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Mutations in the human Superoxide dismutase 1 (hSOD1) gene are well-established cause of the motor neuron disease ALS. Patients and transgenic (Tg) ALS model mice carrying mutant variants develop hSOD1 aggregates in the CNS. We have identified two hSOD1 aggregate strains, which both transmit spreading template-directed aggregation and premature fatal paralysis when inoculated into adult transgenic mice. This prion-like spread of aggregation could be a primary disease mechanism in SOD1-induced ALS. Human SOD1 aggregation has been studied extensively both in cultured cells and under various conditions in vitro. To determine how the structure of aggregates formed in these model systems related to disease-associated aggregates in the CNS, we used a binary epitope-mapping assay to examine aggregates of hSOD1 variants G93A, G85R, A4V, D90A, and G127X formed in vitro, in four different cell lines and in the CNS of Tg mice. We found considerable variability between replicate sets of in vitro-generated aggregates. In contrast, there was a high similarity between replicates of a given hSOD1 mutant in a given cell line, but pronounced variations between different hSOD1 mutants and different cell lines in both structures and amounts of aggregates formed. The aggregates formed in vitro or in cultured cells did not replicate the aggregate strains that arise in the CNS. Our findings suggest that the distinct aggregate morphologies in the CNS could result from a micro-environment with stringent quality control combined with second-order selection by spreading ability. Explorations of pathogenesis and development of therapeutics should be conducted in models that replicate aggregate structures forming in the CNS. (Figure presented.)

Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.

Introduction: Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are two severe neurodegenerative disorders for which the disease mechanisms are poorly understood and reliable biomark ...

A common cause of amyotrophic lateral sclerosis is mutations in superoxide dismutase-1, which provoke the disease by an unknown mechanism. We have previously found that soluble hydrophobic misfolded mutant human superoxide dismutase-1 species are enriched in the vulnerable spinal cords of transgenic model mice. The levels were broadly inversely correlated with life spans, suggesting involvement in the pathogenesis. Here, we used methods based on antihuman superoxide dismutase-1 peptide antibodies specific for misfolded species to explore the composition and amounts of soluble misfolded human superoxide dismutase-1 in tissue extracts. Mice expressing 5 different human superoxide dismutase-1 variants with widely variable structural characteristics were examined. The levels were generally higher in spinal cords than in other tissues. The major portion of misfolded superoxide dismutase-1 was shown to be monomers lacking the C57-C146 disulfide bond with large hydrodynamic volume, indicating a severely disordered structure. The remainder of the misfolded protein appeared to be non-covalently associated in 130- and 250-kDa complexes. The malleable monomers should be prone to aggregate and associate with other cellular components, and should be easily translocated between compartments. They may be the primary cause of toxicity in superoxide dismutase-1-induced amyotrophic lateral sclerosis.

Several of the superoxide dismutase-1 (SOD1) mutations linked to amyotrophic lateral sclerosis (ALS) lead to synthesis of structurally defective molecules, suggesting that any cytotoxic conformatio ...

Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from four different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intra-subunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, four were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.

Antioxidant levels were determined in five patients with the Lesch-Nyhan syndrome. The erythrocyte glutathione peroxidase activity was in average 1.8 times higher in Lesch-Nyhan patients than in controls (1.68 +/- 0.36 versus 0.92 +/- 0.17 mu kat/g hemoglobin, p less than 0.001). Plasma CuZn-superoxide dismutase activity was two times higher (p less than 0.001) and Mn-superoxide dismutase activity was 1.5 times higher (p less than 0.05) than in controls, whilst erythrocyte CuZn-superoxide dismutase, plasma extracellular-superoxide dismutase and lymphocyte superoxide dismutase did not differ between Lesch-Nyhan patients and healthy controls. These data might indicate that Lesch-Nyhan patients are exposed to a higher oxidative load than healthy control persons.

Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.

Mutants of superoxide dismutase-1 (SOD1) cause ALS by an unidentified cytotoxic mechanism. We have previously shown that the stable SOD1 mutants D90A and G93A are abundant and show the highest levels in liver and kidney in transgenic murine ALS models, whereas the unstable G85R and G127X mutants are scarce but enriched in the CNS. These data indicated that minute amounts of misfolded SOD1 enriched in the motor areas might exert the ALS-causing cytotoxicity. A hydrophobic interaction chromatography (HIC) protocol was developed with the aim to determine the abundance of soluble misfolded SOD1 in tissues in vivo . Most G85R and G127X mutant SOD1s bound in the assay, but only minute subfractions of the D90A and G93A mutants. The absolute levels of HIC-binding SOD1 were, however, similar and broadly inversely related to lifespans in the models. They were generally enriched in the susceptible spinal cord. The HIC-binding SOD1 was composed of disulfide-reduced subunits lacking metal ions and also subunits that apparently carried nonnative intrasubunit disulfide bonds. The levels were high from birth until death and were comparable to the amounts of SOD1 that become sequestered in aggregates in the terminal stage. The HIC-binding SOD1 species ranged from monomeric to trimeric in size. These species form a least common denominator amongst SOD1 mutants with widely different molecular characteristics and might be involved in the cytotoxicity that causes ALS.

We provide bioinformatical evidence that protein charge plays a key role in the disease mechanism of amyotrophic lateral sclerosis (ALS). Analysis of 100 ALS-associated mutations in copper/zinc superoxide dismutase (SOD1) shows that these are site-selective with a preference to decrease the proteins' net repulsive charge. For each SOD1 monomer this charge is normally -6. Because biomolecules as a rule maintain net negative charge to assure solubility in the cellular interior, the result lends support to the hypothesis of protein aggregation as an initiating event in the ALS pathogenesis. The strength of the preferential reduction of repulsive charge is higher in SOD1-associated ALS than in other inherited protein disorders.

