Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice

Premature ageing, one of the characteristics of Down syndrome (DS), may involve oxidative stress and impairment of proteasome activity. Transgenic mice overexpressing the human copper/zinc superoxide dismutase (SOD1) gene are one of the first murine models for DS and it has been shown that SOD1 overexpression might be either deleterious or beneficial. Here, we show a reduction in proteasome activities in the cortex of SOD1 transgenic mice and an associated increase in the content of oxidized SOD1 protein. As we demonstrate that in vitro oxidized SOD can inhibit purified proteasome peptidase activities, modified SOD1 might be partially responsible for proteasome inhibition shown in SOD1 transgenic mice.     

The overexpression of major antioxidant enzymes does not extend the lifespan of mice

We evaluated the effect of overexpressing antioxidant enzymes on the lifespans of transgenic mice that overexpress copper zinc superoxide dismutase (CuZnSOD), catalase, or combinations of either CuZnSOD and catalase or CuZnSOD and manganese superoxide dismutase (MnSOD). Our results show that the overexpression of these major antioxidant enzymes, which are known to scavenge superoxide and hydrogen peroxide in the cytosolic and mitochondrial compartments, is insufficient to extend lifespan in mice.     

The amino-acid sequence of copper/zinc superoxide dismutase from swordfish liver. Comparison of copper/zinc superoxide dismutase sequences

The amino acid sequence of copper/zinc superoxide dismutase from swordfish (Xiphias gladius) liver has been determined by alignment of the tryptic peptides according to the known sequence of bovine erythrocyte copper/zinc superoxide dismutase. This alignment has resulted in the ligands to the copper (His-47, 49, 76 and 94) and the zinc (His-76, 85, 134 and Asp-97) being conserved in all the copper/zinc superoxide dismutases sequenced so far. Also conserved in the sequences are the cysteines forming the intrachain disulphide bridge (Cys-58 and 160) and the essential arginine (Arg-157). Comparison of the amino acid sequence of swordfish liver copper/zinc superoxide dismutase with the bovine, human, horse, yeast and Photobacterium leiognathi indicates that the swordfish enzyme has a high homology with the other eukaryotic enzymes. Low homology is, however, observed with the P. leiognathi enzyme.     

Evaluation of superoxide dismutase and glutathione peroxidase enzymes and their cofactors in Egyptian children with Down's syndrome

The present work investigated the activity of Cu/Zn superoxide dismutase enzyme (SOD) in red blood cells and glutathione peroxidase enzyme (GPx) in whole blood by spectrophotometric methods. Plasma levels of the cofactors copper and zinc and whole-blood selenium were evaluated using atomic absorption spectrophotometer. The study included a population of 18 Down's syndrome (DS) patients with complete trisomy 21 (group 1), translocations (group 2), and mosaicism (group 3), and their 15 matched controls. The purpose of this work was to study the gene dosage effect of SOD and its consequence on GPx enzyme and the various cofactors, and to find out correlations with developmental fields. Our results showed that in the population with complete trisomy 21 and translocations, SOD and GPx activities were increased, whereas in cases with mosaicism, the enzymes activities were within normal limits. Plasma copper concentrations were increased, whereas whole-blood selenium concentrations were significantly decreased in the three DS groups. Plasma zinc levels were within normal in all patients. We concluded that changes in trace elements and enzyme activities were not related to age or sex. Also, there was no correlation between the enzyme levels and the developmental activities. Our results are useful tools for identifying nutritional status and guiding antioxidant intervention.     

Immunolocalization of manganese superoxide dismutase in normal and transgenic mice expressing the human enzyme

Summary The localization of manganese superoxide dismutase (MnSOD) was determined using immunohistochemistry of various tissues of normal and transgenic mice which express the human enzyme, with emphasis on studies of mouse kidney and lung. Mouse kidney and lung were studied using both frozen section analysis and paraffin sections following fixation in a variety of fixatives. Formalin fixation resulted in a loss of antigenicity, while fixation in zinc formalin or B5 fixative gave results similar to those from frozen sections. Immunoperoxidase studies using antibodies to MnSOD showed greater staining in transgenic kidney or lung than in identical tissues in normal mice when appropriate fixation was used. In contrast, equal immunostaining was obtained in kidney or lung from normal and transgenic mice when antibodies to catalase or copper zinc superoxide dismutase were utilized. Immunogold ultrastructural analysis of MnSOD localization for lung and kidney was also performed. As compared to normal mice, transgenic mice exhibited greater staining of the mitochondria of kidney interstitial fibroblasts and glomerular, endothelial, and smooth muscle cells. In the lungs of transgenic animals, all cells showed increased staining; smooth muscle cells demonstrated the most marked increase in immunolabelling. The results indicate that these transgenic mice overexpress MnSOD in their mitochondria, and that this occurs selectively in at least some mesenchymal tissues.This study was supported by the Medical Research Service of the Department of Veterans Affairs (TDO), by National Institutes of Health grants No. CA-41267 (LWO), No. HL-39585 and No. HL-44571 (Y-SH), and by the Department of Anesthesiology Research and Development Funds (DBC, HPC).     

Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase.

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid peroxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47; P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 +/- 0.71 mumole MDA/g tissue) than at 2 months (5.69 +/- 0.90) and significantly lower at 28 months (5.13 +/- 0.33) than at 12 months. Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.     

Gene dosage of CuZnSOD and Down''s syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD.     

Analysis of cholinergic markers, biogenic amines, and amino acids in the CNS of two APP overexpression mouse models

Two transgenic mouse models expressing mutated human amyloid precursor protein and previously found to display cognitive and behavioural alterations, reminiscent of Alzheimer patients' symptomatology, were scrutinised for putative brain region-specific changes in neurochemical parameters. Brains of NSE-hAPP751m-57, APP23 and wild-type mice were microdissected to perform brain region-specific neurochemical analyses. Impairment of cholinergic transmission, the prominent neurochemical deficit in Alzheimer brain, was examined; acetylcholinesterase and choline acetyltransferase activity levels were determined as markers of the cholinergic system. Since Alzheimer neurodegeneration is not restricted to the cholinergic system, brain levels of biogenic amines and metabolites, and amino acidergic neurotransmitters and systemic amino acids were analysed as well. Cholinergic dysfunction, reflected in reduced enzymatic activity in the basal forebrain nuclei, was restricted to the APP23 model, which also exhibited more outspoken and more widespread changes in other neurotransmitter systems. Significant changes in compounds of the noradrenergic and serotonergic system were observed, as well as alterations in levels of the inhibitory neurotransmitter glycine and systemic amino acids. These observations were clearly in occurrence with the more pronounced histopathological and behavioural phenotype of the APP23 model. As transgenic models often do not represent an end-stage of the disease, some discrepancies with results from post-mortem human Alzheimer brain analyses were apparent; in particular, no significant alterations in excitatory amino acid levels were detected. Our findings of brain region-specific alterations in compound levels indicate disturbed neurotransmission pathways, and greatly add to the validity of APP23 mice as a model for Alzheimer's disease. Transgenic mouse models may be employed as a tool to study early-stage neurochemical changes, which are often not accessible in Alzheimer brain.     

Cloning,production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.     

Changed serum trace element profile in Down's syndrome

Being cofactors of important antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), which are significantly modified in Down's syndrome (trisomy 21), serum levels of microtrace elements zinc, copper, and selenium and of macroelement magnesium are reported in 16 subjects with Down's syndrome (DS) and their respective well age- and sex-matched controls. Serum zinc and selenium levels were significantly lowered in DS subjects, whereas copper levels were elevated. Consequently, a marked increase (40%) of the copper/zinc ratio in DS persons was observed. There were no differences in serum levels of magnesium between DS and control subjects.     

Two copper-containing proteins from white and gray matter of brain.

The procedure for the isolation of two water soluble copper-containing proteins from the white and gray matter of bovine brain is described. One of the proteins, cerebrocuprein I, is superoxide dismutase; and three molecular forms of this enzyme are to be found in brain. The other protein present in gray and white matter is devoid of superoxide dismutase and amine oxidase activities. The amino acid composition, molecular weight, isoelectric point and copper content of this protein were determined. The effect of some agents, pH and thermal treatment of the optical and EPR spectra of the protein were also studied. The copper of the protein may be removed and the holoprotein reconstituted again from apoprotein and copper. The results obtained led to the conclusion that in brain a new copper protein is discovered, which is named neurocuprein.     

Superoxide Dismutase, Catalase, and Glutathione Peroxidase Activities in Copper/Zinc-Superoxide Dismutase Transgenic Mice

