Polymorphism in the manganese superoxide dismutase gene

Oxidative stress and mitochondrial damage occur in sepsis. Manganese superoxide dismutase (MnSOD) provides the main defence against oxidative stress within mitochondria. Ala9Val is a single nucleotide polymorphism (SNP) in the MnSOD gene, predicted to affect intra-mitochondrial transport of the enzyme. We found a significant difference in the genotype frequency between healthy subjects (n = 100) and patients with sepsis (n = 40, p = 0.009). For assessment of functionality ten healthy subjects of each homozygous genotype (A/A or V/V) were studied. Peripheral blood mononuclear cells were separated and incubated for 18 h with lipopolysaccharide (LPS), followed by analysis of mitochondrial and cytosolic fractions. There was no difference between genotypes in MnSOD activity and cytochrome c concentration, and minor differences in total antioxidant capacity (TAC) and mitochondrial membrane potential, which did not affect response to LPS. Despite predictions from structural enzyme studies that mitochondrial trafficking would be affected by the Ala9Val polymorphism of the MnSOD gene had little functional effect.     

Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri

A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-beta-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361-4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 degrees C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.     

Deficiency of manganese superoxide dismutase in hepatocytes disrupts zonated gene expression in mouse liver

The liver acinus displays a physiological periportal to perivenous oxygen gradient. This gradient was implicated to use reactive oxygen species (ROS) as mediators for the zonal gene expression. Mitochondria use oxygen and produce ROS, therefore they may contribute to the zonation of gene expression. To further elucidate this, we used the Cre-loxP system to generate a hepatocyte-specific null mutation of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) in mice. We found that ROS levels were enhanced in livers of MnSOD(-/-) mice which were reduced in size and displayed signs of liver failure such as intracellular protein droplets, increased apoptotic bodies and Bax levels as well as multinuclear hepatocytes. Further, the zonation of glutamine synthetase, glucokinase and phosphoenolpyruvate carboxykinase was no longer preserved. We conclude that deficiency of mitochondrial MnSOD initiates a dysregulation of zonated gene expression in liver.     

Characterization of the erythrocyte superoxide dismutase allozymes in the deer Cervus elaphus

The cDNA sequences for Cu,Zn superoxide dismutase from two Cervus elaphus subspecies, North American wapiti and European red deer, were determined. The derived amino acid sequences showed two differences: residue 8 was Leu in wapiti and Met in red deer and residue 25 was His in wapiti and Asn in red deer. The extra positive charge at position 25 in the wapiti isoform accounted for its greater mobility towards the cathode during non-denaturing electrophoresis, a procedure widely used in the genetic analysis of deer. There was no difference in specific activity between the two Cu,Zn superoxide dismutase isoforms, but the wapiti isoform was slightly more susceptible to heat denaturation.     

Demonstration and characterization of manganese superoxide dismutase of Providencia alcalifaciens

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and play a role in the pathogenesis of certain invasive bacteria. In this study, we reported for the first time here that Providencia alcalifaciens, a member of the family Enterobacteriaceae, produces a superoxide dismutase (SOD) as a major protein in culture supernatants. This protein was purified by a series of column chromatographic separations. The N-terminal amino acid sequence of the protein was determined to be highly homologous to manganese superoxide dismutase of Escherichia coli or Salmonella reported. The gene (sodA) encoding for SOD of P. alcalifaciens was cloned and sequenced. The sodA-encoded protein has a molecular weight of about 23.5 kDa, and the DNA sequence of P. alcalifaciens sodA gene (627 bp) has about 83% identity to the E. coli SOD gene. We constructed a sodA deletion mutant and its complemented strain of P. alcalifaciens. In J774, a macrophage cell line, the sodA deletion mutant was more susceptible to killing by macrophages than the wildtype strain and its complemented strain. When we injected the mutant strain, its complemented strain and wildtype strain intraperitoneally into DDY strain mice, we found that the sodA deletion mutant proved significantly less virulent while the complemented strain recovered the virulence to the same level of wildtype strain of P. alcalifaciens. These results suggested that manganese superoxide dismutase plays an important role in intracellular survival of P. alcalifaciens.     