Lenses from mice lacking the antioxidant enzyme copper-zinc superoxide dismutase (SOD1) show elevated levels of superoxide radicals and are prone to developing cataract when exposed to high levels of glucose in vitro. As superoxide may react further with nitric oxide, generating cytotoxic reactive nitrogen species, we attempted to evaluate the involvement of nitric oxide in glucose-induced cataract. Lenses from SOD1-null and wild-type mice were incubated with high or normal levels of glucose (55.6 and 5.56 mM). A nitric oxide synthase inhibitor (L-NAME) or a nitric oxide donor (DETA/NO) was added to the culture medium. Cataract development was assessed using digital image analysis of lens photographs and cell damage by analyzing the leakage of lactate dehydrogenase. The levels of superoxide radicals in the lenses were also measured. L-NAME was found to reduce cataract development and cell damage in the SOD1-null lenses exposed to high glucose. On the other hand, DETA/NO accelerated cataract development, especially in the SOD1-null lenses. These lenses also showed a higher leakage of lactate dehydrogenase than wild-type controls. We conclude that a combination of high glucose and absence of SOD1 increases the formation of cataract and that nitric oxide probably contributes to this process.

Mice lacking the secreted extracellular superoxide dismutase (EC-SOD) or the cytosolic copper- and zinc-containing SOD (CuZn-SOD) show relatively mild phenotypes. To explore the possibility that the isoenzymes have partly overlapping functions, single and double knockout mice were examined. The absence of EC-SOD was found to be without effect on the lifespan of mice, and the reduced lifespan of CuZn-SOD knockouts was not further shortened by EC-SOD deficiency. The urinary excretion of isoprostanes was increased in CuZn-SOD knockout mice, and plasma thiobarbituric acid-reactive substances levels were elevated in EC-SOD knockout mice. These oxidant stress markers showed potentiated increases in the absence of both isoenzymes. Other alterations were mainly found in CuZn-SOD knockout mice, such as halved glutathione peroxidase activity in the tissues examined and increased glutathione and iron in the liver. There were no changes in tissue content of the alternative superoxide scavenger ascorbate, but there was a 25% reduction in ascorbate in blood plasma in mice lacking CuZn-SOD. No increase was found in the urinary excretion of the terminal metabolites of NO, nitrite, and nitrate in any of the genotypes. In conclusion, apart from the increases in the global urinary and plasma oxidant stress markers, our phenotype studies revealed no other evidence that the copper- and zinc-containing SOD isoenzymes have overlapping roles.

The purpose of this study was to evaluate the involvement of the superoxide radical in glucose-induced cataract using lenses from mice lacking the cytosolic copper-zinc superoxide dismutase (SOD1). Lenses from wild-type mice and SOD1 null mice were kept in organ culture with either 5.6 or 55.6 mM glucose for 6 days. The cataract formation was followed with digital image analysis and ocular staging. The lens damage was further quantified by analysis of the leakage of lactate dehydrogenase into the medium by the uptake of 86Rb and by determining the water content of the lenses. The formation of superoxide radicals in the lenses was assessed with lucigenin-derived chemiluminescence. Immunohistochemical staining for SOD1 was also performed on murine lenses. The SOD1 null lenses exposed to high glucose developed more cataract showed an increased leakage of lactate dehydrogenase and developed more oedema compared to the control lenses. At 5.6 mM glucose there was no difference between the SOD1 null and wild-type lenses. Staining for SOD1 was seen primarily in the cortex of the wild-type lens. This in vitro model suggests an involvement of the superoxide radical and a protective effect of SOD1 in glucose-induced cataract formation.

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.

Oxygen free radicals apparently play important roles in diseases of the blood vessel wall and increased secretion of superoxide radicals occurs in many situations. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD). The synthesis of the enzyme by the smooth muscle cells (SMC) is modulated by cytokines, growth factors, and vasoactive factors. Here we studied the effects of oxidants (pyrogallol, xanthine oxidase, Cu and Fe), antioxidants (SOD, catalase, and ascorbate), glutathione modulation ( n -acetylcysteine and buthionine sulfoximine) and nitric oxide on EC-SOD expression by human vascular SMCs. Generally, the responses in EC-SOD synthesis were small, and no changes were noted in mRNA levels. High concentrations of some of the agents caused reductions in EC-SOD synthesis, mostly concomitantly with toxic effects on the cells. Cell cultures are normally ascorbate deficient, and addition of ascorbate to approach physiological levels doubled the EC-SOD content. Iron ions up-regulated EC-SOD synthesis but also blocked the secretion of the enzyme. Only down-regulation was found by NO -releasing compounds. In conclusion, there is limited response to oxidant stress of EC-SOD synthesis by SMCs on a cell-autonomous level. The synthesis appears mainly regulated by factors coordinating concerted tissue responses.

