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Biotechnology offers extensive possibilities to incorporate new traits into organisms. Genetically modified (GM) traits relevant for agro-ecosystems include traits such as pest resistance and herbicide tolerance in crop plants, increased growth rate in fish and livestock, and enhanced nitrogen-fixation capabilities of soil microbes. In this review, we evaluated the direct and indirect trait-specific effects of GM plants, microbes, and animals on ecosystem processes and found that most of the effects of genetically modified organisms (GMOs) on ecosystem processes are indirect and are the result of associated changes in management strategy rather than a direct effect of the GMOs. Conflicting results on the performance and effects of GMOs are frequently reported, especially regarding crop yield and impacts on soil organisms. This is partly because methods with different levels of resolution have been used in different ecological contexts. Overall, there is little evidence that the effects of GM traits on ecosystem processes act with different mechanisms from those of traits modified using conventional methods. However, little is known about trait-specific effects of GMOs on ecosystem processes even though GMOs have been used for more than three decades. In particular, studies linking genetically modified traits to ecosystem processes at longer time scales are rare, but needed for evaluating trait effects, especially in an evolutionary context. In addition, biotechnology may provide a unique tool for gaining insights into the links between traits and ecosystem processes when integrated into basic ecological research.

Objectives: Our objectives were to study the relationship between iron stores, HFE genotypes and the risk for first-ever myocardial infarction. Methods: First-ever myocardial infarction cases (n=61 ...

The most common of the amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase-1 (SOD1) mutations, D90A, differs from others in its high structural stability and by the existence of both recessive and dominant inheritance. Here SOD1 in CNS and peripheral organs from five ALS patients homozygous for D90A were compared to controls. In most areas, including ventral horns, there were no significant differences in SOD1 activities and Western blotting patterns between controls and D90A cases. The SOD1 activities in areas vulnerable to mutant SOD1s, ventral horns and precentral gyrus were intermediate among CNS areas and much lower than in kidney and liver. Thus, the vulnerability of motor areas is not explained by high SOD1 content. The findings argue against the idea of expression-reducing protective factors being present near the D90A locus in recessive pedigrees. The similarity to wild-type SOD1 prompts speculations on the involvement of the latter in sporadic ALS.

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

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

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

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

The ability of glycated hemoglobin A(1c) (HbA(1c)) to predict diabetes is unknown, but could be evaluated by analyses on samples stored in biobanks. The stability of HbA(1c) in long-term stored samples is, however, unknown. Moreover, the effect of hemolysis on HbA(1c) in the general population is not assessed. To explore these questions HbA(1c) was determined in 3 groups (n = 717) of samples with storage times at -80 degrees C differing between 10 years and 2 months. The results were compared with HbA(1c) analyzed in fresh blood samples (n = 174). The subjects were free from diabetes and aged 40 to 60 years. HbA(1c) was analyzed by cation exchange high-performance liquid chromatography (HPLC). The mean HbA(1c) results for the fresh and long-term stored samples were 4.25% +/- 0.39 and 4.19% +/- 0.43, respectively (P = not significant [NS]). The HbA(1c) levels in fresh and 2-month stored samples were essentially equal. There was no correlation between HbA(1c) in the fresh samples and the hemolysis parameters reticulocytes, haptoglobin, or bilirubin. HbA(1c) is apparently stable in samples long-term stored at -80 degrees C and is not commonly affected by hemolysis in the general population. HbA(1c) analyzed on biobank samples could be used to assess the predictive value for future diabetes and relationship to other morbidity and mortality.

