into a radical species which is subsequently coupled to NADH to form an INH-NAD(P) adduct. The INH-NAD adduct is a potent inhibitor of Mtb InhA, an enoyl reductase required for the elongation steps in mycolic acid biosynthesis [7,8]. It has been recently suggested that the superoxide reactivity affects antitubercular activity of INH to some extent, and thereby raises the needs to better understand the interaction between INH and superoxide .
elevated levels of the human copper-zinc SOD (CuZnSOD). Upon exposure to hyperoxia (greater than 99% O2, 630 torr) the transgenic CuZnSOD mice showed increased survival, decreased morphologic evidence of lung damage such as edema and hyaline membrane formation, and reduction in the number of lung neutrophils. During continuous exposure to oxygen, both control and transgenic animals who successfully adapted to hyperoxia showed increased activity of lung antioxidant enzymes such as glutathione peroxidase (GPX), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PD), whereas superoxidedismutase activity remained unchanged. The results show that expression of elevated levels of CuZnSOD decreases pulmonary oxygen toxicity and associated histologic damage and mortality.
degeneration, and lipid peroxidation, ultimately leading to cell death. The key antioxidant enzymes, including superoxidedismutase (SOD), catalase (CAT) and glu- tathione peroxidase (GPx), provide a defense system against oxidative stress by removing the OFR, thus pro- tecting cells from oxidative damage [4,5]. However, the endogenous antioxidant activity is severely damaged after ischemia-reperfusion which makes the myocardium extremely vulnerable to OFR . Moreover, exogenous SOD1 and CAT can not be delivered into living cells because of the poor permeability and selectivity of the cell membrane, which has limited its usage in protecting cells/tissues from oxidative stress damage.
Phylogenetic analysis of cvSOD. Phylogenetic analyses pro- duced a surprise in that the Cu-Zn SODs from the phycodnavi- ruses formed a distinct clade from the Cu-Zn SOD-like proteins encoded by the other large dsDNA viruses (Fig. 7). BioNJ (distance method), maximum likelihood, and maximum parsimony tree con- struction methods were used. BioNJ and maximum likelihood meth- ods produced nearly identical tree topologies. Maximum parsimony produced a polytomy, but the clade groupings bracketed on the right in Fig. 7 were the same. The Cu-Zn SOD homologs from the baculo- viruses, poxviruses, and mimiviruses formed their own clade, whereas the phycodnaviruses, including the chloroviruses,Micromo- nas viruses, and the E. huxleyi virus, were in a separate clade. The group phylogenetically closest to the phycodnaviruses was from fungi. Indeed, when the cvSOD sequence from PBCV-1 (NP_048593.1) was blasted against the nonredundant database that excluded all viruses (http://www.ncbi.nlm.nih.gov/Blast.cgi), the best hits were from fungi. The Cu-Zn SOD sequence from PBCV-1 is 187 amino acids in length, similar to those in some fungi, but other fungi such as Aspergillus and Neurospora lack the 30 ⫹ amino acids at the N terminus. These results indicate a phy- logenetic relationship between fungi and the phycodnaviruses and suggest that an ancient horizontal gene transfer event occurred. Indeed, we have long suspected that PBCV-1 has had, or may still have, a host(s) in nature other than just the green alga C. variabilis. Conclusions. Many, but not all, chloroviruses encode a Cu-Zn SOD; however, viruses that encode the enzyme have an extra ⬃ 35 amino acids located at the N-terminal end of the protein com- FIG 6 Measurement of superoxide levels in PBCV-1- and NY-2A virus-in-
changes in AD occurring during cold-acclimation should be observed as a part of newly established IBAT homeostasis that characterizes intensive oxidative metab- olism, uncoupling, lipolysis and decreased rate of apop- tosis [9–11]. Therefore, shown decrease in manganese and copper, zincsuperoxidedismutase (Mn- and CuZn-SOD, respectively, EC 126.96.36.199) activity in rats kept at cold for 45 days, accompanied by an increased UCP1 expression, was explained as adaptive response of enzymatic activities on decreased superoxide (O 2 - ) production by uncoupling