In some families with amyotrophic lateral sclerosis (ALS), the disease is linked to mutations in the gene encoding CuZn-superoxide dismutase. The mutant CuZn-superoxide dismutases appear to cause motor neuron degeneration by a toxic property, suggested to be linked to an altered reactivity of the active-site Cu ions. Asp90Ala mutant CuZn-superoxide dismutase was isolated from six patients with ALS, allowing properties of the mutant enzyme synthesized and conditioned in patients with ALS to be examined. The molecular mass of the Asp90Ala mutant CuZn-superoxide dismutase was 45 Da lower than that of the wild-type enzyme, as expected from the amino acid exchange. The mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was markedly increased, however, suggesting altered properties of the polypeptide. The mutant CuZn-superoxide dismutase showed a minimal reduction in stability but did not differ significantly from the wild-type enzyme in enzymic activity, in content and affinity for active-site Cu ions and in the propensity to catalyze formation of hydroxyl radicals. Our findings suggest that the deleterious effect of mutant CuZn-superoxide dismutases on motor neurons in ALS is not related to altered reactivity of active-site Cu ions, resulting in increased oxidant stress. Attention should therefore also be directed at other mechanisms and properties of the mutant polypeptides and their degradation products.

Mutations in CuZn-superoxide dismutase (CuZn-SOD) have been linked to familial amyotrophic lateral sclerosis (ALS), and motor neurone death is caused by the gain of a toxic property of the mutant protein. Here we determined amounts, activity and molecular forms of CuZn-SOD in CSF from ALS patients carrying the D90A and other CuZn-SOD mutations and patients without such mutations. There were no differences in amount of protein and enzymic activities of CuZn-SOD between 37 neurological controls, 54 sporadic and 12 familial ALS cases, and 10 cases homozygous for the D90A mutation. Three cases heterozygous for the A89V, S105L and G114A CuZn-SOD mutations showed low amounts of CuZn-SOD. There was no evidence for accumulation of inactive protein in any of the groups. Immunoblots showed no evidence for the presence of any precipitates or other molecular forms of CuZn-SOD with higher molecular weight in the groups. About 25% of the CuZn-SOD subunits in CSF from controls shows an N-terminal truncation. This truncated portion does not differ between controls and ALS groups not carrying CuZn-SOD mutations, but is 70% larger in samples from D90A homozygous ALS patients. The findings suggest an essentially normal amount and activity of D90A mutant CuZn-SOD in CNS tissues of ALS cases. The increased occurrence of N-terminally truncated mutant subunits may indicate a difference in degradation routes compared with the wild-type enzyme, resistance against subsequent proteolytic steps and/or a compromised downstream proteolytic machinery. Molecular fragments accumulated to a greater extent from the D90A mutant enzyme might contribute to the motor neurone degeneration. We also determined the other SOD isoenzymes: in the controls, CuZn-SOD contributed 75%, extracellular SOD 25% and Mn-SOD

Chronic cobalt exposure is characterized by severe cardiac insufficiency. Since the mechanisms of cobalt toxicity are not yet clear, we analysed the effects of chronic cobalt exposure on antioxidant enzyme activities and myocardial mitochondrial ATP production rate in a rat model. One group of rats was fed a conventional diet and another a cobalt supplemented diet for 24 weeks. The manganese-superoxide dismutase activity was markedly reduced in the cobalt rats (18+/-4.7 U/mg protein) compared to the control rats (100+/-22 U/mg protein; p

Extracellular superoxide dismutase (EC-SOD) is a secreted antioxidative enzyme with an abundant mRNA expression in kidney and arterial wall. In order to study expression and antioxidative function of EC-SOD, we cloned the rabbit ec-sod cDNA and produced the recombinant protein in cell culture. In vitro studies did not show a direct relationship between the amounts of synthesized mRNA and secreted protein activity, suggesting post-transcriptional regulation. The antiatherogenic role of EC-SOD was studied by determining the effect of EC-SOD on the oxidation (ox) of low density lipoprotein (LDL), and subsequent degradation of oxLDL in RAW 264 macrophages in vitro. It was found that recombinant EC-SOD reduced both the degradation of LDL in RAW 264 macrophages by 28-36% and its electrophoretic mobility caused by endothelial cell-mediated oxidation. It is therefore suggested that EC-SOD can act as a protective enzyme against the development of atherosclerosis.

Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 micrograms/ml) are found circulating in plasma during diverse inflammatory events. The saturable, high affinity binding of extracellular XO to vascular endothelium and the effects of cell binding on both XO catalytic activity and differentiated vascular cell function are reported herein. Xanthine oxidase purified from bovine cream bound specifically and with high affinity (Kd = 6 nM) at 4 degreesC to bovine aortic endothelial cells, increasing cell XO specific activity up to 10-fold. Xanthine oxidase-cell binding was not inhibited by serum or albumin and was partially inhibited by the addition of heparin. Pretreatment of endothelial cells with chondroitinase, but not heparinase or heparitinase, diminished endothelial binding by approximately 50%, suggesting association with chondroitin sulfate proteoglycans. Analysis of rates of superoxide production by soluble and cell-bound XO revealed that endothelial binding did not alter the percentage of univalent reduction of oxygen to superoxide. Comparison of the extent of CuZn-SOD inhibition of native and succinoylated cytochrome c reduction by cell-bound XO indicated that XO-dependent superoxide production was occurring in a cell compartment inaccessible to CuZn-SOD. This was further supported by the observation of a shift of exogenously added XO from extracellular binding sites to intracellular compartments, as indicated by both protease-reversible cell binding and immunocytochemical localization studies. Endothelium-bound XO also inhibited nitric oxide-dependent cGMP production by smooth muscle cell co-cultures in an SOD-resistant manner. This data supports the concept that circulating XO can bind to vascular cells, impairing cell function via oxidative mechanisms, and explains how vascular XO activity diminishes vasodilatory responses to acetylcholine in hypercholesterolemic rabbits and atherosclerotic humans. The ubiquity of cell-XO binding and endocytosis as a fundamental mechanism of oxidative tissue injury is also affirmed by the significant extent of XO binding to human vascular endothelial cells, rat lung type 2 alveolar epthelial cells, and fibroblasts.

