We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA int ) representing approximately 85% of the genes encoding the manganese-dependent superox- ide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA int fragments yields an evolutionary tree having a topology
Ref-1 is a bifunctional protein, serving as a DNA repair enzyme and as a regulator protein for several transcription factors, including AP-1, in a redox-dependent manner. Genetic analysis has identified a cysteine residue at position 65 in the redox regulatory domain of Ref-1 protein that is critical for the redox activation of AP-1 DNA binding (38). This residue is required for the direct interaction between Ref-1 and c-Jun through Cys-272 on c-Jun protein in vitro (36). We observed a decrease in AP-1 DNA binding activity after immunodepletion of Ref-1. Also, this reduction was restored by adding back the immunoprecipitated Ref-1 protein. However, immunopre- cipitated Ref-1 alone did react with the oligonucleotides con- taining the consensus AP-1 binding sequence. These results demonstrate that Ref-1 must be physically present with AP-1 protein in the stimulation of AP-1 activation after adenovirus transfection. In addition, AP-1 activation was also elevated after the nuclear extracts from the control and MnSOD-over- expressing animals had been supplemented with the immuno-
Abstract: We investigated the integrated response of antioxidant defense enzymes (total superoxidedismutase (TotSOD), manganese-containing superoxidedismutase (MnSOD), copper-zinc-containing superoxidedismutase (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
The intensity of oxidative stress, protein expression of antiapoptotic Bcl-2 as well as antioxidant en- zymes manganesesuperoxidedismutase (MnSOD) and glutathione peroxidase (GPx) and their regulator p53 were studied in the mitochondria of rat heart. Sessions of repeated hypoxia/reoxygenation ((h/r), 5 cycles of 10 min hypoxia (5.5% O 2 in N 2 ) alternated with 10 min normoxia, daily) were performed in our study. It was shown that short-term sessions of H/R (during 1-3 days) caused a significant increase in the oxida - tive stress markers (rOS formation and lipid peroxidation), mitochondrial p53 translocation, a decrease in MnSOD protein expression/activity and Bcl-2 protein content, but up-regulated GPx. We have demonstrated that prolonged H/R (7-14 days) induced myocardial tolerance to fluctuation in oxygen levels that was associa- ted with the reduction in mitochondrial p53 protein content, elevation of mitochondrial Bcl-2 protein level, and increase in antioxidant capacity. A close correlation between the mitochondrial p53 accumulation and rOS formation as well as the activity and protein content of MnSOD and GPx allowed us to assume that p53 took an active part in the regulation of prooxidant/antioxidant balance in mitochondria of rat heart during repeated h/r.
SOD, or SOD1) is located in the cytosol and nucleus and to a lesser extent in the mitochondria, and is expressed in almost all cell types (17). Cu,Zn-SOD is reported to dismutate superoxide anions derived from eNOS during NO synthesis (18) and thereby prolongs the half-life of NO (19, 20). Manganese SOD (MnSOD, or SOD2) is located in the mitochondrial matrix and is the primary SOD isoform that dismutates superox- ide anions generated by the respiratory chain (21). Extracellular SOD (ecSOD, or SOD3) is secreted extra- cellularly and is bound to sulfated polysaccharides such as heparan sulfate on cell surfaces (21). Cu,Zn- SOD is a major SOD in blood vessels, accounting for approximately 50–80% of total SOD activity (22, 23). Since we have found that endothelial production of H 2 O 2 /EDHF is reduced in eNOS-knockout (eNOS –/– )
A protocol of 75 lengthening contractions (LCP) administered to skeletal muscles of mice causes an initial force deficit owing to the mechanical disruption of sarcomeres and a reduction in calcium release from the sarcoplasmic reticulum. During the 3 days following the LCP, a ‘sealing off process’ and inflammatory response occurs. The reactive oxygen species (ROS) released by invading inflammatory cells produce a secondary force deficit that is more severe than the initial deficit. The timing of the infiltration of inflammatory cells and increase in force deficit relative to the sealing off process is not well documented. We tested the null hypothesis that following a lifetime of overexpression of the genes for the intracellular antioxidants manganesesuperoxidedismutase, copper zinc superoxidedismutase or catalase in transgenic mice, the force deficits 3 days following the administration of a 75 LCP to in situ extensor digitorum longus muscles are not different from those of wild-type mice. Following the LCP, the force deficits ranged from 39 to 59% for the muscles of transgenic mice that overexpressed the genes for intracellular antioxidants and were not different from the force deficit of 44% observed for muscles of wild-type mice. The results provide evidence that the ROS damage does not occur within the cytosol of the injured fibres. Apparently, the hypercontraction of sarcomeres and accumulation of vesicles seal off and protect the intact portions of damaged fibres, such that the ROS damage and repair occurs in the milieu of the necrotic segments that are continuous with the extracellular matrix.