Increased interest in fullerene C 60 and derivatives in recent years implies an intensification of their environmental spread. Yet, the potential risks for living organisms are largely unknown, including the interaction of C 60 with fungal organisms. This may be especially relevant for mycotoxigenic fungi since C 60 may both scavenge and produce reac- tive oxygen species (ROS), and oxidative stress induces mycotoxin production in fungi. Therefore, this study examined effects of environmentally plausible concentrations of C 60 (0, 10, 50, and 100 ng/mL) on Aspergillus flavus growth and aflatoxin production in culture media. In addition, ROS-dependent oxidative stress biomarkers—thiobarbituric acid reactive substances (TBARS), reduced and oxidised glutathione ratio, superoxidedismutase isoenzymes, cata- lase, glutathione peroxidase, and glutathione reductase were determined in mycelia. Nanoparticles of fullerene C 60 (nC 60 ) did not exhibit strong antifungal activity against A. flavus. At the same time, nC 60 caused an antiaflatoxigenic effect at 10–100 ng/mL, and 50 ng/mL unexpectedly enhanced aflatoxin production. The TBARS content, reduced and oxidised glutathione ratio, and copper, zincsuperoxidedismutase activity suggest that 10 ng/mL nC 60 exerted antioxidative action and reduced aflatoxin B1 production within fungal cells. Detected prooxidative effects of 50 ng/ mL fullerene exceeded cellular defenses and consequently enhanced aflatoxin B1 production. Finally, the results obtained with 100 ng/mL nC 60 point to prooxidative effects, but the absence of increase in aflatoxin output may indi- cate additional, presumably cytotoxic effects of nC 60 . Thus, a range of rather low levels of nC 60 in the environment has a potential to modify aflatoxin production in A. flavus. Due to possible implications, further studies should test these results in environmental conditions.
To identify the site, timing and underlying causes of the events associated with the secondary ROS-induced injury, a 75 LCP was administered to the extensor digitorum longus (EDL) muscles of mice. We tested the null hypothesis that the force deficit at 3 days after the LCP is not affected by a lifetime of overexpression of genes for intracellular antioxidants. The transgenic mice overexpressed a single gene or a combination of genes for manganese superoxidedismutase (MnSOD), copperzincsuperoxidedismutase (CuZnSOD) or catalase (Raineri et al. 2001; Chen et al. 2003, 2004). Superoxidedismutase catalyses the dismutation of superoxide anion to hydrogen peroxide (Chance et al. 1979). Copperzincsuperoxidedismutase is present in the cytosol and the intermembrane space of mitochondria (Sturtz et al. 2001; Okado-Matsumoto & Fridovich, 2001), whereas MnSOD is located within the mitochondrial matrix, a potential source of superoxide anions (McArdle et al. 2004b). Catalase, located in peroxisomes, cellular organelles capable of generating hydrogen peroxide (Chen et al. 2004), converts hydrogen peroxide to oxygen and water (Chance et al. 1979). Since hydrogen peroxide (Chen et al. 2004) and, to a lesser extent, superoxide anions (Han et al. 2003) are able to cross membranes into the cytosol, the overexpression of their corresponding antioxidants, catalase, CuZnSOD and MnSOD, has the
Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the vi- ral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the pro- teome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and con- served in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zincsuperoxidedismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supple- mentation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme’s activation is independent of a chaperone both for di- sulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts.
During our experiments we discovered that CCS protein in WBCs is more susceptible to degradation than CCS in erythrocytes, requiring that WBCs be isolated quickly from blood samples. Samples showing degradation of CCS were excluded from our analyses. Of the 7 rats per diet group analysed, we noticed that the majority of rats fed high Zn were characterised as non-responders for decreased plasma Cu and Cp activity. This over sampling of rats showing normal plasma Cu and Cp activity may account for the absence of a significant increase in WBC CCS in rats fed elevated Zn. Nonetheless, WBC CCS expression was increased in rats fed the Cu-deficient diet and was highly correlated with conventional measures of Cu status. Further, rats characterised as responders for plasma Cu (which represents rats with poorer Cu status) had higher WBC CCS levels than non-responders or Zn- 30 rats, indicating that WBC CCS responds to Cu defi- ciency induced by excess zinc. Because of the slow turno- ver of erythrocytes, WBC CCS may have value as an indicator of early reductions in Cu status. Interestingly, we found that the basal expression level of CCS in WBCs is much higher than in other tissues such as liver, heart and erythrocytes in rats (data not shown).