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.

Quin2, a fluorescent calcium probe, has a low affinity for calcium in comparison to its affinities for transition metal ions. Chelation of ferric ion with quin2 strongly enhanced the formation of oxidizing species in the presence of bolus H2O2 as detected with four assays, electron spin resonance with the spin-trap DMPO, the deoxyribose assay, the DMSO assay, and plasmid DNA strand breakage. In comparison, Fe(III)-EDTA reacted with bolus H2O2 only as detected with electron spin resonance and the deoxyribose assay, but not as detected with the two latter assays. The addition of reductants, like ascorbate or superoxide generated by hypoxanthine/xanthine oxidase, to Fe(III)-EDTA in the presence of H2O2 produced plasmid DNA strand breakage and strong reactivity in both the DMSO and the deoxyribose assays. Our findings suggest that the main oxidizing species produced in Fenton-type reactions is hydroxyl radical. However, the reaction between Fe(III)-EDTA and bolus H2O2 appears to be exceptional and dominated by a nonhydroxyl radical species.

Over 170 mutations in superoxide dismutase-1 (SOD1) have been linked to amyotrophic lateral sclerosis (ALS). The properties of SOD1 mutants differ considerably including copper-binding abilities. Nevertheless, they cause the same disease phenotype, suggesting a common neurotoxic pathway. We have previously reported that copper homeostasis is disturbed in spinal cords of SOD1(G93A) mice. However, it is unknown whether copper dyshomeostasis is induced by other SOD1 mutants. Using the additional mouse strains SOD1(G127insTGGG), SOD1(G85R), and SOD1(D90A), which express SOD1 mutants with different copper-binding abilities, we show that copper dyshomeostasis is common to SOD1 mutants. The SOD1 mutants shifted the copper trafficking systems toward copper accumulation in spinal cords of the mice. Copper contents bound to the SOD1 active site varied considerably between SOD1 mutants. Still, copper bound to other ligands in the spinal cord were markedly increased in all. Zinc was also increased, whereas there were no changes in magnesium, calcium, aluminum, manganese and iron. Further support for a role of copper dyshomeostasis in ALS was gained from results of pharmacological intervention. Ammonium tetrathiomolybdate (TTM), a copper chelating agent, prolonged survival and slowed the disease progression of SOD1(G93A) mice, even when the treatment was started after the disease onset. TTM markedly attenuated pathology, including the loss of motor neurons and axons, and atrophy of skeletal muscles. Additionally, TTM decreased amounts of SOD1 aggregates. We propose that pharmacological agents that are capable of modulating copper dyshomeostasis, such as TTM, might be beneficial for the treatment of ALS caused by SOD1 mutations.

Extracellular superoxide dismutase (EC-SOD) is a secretory SOD isoenzyme. 99% of EC-SOD is anchored to heparan sulfate proteoglycans in the tissue interstitium, and 1% is located in the vasculature in equilibrium between the plasma and the endothelium. Analysis of EC-SOD in plasma samples from 504 random blood donors revealed a common (2.2%) phenotypic variant displaying 10-fold increased plasma EC-SOD content. The EC-SOD in the plasma of these individuals, collected both before and after intravenous injection of heparin, displayed a reduced heparin affinity when compared with samples from normal individuals. The specific enzymatic activity was the same as that of normal enzyme. Nucleotide sequence analyses of two of the affected subjects revealed a nucleotide exchange resulting in a substitution of Arg-213 by Gly. The substitution is located in the center of the carboxyl-terminal cluster of positively charged amino acid residues, which defines the heparin-binding domain. Polymerase chain reaction-single-strand conformational polymorphism and allele-specific polymerase chain reaction showed that all 11 affected individuals are heterozygous, carrying the same single-base mutation. Recombinant EC-SOD containing this mutation had a reduced heparin affinity similar to that of EC-SOD C from variant persons. The high plasma activity can be explained by an accelerated release from the tissue interstitium heparan sulfate to the vasculature and should thus be accompanied by significantly reduced tissue EC-SOD activities.

The objective of this study was to investigate whether quin2, through its metal chelating properties, could affect copper- or iron-driven Fenton reactions. Chelation of ferric ion with quin2 uniformly strongly enhanced the formation of oxidizing species, detected with the DMSO and deoxyribose assays, both by H 2 O 2 and a mixture of superoxide/hydrogen peroxide produced by hypoxanthine/xanthine oxidase. Fe 3+ -EDTA gave the same effects, but lacked reactivity with bolus H 2 O 2 as detected with the DMSO assay. Whereas the formation of oxidizing species with Fe 3+ -EDTA and ferric ions alone were strongly inhibited by superoxide dismutase both in the bolus H 2 O 2 and hypoxanthine/xanthine oxidase systems, such formation in the presence of Fe 3+ -quin2 either did not decrease or decreased only moderately. Fe 3+ -quin2 also strongly enhanced plasmid DNA strand breakage in the presence of H 2 O 2 . Our findings suggest that quin2 as chelator of ferric ion may be a more powerful enhancer of oxidant formation than other chelators so far tested. The formation of oxidizing species from copper ions and bolus H 2 O 2 was found to be fundamentally dependent on the choice of buffer system. We could only detect significant amounts of oxidants in both assays in Hepes buffer, but not in the phosphate, cacodylate or unbuffered systems, which all gave low reactivity in the DMSO assay compared to the deoxyribose assay. Quin2 chelation of cupric ion effectively inhibited the formation of oxidants as well as plasmid DNA strand breakage. To conclude, our results strongly suggest that cellular effects of quin2 may be due to modulations of the reactivities of iron and copper ions, and probably also other transtion metal ions, and should not uncritically be linked to effects on calcium homeostasis.