TNF, increased level of C reactive protein and decreased concentration of anti-inflammatory cytokine IL-10 . This phenomenon was referred to as “inflamm-aging” . The ageing process is also accompanied by chronic oxidative stress that affects all regulatory body systems, including the immune system, and this phenomenon has been called “oxi-inflamm-aging” . Oxidative stress can cause serious cell damage of cells, counteracted by the de- velopment of anti-oxidant protective systems that involve glutathione (GSH), glutathione S-transferase, glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutases (MnSOD, CuZnSOD) . Continuous low- dose oxidative stress during ageing results in adaptive responses based on the activation of NF-κB and subse- quently superoxide dismutases (SODs) with a key role played by the mitochondrial manganesedismutase (SOD2). In this process also heat-shock proteins (HSP70) are in- duced to protect cells from stress-induced molecular damage . HSP70 reveals distinct functions depending on its location. Intracellular HSP70 presents a cytopro- tective, anti-apoptotic and anti-inflammatory function while extracellular HSP70 mediates pro-inflammatory immunological response via Toll-like receptors (TLRs) contributing to a link between innate and adaptive im- mune systems .
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 superoxidedismutase activity, (b) with increases of catalase and glutathione
response the activities of manganese and copper, zinc superoxidedismutase (Mn- and CuZn-SOD, respectively) are decreased, as well. However, molecular mechanisms governing this SODs adaptive response are still unsolved. Besides, knowing that NO reinforces IBAT uncoupling, we wondered whether nitric oxide (NO) is taking part in SODs regulation? Mn- and CuZn-SOD mRNA and protein expression, uncoupling protein 1 (UCP1), nitrotyrosine and nuclear factor-kappa B (NF-jB) immunolabeling, as well as total SOD (tSOD) activity in IBAT of rats subjected to cold (4 ± 1°C) for 1, 3, 7, 12, 21 and 45 days and treated by L -arginine or N x -nitro- L -arginine-methyl ester
dismutase (MnSOD) gene is located at amino ac- id position 16 in the mitochondrial targeting se- quence (MTS) resulting in the replacement of an al- anine with a valine (Ala16Val). This polymorphism has been suggested to alter the conformation of the leader signal and thereby affect the import of Mn- SOD into the mitochondria . Catalase is a homo- tetramer, encoded by a gene consisting of 13 exons separated from each other by 12 introns and located on 11p13 chromosome . Polymorphisms A-21T, C-262T and C-844T, were identified in the 5’-un- translated region of the CAT gene . The CAT A-21T (rs7943316) polymorphism, located inside the promoter region proximal to the start site, is less studied, especially in bronchial asthma . Asth- ma occurrence is dependent on the relationship between antioxidant and inflammatory genes . Higher levels of oxidative stress overwhelm antiox- idant defenses and lead to the induction of many pro-inflammatory factors including tumor necrosis factor α (TNF-α) .