Finally, we determined whether isolates possessed active CuZnSOD. This was achieved by separation of whole-cell ex- tracts by isoelectric focusing (IEF) with pH 3 to 10 Ready gels (Bio-Rad) and SOD activity staining, coupled with differential inhibition, as described previously (15). The copper chelator diethyl dithiocarbamic acid (DEDC, 10 mM) was used as the inhibitor of CuZnSOD activity (3, 15). SOD gels demonstrat- ing the presence of CuZnSOD activity in eight isolates (TIM1, TIM2, TIM4, TIM8, TIM10, TIM12, TIM16, and TIM17, also representative of TIM19 and TIM20), an example of a single strain (TIM18) representative of nine other strains (TIM3, TIM5 to TIM7, TIM9, TIM11, and TIM13 to TIM15), plus the positive control A. pleuropneumoniae 4074, are shown in Fig. 4. In the eight isolates containing CuZnSOD activity (TIM1, TIM2, TIM4, TIM8, TIM10, TIM12, TIM16, and TIM17; Fig. 4, lanes 1 to 8, respectively), two achromatic bands (arrowed) that disappear with DEDC are clearly visible. In contrast, the single band of SOD activity seen in TIM18 was unaffected by DEDC treatment. The positive control (A. pleuropneumoniae 4074) showed three bands of activity, two of which were DEDC inhibitable. Active CuZnSOD (DEDC inhibitable) was found only in those Haemophilus isolates that (i) contained a sodC gene, as indicated by the presence of the 300-bp PCR product by using 3 ⬘ - and 5 ⬘ UNIVSODs as primers or hybridization of the H. parainfluenzae sodC probe in Southern blotting experi- ments, and (ii) produced the CuZnSOD protein, as deter- mined by reactivity with monoclonal antibody HD1. The pres- ence of two bands with CuZnSOD activity in the IEF gels is probably due to the fact that H. haemolyticus SodC is dimeric. FIG. 3. Dot blot showing reactivities of whole-cell protein extracts
concentration and purity were determined from OD260/ 280 readings (ratio > 1.8) using a NanoDrop ND-1000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). After determining the RNA concentration, 1 μg of total RNA was reverse-transcribed into cDNA using PrimeScript™ RT Reagent Kit (TaKaRa Biotechnology, Dalian, Liaoning, China) according to the manufacturer’s guidelines. Real-time PCR was performed on an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA) according to the manufac- turer’s instructions. The primers were designed using the Primer-Blast (http://www.ncbi.nlm.nih.gov). The pri- mer sequences for the target and reference genes (copper- and zinc-containing superoxidedismutase (Cu/Zn-SOD), manganese-containing superoxidedismutase (Mn-SOD), glutathione peroxidase 1 (GPX1), thioredoxin (TXN), thioredoxin 2 (TXN2) and β-actin) are given in Table 2. Briefly, the reaction mixture was prepared using 2 μL of cDNA (50 μg/mL), 0.4 μL of forward primer (20 μmol/L), 0.4 μL of reverse primer (20 μmol/L), 10 μL of SYBR Premix Ex Taq™ (TaKaRa Biotechnology, Dalian, Liaoning, China), 0.4 μL of ROX Reference Dye (TaKaRa Biotech- nology, Dalian, Liaoning, China) and 6.8 μL of double- distilled water. Each sample was tested in duplicate. RT- qPCR consisted of a pre-run at 95 °C for 30 s and 40 cycles of denaturation at 95 °C for 5 s, followed by a 60 °C annealing step for 30 s. The conditions of the melting curve analysis were as follows: one cycle of denaturation at 95 °C for 10 s, followed by an increase in temperature from 65 °C to 95 °C at a rate of 0.5 °C/s. The relative levels of mRNA expression were calculated using the 2 -ΔΔC T method , in which the β-actin gene was
All three strains of laboratory rats used in this study reacted on the injection of Cd, but not on the exposition to Cd in water. Ognjanovic et al. (2003) observed increased activity of antioxidant defense enzymes: copperzinc containing superoxidedismutase, catalase, glutathione peroxidase, gluta- thione reductase, and glutathione S-transferase as a response of male Wistar rats to acute exposure to Cd (0.4 mg Cd/kg of body mass). For glutathione reductase activity, the increase has been observed with the increase of applied Cd concentration for all three strains, in agreement with the literature (Ognjanovic et al. 2003; Flora et al. 2008). In the case of GST activity, a more complex dependence was observed. This could be explained by differ- ent function of these enzymes. In GST measure- ment, strain LE appeared as the most sensitive to Cd exposition – the increase of GST activity was significant already after the intraperitoneal ap- plication of 0.6 mg per 100 g body weight.