We have targeted the expression of recombinant human extracellular superoxide dismutase, a glycosylated tetrameric metalloprotein, to the mammary gland of transgenic mice. This was achieved by using regulatory elements from either the murine whey acidic protein gene or the ovine beta-lactoglobulin gene to control expression of human extracellular superoxide dismutase cDNA. Whey acidic protein regulatory sequences directed high level mammary gland-specific expression of the recombinant gene and secretion of biologically active extracellular superoxide dismutase into the milk. The produced recombinant protein was fully active, it was in tetrameric form, it showed heparin affinity, and its mass was similar to that of the native enzyme. In addition, the in vivo plasma clearance in a rabbit model was similar to the previously studied native and recombinant forms. To our knowledge, this is the first example of efficient production of a tetrameric, protease-susceptible metalloprotein in milk of transgenic animals. Production at equivalent levels in transgenic farm animals would yield sufficient extracellular superoxide dismutase for therapeutic purposes.

A specific biochemical marker for early diagnosing and for monitoring disease progression in amyotrophic lateral sclerosis (ALS) will have important clinical applications. ALS is a heterogeneous syndrome with multiple subtypes with ill-defined borders. A minority of patients carries mutations in the Cu/Zn-superoxide dismutase (SOD1) gene but the disease mechanism remains unknown for all types of ALS. Using a GC-TOFMS platform we studied the cerebrospinal fluid (CSF) metabolome in 16 ALS patients with six different mutations in the SOD1 gene and compared with ALS-patients without such mutations. OPLS-DA was used for classification modeling. We find that patients with a SOD1 mutation have a distinct metabolic profile in the CSF. In particular, the eight patients homozygous for the D90A SOD1 mutation showed a distinctively different signature when modeled against ALS patients with other SOD1 mutations and sporadic and familial ALS patients without a SOD1 gene mutation. This was found irrespective of medication with riluzole and survival time. Among the metabolites that contributed most to the CSF signature were arginine, lysine, ornithine, serine, threonine and pyroglutamic acid, all found to be reduced in patients carrying a D90A SOD1 mutation. ALS-patients with a SOD1 gene mutation appear as a distinct metabolic entity in the CSF, in particular in patients with the D90A mutation, the most frequently identified cause of ALS. The findings suggest that metabolomic profiling using GC-TOFMS and multivariate data analysis may be a future tool for diagnosing and monitoring disease progression, and may cast light on the disease mechanisms in ALS.

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.

Background/Aim The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF. Methodology Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls. Principal Findings The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar. Conclusions/Significance Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.

The tetrameric extracellular superoxide dismutase (EC-SOD) in human tissues and plasma has previously been found to be heterogenous with regard to heparin affinity and could be divided into at least three classes: A, lacking heparin affinity; B, with weak affinity; and C, with strong affinity. Using rigorous extraction conditions and an extensive set of anti-proteolytic agents, tissue EC-SOD is now shown to be almost exclusively of native homotetrameric C-class. Plasma EC-SOD on the other hand is shown to be mainly composed of a complex mixture of heterotetramers with modifications probably residing in the C-terminal heparin-binding domain. Proteolytic truncations appear to be a major cause of this heterogeneity. The findings suggest that, since 99% of the EC-SOD in the human body exists in the extravascular space of tissue, EC-SOD is primarily synthesized in tissues and secreted as homotetrameric native EC-SOD C. This tissue EC-SOD C should exist almost completely sequestered by heparin sulphate proteoglycans. C-terminal modifications subsequently occurring in the EC-SOD C would weaken the binding to heparan sulphate proteoglycan, facilitate entrance to the vasculature through capillaries and lymph flow, and finally result in the heterogeneous plasma EC-SOD pattern. With the new extraction and analysis procedure, the tissue content of EC-SOD is found to be higher than previously reported. It is found, for example, when compared with Mn-SOD, to be higher in umbilical cord and uterus, about equal in placenta and testis and as high as that of CuZn-SOD in umbilical cord. The findings suggest that the protection level against superoxide radicals provided by EC-SOD in the tissue interstitial space, given the small distribution volume, is not much less prominent than that bestowed on the intracellular space by CuZn-SOD and Mn-SOD.

Comparison of amino acid sequences have suggested similarities between the active site portions of the tetrameric, Cu- and Zn-containing glycoprotein, extracellular superoxide dismutase (EC-SOD) and the dimeric CuZn-SOD. In the present study spectral and physical properties of EC-SOD were analyzed to further knowledge about the enzyme and to extend the comparison with CuZn-SOD. EC-SOD displays an absorbance peak at 652 nm, blue-shifted some 30 nm compared with CuZn-SOD. The molar extinction coefficient, 167 M-1 cm-1 per copper atom, is similar to those of CuZn-SODs. In contrast to bovine and human CuZn-SOD, EC-SOD has a strong uv absorption originating from the rich content of aromatic amino acids. The extinction coefficient at 280 nm is 1.73 ml mg-1 cm-1. The electron paramagnetic resonance spectrum is also similar to those of bovine and human CuZn-SOD, but shows a slightly higher Az and a smaller difference between gx and gy. These spectral findings support the notion that the active sites of the SOD iso-enzymes are similar, but with a higher tetragonal symmetry of the copper ligands in EC-SOD. The circular dichroism spectrum of EC-SOD in the ultraviolet indicates a high alpha-helix content of the amino-terminal and carboxy-terminal parts flanking the central homologous active site portion. Similar to bovine CuZn-SOD, EC-SOD displays a marked physical resistance toward high temperature, pH extremes, and high urea and guanidinium chloride concentrations. This similarity in physical resistance suggests a central role of the homologous active site portion for the structural integrity of the SOD iso-enzymes.