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

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

Objectives With the present studies we sought to determine how treatment with nitroglycerin (NTG) affects endothelial function, oxidative stress and nitric oxide (NO)-downstream signaling in Watanabe heritable hyperlipidemic rabbits (WHHL). Background In vitro experiments have demonstrated potent antiatherosclerotic effects of NO suggesting that treatment with NO-donors such as NTG could compensate for the diminished availability of endothelial NO. Nitric oxide may, however, not only be scavenged by reaction with endothelium-derived superoxide but also form the potent oxidant and inhibitor of vascular function, peroxynitrite (ONOO-). Methods Watanabe heritable hyperlipidemic rabbits were treated for three days with NTG patches. Normolipidemic New Zealand White rabbits (NZWR) served as controls. Endothelial function was assessed ex vivo with organ chamber experiments and vascular superoxide was quantified using lucigenin (5 and 250 μM) and CLA-enhanced chemiluminescence. Vascular ONOO- formation was determined using nitrotyrosine antibodies. The activity of the cGMP-dependent kinase (cGK-I) was assessed by determining the phosphorylation of vasodilator-stimulated phosphoprotein VASP (P-VASP). Results Nitroglycerin treatment caused endothelial dysfunction in NZWR and WHHL, associated with an increase in superoxide and ONOO- production and a substantial drop in cGK-I activity. In vivo NTG-treatment decreased lipophilic antioxidants (α- and β-carotene) in NZWR and WHHL. Treatment of NZWR with NTG also decreased plasma extracellular superoxide dismutase (EC-SOD)-activity. Conclusions Nitroglycerin treatment of WHHL with exogenous NO worsens rather than improves endothelial dysfunction secondary to increased formation of superoxide and/or peroxynitrite leading to decreased cGK-I activity. The decrease in plasma levels of α- and β-carotene may be at least in part due to a decrease in EC-SOD activity.

Extracellular-superoxide dismutase (EC-SOD) exists primarily in the tissue interstitium and the lung contains particularly large amounts of the enzyme. To determine the roles of EC-SOD and extracellularly formed superoxide radicals in the pulmonary response to the common air pollutant ozone, wild-type mice and mice lacking EC-SOD were exposed to 1·5 ppm ozone for 48 h. The exposure resulted in a marked neutrophilic inflammatory reaction observed both in the bronchoalveolar lavage fluid (BALF) and by histopathology of the lungs, which was much stronger in the mice lacking EC-SOD. Unlike the wild-type mice, the null mutants also showed increased levels of interleukin-6 in the BALF. The ozone exposure also resulted in increased airway mucosal permeability and cell damage as indicated by increased protein and lactate dehydrogenase in the BALF. There was, however, no difference between the two groups of mice. The results suggest that extracellular superoxide radicals are important inflammatory mediators in the pulmonary response to ozone, but in the present model, the radical and the infiltrating neutrophils contributed little to the pulmonary injury. The data, together with previous findings, support a role for EC-SOD as a modulator of inflammatory reactions.

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

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

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) Type I with mutations in both transthyretin (TTR) and extracellular superoxide dismutase (EC-SOD). This patient started to develop peripheral neuropathy at age 25, followed by cardiac, renal, and autonomic nervous system failure due to massive amyloid deposition. Thirteen years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, multiple abscesses in the bile ducts and urinary tract, and more marked amyloid deposition than commonly seen in FAP Amyloid deposition occurred in various organs and tissues, especially prominently around blood vessels and in interstitial tissues, and was demonstrated immunohistochemically to be composed of TTR but not amyloid A (AA) and not amyloid L (AL) proteins. The serum EC-SOD content of the patient was 10 fold higher than those seen often in other FAP patients and in healthy controls. Genetic analysis demonstrated the single amino acid substitutions in Val30Met TTR and Arg213G1y EC-SOD. Since these data suggest the dissociation of EC-SOD from the vascular wall, massive amyloid deposition in the present case may be related to increased oxidative stress in loco.

Mutations of the SOD1 gene, which encodes the enzyme copper/zinc superoxide dismutase, are associated with familial amyotrophic lateral sclerosis (ALS). SOD1 consists of five exons, and over 50 different mutations have been described involving exons 1,2,4 and 5. The absence of mutations in exon 3 has been attributed to a critical function of this exon, its integrity being necessary for the toxic effect of mutant SOD1, and it has been suggested that such mutations may be lethal rather than leading to adult onset disease. We identified the heterozygote mutation Gly72Ser (exon 3) in a family with two individuals affected by ALS. SOD enzyme activity was reduced by 45% when measured in erythrocytes indicating reduced enzyme activity, or reduced stability of the mutant protein. These findings indicate that exon 3 is not a privileged region from mutation; that all five exons should be investigated when seeking SOD1 mutations in human disease; and may help in a better understanding of the pathogenicity of these mutations in ALS.

