manganese-containing superoxide dismutase

NF-␬B and AP-1 are two transcriptional factors known to respond directly to oxidative stress. Oxidative stress has been linked to pathological cell death from many insults, such as ischemia-reperfusion, trauma, and hyperthermia, concomitant with induction of NF-␬B and AP-1 activation in these condi- tions (3). Therefore, oxidative stress may also participate in the adenovirus-induced stress response. To study this possibility, we manipulated the in vivo levels of manganese-containing superoxide dismutase (MnSOD) and modified the activation of AP-1 and NF-␬B. MnSOD, which is found in mitochondria, is one of the primary antioxidant enzymes that catalyze the dis- mutation of superoxide to hydrogen peroxide. Hydrogen per- oxide is further reduced to water by catalase or one of the peroxidases. Thus, overexpression of MnSOD creates a mech- anism for reactive oxygen species (ROS) removal and, in turn, modulates intracellular redox status.

Abstract: We investigated the integrated response of antioxidant defense enzymes (total superoxide dismutase (TotSOD), manganese-containing superoxide dismutase (MnSOD), copper-zinc-containing superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and phase II biotransformation enzyme, glutathione- S-transferase (GST)) in the liver and white muscle of females of European hake (Merluccius merluccius L.) from the Adriatic Sea (Montenegro) in winter and spring. The activity of GSH-Px in the liver was significantly increased, while GST activity was decreased in spring compared to the winter. In white muscle, the activities of TotSOD and CuZnSOD were increased, while the activities of MnSOD, CAT, GSH-Px, GR and GST were decreased in spring when compared to the matching values in winter. The activities of TotSOD and CuZnSOD in winter were markedly lower in the muscle than in the liver, while the activity of MnSOD in the muscle was higher when compared to the liver. Principal component analysis (PCA) revealed clear separation of the investigated antioxidant biomarkers between tissues and seasons, while the integrated biomarker response (IBR) showed that the most intensive antioxidant biomarker response was in the liver in spring. Star plots of IBR showed a dominant contribution of glutathione-dependent biomarkers (GSH-Px, GR and GST) and CAT in both tissues and seasons with respect to SOD isoenzymes. All enzyme activities (except MnSOD) were greater in the liver in comparison to the white muscle. Our results show that the liver possesses a greater capacity to establish and maintain homeostasis under changing environmental conditions in winter and spring. At the same time, seasonal effects are more pronounced in muscle tissue. Key words: antioxidant enzymes; marine fish; oxidative stress; seasonal; tissues

SVP level. The active site surface is mainly positive in character, which is due to the positive formal (+ +1) charge of the metal cofactor. The surplus charge is not limited to the metal ion as it smears into the ligands coordinated to the metal. Figure 3 shows the results of these calculations for the cambialistic mutant MnSOD-3[Q142H]. We have observed and analysed similar electrostatics for the wild-type and iron-containing MnSOD-3 and for the manganese- and iron-substituted mutant. We have looked at the overall electrostatic potentials generated at the protein surface as well as within the active site (Figure 3 and Supporting Information) and have found little difference between the proteins.

7RSUHSDUH51$SUREHVIRUin situ K\EULGL]DWLRQDS*(07HDV\SODVPLG (Promega, Madison, WI, USA) containing the mouse MnSOD cDNA clones (accession number: NM_013671, fragment; 287 bp) was linearized with SpeI or NcoI restriction enzymes. Digoxigenin- (DIG; Roche, Penzberg, Germany) labeled sense or antisense riboprobes for MnSOD were generated via in vitro WUDQVFULSWLRQLQWKHSUHVHQFHRI7RU6S51$SRO\PHUDVH7DNDUD6KLJD Japan) at 37°C for 60 min. For whole-mount in situ hybridization of embryos, ('VPRXVHHPEU\RVZHUHÀ[HGLQSDUDIRUPDOGHK\GHLQSKRVSKDWH EXIIHUHGVDOLQH3%6RYHUQLJKWDQGGHK\GUDWHGLQPHWKDQRO7KHJHQHUDOin situ hybridization procedure for tissue sections (EDs 13.5-18.5) was carried out as previously described (Baek et al., 2005). Hybridization signals were detected using an alkaline phosphatase-conjugated antibody against DIG (Roche) and 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium solution (Roche) substrate.