Excessive amounts of cobalt are cardiotoxic, although the mechanism for this toxicity remains unclear. We studied the effects of acute cobalt exposure on the activities of free radical scavengers in the myocardium in 5 groups of rats. Six rats served as a control group and were given a daily subcutaneous injection of 1 ml saline for 8 days. The other 4 groups of rats received a daily injection subcutaneously of cobalt chloride in doses of 1 mg/kg bw, 5 mg/kg bw, 20 mg/kg bw and 50 mg/kg bw, respectively for 8 days. There was a marked and dose-dependent accumulation of cobalt in the myocardium of the cobalt exposed rats. Creatine kinase, copper-zinc superoxide dismutase (CuZn-SOD) and alpha-tocopherol content did not differ between the control and the cobalt exposed groups. The activity of glutathione peroxidase increased, while the activity of manganese-superoxide dismutase (Mn-SOD) was significantly reduced in the cobalt exposed groups. There was an inverse relationship (r = 0.60, P0.0001) between the cobalt content and Mn-SOD activity in the myocardium. These results suggest that acute cobalt cardiotoxicity may involve a reduction of intrinsic scavengers resulting in an increased vulnerability to oxygen free radical toxicity.

The heparin-binding affinity of the tetrameric extracellular superoxide dismutase (EC-SOD) is a result of the cooperative effect of the heparin-binding domains of the subunits, located in the hydrophilic, strongly positively charged C-terminal ends. EC-SOD C, the high-heparin-affinity type, exposed to immobilized trypsin and plasmin was found to rapidly lose its affinity for heparin, without any loss of enzymic activity or major change in molecular mass as judged by size-exclusion chromatography. Heparin and dextran sulphate 5000 inhibited the proteolysis, suggesting that EC-SOD C sequestered by heparan sulphate proteoglycan in vivo is partially protected against proteolysis. The loss of heparin-affinity occurred with the stepwise formation of intermediates, and the pattern upon chromatography on heparin-Sepharose and subsequent immunoblotting was compatible with the notion that the changes are due to sequential truncations of heparin-binding domains from subunits composing the EC-SOD tetramers. A similar pattern with intermediates and apparent truncations has previously been found with EC-SOD of human plasma. The findings show that the unique design of the heparin-binding domain of EC-SOD allows easy modification of the heparin-affinity by means of limited proteolysis, and suggest that such proteolysis is a major contributor to the heterogeneity in heparin-affinity of EC-SOD in mammalian plasma.

Extracellular-superoxide dismutase C (EC-SOD C) is a secretory tetrameric Cu- and Zn-containing glycoprotein which has high affinity for heparin and heparan sulfate. Upon intravenous injection into rabbits, recombinant human (rh) EC-SOD C was found to be rapidly 97-98% sequestered to the vascular wall, forming an equilibrium with the plasma phase. Recombinant EC-SOD truncation variants with reduced, T216, and without, T213, heparin affinity were found to be sequestered to a reduced extent and not at all, respectively, establishing the importance of the heparin affinity for this behaviour. The halflife of rhEC-SOD C in the vasculature was of the order of 20 h. Injection of large doses resulted in saturation of the binding of rhEC-SOD C to the vascular wall. Scatchard analysis revealed a heterogeneity in affinity of the ligands on the vascular wall. The maximal binding capacity was very high. The equilibration of rhEC-SOD C to the vascular wall of an organ, clamped during enzyme injection, and the primary equilibration phase was studied by comparing binding to a clamped and reperfused kidney with binding to the contralateral control kidney. rhEC-SOD C injected in a low dose was found to equilibrate very slowly to the reperfused kidney with a halftime of about 2 h. With higher rhEC-SOD C doses, at which evidence for saturation is seen, and with the variant rhEC-SOD with reduced heparin affinity. T216, very rapid equilibrations were found.(ABSTRACT TRUNCATED AT 250 WORDS)

The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.

The secretory enzyme extracellular-superoxide dismutase (EC-SOD) has affinity for heparin and some other sulfated glycosaminoglycans and is in vivo bound to heparan sulfate proteoglycan. Nonenymic glycation of EC-SOD, both in vivo and in vitro, is associated with a reduction in heparin affinity, whereas the enzymic activity is not affected. The glycation sites in EC-SOD are further studied in the present article. It is shown that the modification of a few of the five lysyl residues of the subunits of the enzyme with trinitrobenzyme sulfonic acid nearly abolishes the in vitro glycations susceptibility. From a chymotryptic digest of in vitro glycated EC-SOD, two peptides with affinity for boronate could be isolated. Amino acid sequence analysis showed that both encompassed the carboxyterminal end. ϵ-Glucitol lysine was identified in both peptides at positrons 211 and 212. The primary glycation sites in EC-SOD are thus lysine-211 and lysine-212 in the putative heparin-binding domaine in the carboxyterminal end.