Pseudomonas aeruginosa infection in patients with cystic fibrosis and bronchiectasis is characterised by a profound neutrophilic inflamma- tion, with neutrophil products (for example, elastases) implicated in tis- sue injury. We have shown that pyocyanin, a pseudomonas exotoxin, accelerates neutrophil apoptosis (programmed cell death) potentially promoting bacterial persistence (Usher, et al. J Immunol 2002;168:1861–8). We hypothesised that accelerated neutrophil death may result in further tissue injury if apoptotic cell clearance was impaired. We therefore studied the effects of pyocyanin on the inges- tion of apoptotic neutrophils (APMN) by monocyte-derived macro- phages (MDM). All data are expressed as controls versus 24 hour pyocyanin pretreatment. We have observed a time (100 (0%) v 22.7 (7.1%), p<0.001) and concentration dependent reduction in MDM ingestion of APMN in the presence of pyocyanin. We have shown that the reduced interaction is not due to loss of viability (23.2 (2.5) v 22.7 (1.2) cells/x400 field, p=0.440) or induction of MDM apoptosis by physiological concentrations of pyocyanin (0.6 (0.15%) v 0.93 (0.41%), p=0.108). Similarly we have demonstrated no loss in MDM function as assessed by basal and lipopolysaccharide induced MDM cytokine production. The impairment of phagocytosis has been shown to be specific to APMN as MDM phagocytosis of opsonised latex bead is not impaired (33.3 (4.2%) v 29.7 (2.3%), p=0.312). Also we have determined that the pyocyanin-induced defect is specific to the MDM—APMN killed by constitutive ageing or following exposure to pyocyanin are ingested similarly by healthy MDM. Using flow cytometry we have shown that pyocyanin is capable of inducing high levels of reactive oxygen species (ROS) within the MDM—however it Abstract P106
Background Manganesesuperoxidedismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species, constituting a major cellular defense mechanism against agents that induce oxidative stress. A genetic polymorphism in the mitochondrial targeting sequence of this gene has been associated with increased cancer risk and survival in breast cancer. This base pair transition (-9 T > C) leads to a valine to alanine amino acid change in the mitochondrial targeting sequence. A polymorphism has also been identified in the proximal region of the promoter (-102 C>T) that alters the recognition sequence of the AP-2 transcription factor, leading to a reduction in transcriptional activity. The aim of our study was to investigate possible associations of the -102 C>T polymorphism with overall and relapse-free breast cancer survival in a hospital-based case-only study.
characteristics of a model manganese-requiring NADPH-oxidizing system that employs a defined O2-generator have now been compared to the original neutrophil-granule system. With respect to pH dependence, cyanide sensitivity, and reduced pyridine nucleotide specificity, the properties of the two systems are very similar. Additional information has been obtained concerning cation specificity and the kinetics of the metal-catalyzed NADPH oxidation. From the similarities between the properties of the model and neutrophil particle systems, we postulate that the manganese-dependent NADPH oxidation observed in the presence of neutrophil granules represents in large part of nonenzymatic free radical chain involving the oxidation of NADPH to NADP, with O2- as both the chain initiator and one of the propagating species. In this reaction, the neutrophil particles serve only as a source of O2-. Further, the same changes in kinetics (decrease in apparent Km for NADPH) observed previously when granules from phagocytizing rather than resting cells were employed could be mimicked by varying the rate of O2-generation by the model system. We conclude from […]
iron-substituted MnSOD was due to the manganese present (0.13 g atom per monomer) we would expect an activity of nearly 500. It may be assumed therefore that any residual man- ganese might be randomly distributed throughout the four subunits of each tetramer. It has previously been assumed that the minimal unit to provide enzyme activity is a fully metalated dimer within a tetramer (see Figure 1). This explains the very low, almost undetectable activity of the iron-substituted MnSOD-3, despite some manganese being present. Enzyme ac- tivity of the MnSOD-3[Q142H] mutant was found to be 900 : 30 U mg @1 of protein when isolated from manganese-supple-
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.
79. Chenghong Lan,1,2 Pancy O. S. Tam,1 Sylvia W. Y. Chiang,1 Carmen K. M. Chan,1 Fiona O. J. Luk,1 Gary K. Y. Lee,1 Jasmine W. S. Ngai,1 Jason S. S. Law,1 Dennis S. C. Lam,1,2 Chi-Pui Pang,1,2 and Timothy Y. Y. Lai1Manganese SuperoxideDismutase and Chemokine Genes Polymorphisms in Chinese Patients with Anterior Uveitis. Investigative Ophthalmology & Visual Science, December 2009, Vol. 50, No. 12
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 manganesesuperoxidedismutase (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.
Superoxidedismutase 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.
SOD2 expression was semi-quantitatively evaluated in 61 primary tumors of EAOC. Figure 1a – d show exam- ples of mitochondrial superoxidedismutase (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.