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The melanocortin-4 receptor (MC4R), a G-protein coupled receptor, is implicated in mediating the effect of leptin on food intake and energy balance. A previous candidate gene study reported an association between an MC4R missense mutation (Asp298Asn) and fatness, growth and feed intake in pigs. To assess this association further, we analysed the segregation of this missense mutation in relation to variation in fatness traits using a Wild Boar x Large White intercross. The Wild Boar and Large White founders were homozygous for different MC4R alleles. The MC4R was assigned to the expected region on pig chromosome 1. The statistical evaluation did not reveal any indication of a significant effect on fatness related traits in this pedigree.

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.

Motor nerve excitability studies by "threshold tracking" in amyotrophic lateral sclerosis (ALS) revealed heterogeneous abnormalities in motor axon membrane function possibly depending on disease stage. It remains unclear to which extent the excitability deviations reflect a pathogenic mechanism in ALS or are merely a consequence of axonal degeneration. We investigated motor axon excitability in presymptomatic and symptomatic SOD1(G127X) mutants, a mouse model of ALS with late clinical onset and rapid disease progression. After clinical onset, there was a rapid loss of functional motor units associated with an increase in rheobase and strength-duration time constant, an increase in refractoriness at the expense of the superexcitability, larger than normal threshold deviations during both depolarizing and hyperpolarizing threshold electrotonus with impaired accommodation and reduction of the input conductance. These abnormalities progressed rapidly over a few days and were associated with morphological evidence of ongoing axonal degeneration. Presymptomatic mice with unaltered motor performance at rotor-rod measurement also had an increase in refractoriness at the expense of the superexcitability during the recovery cycle. This was, however, associated with smaller than normal deviations during threshold electrotonus, and a steeper resting current-threshold slope indicating slight axonal depolarization in agreement with motoneuronal hyperexcitability indicated by enhanced F-waves. Our data suggest that SOD1(G127X) motor axons undergo a state of membrane depolarization; however, during rapid motoneuron loss disease-specific nerve excitability measures are confounded by excitability changes in degenerating but still conducting axons. These findings should be considered in the interpretation of disease-stage-related nerve excitability changes 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.

Response to letter regarding article "Plasma bilirubin and UGT1A1*28 are not protective factors against first-time myocardial infarction in a prospective nested case-referent setting"

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 conformational species common for all mutations should be misfolded. SOD1 can be secreted and evidence from ALS model systems suggests that extracellular SOD1 may be involved in cytotoxicity. Three ELISAs specifically reacting with different sequence segments in misfolded SOD1 species were used for analysis of CSF from 38 neurological controls and from 96 ALS patients, 57 of whom were sporadic cases and 39 familial, including 22 patients carrying SOD1 mutations. Misfolded SOD1 was found in all samples. There were, however, no significant differences between patients with and without mutations, and between all the ALS patients and the controls. The estimated concentration of misfolded SOD1 in the interstitium of the CNS is a 1000 times lower than that required for appreciable cytotoxicity in model systems. The results argue against a direct cytotoxic role of extracellular misfolded SOD1 in ALS. Misfolded SOD1 in CSF cannot be used as a biomarker of ALS in patients with and without mutations in the enzyme.

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)

