Taken together, this ex vivo study has established that allylpyrocatechol has potent scavenging activity and could be considered as an add-on therapy in the treatment of inflammation-associated disorders like Rheumatoid Arthritis.     

Effect of exercise on bone and articular cartilage in heterozygous manganese superoxide dismutase (SOD2) deficient mice

Abstract

Reactive oxygen species (ROS) are involved in both bone and cartilage physiology and play an important role in the pathogenesis of osteoporosis and osteoarthritis. The present study investigated the effect of running exercise on bone and cartilage in heterozygous manganese superoxide dismutase (SOD2)-deficient mice. It was hypothesized that exercise might induce an increased production of ROS in these tissues. Heterozygous SOD2-deficient mice should exhibit an impaired capability to compensate, resulting in an increased oxidative stress in cartilage and bone. Thirteen female wild type and 20 SOD2^+/? mice (aged 16 weeks) were randomly assigned to a non-active wild type (SOD2^+/+Con, n = 7), a trained wild type (SOD2^+/+Run, n = 6), a non-active SOD2^+/? (SOD2^+/?Con, n = 9) and a trained SOD2^+/? (SOD2^+/?Run, n = 11) group. Training groups underwent running exercise on a treadmill for 8 weeks. In SOD2^+/? mice elevated levels of 15-F_2t-isoprostane and nitrotyrosine were detected in bone and articular cartilage compared to wild type littermates. In osteocytes the elevated levels of these molecules were found to be reduced after exercise while in chondrocytes they were increased by aerobic running exercise. The observed changes in oxidative and nitrosative stress did neither affect morphological, structural nor mechanical properties of both tissues. These results demonstrate that exercise might protect bone against oxidative stress in heterozygous SOD2-deficient mice.     

Low rates of hydrogen peroxide production by isolated heart mitochondria associate with long maximum lifespan in vertebrate homeotherms

An inverse correlation between free radical production by isolated mitochondria and longevity in homeotherms has been reported, but previous comparative studies ignored possible confounding effects of body mass and phylogeny. We investigated this correlation by comparing rates of hydrogen peroxide (H(2)O(2)) production by heart mitochondria isolated from groups or pairs of species selected to have very different maximum lifespans but similar body masses (small mammals, medium-sized mammals, birds). During succinate oxidation, H(2)O(2) production rates were generally lower in the longer-lived species; the differences arose at complex I of the electron transport chain during reverse electron transport. Additional data were obtained from large species and the final dataset comprised mouse, rat, white-footed mouse, naked mole-rat, Damara mole-rat, guinea pig, baboon, little brown bat, Brazilian free-tailed bat, ox, pigeon and quail. In this dataset, maximum lifespan was negatively correlated with H(2)O(2) production at complex I during reverse electron transport. Analysis of residual maximum lifespan and residual H(2)O(2) production revealed that this correlation was even more significant after correction for effects of body mass. To remove effects of phylogeny, independent phylogenetic contrasts were obtained from the residuals. These revealed an inverse association between maximum lifespan and H(2)O(2) production that was significant by sign test, but fell short of significance by regression analysis. These findings indicate that enhanced longevity may be causally associated with low free radical production by mitochondria across species over two classes of vertebrate homeotherms.     

Production of a thermostable archaeal superoxide reductase in plant cells

Pyrococcus furiosus superoxide reductase (SOR) is a thermostable archaeal enzyme that reduces superoxide without producing oxygen. When produced as a fusion protein with the green fluorescent protein in plant cells, P. furiosus SOR is located in the cytosol and nucleus. The recombinant SOR enzyme retains its function and heat stability when assayed in vitro. Importantly, expressing SOR in plant cells enhances their survival at high temperature indicating that it functions in vivo. The archaeal SOR provides a novel mechanism to reduce superoxide and demonstrates the potential for using archaeal genes to alter eukaryotic metabolism.     

Oxidative stress as a potential biomarker for determining disease activity in patients with Rheumatoid Arthritis

Rheumatoid arthritis is an inflammatory, autoimmune disease where oxidative stress has been proposed to contribute to the joint tissue damage. To establish whether measurement of the redox status in blood mirrors the oxidant status at sites of inflammation in patients with rheumatoid arthritis, we concomitantly examined their oxidant status by spectrophotometry and/or flow cytometry. The basal levels of total reactive oxygen species (ROS), superoxide and hydroxyl radicals were significantly raised in neutrophils sourced from peripheral blood and synovial infiltrate, as also showed a strong positive correlation; however, there was no major increase in the reactive nitrogen species RNS generated in monocytes from both sources. Furthermore, raised levels of superoxide in neutrophils of synovial infiltrate showed a positive correlation with NADPH oxidase activity in synovial fluid. Additionally, as ROS generated in both peripheral blood and synovial infiltrate correlated positively with both DAS 28 and CRP/anti-CCP levels, its measurement can serve as an indirect measure of the degree of inflammation in patients with RA.     

Estrogen regulation of superoxide dismutase in normal rat mammary tissues and mammary tumors

Multiple electrophoretic molecular variants of superoxide dismutase were demonstrated in normal rat mammary tissues and DMBA-induced rat mammary tumors. The specific activities of $\text{Cu}\text{Zu}$ superoxide dismutase in mammary tumors of estrogen-treated rats were not significantly different from those activities seen in normal rat mammary tissues. However, the enzyme activities of mammary tumors from untreated rats (no estrogen) were significantly lower than the activities of normal rat mammary tissues. Exogenous estrogen appeared to raise superoxide dismutase levels in DMBA-induced rat mammary tumors to those levels seen in normal rat mammary tissues.     

The role of a cytosolic superoxide dismutase in barley–pathogen interactions

Reactive oxygen species (ROS), including superoxide ( / ) and hydrogen peroxide (H₂O₂), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi‐biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of / to H₂O₂, regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi‐biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up‐regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild‐type plants, suggesting that cytosolic / contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi‐biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.