Progestin stimulation of manganese superoxide dismutase and invasive properties in T47D human breast cancer cells

Superoxide dismutase (SOD) occurs in two intracellular forms in mammals, copper–zinc SOD (CuZnSOD), found in the cytoplasm, mitochondria and nucleus, and manganese superoxide dismutase (MnSOD), in mitochondria. Changes in MnSOD expression (as compared to normal cells) have been reported in several forms of cancer, and these changes have been associated with regulation of cell proliferation, cell death, and metastasis. We have found that progestins stimulate MnSOD in T47D human breast cancer cells in a time and physiological concentration-dependent manner, exhibiting specificity for progestins and inhibition by the antiprogestin RU486. Progestin stimulation occurs at the level of mRNA, protein, and enzyme activity. Cycloheximide inhibits stimulation at the mRNA level, suggesting that progestin induction of MnSOD mRNA depends on synthesis of protein. Experiments with the MEK inhibitor UO126 suggest involvement of the MAP kinase signal transduction pathway. Finally, MnSOD-directed siRNA lowers progestin-stimulated MnSOD and inhibits progestin stimulation of migration and invasion, suggesting that up-regulation of MnSOD may be involved in the mechanism of progestin stimulation of invasive properties. To our knowledge, this is the first characterization of progestin stimulation of MnSOD in human breast cancer cells.     

RNA interference mediated knockdown of apolipophorin-III leads to knockdown of manganese superoxide dismutase in Hyphantria cunea

Apolipophorin-III (apoLp-III), a hemolymph protein that facilitates lipid transport in aqueous media in insects was recently shown to play a role in insect immune activation. Here, we report another novel possible function of apoLp-III in insects. To identify genes affected by apoLp-III expression in larvae, we decreased endogenous apoLp-III mRNA in Hyphantria cunea (Hc) through RNA interference; subsequently, we observed lower levels of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), glutathione S-transferase, and immune proteins. Knockdown of Hc apoLp-III led to decreased MnSOD expression in fat body tissues and elevated superoxide anion levels in Hc fat body cells, suggesting that Hc apoLp-III is involved in the action and/or expression of antioxidant enzymes, especially MnSOD.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Decreased accumulation of superoxide dismutase 2 within mitochondria in the yeast model of Shwachman-Diamond syndrome

Mutations in the human SBDS gene is the most common cause of Shwachman-Diamond syndrome (SDS). The SBDS protein participates in ribosome biogenesis; however, effects beyond reduced translation efficiency are thought to be involved in SDS progression. Impaired mitochondrial function has been reported for cells lacking either SBDS or Sdo1p, the Saccharomyces cerevisiae SBDS ortholog. To better understand how the loss of SBDS/Sdo1p leads to mitochondria damage, we utilized the S. cerevisiae model of SDS. Yeast deleted for SDO1 show increased oxidative damage to mitochondrial proteins and a marked decrease in protein levels and activity of mitochondrial superoxide dismutase 2 (Sod2p), a key enzyme involved in defense against oxidants. Immature forms of Sod2p are observed in sdo1∆ cells suggesting a defect in proteolysis of the presequence. Yeast deleted for CYM1, encoding a presequence protease, display a similar reduction in Sod2p activity as sdo1∆ cells, as well as elevated oxidative damage, to mitochondrial proteins. Sod2p protein levels and activity are largely restored in a por1∆ sdo1∆ strain, lacking the major mitochondrial voltage-dependent anion channel. Together these results indicate that mitochondrial insufficiency in sdo1∆ cells may be linked to the accumulation of immature presequence containing proteins and this effect is a consequence, at least in part, from loss of counter-regulation of Por1p by Sdo1p.     

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.     

Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation

Numerous sequence variants of wheat (Triticum aestivum L.) manganese superoxide dismutase (MnSOD) genes have been found. Quantitative real-time PCR was used to measure the expression levels of three MnSOD genes distinguished by a variable amino acid, and three genes distinguished by sequence variation in the 3′ untranslated region (3′ UTR), in wheat plants grown at 20°C and cold-acclimated for 1–4 weeks at 2°C. The amino acid variants did not differ significantly in expression levels, however, differential expression of genes differing in the 3′ UTR was observed. Diploid wheat-related species also carried sequence variants of MnSOD, with differing levels of expression, suggesting diversification of the MnSOD gene family occurred prior to the polyploidization events of hexaploid wheat.     