For real-time RT-qPCR determinations, fermentation sam- ples were immediately poured on an ice-cooled tube containing RNAlater solution (Ambion Inc. Austin, TX). Total RNA extraction was performed using mirVana miRNA isolation kit (Ambion Inc. Austin, TX). Total RNA concentration was estimated by measuring optical density at 260 nm using NanoDrop 2000/2000c spectrophotom- eter (Thermo Fisher Scientific, Wilmington, DE) and integ- rity was visualized on a 2% agarose gels. To reduce genomic DNA contamination, isolated RNA was treated with turbo DNase kit (Ambion Inc. Austin, TX). cDNA was generated by using 2 μg RNA in a total volume of 20 μl with 250 nM of specific DNA primers (antisense primers in Additional file 1: Table S1) according to the protocol of Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA). Real-time qPCR was performed using an ABI Prism 7900H Sequence Detection System (Applied Biosystems, Foster City, CA) with 40 amplification cycles using SYBR Green PCR Mas- ter Mix as signal reporter. Each reaction composed of 6 ng cDNA, 400 nM sense and antisense primers in a total vol- ume of 20 μl. RT-qPCR was done in a 96-well microtiter PCR plates using the following amplification conditions: 1 - cycle 10 min at 95°C; and 40 two-step cycle at 95°C for 15 - seconds and 60°C for 60 seconds. Each sample was done in triplicate. To assess for reagent and genomic DNA contam- ination, no template and no reverse transcriptase controls were included. Data were analyzed using 2 -ΔΔCT method described by Livak and Schmittgen [35]. The expression of the ssrA gene was used as an endogenous control to normalize the amount of mRNA obtained from a target gene [36]. Expression data obtained for each time-point were normalized to the expression of each gene obtained at time zero of the oxygen switch.

DNA manipulations. Rapid extraction of bacterial genomic DNA was per- formed as described previously (6), and primers d1 (59-CCITAYICITAYGAYG CIYTIGARCC-39) and d2 (59-ARRTARTAIGCRTGYTCCCAIACRTC-39) were used to amplify an internal fragment representing approximately 85% of the sodA genes of the bacterial strains. PCRs were performed with a Gene Amp System 9600 instrument (Perkin-Elmer Cetus, Roissy, France) in a final volume of 50 ml containing 250 ng of DNA as template, 0.25 mM (each) primer, 200 mM (each) deoxynucleoside triphosphate, and 1 U of Taq DNA polymerase in a 13 amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl 2 ).

The second hypothesis (Figure 7B) is that the SpMnSOD enzymes are able to further reduce peroxide to hydroxyl radical, which is the reactive oxidant to attach lignin. It is reported that bovine erythrocyte Cu/Zn superoxide dismutase can generate hydroxyl radical, but that E. coli MnSOD, which in our hands shows very little lignin oxidation activity, does not generate hydroxyl radical. 35 The observation of oxidation products containing additional phenolic hydroxyl groups (e.g. products 3, 6, 7) is consistent with the known ability of hydroxyl radical to carry out phenolic hydroxylation, 36 hence this appears to be a possible mechanism for lignin oxidation via these enzymes. We note that Nature uses hydroxyl radical to attack lignin in a different context: brown rot fungi utilise Fenton chemistry to generate hydroxyl radical to attack lignin. 37,38 There are also literature reports of the production of hydroxyl radical in white rot fungus Phanerochaete chrysosporium, 39-41 though subsequent data implied that this is not a major contributing mechanism in white-rot fungal lignin degradation. 42

Tissue microarray (Shanghai Outdo Biotech Co., Ltd., n = 118) were deparaffinized and dehydrated with graded alcohol. The samples were pretreated with 0.01 M citrate buffer (pH 6.0) for 2 min at 100˚C in an autoclave; then the slides were allowed to cool to room temperature. Endogenous peroxidase activity was quenched by incubation in methanol containing 3% H 2 O 2 for 10 min at room temperature. After several washes in Phosphate- Buffered Saline Tween-20 (PBST) (pH 7.2), the sections were blocked with goat serum for 60 min at room temperature and then incubated with MnSOD antibody (BD Biosciences) overnight at 4°C in a humidified chamber. After a brief rinse in PBST, sections were incubated for 40 min at 37˚C with a biotin-conjugated secondary antibody (mouse) followed by incubation with DAB for 15 s. After rinsing with distilled water, sections were counterstained with hematoxylin. As a negative control, slides were incubated in PBS in place of primary antibody. The IHC intensity of MnSOD was analyzed using ImageJ.