Background Next-generation sequencing technologies are widely used for detection of millions of Single Nucleotide Polymorphisms (SNPs) and also provide a means of assessing their variation. This information is useful for composing subsets of highly informative SNPs for region-specific or genome-wide analysis and to identify mutations regulating phenotypic differences within or between populations. In this study, we investigated the sensitivity of SNP detection and introduced the flanking SNPs value (FSV) as a novel measure for predicting SNP-variability using ~5X genome resequencing with ABI SOLID and DNA pools from two chicken lines divergently selected for juvenile bodyweight. Results Genotyping with a 60 K SNP chip revealed polymorphisms within or between two divergently selected chicken lines for 31 363 SNPs, 48% of which were also detected using resequencing of DNA pools. SNP detection using resequencing was more powerful for positions with larger differences in allele frequency between the lines. About 50% of the SNPs with non-reference allele frequencies in the range 0.5-0.6 and 67% of those with frequencies > 0.9 could be detected. On average, ~3.7 SNPs/kb were detected by resequencing, with about 5% lower density on microchromosomes than on macrochromosomes. There was a positive correlation between the observed between-line SNP variation from the 60 K chip analysis and our proposed FSV score computed from the genome resequencing data. The strongest correlations on macrochromosomes and microchromosomes were observed when the FSV was calculated with total flanking regions of 62 kb (correlation 0.55) and 38 kb (correlation 0.45), respectively. Conclusions Genome resequencing with limited coverage (~5X) using pooled DNA samples and three non-reference reads as a threshold for SNP detection, identified 50 - 67% of the 60 K SNPs with a non-reference allele frequency larger than 0.5. The SNP density was around 5% lower on the microchromosomes, most likely because of their higher gene content. Our proposed method to estimate the SNP variation (FSV) uses additional sequence information to better predict SNP informativity. The FSV scores showed higher correlations for SNPs with a larger difference in allele frequency between the populations. The correlation was strongest on macrochromosomes, probably due to a lower recombination rate.

Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA) patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.

Background— Bilirubin, an effective antioxidant, shows a large variation in levels between individuals and has been positively associated with reduced cardiovascular disease risk. A major reason for the variability is a common promoter polymorphism, UGT1A1*28, which reduces the transcription of the enzyme that conjugates bilirubin, UDP-glucuronosyltransferase 1A1. The aim of the study was to evaluate a possible protective effect of plasma bilirubin and the UGT1A1*28 polymorphism against myocardial infarction in a prospective case-referent setting. Methods and Results— Subjects (n=618) with a first-ever myocardial infarction (median event age, 60.5 years; median lag time, 3.5 years) and 1184 matched referents were studied. Plasma bilirubin was lower in cases versus referents. Despite a strong gene-dosage effect on bilirubin levels in both cases and referents, the UGT1A1*28 polymorphism did not influence the risk of myocardial infarction. Among multiple other variables, serum iron showed one of the strongest associations with bilirubin levels. Conclusions— We found no evidence for a protective effect of the UGT1A1*28 polymorphism against myocardial infarction and consequently neither for bilirubin. The lower bilirubin levels in cases might be caused by decreased production, increased degradation, or increased elimination.

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.

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.

Extensive preparative surgery and lengthy experimentation may lead to high rate of complications and mortality in myocardial ischaemia studies. These problems are particularly common when pigs are used as the subject as they are prone to develop lethal ventricular arrhythmias. Here, a closed-chest model is presented, in which the trauma of major preparative surgery is avoided. One-hundred and twelve pentobarbital-anaesthetized, mechanically ventilated pigs were used. Coronary occlusion was produced by injection of a 2 mm diameter ball via a modified coronary angiography catheter. Reperfusion was induced by retraction of the ball via a thin filament attached to the ball. The amount of the myocardium at risk (MAR) was 8.23 +/- 2.41% (mean +/- SD) of the left plus right ventricular weight. It was possible to carry out scheduled 24 h experiments in 87 out of 93 animals (93.5%). Preparative mortality was 1.8% and 24 h mortality 6.5%. Ventricular fibrillation (VF) occurred during preparation in 3.6%, during coronary occlusion in 7.3% and during reperfusion in 5.0% of the animals. VF was significantly related to a large zone of MAR and insufficient premedication. Catheter- or ball-induced complications were found in 10.7%. Mortality and incidence of VF are considerably lower in this closed-chest model than in a previously reported open-chest pig preparation.

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.