The importance of early microcirculatory changes in the rat colon after exposure to acetic acid was investigated. Administration of 4% acetic acid for 15 sec into an exteriorized colonic segment induced a marked, transient (starting 2 min after the challenge with acetic acid and persisting for 15 min) decrease in the colonic blood flow as estimated by a laser–Doppler flowmeter. Four days after acetic acid administration, a uniform colitis had developed in the exteriorized colonic segment with a total morphological score (TMS) of 15.1 ± 0.8, myeloperoxidase activity (MPO) increased more than threefold, and plasma exudation into the colonic lumen increased sevenfold. Administration of hydrochloric acid (HCl) with the same pH as the acetic acid or sodium acetate (pH 7.0) did not affect colonic blood flow or produce colitis. Mechanical colonic ischemia, induced by a controlled increase in the intraluminal pressure, resulted in several pathological features of colitis with a TMS of 7.3 ± 0.2, combined with a significant increase in MPO activity. The TMS and MPO were further increased when mechanical colonic ischemia was combined with HCl or sodium acetate. Pretreatment with SOD and catalase 5 or 15 min before acetic acid administration did not affect the transient ischemia immediately following acetic acid administration. However, it partially prevented the development of colitis. It is concluded that immediate transient ischemia accompanied by the generation of oxygen free radicals might be of importance in the pathogenesis of acetic acid-induced colitis in the rat.

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

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

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

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.

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

The secretory 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 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.

BACKGROUND: We have developed a formulation of polycationic liposomes that has been shown to be non-toxic, highly stable in the bloodsteam, and very effective in liver gene transfer in mice (Gene Therapy 2003; 10:180–187). Extracellular superoxide dismutase (EC-SOD) inhibits the generation of superoxide anions in the interstitial space of tissues, and may play a role in minimizing oxidative stress in liver injury. The aim of the present study is to investigate whether our polycationic liposome-mediated human EC-SOD gene delivery will protect against lipopolysaccharide (LPS)-induced liver toxicity in D-galactosamine (GalN)-sensitized mice. METHODS: Polycationic liposomes were generated from polycationic lipid (PCL) and cholesterol (Chol). Lipoplexes were formed by complexing liposomes with control plasmid (pEGFP-C1) or EC-SOD plasmid (pEGFP-C1-ECSOD) before use. Mice were injected with thyroid hormone (T3, 4 mg/kg, s.c.) two days before lipoplexes were injected via the portal vein. One day following lipoplex injection, mice were treated with GalN (500 mg/kg, i.p.) plus LPS (25 mg/kg, i.p.). Serum alanine aminotransferase (ALT), SOD activity, liver histology, glutathione (GSH) content and lipid peroxidation were evaluated one day after GalN/LPS exposure. Human EC-SOD gene expression in mouse liver tissue was determined by quantitative RT-PCR two days after lipoplex injection. RESULTS: somes and the control lipoplexes were stable in their size for three months at 4°C. Injection of T3, PCL-Chol liposomes or control lipoplexes did not cause a significant change in serum ALT levels. Real time quantitative RT-PCR analysis showed that human EC-SOD mRNA levels in mouse liver tissue in EC-SOD lipoplex-injected group were 55-fold higher than saline, liposome or control lipoplex controls when mouse -actin was employed as a house-keeping control gene. Serum ALT levels in animals receiving portal vein injections of EC-SOD lipoplexes plus GalN/LPS exposure were much lower than in those receiving normal saline, liposomes alone, or control lipoplexes plus GalN/LPS exposure (62583 vs. 150865, 153194, 1880520 units/ml, p

A simple, inexpensive and specific method for the determination of hemoglobin in tissue homogenates is described. It is based on the specific binding of hemoglobin by haptoglobin and the stabilizing effect of this binding on the peroxidase activity of hemoglobin. The haptoglobin is added as serum and no purification is needed. The method gives reliable estimates of hemoglobin contents above 10 mg/l.