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 activity of SOD is stimulated by an increased superoxide production, which may be reﬂ ected in changes of oxidation parameters. A small amount of superoxide formed during malting generates a highly reactive hydroxyl radical (Meng et al., 2007). This hydroxyl radical is considered a principal initiator of harmful eﬀ ects on the biochemical systems of cells; it reduces stability of organoleptic beer characters (Bamforth and Parsons, 1985). The presence of superoxide anion radical in a barley caryopsis or malt and beer may aﬀ ect lipide peroxidation, polysaccharide degradation, enzyme inactivation and it can result in the reduction of yeast vitality, decline in colloidal stability, change of color, formation of undesirable beer oﬀ -ﬂ avors developed during storage (Boivin, 2001; Havlová, 1999; Meng et al., 2007). SOD content in grain is suﬃ cient for the prevention of free radicals during germination, drying and malting (Bamforth and Parsons, 1985). Reduced SOD activity results in the insuﬃ cient removal of the superoxide, which leads to damage of the organism by reactive oxygen species (Racek and Holeček, 1999). Březinová Belcredi et al. (2007) studied the eﬀ ect of application of zinc in a form of zinc sulphate and zinc oxide on SOD activity in barley grain.
A key cellular response to oxidative stress is mediated via the translocation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus, where Nrf2 binds to antioxidant-responsive elements (AREs) in the promoter region of antioxidant genes and promotes their transcription (Joshi & Johnson 2012). Genes under Nrf2-ARE control include enzymes involved in redox regulation such as superoxidedismutase, catalase, peroxiredoxin, thioredoxin, sulfiredoxin, and enzymes for glutathione synthesis (Joshi & Johnson 2012). The antioxidant protein metallothionein (MT), while containing an ARE sequence in its promoter region, is activated more strongly by the related Nrf1 transcription factor than by Nrf2 (Ohtsuji et al. 2008). In ALS patient spinal cord motor neurons, Nrf2 mRNA and protein levels were reduced compared with those of controls (Sarlette et al. 2008). Expression of mutant SOD1 in embryonic neurons depleted Nrf2-controlled glutathione synthesis enzymes and increased susceptibility to apoptosis induced by nerve growth factor/p75 signalling (Pehar et al. 2007). Interestingly, expression of mutant TDP43 in NSC34 cells prevented Nrf2-ARE-mediated induction of antioxidant genes (Duan et al. 2010), indicating that reduced oxidative stress responses may contribute to cell death in ALS. It is worth noting that the Nrf2-ARE pathways are activated more strongly in astrocytes than in neurons, but induction of Nrf2-ARE-mediated genes in astrocytes confers protection to neurons (Johnson et al. 2008). The role of astrocytes in ALS will be discussed further in section 1.3.3 on glia and neuroinflammation.