Background: Bilirubin, an antioxidant, has been associated with reduced cardiovascular disease risk. A major cause of elevated plasma bilirubin is the common UGT1A1*28 promoter polymorphism in the gene of the bilirubin-conjugating enzyme UDP-glucuronosyltransferase 1A1, which reduces transcription by 70%. Earlier studies reporting a protective effect of bilirubin on stroke have not included analysis of UGT1A1*28. The purpose of this study is to investigate if bilirubin and UGT1A1*28 are protective against ischemic stroke in a prospective case-referent setting. Methods: Cases with first-ever ischemic stroke (n = 231; median lag time 4.9 years) and 462 matched referents from the Northern Sweden Health and Disease Study Cohort were included. Plasma bilirubin was measured and UGT1A1*28 was analyzed by fragment analysis. Results: Plasma bilirubin was lower in cases than in referents, but the difference reached significance only for women. The UGT1A1*28 polymorphism (allele frequency 30%) showed a strong gene-dose relationship with bilirubin levels both among cases and referents, but was not associated with risk for stroke. Among multiple other variables analyzed, the strongest correlation with bilirubin was found for plasma iron. Conclusions: There was no evidence for a protective effect of the UGT1A1*28 polymorphism against stroke and consequently neither for bilirubin. The findings suggest that other factors influencing the risk for stroke might also affect bilirubin levels.

Our objectives were to study the relationship between iron stores, HFE genotypes and the risk for first-ever myocardial infarction.First-ever myocardial infarction cases (n=618) and double matched referents from the Northern Sweden Health and Disease Cohort Study were studied in a prospective nested case-referent setting. Plasma iron, total iron binding capacity, transferrin iron saturation and ferritin were analyzed, as well as several confounders. HFE C282Y and H63D genotypes were determined.There was an inverse risk association for myocardial infarction in the highest quartiles of iron (OR 0.68; 95% CI 0.48-0.96) and transferrin iron saturation (OR 0.62; 95% CI 0.42-0.89) in men. This association, however, was lost after adjusting for C-reactive protein. Women homozygous for H63D had a higher risk for myocardial infarction.No risk association between high iron stores and first-ever myocardial infarction was found. The higher risk in female H63D homozygotes is probably not related to iron metabolism.

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.

Mutant superoxide dismutases type 1 (SOD1s) cause amyotrophic lateral sclerosis by an unidentified toxic property. In a patient carrying the G127X truncation mutation, minute amounts of SOD1 were found in ventral horns using a mutant-specific antibody. Still, both absolute levels and ratios versus wild-type SOD1 were considerably greater than in other central nervous system areas and peripheral organs. Inclusions of mutant SOD1 were abundant in motoneurons but were also seen in hepatocytes and kidney epithelium. This first examination of mutant SOD1 in both central nervous system and peripheral organs supports the notion that enrichment of misfolded SOD1s might explain the particular vulnerability of motor areas.

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 purpose of this investigation was to elucidate the regulation of corneal extracellular superoxide dismutase (SOD3) synthesis in keratoconus. We compared the basal and cytokine-regulated SOD3 synthesis in cultured human stromal cells from keratoconus corneas to stromal cells from normal and bullous keratopathy corneas.Keratocyte cultures were obtained from patients undergoing corneal transplantation for keratoconus and bullous keratopathy, and from healthy donor corneas. The cell lines obtained were cultured until near confluence and interleukin-1alpha, interleukin-6, transforming growth factor beta, or platelet derived growth factor were added to the media. The phenotypes of the cultured cells were assessed by immunocytochemical expression of alpha-smooth muscle actin and CD34. SOD3 protein contents were determined in the culture media with ELISA after 24, 48, 72, and 96 h.Interleukin-1alpha had an inhibitory effect on SOD3 synthesis exclusively in the keratoconus cultures (p0.01). Platelet derived growth factor induced a reduction in SOD3 synthesis in all groups (p0.05).Here, we demonstrate that cultured keratoconus stromal cells respond with a reduced SOD3 synthesis to interleukin-1alpha, which is not the case in corresponding normal or bullous keratopathy cells. Since interleukin-1alpha is upregulated in corneal trauma and inflammation, keratoconus corneas may muster an insufficient oxidative defense under such conditions.

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.