Intravenous heparin has previously been shown to release the high-heparin-affinity fraction C of extracellular-superoxide dismutase (EC-SOD, EC 1.15.1.1) to plasma in man and other mammals. This paper reports on further studies of the phenomenon in the pig. A dose-response curve of the effect of heparin revealed that 1000 IU/kg body weight is needed for maximal release of EC-SOD C. This dose is an order of magnitude larger than that needed for the maximal release to plasma of factors such as lipoprotein lipase, hepatic lipase, and diamine oxidase, which are distributed between plasma and endothelium similarly to EC-SOD C. Thus EC-SOD C appears to have an unusually high affinity for endothelial cell-surface sulfated glycosaminoglycans relative to the affinity for heparin. There was no significant difference in releasing potency between unfractionated heparin and heparin subfractions with high or low affinity for antithrombin III. The heparin structure conferring high-affinity binding to antithrombin III is thus not specifically involved in binding to EC-SOD C. The non-biosynthetic compound dextran sulfate 5000 was an order of magnitude more efficient than heparin. Protamine displayed dual effects. Given alone in high dose it released EC-SOD to plasma, probably due to binding to endothelial cell-surface sulfated glycosaminoglycans displacing fraction C of the enzyme. Whe given after heparin, in a dose just below that expected to neutralize the heparin, protamine reversed the heparin-induced EC-SOD release.

Serum from healthy volunteers contained very little CuZn Superoxide dismutase. Larger amounts were found in serum from patients with impaired renal function, and there was a good correlation between serum creatinine and serum CuZn superoxide dismutase content. Almost all superoxide dismutase activity of human serum was cyanide sensitive. and was found to be given by a factor(s) with a molecular mass of approximately 130 000. The factor was found in all human extracellular fluids investigated: plasma, serum, lymph, ascites and cerebrospinal fluid. The factor was not recognized by radioimmunoassay for human CuZn superoxide dismutase, and it was not inhibited by rabbit antibodies against human CuZn superoxide dismutase. A similar high-molecular mass factor was found in plasma from all mammals investigated: horse, cow, pig, dog, cat, rabbit, rat and mouse. The activities differed much among species and were mostly higher than those found in human serum/plasma.

Alloxan diabetes may be mediated by an iron-catalyzed formation of hydroxyl radicals. The iron chelators desferrioxamine and diethylenetriaminepentacetic acid (DETAPAC) which inhibit hydroxyl radical formation in vitro were tested against alloxan diabetes in mice. DETAPAC inhibited while desferrioxamine stimulated the hyperglycemic response to alloxan. The diverging treatment results are discussed.

In various types of autoimmune disease, an increased frequency of spontaneous chromosome breaks has been reported. Plasma from such patients induces chromosome breaks in normal cells. Exposure of plasma to superoxide radicals increases the breakage activity, and addition of superoxide dismutase protects against it. The New Zealand black mouse is an animal model of autoimmune disease which displays the breakage phenomenon. To test for the possibility that the breakage is related to deficient protection against superoxide radicals, the activities of superoxide dismutase isoenzymes were determined in tissues and blood from New Zealand black mice and compared with the activities of normal BALB/c mice. No differences between the strains were revealed in tissue EC-superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase activity. The erythrocyte superoxide dismutase activities were also equal. The plasma EC-superoxide dismutase activity was 35% lower in the New Zealand black mice than in the BALB/c mice. Between euthymic BALB/c mice and nude mice, previously reported to be deficient in tissue superoxide dismutase activity, no difference could be demonstrated.

The effect of genetically determined glutathione deficiency on the fibroblast content of CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase was investigated. No significant differences between glutathione-deficient and -proficient human fibroblasts were revealed. There was a large variation in the content of the investigated enzymes in fibroblasts grown and analysed on different occasions. Whereas the contents of CuZn superoxide dismutase, catalase and glutathione peroxidase did not deviate much from what has been found in other human cell-lines and tissues, the fibroblasts were found to contain exceptional amounts of Mn superoxide dismutase.

