Spectral and physical properties of human extracellular superoxide dismutase: a comparison with CuZn superoxide dismutase

A mouse monoclonal IgG1 antibody, referred to as NI-58, has been produced. In immunofluorescence assay, this antibody reacted with myelomonocytes, EBV-B cells, Burkitt's lymphoma cells, T cell leukemia cells and peripheral blood mononuclear cells, but not with erythroid cells. The surface antigen on U937 cells recognized by NI-58 had a molecular size of 65 kDa as determined by immunoblotting analysis. As a biological function, NI-58 strongly inhibited the homotypic cell adhesion of LPS-stimulated U937 cells. It was found that the antigen defined by NI-58 was distinct from CD54 (intercellular adhesion molecule-1) in it's pattern of cellular expression and molecular weight, suggesting that NI-58 recognizes a new adhesion molecule and inhibits the homotypic cell adhesion of LPS-stimulated U937 cells.     

Purification and subunit structure of extracellular superoxide dismutase from mouse lung tissue

The first purification of mouse extracellular superoxide dismutase (EC-SOD) and the analysis of the native enzyme are described. Mouse EC-SOD was purified from lung tissues with a high recovery (41%) and a specific polyclonal antibody against the purified enzyme was obtained. The purified enzyme had a strong affinity for, heparin and a molecular mass of 150 kDa (estimated by a gel filtration chromatography). The native mouse EC-SOD was composed of two different sizes of subunits, a M(r) of 33 and 35 kDa (determined by SDS-PAGE). The 35-kDa subunit had an interchain disulfide bond at the C-terminus and existed as a covalent dimer in the molecule, whereas the 33-kDa subunit resulted from the 35-kDa subunit by truncating its C-terminus as a posttranslational modification, with resultant loss of the interchain disulfide bond. These results suggest that the native mouse EC-SOD is a heterotetramer composed of two different dimers, with or without a covalent bond.     

Gene therapy with extracellular superoxide dismutase attenuates myocardial stunning in conscious rabbits

BACKGROUND: Administration of Cu/Zn superoxide dismutase (SOD) without catalase fails to alleviate myocardial stunning, but extracellular SOD (Ec-SOD) may be more effective because it binds to heparan sulfate proteoglycans on the cellular glycocalyx. We therefore used in vivo gene transfer to increase systemic levels of Ec-SOD and determined whether this gene therapy protects against myocardial stunning. METHODS and RESULTS: The cDNA for human Ec-SOD was cloned behind the cytomegalovirus (CMV) promoter and incorporated into a replication-deficient adenovirus (Ad5/CMV/Ec-SOD). Injection of this virus (2x10(8) pfu/kg IV) produced high levels of Ec-SOD in the liver, which could be redistributed to the heart and other organs by injection of heparin. Conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days starting 3 days after intravenous injection of Ad5/CMV/Ec-SOD or Ad5/CMV/nls/LacZ (negative control). Both groups were given heparin (2000 U/kg IV) 2 hours before the first sequence of occlusions. The severity of myocardial stunning was measured as the total deficit of LV wall thickening after the last reperfusion. On day 1, the total deficit of wall thickening was markedly decreased in Ad5/CMV/Ec-SOD rabbits versus controls and similar to that seen on days 2 and 3 in controls. CONCLUSIONS: The results demonstrate that in vivo gene transfer of the cDNA encoding Ec-SOD provides the heart with substantial protection against myocardial stunning without the need for concomitant administration of catalase. The present observations provide the basis for controlling gene therapy at the posttranslational level and for simultaneously protecting multiple organs from oxidant stress.     

Plasma extracellular superoxide dismutase levels in an Australian population with coronary artery disease

