Mn and Cu/Zn SOD expression in cells from LPS-sensitive and LPS-resistant mice.

We examined the effect of lipopolysaccharide (LPS) treatment on the expression of manganese and copper/zinc superoxide dismutase (MnSOD and Cu/ZnSOD) mRNA and protein in resident peritoneal macrophages and lung endothelial cells derived from LPS-sensitive (LPS-s) and LPS-resistant (LPS-r) mice. Macrophages from both LPS-s and LPS-r mice treated with LPS for 24 h produced increased levels of MnSOD mRNA and protein. In contrast, levels of lung endothelial cell MnSOD mRNA and protein from LPS-s mice were increased by LPS treatment, while no increases in these parameters were observed in endothelial cells from LPS-r mice. Tumor necrosis factor-alpha (TNF alpha) treatment, however, did increase levels of MnSOD mRNA in both LPS-s and LPS-r endothelial cells to an equal extent. Both macrophage and endothelial cell Cu/ZnSOD mRNA and protein levels were not significantly affected by LPS treatment. These results demonstrate that the mutation that affects susceptibility to LPS in LPS-r mice exerts a differential influence on MnSOD inducibility in a cell specific manner.     

Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases.

Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.     

Water restriction increases renal inner medullary manganese superoxide dismutase (MnSOD)

Oxidative stress damages cells. NaCl and urea are high in renal medullary interstitial fluid, which is necessary to concentrate urine, but which causes oxidative stress by elevating reactive oxygen species (ROS). Here, we measured the antioxidant enzyme superoxide dismutases (SODs, MnSOD, and Cu/ZnSOD) and catalase in mouse kidney that might mitigate the oxidative stress. MnSOD protein increases progressively from the cortex to the inner medulla, following the gradient of increasing NaCl and urea. MnSOD activity increases proportionately, but MnSOD mRNA does not. Water restriction, which elevates renal medullary NaCl and urea, increases MnSOD protein, accompanied by a proportionate increase in MnSOD enzymatic activity in the inner medulla, but not in the cortex or the outer medulla. In contrast, Cu/ZnSOD and TNF-α (an important regulator of MnSOD) do not vary between the regions of the kidney, and expression of catalase protein actually decreases from the cortex to the inner medulla. Water restriction increases activity of mitochondrial enzymes that catalyze production of ROS in the inner medulla, but reduces NADPH oxidase activity there. We also examined the effect of high NaCl and urea on MnSOD in Madin-Darby canine kidney (MDCK) cells. High NaCl and high urea both increase MnSOD in MDCK cells. This increase in MnSOD protein apparently depends on the elevation of ROS since it is eliminated by the antioxidant N-acetylcysteine, and it occurs without raising osmolality when ROS are elevated by antimycin A or xanthine oxidase plus xanthine. We conclude that ROS, induced by high NaCl and urea, increase MnSOD activity in the renal inner medulla, which moderates oxidative stress.     

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H₂O₂ outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H₂O₂ further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H₂O₂ to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.     

Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts.

A chimeric gene consisting of the coding sequence for chloroplastic Fe superoxide dismutase (FeSOD) from Arabidopsis thaliana, coupled to the chloroplast targeting sequence from the pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, was expressed in Nicotiana tabacum cv Petit Havana SR1. Expression of the transgenic FeSOD protected both the plasmalemma and photosystem II against superoxide generated during illumination of leaf discs impregnated with methyl viologen. By contrast, overproduction of a mitochondrial MnSOD from Nicotiana plumbaginifolia in the chloroplasts of cv SR1 protected only the plasmalemma, but not photosystem II, against methyl viologen (L. Slooten, K. Capiau, W. Van Camp, M. Van Montagu, C. Sybesma, D. Inzé [1995] Plant Physiol 107: 737-750). The difference in effectiveness correlates with different membrane affinities of the transgenic FeSOD and MnSOD. Overproduction of FeSOD does not confer tolerance to H2O2, singlet oxygen, chilling-induced photoinhibition in leaf disc assays, or to salt stress at the whole plant level. In nontransgenic plants, salt stress led to a 2- to 3-fold increase in activity, on a protein basis, of FeSOD, cytosolic and chloroplastic Cu/ZnSOD, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In FeSOD-overproducing plants under salt stress, the induction of cytosolic and chloroplastic Cu/ZnSOD was suppressed, whereas induction of a water-soluble chloroplastic ascorbate peroxidase isozyme was promoted.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Protein turnover in the extreme thermophile Thermus aquaticus.