Smoking and COPD increase sputum levels of extracellular superoxide dismutase

Extracellular superoxide dismutase (ECSOD) is the major superoxide-scavenging enzyme in the lung. Certain ECSOD polymorphisms are protective against COPD. We postulated that smokers and COPD subjects would have altered levels of ECSOD in the lung, airway secretions, and/or plasma. Lung tissue ECSOD was evaluated from nonsmokers, smokers, and subjects with mild to very severe COPD by Western blot, immunohistochemistry, and ELISA. ECSOD levels in plasma, bronchoalveolar lavage fluid (BALF), and induced-sputum supernatants were analyzed by ELISA and correlated with smoking history and disease status. Immunohistochemistry identified ECSOD in extracellular matrix around bronchioles, arteries, and alveolar walls, with decreases seen in the interstitium and vessels of severe COPD subjects using digital image analysis. Plasma ECSOD did not differ between COPD subjects and controls nor based on smoking status. ECSOD levels in induced sputum supernatants were elevated in current smokers and especially in COPD subjects compared to nonsmokers, whereas corresponding changes could not be seen in the BALF. ECSOD expression was reduced around vessels and bronchioles in COPD lungs. Substantial increases in sputum ECSOD in smokers and COPD is interpreted as an adaptive response to increased oxidative stress and may be a useful biomarker of disease activity in COPD.     

Inactivation of genes encoding superoxide dismutase modifies yeast response to S-nitrosoglutathione-induced stress

Antioxidant enzymes can modify cell response to nitrosative stress induced, for example, by nitric oxide or compounds decomposing with its formation. Therefore, we investigated the effects of S-nitrosoglutathione (GSNO) on cell survival, activity of antioxidant enzymes, and concentrations of reduced and oxidized glutathione in parental and isogenic strains defective in Cu,Zn- or Mn-superoxide dismutases (Cu,Zn-SOD and Mn-SOD, respectively), or in both of them. Stress was induced by incubation of the yeast with 1–20 mM GSNO. The strains used demonstrated different sensitivity to GSNO. A Cu,Zn-SOD-defective strain survived the stress better than the parental strain, while the double mutant was the most sensitive to GSNO. The ^·NO-donor at low concentrations (1–5 mM) increased SOD activity, but its high concentrations (10 and 20 mM) decreased it. The activity of catalase in all strains was enhanced by GSNO. Inhibition of protein synthesis by cycloheximide did not prevent the activation of SOD, but it prevented the activation of catalase. These facts suggest that SOD was activated at a posttranslational level and catalase activity was enhanced via de novo synthesis. A GSNO-induced increase in oxidized glutathione level in the studied yeast strains might account for cell killing by GSNO due to the development of oxidative/nitrosative stress. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 4, pp. 550–557.     

Extracellular superoxide dismutase

The extracellular space is protected from oxidant stress by the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), which is highly expressed in selected tissues including blood vessels, heart, lungs, kidney and placenta. EC-SOD contains a unique heparin-binding domain at its carboxy-terminus that establishes localization to the extracellular matrix where the enzyme scavenges superoxide anion. The EC-SOD heparin-binding domain can be removed by proteolytic cleavage, releasing active enzyme into the extracellular fluid. In addition to protecting against extracellular oxidative damage, EC-SOD, by scavenging superoxide, preserves nitric oxide bioactivity and facilitates hypoxia-induced gene expression. Loss of EC-SOD activity contributes to the pathogenesis of a number of diseases involving tissues with high levels of constitutive extracellular superoxide dismutase expression. A thorough understanding of the biological role of EC-SOD will be invaluable for developing novel therapies to prevent stress by extracellular oxidants.     

Oxygen toxicity and superoxide dismutase as an antioxidant in physiological stress

Although oxygen is vital for all aerobic life forms, excessive levels of oxygen free radical production can lead to potentially lethal oxidative reactions. The role of superoxide dismutase is examined as an integral part of the defence against oxidative injury resulting from various physiological stresses.