Superoxide dismutase activity has been identified in both human neutrophils and rabbit alveolar macrophages by two distinct assay procedures. The enzyme is insensitive to both cyanide and azide and is present in the cytosol of the cell. The identification of this enzyme in phagocytic cells is compatible with the theory that superoxide anion might be involved in the bactericidal activity of the cell. It is proposed that the enzyme functions to protect the cell against superoxide generated during the phagocytic process.

Background: NK cells are key effector lymphocytes of innate immunity provided with constitutive cytolytic activity, however, their role in human ageing is not entirely understood. The study aimed to analyze the expression of proteins involved in cellular stress response sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in non-stimulated NK cells of the oldest seniors ( n = 25; aged over 85; mean age 88 years) and compare with NK cells of the old ( n = 30; aged under 85; mean age 76 years) and the young ( n = 32; mean age 21 years) to find potential relationships between the level of expression of these proteins in NK cells and longevity. The concentration of carbonyl groups and 8-isoprostanes in NK cell lysates reflecting the level of oxidative stress was also measured.

SOD2 expression was semi-quantitatively evaluated in 61 primary tumors of EAOC. Figure 1a – d show exam- ples of mitochondrial superoxide dismutase (SOD2) ex- pression in endometriosis-associated ovarian cancers on immunohistochemical analysis. In both endometrioid and clear cell carcinomas, SOD2 positivity was seen as strong dot-like structures in the cytoplasm, suggesting mitochondrial expression. SOD2 reactivity of normal ovarian stromal cells was used as an internal control of each histological section. Cases with stronger SOD2 staining of the tumor cells than normal ovarian stromal cells were categorized as high SOD2 cases. Among 61 tumors, 46 (75%) tumors expressed high levels of SOD2.

Amputation as a result of impaired wound healing is a serious complication of diabetes. Inadequate angiogen- esis contributes to poor wound healing in diabetic patients. Endothelial progenitor cells (EPCs) normally aug- ment angiogenesis and wound repair but are functionally impaired in diabetics. Here we report that decreased expression of manganese superoxide dismutase (MnSOD) in EPCs contributes to impaired would healing in a mouse model of type 2 diabetes. A decreased frequency of circulating EPCs was detected in type 2 diabetic (db/db) mice, and when isolated, these cells exhibited decreased expression and activity of MnSOD. Wound healing and angiogenesis were markedly delayed in diabetic mice compared with normal controls. For cell therapy, topical transplantation of EPCs onto excisional wounds in diabetic mice demonstrated that diabetic EPCs were less effective than normal EPCs at accelerating wound closure. Transplantation of diabetic EPCs after MnSOD gene therapy restored their ability to mediate angiogenesis and wound repair. Conversely, siRNA- mediated knockdown of MnSOD in normal EPCs reduced their activity in diabetic wound healing assays. Increasing the number of transplanted diabetic EPCs also improved the rate of wound closure. Our findings demonstrate that cell therapy using diabetic EPCs after ex vivo MnSOD gene transfer accelerates their ability to heal wounds in a mouse model of type 2 diabetes.

dismutase (MnSOD) activity fell approximately 50% despite a threefold increase of MnSOD mRNA concentration; addition of a reducing agent to lung extracts from O2-exposed rats partially restored MnSOD activity. Endotoxin induced tolerance to O2 (a) without elevating Cu,Zn superoxide dismutase activity, (b) with increases of catalase and glutathione

38. Nilakantan V, Halligan NL, Nguyen TK, Hilton G, Khanna AK, Roza AM, Johnson CP, Adams MB, Griffith OW, Pieper GM (2005) Post-translational modification of manganese superoxide dismutase in acutely rejecting cardiac transplants: role of induc- ible nitric oxide synthase. J Heart Lung Transplant 24:1591–1599 39. Govindaraju K, Shan J, Levesque K, Hussain SN, Powell WS, Eidelman DH (2008) Nitration of respiratory epithelial cells by myeloperoxidase depends on extracellular nitrite. Nitric Oxide 18:184–194