In vitro experiments suggest that free radicals may contribute importantly to atherogenesis. Superoxide dismutase (SOD), particularly extracellular SOD (EC-SOD), which accounts for the majority of SOD biological activity, is a major superoxide scavenger. We explored factors that may affect plasma EC-SOD levels measured by ELISA and assessed the association between plasma EC-SOD and coronary artery disease documented angiographically in 590 white Australian patients 400 ng/mL were heterozygous for the Arg213-->Gly mutation at the EC-SOD gene; there was also a positive correlation with age (r=0.131, P=0.0016). Plasma EC-SOD in current smokers (75. 0+/-9.3 ng/mL) was much lower than in nonsmokers (111.7+/-8.2 ng/mL, P<0.01), and ex-smokers had intermediate levels (84.3+/-7.1 ng/mL). Levels were significantly lower in patients with than in those without a history of acute myocardial infarction (MI) (76.1+/-7.5 versus 110.1+/-6.0 ng/mL, P<0.05), and low plasma EC-SOD was independently associated with an increased likelihood of a history of MI (OR, 2.04; 95% CI, 1.10 to 3.82); higher EC-SOD levels also tended to be associated with delayed onset of MI. In conclusion, our study establishes that in patients assessed by coronary angiography, circulating EC-SOD is lower in men than in women and in smokers of each sex and that low levels are independently associated with a history of MI. These findings are consistent with EC-SOD's being protective and contributing to reduced coronary risk.     

Rat testicular extracellular superoxide dismutase: its purification, cellular distribution, and regulation

Rehabilitative measures for stroke are not generally based on basic neurobiological principles, despite evidence from animal models that certain anatomical and pharmacological changes correlate with recovery. In this report, we use functional magnetic resonance imaging (fMRI) to study in vivo human brain reorganization in a right handed patient with an acquired reading disorder from stroke. With phonological dyslexia, her whole-word (lexical) reading approach included inability to read nonwords and poor reading of function words. Following therapy, she was able to read nonwords and function words, and preferred a decompositional (sub-lexical) strategy in general. fMRI was performed during a reading task before and after treatment. Prior to therapy, her main focus of brain activation was in the left angular gyrus (area 39). After therapy, it was instead in the left lingual gyrus (area 18). This result suggests first that it is possible to alter brain physiology with therapy for acquired language disorders, and second, that two reading strategies commonly used in normal reading use distinct neural circuits, possibly reconciling several conflicting neuroimaging studies of reading.     

Familial amyloidotic polyneuropathy type I with extracellular superoxide dismutase mutation: a case report

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) Type I with mutations in both transthyretin (TTR) and extracellular superoxide dismutase (EC-SOD). This patient started to develop peripheral neuropathy at age 25, followed by cardiac, renal, and autonomic nervous system failure due to massive amyloid deposition. Thirteen years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, multiple abscesses in the bile ducts and urinary tract, and more marked amyloid deposition than commonly seen in FAP. Amyloid deposition occurred in various organs and tissues, especially prominently around blood vessels and in interstitial tissues, and was demonstrated immunohistochemically to be composed of TTR but not amyloid A (AA) and not amyloid L (AL) proteins. The serum EC-SOD content of the patient was 10 fold higher than those seen often in other FAP patients and in healthy controls. Genetic analysis demonstrated the single amino acid substitutions in Val30Met TIR and Arg213Gly EC-SOD. Since these data suggest the dissociation of EC-SOD from the vascular wall, massive amyloid deposition in the present case may be related to increased oxidative stress in loco.     

Expression of superoxide dismutase following axotomy

Genomic clones for a chitinolytic enzyme were isolated from a library of Sau 3A digested DNA from the tobacco hornworm, Manduca sexta, using a previously isolated chitinase cDNA clone as a probe [Kramer et al., Insect Biochem. Molec. Biol. 23, 691-701 (1993)]. Restriction enzyme mapping and Southern blot analysis of four genomic clones suggested that these are overlapping clones. Sequence analysis of the genomic clones and Southern blot analysis of total genomic DNA also suggest that the M. sexta genome has only one chitinase gene detectable by the cDNA probe. This gene is organized into at least 11 exons in a region spanning > 11 kb. The sequenced M. sexta chitinase gene has a series of exons corresponding to identifiable structural/functional regions of the protein. Similarities in structure and organization between the M. sexta chitinase gene and chitinase genes from other sources are described.     

The copper chaperone for superoxide dismutase

Copper is distributed to distinct localizations in the cell through diverse pathways. We demonstrate here that the delivery of copper to copper/zinc superoxide dismutase (SOD1) is mediated through a soluble factor identified as Saccharomyces cerevisiae LYS7 and human CCS (copper chaperone for SOD). This factor is specific for SOD1 and does not deliver copper to proteins in the mitochondria, nucleus, or secretory pathway. Yeast cells containing a lys7Delta null mutation have normal levels of SOD1 protein, but fail to incorporate copper into SOD1, which is therefore devoid of superoxide scavenging activity. LYS7 and CCS specifically restore the biosynthesis of holoSOD1 in vivo. Elucidation of the CCS copper delivery pathway may permit development of novel therapeutic approaches to human diseases that involve SOD1, including amyotrophic lateral sclerosis.     

