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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.)

Objective: Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous p ...

Background: As the lens is constantly exposed to light and oxygen that generate harmful reactive oxygen species, the importance of the intracellular antioxidant enzyme copper-zinc superoxide dismut ...

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 ...

We demonstrated that, in vivo, at resting membrane potential, spinal motor neurones of the adult G127X mice do not show an increased excitability. However, when depolarized they show evidence of an ...

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.

Mucosal tissue damage and dysfunction in chronic inflammatory bowel disease (IBD) are partly caused by an enduring exposure to excessive amounts of reactive oxygen metabolites (ROMs). Although the three human isoforms of superoxide dismutase (SOD), copper/zinc (Cu/Zn)-SOD, manganese (Mn)-SOD, and extracellular (EC)-SOD, form the primary endogenous defence against ROMs, their expression levels and cellular localization in IBD mucosa are largely unknown. The present study used enzyme-linked immunosorbent assays (ELISAs), spectrophotometric activity assays, and immunohistochemistry to evaluate the protein concentration, enzymatic activity, and distribution of Cu/Zn-, Mn-, and EC-SOD in paired inflamed and non-inflamed mucosal resection specimens of patients with Crohn's disease (CD) or ulcerative colitis (UC) and compared these with the levels obtained in normal control mucosa. Gut mucosal SOD isoform expression was found to be differentially affected in IBD patients, without major differences between CD and UC. A marked step-wise increase in Mn-SOD protein levels was observed in non-inflamed and inflamed IBD mucosae, whereas the Cu/Zn-SOD content decreased with inflammation. EC-SOD was only found in low amounts, which tended to be decreased in IBD patients. Immunohistochemical evaluation confirmed these observations. Mn-SOD and Cu/Zn-SOD were both predominantly expressed in intestinal epithelial cells and the percentage of epithelial cells positive for Mn-SOD was considerably increased in IBD, whereas epithelial Cu/Zn-SOD expression was much less affected. Within the lamina propria, SOD expression was much lower. Cu/Zn-SOD and Mn-SOD were prominently present in neutrophils and macrophages, and EC-SOD was mainly localized in small vessels, stromal cells, and neutrophils. The percentage of lamina propria cells positive for Cu/Zn-, Mn-, or EC-SOD was not affected by inflammation. Enzyme activity measurements showed consistent results for Cu/Zn-SOD and EC-SOD, but the activity of Mn-SOD did not concordantly increase with the immunological assessments, which may indicate that a proportion of the Mn-SOD in IBD is present in an enzymatically inactive form. This study reveals remarkable changes in the expression levels of the three SOD isoforms in IBD, particularly in the epithelium. Disturbances in the carefully orchestrated mucosal antioxidant cascade may contribute to the induction and perpetuation of intestinal inflammation in IBD, and may have important implications for the development of antioxidant treatment of IBD patients.

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.

The cytochrome P-450, subfamily I, polypeptide 1 (CYP1A1), somatostatin receptor 1 (SSTR1) and transcription termination factor 1 (TTF1) genes, predicted to reside on SSC7q based on chromosome painting studies, were selected for genetic mapping in the pig. Porcine sequence tagged sites (STSs) spanning 0.9-1.5 kb genomic DNA sequences were generated and all genes were physically assigned to SSC7q12-q23, q26 using a pig-rodent somatic cell hybrid panel. Direct sequencing of PCR products from four different breeds revealed single nucleotide polymorphisms (SNPs) in all genes. SNPs in the SSTR1 and CYP1A1 genes allowed simple genotyping using PCR-restriction fragment length polymorphism (RFLP) analysis and linkage mapping with the PiGMaP reference families. SSTR1 was found to be closely linked to CYP1A1 (0.0 cM; LOD = 11.1) and ANPEP (0.0 cM; LOD = 9.0). This refined the localization of a previously identified breakpoint in the linkage map between regions with orthology to human chromosome 15q (represented by CYP1A1 and ANPEP) and 14q (represented by SSTR1). Moreover, multipoint linkage data suggest inverted segments of orthology between HSA14q11-q13 and SSC7q. The TTF1 SNP was confirmed by allele-specific PCR but was not informative in any of the families used for linkage analysis.

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.

The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6. The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− -independent mechanisms rapidly become dominant. Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism. A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol. A plausible explanation is given for the non-competitive part of the inhibition of catechol O-methyltransferase brought about by pyrogallol.

Enzyme secretion from incubated guinea pig submandibular gland slices was induced by adrenaline, noradrenaline, dopamine, 5-hydroxytryptamine or dibutyryl cyclic AMP combined with theophylline. Effects of alpha- and beta-blocking agents on stimulated peroxidase and amylase discharge were studied. Propranolol (beta-blocker), but not phenoxybenzamine (alpha-blocker), markedly inhibited the secretion of both enzymes induced by adrenaline or noradrenaline. Dopamine and 5-hydroxytryptamine were found to be potent enzyme secretagogues. Both propranolol and phenoxybenzamine antagonized the effects of these two amines. The secretagogic effect of dibutyryl cyclic AMP in combination with theophylline was not affected by either propranolol or phenoxybenzamine. L-DOPA and 5-hydroxytryptophan were without effect on enzyme secretion. It is concluded that peroxidase and amylase are secreted in parallel from guinea pig submandibular gland and that enzyme release is mainly mediated via the beta-adrenergic receptor.