High iron levels can increase the formation of noxious oxygen radicals, which are thought to contribute to cerebrovascular disease. The aim of this prospective study was to determine if iron status and HFE genotypes constitute risk factors for stroke.First-ever stroke cases (231 ischemic and 42 hemorrhagic) and matched double referents from the population-based Northern Sweden cohorts were studied in a nested case-referent setting.For total iron binding capacity, an increased risk of ischemic stroke was seen in the highest quartile (OR 1.80; 95% CI 1.14-2.83; p for trend 0.012). The highest quartile of transferrin iron saturation showed a decreased risk of ischemic stroke in men (OR 0.44; 95% CI 0.22-0.87; p for trend 0.028), but not in women. There was an increased risk of hemorrhagic stroke in the second (OR 4.07; 95% CI 1.09-15.20) and third quartile (OR 4.22; 95% CI 1.08-16.42) of ferritin. Neither quartiles of plasma iron concentrations nor the HFE C282Y and H63D genotypes were associated with ischemic or hemorrhagic stroke.Iron stores were not positively related to increased risk of ischemic stroke. Furthermore, HFE genotypes did not influence the risk of ischemic or hemorrhagic stroke.

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.

A 71-year-old woman with a family history of amyotrophic lateral sclerosis (ALS) was investigated for symmetrical, proximal limb and abdominal muscle weakness. Initial examination showed mild proximal muscle weakness in the arms and legs, slightly elevated serum creatine kinase (CK) level, and normal electromyographic (EMG) findings. A myopathy was the presumed diagnosis. Over the next year, weakness became severe and tendon reflexes became unelicitable; no upper motor signs were present. EMG then showed acute and chronic denervation and a muscle biopsy showed target fibers and grouped atrophy. DNA analysis revealed a G72C CuZn-superoxide dismutase (SOD1) mutation. Fasciculations were absent throughout the disease. The patient died 53 months after symptom onset and autopsy revealed loss of lower motor neurons (LMN) and SOD1-positive inclusions. This case expands the phenotypic spectrum of ALS associated with SOD1 mutations to include presenting features that mimic a myopathy.

Analysis of the entire set of low molecular weight compounds (LMC), the metabolome, could provide deeper insights into mechanisms of disease and novel markers for diagnosis. In the investigation, we developed an extraction and derivatization protocol, using experimental design theory (design of experiment), for analyzing the human blood plasma metabolome by GC/MS. The protocol was optimized by evaluating the data for more than 500 resolved peaks using multivariate statistical tools including principal component analysis and partial least-squares projections to latent structures (PLS). The performance of five organic solvents (methanol, ethanol, acetonitrile, acetone, chloroform), singly and in combination, was investigated to optimize the LMC extraction. PLS analysis demonstrated that methanol extraction was particularly efficient and highly reproducible. The extraction and derivatization conditions were also optimized. Quantitative data for 32 endogenous compounds showed good precision and linearity. In addition, the determined amounts of eight selected compounds agreed well with analyses by independent methods in accredited laboratories, and most of the compounds could be detected at absolute levels of approximately 0.1 pmol injected, corresponding to plasma concentrations between 0.1 and 1 microM. The results suggest that the method could be usefully integrated into metabolomic studies for various purposes, e.g., for identifying biological markers related to diseases.

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.

One cause of amyotrophic lateral sclerosis (ALS) is mutation in ubiquitously expressed copper/zinc superoxide dismutase (SOD1), but the mechanism of toxicity to motor neurons is unknown. Multiple disease-causing mutants, but not wild-type SOD1, are now demonstrated to be recruited to mitochondria, but only in affected tissues. This is independent of the copper chaperone for SOD1 and dismutase activity. Highly preferential association with spinal cord mitochondria is seen in human ALS for a mutant SOD1 that accumulates only to trace cytoplasmic levels. Despite variable proportions that are successfully imported, nearly constant amounts of SOD1 mutants and covalently damaged adducts of them accumulate as apparent import intermediates and/or are tightly aggregated or crosslinked onto integral membrane components on the cytoplasmic face of those mitochondria. These findings implicate damage from action of spinal cord-specific factors that recruit mutant SOD1 to spinal mitochondria as the basis for their selective toxicity in ALS.

Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.