Copper/zinc-superoxide dismutase (CuZn-SOD) transgenic mice overexpress the gene for human CuZn-SOD. To assess the effects of the overexpression of CuZn-SOD on the brain scavenging systems, we have measured the activities of manganese-SOD (Mn-SOD), catalase, and glutathione peroxidase (GSH-Px) in various regions of the mouse brain. In nontransgenic mice, cytosolic CuZn-SOD activity was highest in the caudate-putamen complex; this was followed by the brainstem and the hippocampus. The lowest activity was observed in the cerebellum. In transgenic mice, there were significant increases of cytosolic CuZn-SOD activity in all of these regions, with ratios varying from a twofold increase in the brainstem to 3.42-fold in the cerebellum in comparison with nontransgenic mice. Particulate Mn-SOD was similarly distributed in all brain regions, and its levels also were significantly increased in superoxide dismutase (SOD)-transgenic mice. In the brains of nontransgenic mice, cytosolic catalase activity was similar in all brain regions except the cortex, which showed less than 50% of the activity observed in the other regions. In transgenic mice, cytosolic catalase activity was significantly increased, with the cortex showing the greatest changes (133%) in comparison with nontransgenic mice. The smallest increases were observed in the hippocampus (34%). In contrast to what was observed for SOD and catalase, there were no significant changes in cytosolic GSH-Px activity in any of the brain regions examined. The present results indicate that, in addition to displaying marked increases in the levels of brain CuZn-SOD activity, SOD-transgenic mice also exhibit increases in other enzymes that scavenge oxygen-based radicals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparisons of copper deficiency states in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice.

The activity of two copper-dependent enzymes, cytochrome c oxidase and copper, zinc-superoxide dismutase, was determined in six tissues of age-matched (13-day-old) copper-deficient mutant and normal mice. In the two mutants 'brindled' and 'blotchy', brain, heart and skeletal muscle had significant enzyme deficiencies. Cytochrome c oxidase was more severely affected than was superoxide dismutase. In these three tissues the degree of deficiency could be correlated with decreased copper concentration; however, enzyme activity was normal in liver, kidney and lung, despite abnormal copper concentrations in these tissues. In nutritionally copper-deficient mice, all six tissues showed decreased enzyme activity, which was most marked in brain, heart and skeletal muscle, the tissues which showed enzyme deficiencies in the mutants. Analysis in vitro of cytochrome c oxidase (temperature coefficient = 2) at a single temperature was found to underestimate the deficiency of this enzyme in hypothermic copper-deficient animals. Cytochrome c oxidase deficiency may therefore be sufficiently severe in vivo to account for the clinical manifestations of copper deficiency. An injection of copper (50 micrograms of Cu+) at 7 days increased cytochrome c oxidase activity by 13 days in all deficient tissues of brindled mice, and in brain and heart from blotchy mice. However, skeletal-muscle cytochrome c oxidase in blotchy mutants did not respond to copper injection. Cytochrome c oxidase activity increased to normal in all tissues of nutritionally copper-deficient mice after copper injection, except in the liver. Hepatic enzyme activity remained severely deficient despite a liver copper concentration three times that found in copper-replete controls. Superoxide dismutase activity did not increase with treatment in either mutant, but its activity was higher than control levels in nutritionally deficient mice after injection. This difference is probably due to sequestration of copper in mutant tissue such as kidney, but a defect in the copper transport pathway to superoxide dismutase cannot be excluded.     

Regional distribution of biogenic amines, amino acids and cholinergic markers in the CNS of the C57BL/6 strain

Summary. A reliable extrapolation of neurochemical alterations from a mouse model to human metabolic brain disease requires knowledge of neurotransmitter levels and related compounds in control mouse brain. C57BL/6 is a widely used background strain for knockout and transgenic mouse models. A prerequisite for reliable extrapolation from mouse brain to the human condition is the existence of analogous distribution patterns of neurotransmitters and related compounds in control mouse and human brain. We analysed regional distribution patterns of biogenic amines, neurotransmitter and non-neurotransmitter amino acids, and cholinergic markers. Distribution patterns were compared with known neurotransmitter pathways in human brain. The present study provides a reference work for future analyses of neurotransmitters and related compounds in mouse models bred in a C57BL/6 background strain.     

Photosynthetic apparatus of chilling-sensitive plants. XI. Reversibility by light of cold- and dark-induced inactivation of cyanide-sensitive superoxide dismutase activity in tomato leaf chloroplasts

(1) The inactivation of cyanide-sensitive, copper- and zinc-containing superoxide dismutase activity in chloroplasts following cold and dark storage of both detached leaves and growing tomato plants is accompanied by a decrease in copper and zinc content in both chloroplast preparations and butanol extracts of the enzyme. In contrast, this treatment of chloroplast preparations affects neither superoxide dismutase activity nor copper and zinc content. (2) Copper- and zinc-containing superoxide dismutase is not reactivated following the 2–3 h illumination of cold- and dark-stored detached leaves. However, prolonged illumination of growing seedlings results in the restoration of both the enzyme activity and copper and zinc content in chloroplasts. (3) The data suggest that the dissociation of copper, and probably of zinc, from the enzyme during cold and dark treatment of either detached leaves or growing plants and reincorporation of the metals following the illumination of intact plants are responsible for the reversible inactivation of chloroplast cyanide-sensitive superoxide dismutase of chilling-sensitive plants.     

Hematopoietic progenitor cells of transgenic mice with increased copper/zinc-superoxide dismutase activity are resistant to tumor necrosis factor

The mechanism of growth inhibition mediated by tumor necrosis factor (TNF) is unclear. Since recent data strongly suggested that generation of superoxide is a key step in cytotoxicity of TNF, we reasoned that cells expressing high levels of enzymes that degrade superoxide radicals would be resistant to TNF. Therefore, we examined the TNF-sensitivity of bone marrow progenitor cells of transgenic mice that expressed the gene for human copper zinc-superoxide dismutase (CuZn-SOD). The CuZn-SOD is a key enzyme in the metabolism of superoxide radicals. Heterozygous and homozygous transgenic mice had 3- and 5-fold increased levels of CuZn-SOD activity, respectively. Bone marrow cells of transgenic and nontransgenic mice were plated in soft gel culture with TNF (0.01–100 ng/ml). TNF inhibited myeloid colony formation supported by either granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF from nontransgenic mice in a dose-dependent manner. In contrast, the myeloid clonal growth of homozygote transgenic mice was not inhibited by TNF at concentrations up to 100 ng/ml. As expected, the effects of TNF on erythroid clonogenic cells, which do not produce superoxide, and the action of transforming growth factor-β on myeloid progenitor cells, were similar in both transgenic and nontransgenic mice. These results suggest that the mechanism of TNF-mediated growth inhibition of hematopoietic cells occurs through production of superoxide. © 1994 Wiley-Liss, Inc.     

The primary structure of human liver manganese superoxide dismutase

The complete amino acid sequence of manganese superoxide dismutase from human liver was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus digests of the apoprotein. Chemical cleavage with dimethyl sulfoxide-hydrobromic acid was also carried out. The amino acid sequence listed below is made up of 196 amino acids and the two subunit polypeptides in the native enzyme appear to be identical. No homology was observed with copper/zinc containing class of superoxide dismutase. Lys-His-Ser-Leu-Pro-Asp-Leu-Pro-Tyr-Asp-Tyr-Gly-Ala-Leu-Glu-Pro-His-Il e -Asn-Ala-Gln-Ile-Met-Gln-Leu-His-His-Ser-Lys-His-His-Ala-Ala-Tyr-Val-Asn -Asn-Leu-Asn-Val-Thr-Gln-Glu-Lys-Tyr-Gln-Glu-Ala-Leu-Ala-Lys-Gly-Asp-Val -Thr-Ala-Gln-Ile-Ala-Leu-Gln-Pro-Ala-Leu-Lys-Phe-Asn-Gly-Gly-Gly-His-Ile -Asn-His-Ser-Ile-Phe-Trp-Thr-Asn-Leu-Ser-Pro-Asn-Gly-Gly-Gly-Gln-Pro-Lys -Gly-Glu-Leu-Leu-Glu-Ala-Ile-Lys-Arg-Asp-Phe-Gly-Ser-Phe-Asp-Lys-Phe-Lys -Gln-Lys-Leu-Thr-Ala-Ala-Ser-Val-Gly-Val-Gln-Gly-Ser-Gly-Trp-Leu-Gly-Phe -Asn-Lys-Gln-Arg-Gly-His-Leu-Gln-Ile-Ala-Ala-Cys-Pro-Asn-Gln-Asp-Pro-Leu -Gln-Gly-Thr-Thr-Gly-Leu-Ile-Pro-Leu-Leu-Gly-Ile-Asp-Val-Trp-Glu-His-Ala -Tyr-Tyr-Leu-Gln-Tyr-Lys-Asn-Val-Arg-Pro-Asp-Tyr-Leu-Lys-Ala-Ile-Trp-Asn -Val-Ile-Asn-Trp-Glu-Asn-Val-Thr-Glu-Arg-Tyr-Met-Ala-Cys-Lys-Lys.     

Mice that overexpress Cu/Zn superoxide dismutase are resistant to allergen-induced changes in airway control

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M(2) muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.     

Effect of Gamma Radiation on Growth Factors, Biochemical Parameters, and Accumulation of Trace Elements in Soybean Plants (Glycine max L. Merrill)

The toxicity of iron, copper, and zinc was studied in soybean seeds of the NE 3297 variety irradiated at different dosages of gamma rays. After cultivating in plastic boxes for 14 days, the average plant heights, fresh weight, and chlorophyll content decreased in inverse proportion to radiation dose. As the radiation dose increased, the concentrations of iron, copper, zinc, soluble protein, and malondialdehyde increased, but the activities of superoxide dismutase and peroxidase enzymes activities were significantly decreased. The activities and the number of the superoxide dismutase isoenzymes also varied depending on the irradiation dosages.     

Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.