We investigated if superoxide dismutase and catalase could reduce myocardial infarct size in an open chest occlusion-reperfusion model. Thirty pigs were used for the experiment. The left anterior descending artery was ligated for 60 min followed by a 5 h reperfusion period. After randomisation and blinding the two enzymes or placebo were injected into the left atrium as a bolus immediately before and at the end of the occlusion and as a continuous infusion over the first hour of the reperfusion period. The total dose for each enzyme was 8 mg/kg bw. Tetrazolium staining was used to determine infarct size. The study code was not broken until all calculations and exclusions had been made. Nine animals died from intractable ventricular fibrillation, most commonly during the occlusion. Another three were excluded for technical reasons. We found that superoxide dismutase and catalase reduced infarct size in relation to myocardium at risk from a mean of 89% to 63% (P less than 0.01). Initial plasma half life for the two enzymes after the bolus infusions were calculated to be 30 min.

The high heparin affinity subtype C of the secretory enzyme extracellular superoxide dismutase (EC-SOD) exists in the body mainly complexed with extracellular sulfated glycosaminoglycans (SGAGs). Addition of sulfated polysaccharides to EC-SOD C resulted in a prompt partial inhibition of the enzymic activity, in most cases amounting to 10-17%, but with the large dextran sulfate 500,000 amounting to 35%. Complex formation between heparin and EC-SOD C could also be observed as increases in apparent molecular weight of the enzyme. The findings suggest that the binding sites for SGAGs on EC-SOD C are localized far from the active site and that EC-SOD in vivo associated with SGAGs should retain the major part of its enzymic activity. Studies with amino acid-specific reagents suggested that both lysine and arginine residues are involved in the binding of SGAGs. In particular, modification of only a few lysine residues/subunit resulted in loss of high SGAG affinity, whereas arginine modification resulted in loss of not only SGAG affinity but also enzymic activity. We propose that this is due to modification of Arg-186, which is homologous to the highly conserved arginine in the entrance to the active site of the copperzinc-SODs.

1. 1. The catalytic properties of two horseradish peroxidases, isoenzymes A2 and C2, with p I 3.9 and 8.8, have been compared. 2. 2. The rate of formation of Compound I has been determined for the hydrogen, methyl, ethyl, n -propyl, and hydroxymethyl hydroperoxides and for p -nitroperoxybenzoic acid. The elongation of the alkyl group hampered the reaction with the acidic peroxidase and stimulated the reaction with the basic peroxidase. The acidic peroxidase generally reacted more slowly. Its reaction with H 2 O 2 showed a higher energy of activation than that of the slightly basic peroxidase. 3. 3. The rate of reaction with hydrogen peroxide was slightly but definitely influenced by pH for both peroxidases. 4. 4. The rates of reaction of twelve hydrogen donors (phenols, aromatic amines, acidic substances) with the secondary peroxidase-peroxide complex of the two peroxidases could be arranged in three groups according to the chemical nature of the donors. The acidic peroxidase was consistently more active at pH 4.5 than at pH 7.0 whereas the slightly basic peroxidase showed the reverse behavior in the presence of phenols. 5. 5. It is concluded that the two peroxidases are kinetically different with distinct substrate profiles and that they may fulfill different physiological functions.

The neuronal ceroid-lipofuscinoses are characterized by a widespread deposit in the body of pigments believed to be end products of lipid peroxidation damaged organelles. CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase, enzymes which conceivably might protect against lipid peroxidation, were investigated in blood cells from patients afflicted with infantile, late infantile and juvenile neuronal ceroid-lipofuscinosis. In some cases the enzymic activities were slightly lower than the activities of the controls, but no deficiencies which might be of etiological importance were revealed.