Elevated superoxide dismutase in black alcoholics

Superoxide dismutase concentrations in lysates of erythrocytes from black alcoholics were higher than those of white alcoholics and of nonalcoholics of both races. Higher concentrations of enzyme protein, as determined by competition radioimmunoassay, correspond to proportionately higher enzyme activity. Elevated superoxide dismutase levels were not related to any other clinical, historical, or demographic variables. Increased superoxide dismutase levels may delay or prevent some of the pathological sequelae of alcoholism and may be a useful biological marker for alcohol abuse.     

Constitutive overexpression of Cu/Zn superoxide dismutase exacerbates kainic acid-induced apoptosis of transgenic-Cu/Zn superoxide dismutase neurons

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.     

Malonaldehyde, superoxide dismutase and human cataract

UV-spectrophotometry and fluorometry were used to study Malonaldehyde (MDA) and Superoxide Dismutase (SOD) in normal, cataractous human lenses and red blood cells of the patients with cataract. MDA content of senile and complicated cataractous lenses was significantly higher than that of normal human lenses, while that of complicated cataract was significantly higher than that of senile cataract. SOD activity of senile and complicated cataractous lenses was significantly lower than that of normal human lenses, while there was no marked difference between senile and complicated cataractous lenses. Significant correlation between cataractous lenses and red blood cells was not found in MDA content and SOD activity. There was a negative correlation between SOD and MDA in normal human lenses, but no correlation between SOD and MDA in cataractous lenses. The study shows that lipid peroxidation may be one of the possible mechanisms of cataractogenesis in human, and emphasizes the role of SOD in prevention of photoperoxidative damages to the tissues.     

Identification of superoxide dismutase activity in Borrelia burgdorferi

Infective and noninfective strains of Borrelia burgdorferi, along with Borrelia afzelii and Borrelia garinii, possessed a single iron-containing superoxide dismutase (SOD). None of the Lyme disease spirochetes tested possessed catalase or peroxidase activities. The borrelial SOD was not inducible by growth with increased oxygen concentrations and thus appeared to be produced constitutively.     

Autoxidation of ubiquinol-6 is independent of superoxide dismutase

Ubiquinone (Q) is an essential, lipid soluble, redox component of the mitochondrial respiratory chain. Much evidence suggests that ubiquinol (QH2) functions as an effective antioxidant in a number of membrane and biological systems by preventing peroxidative damage to lipids. It has been proposed that superoxide dismutase (SOD) may protect QH2 form autoxidation by acting either directly as a superoxide-semiquinone oxidoreductase or indirectly by scavenging superoxide. In this study, such an interaction between QH2 and SOD was tested by monitoring the fluorescence of cis-parinaric acid (cPN) incorporated phosphatidylcholine (PC) liposomes. Q6H2 was found to prevent both fluorescence decay and generation of lipid peroxides (LOOH) when peroxidation was initiated by the lipid-soluble azo initiator DAMP, dimethyl 2,2'-azobis (2-methylpropionate), while Q6 or SOD alone had no inhibitory effect. Addition of either SOD or catalase to Q6H2-containing liposomes had little effect on the rate of peroxidation even when incubated in 100% O2. Hence, the autoxidation of QH2 is a competing reaction that reduces the effectiveness of QH2 as an antioxidant and was not slowed by either SOD or catalase. The in vivo interaction of SOD and QH2 was also tested by employing yeast mutant strains harboring deletions in either CuZnSOD and/or MnSOD. The sod mutant yeast strains contained the same percent Q6H2 per cell as wild-type cells. These results indicate that the autoxidation of QH2 is independent of SOD.     