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate+bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 +/- 24%; p less than .05) than in groups A (42 +/- 40%), C (47 +/- 36%), D (44 +/- 33%) and E (58 +/- 25%). Likewise, percent recovery of the double product (heart rate x systolic left ventricular pressure) was better in group B (104 +/- 18%; p less than .05) than in the other groups (A: 47 +/- 37%, C: 49 +/- 36%, D: 50 +/- 35%, E: 69 +/- 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.

The influence of cytokines on extracellular superoxide dismutase (EC-SOD) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by interferon-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 alpha (IL-1 alpha), was intermediately depressed by tumor necrosis factor-alpha (TNF-alpha), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-alpha, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 alpha. The ratio between the maximal stimulation and depression observed was around 30-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-alpha, IL-2, IL-3, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-SOD expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-SOD response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-SOD is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-SOD activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-SOD activity was depressed by TGF-beta. TNF-alpha, IL-1 alpha, and IFN-gamma stimulated the Mn-SOD activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three SOD isoenzymes illustrate their different physiological roles.

The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.

The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.

Extracellular superoxide dismutase (SOD3) is a homotetrameric copper- and zinc-containing glycoprotein with affinity for heparin. The level of SOD3 is particularly high in blood vessel walls and in the lungs. The enzyme has multiple roles including protection of the lungs against hyperoxia and preservation of nitric oxide. The common mutation R213G, which reduces the heparin affinity of SOD3, is associated with increased risk of myocardial infarctions and stroke. We report the first crystal structure of human SOD3 at 1.7 A resolution. The overall subunit fold and the subunit-subunit interface of the SOD3 dimer are similar to the corresponding structures in Cu-Zn SOD (SOD1). The metal-binding sites are similar to those found in SOD1, but with Asn180 replacing Thr137 at the Cu-binding site and a much shorter loop at the zinc-binding site. The dimers form a functional homotetramer that is fashioned through contacts between two extended loops on each subunit. The N- and C-terminal end regions required for tetramerisation and heparin binding, respectively, are highly flexible. Two grooves fashioned by the tetramer interface are suggestive as the probable sites for heparin and collagen binding.

The selenium-dependent glutathione peroxidase activities of two human cell lines, the colon carcinoma HT29 and the mesothelioma P31, cultured in medium containing 2% serum, increased from 195 to 541 and from 94 to 361 units/mg of protein respectively after supplementation with 100 nM-selenite. The catalase activity remained unchanged by this treatment. The effects of the obtained variation in glutathione peroxidase activities were investigated by exposing cells to H2O2 and t-butyl hydroperoxide. Selenite supplementation resulted in a decrease in H2O2-induced DNA single-strand breaks in both HT29 and P31 cells. A small, but significant, decrease in the number of DNA single-strand breaks for low doses (10-50 microM) of t-butyl hydroperoxide was found only in P31 cells and not in HT29 cells. We could detect neither induction of double-strand breaks (detection limit approx. 1000 breaks per cell) nor DNA-protein cross-links after exposing the cells to the two peroxides. In spite of the apparent protective effect of increased glutathione peroxidase activity on DNA single-strand break formation, there were no differences between selenite-supplemented and non-supplemented cells in cell survival after exposure to peroxide.

Study objective – The aim of the study was to determine whether recombinant human extracellular superoxide dismutase type C (rh-EC-SOD C) has antioxidant effects similar to those of the previously studied Cu-Zn superoxide dismutase. Design – Electron paramagnetic resonance spectroscopy was used for quantification of free radical generation during reperfusion of postischaemic isolated rat hearts. The hearts were perfused with Krebs-Henseleit buffer and then subjected to 15 min global ischaemia. N- tert -butyl-α-phenylnitrone (PBN) was added as a spin trapping agent to the perfusate. Experimental material – 18 hearts from Sprague-Dawley rats (280-400 g) were used. Measurements and main results – Reperfusion resulted in a burst of radical formation. In presence of rh-EC-SOD C plus catalase, the PBN spin adduct concentration was only 18% (p

Extracellular superoxide dismutase (EC-SOD) is the major SOD isoenzyme in extracellular fluids, but occurs also in tissues. The sites and characteristics of the synthesis of the enzyme are unknown. The occurrence of EC-SOD in cultures of a large panel of human cell lines was assayed by means of an e.l.i.s.a. Unlike the situation for the intracellular isoenzymes CuZn-SOD and Mn-SOD, expression of EC-SOD occurs in only a few cell types. None of the ten investigated suspension-growing cell lines produced EC-SOD. Among normal diploid anchorage-dependent cell lines, expression was found in all 25 investigated fibroblast cell lines, in the two glia-cell lines, but not in six endothelial-cell lines, two epithelial-cell lines or in two amnion-derived lines. Among neoplastic anchorage-dependent cell lines expression was found in 13 out of 29. EC-SOD was secreted into the culture medium by cell lines expressing the enzyme. The rate of EC-SOD synthesis varied by nearly 100-fold among the fibroblast lines and remained essentially constant in the individual lines during long-term culture. In the nine investigated cases, the secreted EC-SOD was of the high-heparin-affinity C type. It is suggested that tissue EC-SOD is secreted by a few well-dispersed cell types, such as fibroblasts and glia cells, to diffuse subsequently around and reversibly bind to heparan sulphate proteoglycan ligands in the glycocalyx of the surface of most tissue cell types and in the interstitial matrix.