Nrf2 downstream genes such as AOEs are be- lieved to have an important role in cellular prolif- eration, tumor infiltration, and in the development of chemo-resistance by malignant cells [5, 17]. SODs reduce superoxide anion into hydrogen peroxide. There are three SODs, all of which are expressed also in human lung. SOD2 (MnSOD) is mitochondrial, SOD1 (CuZnSOD) cytosolic and extracellular . CAT and GPx, convert hydro- gen peroxide to water. GPx, a selenoenzyme, de- toxifies hydrogen and fatty acid peroxides by using glutathione (GSH) as a hydrogen donor . GSTs catalyze the conjugation of electrophilic metabo- lites or drugs to GSH to facilitate their detoxica- tion in the cells . The cellular thiols such as GSH and metallothioneins have been shown to play an important role in detoxication of platinum-based anticancer agents. Increased thiol contents have been often observed in many types of CPT-resis- tant human cancer .
100% ethanol and epoxy resin (the latter consisting o f agar 100, dodecenyl succinic anhydride, methyl nadic anhydride and 2,4,6-tri(dimethyl aminomethyl) phenol) (at a ratio o f 1:1) was then added to the cells for a 30 minute period. This was replaced with 100% epoxy resin, and left for a further 3 hours at room temperature. Thereafter, the resin was incubated at 60°C over-night, for polymerization to take place. Areas for electron microscope study were selected from samples o f 1pm survey sections, stained with toluidine blue/1% borax. 70nm-thin sections o f cells contained within the resin were then cut on an RMC MT6000 ultramicrotome, using a Diatome diamond knife. Cut sections were collected on 3mm mesh copper grids, and stained with 25% uranyl acetate in 50% methyl alcohol over a 20 minute period. The sections were washed in methanol, and transferred to aqueous Reynold’s lead citrate for a 20 minute period. After further washing in distilled water, the stained cells were examined in a JOEL 1200EX electron microscope.
Increased ecSOD expression in atherosclerotic vessels and in particular by foam cells may have several important implica- tions. An obvious explanation for this finding is that the ex- pression of ecSOD is increased in response to the oxidative en- vironment of the macrophage and atherosclerotic vessel. This would represent a compensatory adaptive function. It is possi- ble that it serves to scavenge superoxide produced by the acti- vated macrophage. If this were the case, then the principal re- active oxygen species released by these cells would likely not be superoxide, but hydrogen peroxide. This could have impor- tant implications regarding the distribution of macrophage- derived reactive oxygen species. Since hydrogen peroxide is more stable than superoxide and is uncharged, it is more likely to enter adjacent cells. Further, the potential redox reactions of superoxide and hydrogen peroxide are quite different. Su- peroxide is more capable of acting as a reducing agent, whereas hydrogen peroxide and its Fenton product hydroxyl radical are more likely to act as oxidizing agents. In addition, SODs have been shown to catalyze the nitration of tyrosines by peroxynitrite, which is produced by activated macrophages (38, 39). It has been reported that atherosclerotic lesions pos- sess increased peroxidase activity (40), although the precise enzyme involved in this activity has not been identified. Fi- nally, SODs can have peroxidative properties (41–44), in which hydrogen peroxide is used as a source of electrons and are transferred to target molecules. In this fashion, increased levels of ecSOD in the atherosclerotic vessels could participate in propagation of oxidation reactions. Such a phenomenon might contribute to an increase in fatty streak formation re- ported recently in fat-fed mice overexpressing Cu/Zn SOD (45).
The aim of the work was modification of superoxidedismutase enzyme (SOD, EC 188.8.131.52) activity analysis in barley grain and identical malts with using of the Ransod set. This set from company Ran- dox were used for enzyme determination in blood samples. This method employs xanthine and xanthi- ne oxidase to generate superoxide radicals, which react with tetrazolium chloride to form a red forma- zan dye. SOD is classified as natural antioxidants and enzyme plays a significant role at detoxication of products of molecular oxygen degradation. The largest rate of SOD occurs in embryo of barley grain. Its presence in barley grain and malt thus inhibits rancidity of grain during storage and undesirable beer flavour. The line Wabet x Washonubet (in grain-104,93 and malt 152,42 U/g dry matter) and the variety Annabell (104,65 a 147,21 U/g dry matter) had the highest activity of SOD in grain and malt of barley while the lowest activity was measured in the line KM 1910 (73,15 a 88,16 U/g dry matter) and variety Tolar (74,34 a 96,44 U/g dry matter).