Mutant forms of superoxide dismutase-1 (SOD1) cause amyotrophic lateral sclerosis (ALS) by an unknown noxious mechanism. Using an antibody against a novel epitope in the G127insTGGG mutation, mutant SOD1 was studied for the first time in spinal cord and brain of an ALS patient. The level was below 0.5% of the SOD1 level in controls. In corresponding transgenic mice the content of mutant SOD1 was also low, although it was enriched in spinal cord and brain compared with other tissues. In the mice the misfolded mutant SOD1 aggregated rapidly and 20% occurred in steady state as detergent-soluble protoaggregates. The misfolded SOD1 and the protoaggregates form, from birth until death, a potentially noxious burden that may induce the motor neuron injury. Detergent-resistant aggregates, as well as inclusions of mutant SOD1 in motor neurons and astrocytes, accumulated in spinal cord ventral horns of the patient and mice with terminal disease. The inclusions and aggregates may serve as terminal markers of long-term assault by misfolded SOD1 and protoaggregates.

The bioavailability of nitric oxide (NO) within the vascular wall is limited by superoxide anions (O 2 ·− ). The relevance of extracellular superoxide dismutase (ecSOD) for the detoxification of vascular O 2 ·− is unknown. We determined the involvement of ecSOD in the control of blood pressure and endothelium-dependent responses in angiotensin II–induced hypertension and renovascular hypertension induced by the two-kidney, one-clip model in wild-type mice and mice lacking the ecSOD gene. Blood pressure was identical in sham-operated ecSOD +/+ and ecSOD −/− mice. After 6 days of angiotensin II–treatment and 2 and 4 weeks after renal artery clipping, blood pressure was significantly higher in ecSOD −/− than ecSOD +/+ mice. Recombinant ecSOD selectively decreased blood pressure in hypertensive ecSOD −/− mice, whereas ecSOD had no effect in normotensive and hypertensive ecSOD +/+ mice. Compared with sham-operated ecSOD +/+ mice, sham-operated ecSOD −/− mice exhibited attenuated acetylcholine-induced relaxations. These responses were further depressed in vessels from clipped animals. Vascular O 2 ·− , as measured by lucigenin chemiluminescence, was higher in ecSOD −/− compared with ecSOD +/+ mice and was increased by clipping. The antioxidant tiron normalized relaxations in vessels from sham-operated and clipped ecSOD −/− , as well as from clipped ecSOD +/+ mice. In contrast, in vivo application of ecSOD selectively enhanced endothelium-dependent relaxation in vessels from ecSOD −/− mice. These data reveal that endogenous ecSOD is a major antagonistic principle to vascular O 2 ·− , controlling blood pressure and vascular function in angiotensin II–dependent models of hypertension. ecSOD is expressed in such an abundance that even in situations of high oxidative stress no relative lack of enzyme activity occurs.

The PRKAG3 gene encodes a muscle-specific isoform of the regulatory gamma subunit of AMP-activated protein kinase (AMPK). A major part of the coding PRKAG3 sequence was isolated from horse muscle cDNA using reverse-transcriptase (RT)-PCR analysis. Horse-specific primers were used to amplify genomic fragments containing 12 exons. Comparative sequence analysis of horse, pig, mouse, human, Fugu, and zebrafish was performed to establish the exon/intron organization of horse PRKAG3 and to study the homology among different isoforms of AMPK gamma genes in vertebrates. The results showed conclusively that the three different isoforms (gamma1, gamma2, and gamma3) were established already in bony fishes. Seven single nucleotide polymorphisms (SNPs), five causing amino acid substitutions, were identified in a screening across horse breeds with widely different phenotypes as regards muscle development and intended performance. The screening of a major part of the PRKAG3 coding sequence in a small case/control material of horses affected with polysaccharide storage myopathy did not reveal any mutation that was exclusively associated with this muscle storage disease. The breed comparison revealed several potentially interesting SNPs. One of these (Pro258Leu) occurs at a residue that is highly conserved among AMPK gamma genes. In an SNP screening, the variant allele was only found in horse breeds that can be classified as heavy (Belgian) or moderately heavy (North Swedish Trotter, Fjord, and Swedish Warmblood) but not in light horse breeds selected for speed or racing performance (Standardbred, Thoroughbred, and Quarter horse) or in ponies (Icelandic horses and Shetland pony). The results will facilitate future studies of the possible functional significance of PRKAG3 polymorphisms in horses.