Protein turnover in the extreme bacterial thermophile Thermus aquaticus was examined in exponential cultures at 75 degrees C. The relative amount of [3H]leucine incorporated into trichloroacetic acid-insoluble material was stable in pulse-chase experiments assayed over 2.5 h. The trichloroacetic acid-insoluble radioactive leucine was stable upon the addition of chloramphenicol, which blocks protein synthesis in T. aquaticus. The specific activity of a phosphate-repressible alkaline phosphatase, investigated in the presence of chloramphenicol, did not decrease. The addition of excess orthophosphate to cultures derepressed for the alkaline phosphatase did not show a marked effect on the specific activity over a 2-h period. On the basis of these four experiments, it does not appear that a high protein turnover rate is essential for the thermophily of T. aquaticus at 75 degrees C.     

Improved procedure for detection of superoxide dismutase isoforms in potato,Solanum tuberosum L.

Superoxide dismutase (SOD) in-gel activity assay with selective inhibitors (KCN and H₂O₂) is one of the most commonly used methods for identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and evaluation of oxidative stress response in plants. However, there are potential pitfalls that surround this assay, such as problem to detect isoforms with low activity, comigration of SOD isoforms or application of inappropriate inhibitor concentration. We propose an improved method based on the combination of in-gel analysis of SOD activity and native-PAGE immunoblotting for identification of isoforms and determination of SOD isoenzyme activity pattern in potato. Depending on cultivar and growing conditions, one MnSOD, 3 FeSOD and 5–6 Cu/ZnSOD isoforms were identified in potato leaves. The most important qualitative difference between ex vitro- and in vitro-grown plants was the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets grown in vitro. Compared with results of in-gel activity assay with selective inhibitors, new method allowed accurate identification of comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of ambiguous identities. Potato SODs were also characterized by SDS-PAGE immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides (17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were detected in leaves of four examined cultivars. The difference in the number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE and SDS-PAGE immunoblots suggests that SOD proteins may have undergone post-translational modifications affecting protein mobility or existence of isoforms that differ from each other in total protein charge, but not in molecular weight.     

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.     

Superoxide Dismutase Activity During Dimethylhydrazine Colon Carcinogenesis and the Effects of Cholic Acid and Indole

Copper, zinc superoxide dismutase (Cu, ZnSOD) and manganese superoxide dismutase (MnSOD) activities were measured in mouse large intestinal mucosa during dimethylhydrazine (DMH) carcinogenesis. Mice were divided into five groups. Group A was subcutaneously injected with DMH (20mg/kg) weekly and fed with a diet containing 0.2% cholic acid (C) and 0.8% indole (I). Group B was injected with DMH and given indole feeding. Group C was treated with DMH injection and cholic acid feeding. Group D was given DMH injection alone. Group E was an age-matched control group given 0.9% NaCl injection. The experiment last 21 weeks. The Cu, ZnSOD activity of intestinal mucosa in group A animals began to increase significantly at the 7th week of the experiment. In groups B, C and D, however, this enzyme was not elevated statistically until the 16th week, and then each of these groups kept an increased Cu, ZnSOD level the rest of the experimental period. MnSOD activity was elevated statistically in group C animals at the 7th week. The enzyme activity in group A and D animals increased at the 9th week, but the enzyme activity did not increase statistically until the 11th week in group B. After the 16th week of the experiment the increased activity of MnSOD in all experimental groups returned to the level of the control group. Large intestinal cancer tissues had increased Cu, ZnSOD activity and decreased MnSOD activity.     