BAL fluid contains detectable superoxide dismutase 1 activity

We retrospectively analyzed the prognostic significance of angiogenesis and the relationships between tumor angiogenesis and clinopathological variables in a series of 127 patients with primary esophageal squamous cell carcinoma which were curatively resected. Vessels were stained with anti-factor VIII polyclonal antibody and areas with the highest number of microvessels were counted in a x400 field. Microvessel counts were significantly correlated with pN category, pM category, venous invasion and recurrence (p=0.002, p=0.040, p=0.016 and p=0.0013, respectively). The proportion of patients with recurrence increased in proportion to the number of microvessels. multivariate analysis using Cox's proportional hazard modelling showed angiogenesis assessed by microvessel count affected the poorer prognosis of patients with esophageal squamous cell carcinoma (hazard ratio, 1.03; 95%CI, 1.00-1.07), although it was not a significant independent prognostic factor (P=0.088). This study suggest that angiogenesis assessed by microvessel count is a marker for relapse and prognosis in patients with esophageal squamous cell carcinoma.     

Mitochondrial disease in superoxide dismutase 2 mutant mice

Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissue-specific inhibition of the respiratory chain enzymes NADH-dehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.     

Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum

The characterization of a new mAb, named 2F4/11, specific for porcine myelomonocytic cells is described. This mAb immunoprecipitates a non-covalently linked heterodimer of 155,000/95,000, which is expressed by granulocytes, monocytes and tissue macrophages but not by lymphocytes, erythrocytes or platelets. Immunoblot analysis localizes the 2F4/11 epitope on the largest subunit of the heterodimer. Mab 2F4/11 is able to block phagocytosis of complement-opsonized zymosan particles by PMN granulocytes and alveolar macrophages, as well as adherence to plastic surfaces of PMA-activated PMN. Together, these results suggest that mAb 2F4/11 recognizes the CD11b or alpha chain of the porcine complement type 3 receptor (CR3).     

In vivo architecture of the manganese superoxide dismutase promoter

Mitochondrial manganese superoxide dismutase (Mn-SOD) is the primary cellular defense against damaging superoxide radicals generated by aerobic metabolism and as a consequence of inflammatory disease. Elevated expression of Mn-SOD therefore provides a potent cytoprotective advantage during acute inflammation. Mn-SOD contains a GC-rich and TATA/CAAT-less promoter characteristic of a housekeeping gene. In contrast, however, Mn-SOD expression is dramatically regulated in a variety of cells by numerous proinflammatory mediators, including lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1. To understand the underlying regulatory mechanisms controlling Mn-SOD expression, we utilized DNase I-hypersensitive (HS) site analysis, which revealed seven hypersensitive sites throughout the gene. Following high resolution DNase I HS site analysis, the promoter was found to contain five HS subsites, including a subsite that only appears following stimulus treatment. Dimethyl sulfate in vivo footprinting identified 10 putative constitutive protein-DNA binding sites in the proximal Mn-SOD promoter as well as two stimulus-specific enhanced guanine residues possibly due to alterations in chromatin structure. In vitro footprinting data implied that five of the binding sites may be occupied by a combination of Sp1 and gut-enriched Kr uppel-like factor. These studies have revealed the complex promoter architecture of a highly regulated cytoprotective gene.     

Thiol-dependent metal-catalyzed oxidation of copper, zinc superoxide dismutase

Superoxide dismutase (SOD) is a key enzyme in the antioxidant system of the cells. When exposed to a metal-catalyzed oxidation (MCO) system composed of Fe3+, O2, and thiol as an electron donor copper, zinc SOD (CuZnSOD) was susceptible to oxidative modification and damage as indicated by the loss of activity, fragmentation and aggregation of peptide as well as by the formation of carbonyl groups. Oxidative damage to CuZnSOD was inhibited by diethylenetriaminepentaacetic acid as well as by free radical scavengers and spin-trapping agents. The results of the present study indicate that hydrogen peroxide may be generated from a thiol/Fe3+/O2 system and that hydroxyl free radicals, produced by metal-catalyzed Fenton reactions, may be the ultimate species mediating the SOD damage. Incubation with the MCO system resulted in the release of Cu ions from CuZnSOD. Incubation with the thiol-MCO did not significantly increase the formation of 2-oxohistidine in CuZnSOD. The lack of formation of 2-oxohistidine, as well as the pronounced preventive effect of spin-traps on the thiol-MCO-mediated damage to CuZnSOD, indicates that inactivation might actually be predominantly due to global oxidation rather than a site-specific oxidation. The thiol-MCO-mediated damage to SOD may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.