Fish-odor syndrome or Trimethylaminuria (OMIM #602079) in humans is an inborn error of metabolism associated with a characteristic fishy body odor due to elevated levels of trimethylamine (TMA) in body fluids. It is caused by loss-of-function mutations in FMO3encoding flavin-containing mono-oxygenase 3. A fishy off-flavor is occasionally observed in cow's milk and it has been established recently that this phenotype is due to elevated TMA levels. Here, we report that fishy off-flavor in cow's milk is caused by a nonsense mutation (R238X) in the bovine FMO3 ortholog. RT–PCR analysis indicated that the mutant transcript is present in a very low amount. The mutation was found to be surprisingly common (q = 0.155) in one breed of cattle.[The sequence data described in this paper have been submitted to GenBank with accession nos.AF488417–AF488422. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: K. Sandberg and I. Hansson.]

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.

The lung is exposed to high oxygen tension and oxygen free radicals have been implicated in many pathologies of the organ. Extracellular superoxide dismutase occurs in high concentration in the lung and protects against hyperoxia-induced inflammation. We hypothesized that the enzyme might ameliorate other types of inflammation as well as aging-related changes of the organ. Tracheal instillation of endotoxin plus zymosan into extracellular superoxide dismutase knockout and wild-type mice resulted in a marked neutrophilic inflammation and increases in inflammatory cytokines, protein, and lactate dehydrogenase activity in the bronchoalveolar lavage fluid. There were no significant differences between the genotypes. Repeated challenges with ovalbumin caused an allergic inflammation with increases in eosinophils, interleukin-5, protein, and lactate dehydrogenase activity in the bronchoalveolar lavage fluid. Only minimal differences between the genotypes were found. In lungs from 2-year-old mice, marginal increases in inflammatory variables and fibrosis were found in the knockout mice. In conclusion, extracellular superoxide dismutase had a negligible role in the present inflammation and allergy models and for the long-term integrity of the organ.

CSF from patients with motor neurone disease (MND) has been reported to be toxic to cultured primary neurones. We found that CSF from MND patients homozygous for the D90A CuZn-superoxide dismutase (CuZn-SOD) mutation, patients with sporadic MND and patients with familial MND without CuZn-SOD mutations significantly increased apoptosis and reduced phosphorylation of neurofilaments in cultured spinal cord neurones when compared with the effects of CSF from patients with other neurological diseases. Exposure of spinal cord cultures to MND CSF also triggered microglial activation. The toxicity of MND CSF was independent of the presence of the CuZn-SOD mutation, and it did not correlate with gelatinase activity or the presence of immunoglobulin G autoantibodies in the CSF. The concentrations of glutamate, aspartate and glycine in MND CSF were not elevated. Antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptors prevented the toxic CSF-induced neuronal death but not microglial activation, whereas minocycline, a tetracycline derivative with anti-inflammatory potential independent of antimicrobial activity, reduced both the apoptotic neuronal death and microglial activation. We conclude that the cytotoxic action of CSF is prevalent in all MND cases and that microglia may mediate the toxicity of CSF by releasing excitotoxicity-enhancing factors.

The Dominant White (I/KIT) locus is one of the major coat color loci in the pig. Previous studies showed that the Dominant White (I) and Patch (IP) alleles are both associated with a duplication including the entire KIT coding sequence. We have now constructed a BAC contig spanning the three closely linked tyrosine kinase receptor genes PDGFRA-KIT-KDR. The size of the duplication was estimated at about 450 kb and includes KIT, but not PDGFRA and KDR. Sequence analysis revealed that the duplication arose by unequal homologous recombination between two LINE elements flanking KIT. The same unique duplication breakpoint was identified in animals carrying the I and IP alleles across breeds, implying that Dominant White and Patch alleles are descendants of a single duplication event. An unexpected finding was that Pietrain pigs carry the KIT duplication, since this breed was previously assumed to be wild type at this locus. Comparative sequence analysis indicated that the distinct phenotypic effect of the duplication occurs because the duplicated copy lacks some regulatory elements located more than 150 kb upstream of KIT exon 1 and necessary for normal KIT expression.