Genomic DNA was isolated from the whole blood samples using a QIAamp DNA Blood Mini Kit (Quiagen GmbH, Hilden, Germany). The polymor- phisms were determined using the polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) technique. Polymerase chain reaction was performed in a final volume of 25 μL containing 20 ng of DNA, 12.5 μL KAPA2G Fast HotStart Read- yMix (Kapa Biosystems, Inc, USA) and 20 pmol of each primer. The primer sequences used in this study are summarized in Table 1.

obtain the rest of the antioxidants it needs . These exogenous antioxidants are commonly called dietary antioxidants and those are found in fruits, vegetables, and grains 19 . The in vivo antioxidant assay showed that the extract increased the activity of serum superoxide dismutase (SOD) and catalase and decreased the serum level of TBARS. Catalase is a ubiquitous enzyme that catalyzes the decomposition of hydrogen peroxide, a reactive oxygen species, which is a toxic product of both normal aerobic metabolism and pathogenic ROS production 20 . The SOD catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen, thereby reducing the likelihood of superoxide anion reacting with nitric oxide to form reactive peroxynitrite 21 . The increased serum activities of catalase and SOD as observed in this study suggest that the extract has an in vivo antioxidant activity and is capable of ameliorating the effect of ROS in biologic system 22 .Also, ROS react with all biological substance; however, the most susceptible ones are polyunsaturated fatty acids. Reactions with these cell membrane constituents lead to lipid peroxidation (LPO) 23 . Increased LPO impairs membrane function by decreasing membrane fluidity and changing the activity of membrane- bound enzymes and receptor 24 . Thiobarbituric acid reactive substance (TBARS) levels were measured as a marker of LPO and malondialdehyde (MDA) production. Malondialdehyde is an endogenous genotoxic product of enzymatic and ROS- induced LPO whose adducts are known to exist in DNA isolated from healthy human being 25 . In our study, the level of TBARS in the extract treated groups decreased in a dose dependent manner when compared to control. This decrease in the TBARS levels may indicate increase in the activities of glutathione peroxidase

mitochondrial targeting sequence of the manganese superoxide dismutase enzyme is seen. This polymorphism is present in exon 2 of the gene where normal GCT is mutated to GTT. This results in creation of a restriction site(rs 4880) as well as change of aminoacid from alanine to valine at 16 th position. This enzyme otherwise called as manganese superoxide dismutase is present inside mitochondria. This polymorphism in mitochondrial targeting sequence of the enzyme causes impaired targeting of the SOD 2 enzyme resulting in reduced activity of the enzyme inside mitochondria. A study has shown that individuals with this polymorphism had thickened carotid wall making them prone for cardiac complications. 6 . This polymorphism causes defective targeting of superoxide dismutase to mitochondria where it is required to combat oxidative stress, which can lead to development of oxidized LDL and accelerated foam cell formation in atherosclerosis 7 .

Activation increases the levels of ROS in T cells: ROS may play a role in activated T cell death Activation increases the amount of ROS in T cells (5–7), although it is unclear how these extra ROS are created. T cells lack the conventional NADPH oxidase enzymes used by granulocytes for oxidative bursts. However, other mechanisms for producing ROS have been described and might occur in T cells. One such mechanism could be driven by the increased demands for ATP production imposed on T cells by their con- version from a resting condition to the state of rapid cell division that accompanies activation. This is exem- plified by experiments in which thymocytes are stimu- lated with phorbol myristate acetate and ionomycin, leading to rapid glucose consumption followed by increased oxidative phosphorylation and a subsequent increase in ROS production (8). This production of ROS probably occurs as a consequence of direct inter- action of electrons shed from the respiratory chain with molecular oxygen, resulting in the formation of superoxide (9). Increased demands on mitochondrial electron transport for energy can therefore lead to the increased levels of superoxide within cells. For T cells this is evidenced by increased alkalinization of the cell cytosol (which is indicative of increased respiratory activity) rather than increased acidification (which would be evidence of increased glycolytic activity) (10). Thus, mitochondria can be a major source of ROS within cells, and increased energy demands, such as those seen with rapid T cell proliferation, can increase levels of mitochondrially derived ROS.

Copper chaperone for superoxide dismutase co-aggregates with superoxide dismutase 1 (SOD1) in neuronal Lewy body-like hyaline inclusions: an immu- nohistochemical study [r]

Downloaded from thorax.bmj.com on July 12, 2010 - Published by group.bmj.com iii52 Poster presentations have highlighted the difficulty of predicting mortality in this patient group from[r]