CuZn superoxide dismutase and Mn superoxide dismutase were analysed in hypothalamus, nucleus caudatus, hippocampus and cortex gyrus cinguli from 14 patients with dementia of Alzheimer type and from 16 controls. Ample amounts of both enzymes were demonstrated in the brains and there was little difference between the various parts of the brain. There were only small differences in the enzymic activities between the two groups indicating that deficiencies of these enzymes are not a very likely cause of brain degeneration in dementias of Alzheimer type.

Ceruloplasmin and extracellular-superoxide dismutase are similar in physical properties. Both are found in extracellular fluids and both are scavengers of the superoxide radical. The relationship between the two proteins was further explored in the present investigation. Ceruloplasmin preparations were found to be commonly contaminated with extracellular-superoxide dismutase. In one preparation, 80% of the superoxide dismutase activity was due to extracellular-superoxide dismutase. Ceruloplasmin, freed from contaminating superoxide dismutase, was found to catalytically dismute the superoxide anion radical with a rate constant of about 1.0 × 10 4 M − s −1 per copper atom. Under physiological conditions with a low rate of superoxide production, ceruloplasmin preferentially reacts stoichiometrically with the superoxide radical with a rate constant of about 2 × 10 5 M −1 s −1 per copper atom. Under such conditions, the reaction does not result in hydrogen peroxide formation. From the kinetic data obtained it was calculated that in normal human plasma, extracellular-superoxide dismutase will scavenge about twice as much superoxide as ceruloplasmin. Using immobilized antibodies toward extracellular superoxide dismutase and ceruloplasmin, no antigenic cross-reactivity between the two proteins could be detected.

Hydrogen peroxide efficiently protects horseradish peroxidase against inactivation by hydroxymethylhydroperoxide, whereas the hydrogen donor substrate guaiacol has little protective effect. These results, and direct studies of the effects of hydroxymethylhydroperoxide on peroxidase compound II, indicate that compound II and possibly also compound I are quite resistant to hydroxymethylhydroperoxide. The inactivation of peroxidase caused by hydroxymethylhydroperoxide seems to be due to a reaction of the peroxide with free Fe 3+ peroxidase. Albumin, which protects thiol enzymes against hydroxymethylhydroperoxide, does not protect peroxidase. Neither does 2-mercaptoethanol, which also protects thiol enzymes, appear to hamper the inactivation reaction. This seems to indicate that the inactivation is not caused by a free radical released from hydroxymethylhydroperoxide. Hydroxymethylhydroperoxide inactivates peroxidase by attacking the hematin group, the ring of which is opened by way of at least three intermediates. The most stable intermediate is green (“compound IV”). H 2 C 2 attacks the methemoglobin-peroxide compound faster than hydroxymethylhydroperoxide does. These reactions are much slower than the attack of hydroxymethylhydroperoxide on peroxidase. It is suggested that the inactivation of peroxidase takes place as a side reaction in the process of forming compound I from hydroxymethylhydroperoxide and peroxidase.

1. 1.|Many reports attribute biochemical actions to bis(hydroxymethyl)peroxide. Mixtures of hydrogen peroxide and formaldehyde are known to exert a greater effect on biochemical systems than either substance alone. In both cases the effects are probably caused by hydroxymethylhydroperoxide, formed from bis(hydroxymethyl)-peroxide by hydrolysis or from hydrogen peroxide and formaldehyde by a condensation. 2. 2.|Hydroxymethylhydroperoxide is a fairly potent inhibitor of fumarate hydratase ( l -malate hydro-lyase, EC 4.2.1.2), lactate dehydrogenase ( l -lactate:NAD+ oxidoreductase, EC 1.1.1.27), and aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), whereas glucose oxidase (β- d -glucose:O2 oxidoreductase, EC 1.1.3.4) and especially acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) are less affected. 3. 3.|2-Mercaptoethanol and albumin protect against the inhibitory action of hydroxymethylhydroperoxide without reducing the amount of peroxide. 4. 4.|Fumarate hydratase and lactate dehydrogenase, exposed to hydroxymethyl-hydroperoxide, are not reactivated by 2-mercaptoethanol, whereas aspartate amino-transferase slowly partially regains its activity.