The purpose was to elucidate the involvement od superoxide radical (O 2 − ·) in the postischemic increase in the vascular permeability in the hamster cheek pouch. Cheek pouches of anesthetized hamsters were everted, prepared for intravital microscopy, and superfused with a bicarbonate buffered saline solution. Local ischemia for 30 min was obtained using a cuff placed around the proximal part of the cheek pouch. The vascular permeability in the postcapillary venules was quantified as leakage of intravenously injected fluorescein labeled dextran (FITC-dextran, Mw 150 000), using intravital microscopy and fluorimetry. There was a significant and reversible permeability increase after the reperfusion started. In the first series of experiments, combined intravenous infusion and topical application of human recombinant extracellular superoxide dismutase C (EC-SOD C) reduced the postischemic permeability response by 80%. Bovine CuZn-SOD given in exactly the same way reduced the response by 60%. In the second series of experiments, inactivated EC-SOD C was given to the control animals and active EC-SOD C was given to the treated animals. The topical treatment was excluded. Only active EC-SOD C reduced significantly the postischemic permeability increase when present during the ischemic period. Treatment with mannitol (i.v.) did not alter the postischemic response. Since active EC-SOD C and CuZn-SOD but not inactivated EC-SOD C effectively inhibited the response, we suggest that the superoxide anion is involved in the mediation of the postischemic permeability increase in the hamster.

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.

Background We have previously shown that the protective effect of inhaled nitric oxide ( i NO) against methacholine-induced bronchoconstriction is negated in airways subjected to hyperosmotic stress. In this study, hypothesizing that the impaired efficiency of i NO was caused by release of reactive oxygen radicals, we examined the effect of the radical scavenging enzyme superoxide dismutase (SOD). Methods Hemodynamic and respiratory measurements were performed on anesthetized rabbits after (1) inhalation of methacholine (MCh), (2) i NO (80 ppm), followed by MCh, (3) inhalation of hypertonic saline (HS), followed by i NO and MCh and (4) pre-treatment with inhalation of SOD, followed by HS, i NO and MCh. We analyzed plasma for a marker of oxidative stress, 8-iso-prostaglandin (PG)F 2α and for a marker of activation of COX-mediated inflammatory cascades, PGF 2α metabolite. Results Pre-treatment with SOD restored the bronchoprotective response to i NO in hyperosmotic airways. No direct effect was seen by SOD treatment on levels of 8-iso-PGF 2α , but this marker of oxidative stress correlated positively with increased bronchoconstriction. Hyperosmotic challenge elevated levels of PGF 2α metabolite, and pre-treatment with SOD protected against this activation of the inflammatory cascade. Conclusion SOD pre-treatment restores the relaxant effects of i NO in hyperosmotically challenged airways by attenuating oxidative stress and activation of COX-mediated inflammatory cascades.

This study tested the hypothesis that human macrophages have the ability to modify oxidation products in LDL and oxidized LDL (oxLDL) via a cellular antioxidant defence system. While many studies have focused on macrophage LDL oxidation in atherosclerosis development, less attention has been given to the cellular antioxidant capacity of these cells. Compared to cell-free controls (6.2 ± 0.7 nmol/mg LDL), macrophages reduced TBARS to 4.42 ± 0.4 nmol/mg LDL after 24 h incubation with LDL (P = 0.022). After 2 h incubation with oxLDL, TBARS were 3.69 ± 0.5 nmol/mg LDL in cell-free media, and 2.48 ± 0.9 nmol/mg LDL in the presence of macrophages (P = 0.034). A reduction of lipid peroxides in LDL (33.7 ± 6.6 nmol/mg LDL) was found in the presence of cells after 24 h compared to cell-free incubation (105.0 ± 14.1 nmol/mg LDL) (P = 0.005). The levels of lipid peroxides in oxLDL were 137.9 ± 59.9 nmol/mg LDL and in cell-free media 242 ± 60.0 nmol/mg LDL (P = 0.012). Similar results were obtained for hydrogen peroxide. Reactive oxygen species were detected in LDL, acetylated LDL, and oxLDL by isoluminol-enhanced chemiluminescence (CL). Interestingly, oxLDL alone gives a high CL signal. Macrophages reduced the CL response in oxLDL by 45% (P = 0.0016). The increased levels of glutathione in oxLDL-treated macrophages were accompanied by enhanced catalase and glutathione peroxidase activities. Our results suggest that macrophages respond to oxidative stress by endogenous antioxidant activity, which is sufficient to decrease reactive oxygen species both in LDL and oxLDL. This may suggest that the antioxidant activity is insufficient during atherosclerosis development. Thus, macrophages may play a dual role in atherogenesis, i.e. both by promoting and limiting LDL-oxidation.

Mice of the nonobese diabetic strain develop a progressive insulitis resulting in beta-cell destruction and diabetes. Superoxide radicals are abundantly formed by leukocytes and other mechanisms in inflammatory reactions. We here aimed to determine whether superoxide radicals contribute to the beta cell destruction in the mouse model. Transgenic nonobese diabetic mice secreting extracellular-superoxide dismutase under control of the insulin promoter were generated and the development of glucosuria monitored. The overexpression of extracellular-superoxide dismutase resulted in a 6-fold increase in the total superoxide dismutase activity of the islets. The incidence of diabetes of the transgenic mice was, however, not modified. The results suggest that superoxide radicals secreted to the extracellular space do not contribute to the beta cell destruction in the nonobese diabetic mouse model.