Superoxide Dismutase Activity During Dimethylhydrazine Colon Carcinogenesis and the Effects of Cholic Acid and Indole

Copper, zinc superoxide dismutase (Cu, ZnSOD) and manganese superoxide dismutase (MnSOD) activities were measured in mouse large intestinal mucosa during dimethylhydrazine (DMH) carcinogenesis. Mice were divided into five groups. Group A was subcutaneously injected with DMH (20mg/kg) weekly and fed with a diet containing 0.2% cholic acid (C) and 0.8% indole (I). Group B was injected with DMH and given indole feeding. Group C was treated with DMH injection and cholic acid feeding. Group D was given DMH injection alone. Group E was an age-matched control group given 0.9% NaCl injection. The experiment last 21 weeks. The Cu, ZnSOD activity of intestinal mucosa in group A animals began to increase significantly at the 7th week of the experiment. In groups B, C and D, however, this enzyme was not elevated statistically until the 16th week, and then each of these groups kept an increased Cu, ZnSOD level the rest of the experimental period. MnSOD activity was elevated statistically in group C animals at the 7th week. The enzyme activity in group A and D animals increased at the 9th week, but the enzyme activity did not increase statistically until the 11th week in group B. After the 16th week of the experiment the increased activity of MnSOD in all experimental groups returned to the level of the control group. Large intestinal cancer tissues had increased Cu, ZnSOD activity and decreased MnSOD activity.     

Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities ofsuperoxide dismutase (SOD) was studied in hydroponically-grownseedlings of transgenic tobacco (Nicotiana tabacum L.) engineeredto overexpress FeSOD in chloroplasts or MnSOD in chloroplastsor mitochondria. In comparison to the non-transgenic parentalline, the activity of MnSOD in the lines overproducing MnSODwas 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducinglines was 3.2-fold greater, regardless of the Mn treatment (deficientor sufficient). The MnSOD activities decreased due to Mn deficiency,while activities of FeSOD and Cu/ZnSOD remained unaffected 25d after transplanting (DAT). With an increased duration of theMn deficiency stress (45 DAT), FeSOD activity decreased, andthat of MnSOD continued to decrease, while Cu/ZnSOD activitysimultaneously increased. Under Mn sufficiency, non-transgenicparental plants had greater shoot biomass than the transgenics;however, when subjected to Mn deficiency stress, non-transgenicparents suffered a proportionally greater growth reduction thantransgenic lines. Thus, overproduction of MnSOD in chloroplastsmay provide protection from oxidative stress caused by Mn deficiency.Copyright 1999 Annals of Botany Company Manganese deficiency, Nicotiana tabacum, superoxide dismutase (SOD), transgenic tobacco.     

A manganese superoxide dismutase (MnSOD) from Ruditapes philippinarum: comparative structural- and expressional-analysis with copper/zinc superoxide dismutase (Cu/ZnSOD) and biochemical analysis of its antioxidant activities

Superoxide dismutases (SODs), antioxidant metalloenzymes, represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ROS), in particular O(2)(?-). Two distinct members of SOD family were identified from Manila clam Ruditapes philippinarum (abbreviated as RpMnSOD and RpCu/ZnSOD). The structural analysis revealed all common characteristics of SOD family in both RpSODs from primary to tertiary levels, including three MnSOD signatures and two Cu/ZnSOD signatures as well as invariant Mn(2+)- and Cu/Zn(2+)-binding sites in RpMnSOD and RpCu/ZnSOD, respectively. Putative RpMnSOD and RpCu/ZnSOD proteins were predicted to be localized in mitochondrial matrix and cytosol, respectively. They shared 65.2% and 63.9% of identity with human MnSOD and Cu/ZnSOD, respectively. Phylogentic evidences indicated the emergence of RpSODs within molluscan monophyletic clade. The analogous spatial expression profiles of RpSODs demonstrated their higher mRNA levels in hemocytes and gills. The experimental challenges with poly I:C, lipopolysaccharide and Vibrio tapetis illustrated the time-dependent dynamic expression of RpSODs in hemocytes and gills. The recombinant RpMnSOD was expressed in a prokaryotic system and its antioxidant property was studied. The rRpMnSOD exhibited its optimum activity at 20?°C, under alkaline condition (pH 9) with a specific activity of 3299?U?mg(-1). These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam.