Molecular cloning,characterization and expression analysis of cytoplasmic Cu/Zn-superoxid dismutase (SOD) from pearl oyster Pinctada fucata

Because of its capacity to rapidly convert superoxide to hydrogen peroxide, superoxide dismutase (SOD) is crucial in both intracellular signalling and regulation of oxidative stress. In this paper we report the cloning of a Cu/Zn SOD (designated as pfSOD) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this Cu/Zn SOD contains an open reading frame (ORF) of 471 bp coding for 156 amino acids. No signal peptide was identified at the N-terminal amino acid sequence of Cu/Zn SOD indicating that this pfSOD encodes a cytoplasmic Cu/Zn SOD. This is supported by the presence of conserved amino acids required for binding copper and zinc. Semi-quantitative analysis in adult tissues showed that the pfSOD mRNA was abundantly expressed in haemocytes and gill and scarcely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfSOD mRNA in haemocytes was increased, reaching the highest level at 8 h, then dropping to basal levels at 36 h. These results suggest that Cu/Zn SOD might be used as a bioindicator of the aquatic environmental pollution and cellular stress in pearl oyster.     

Characterization and expression of superoxide dismutase genes in Chironomus riparius (Diptera, Chironomidae) larvae as a potential biomarker of ecotoxicity

Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme involved in the scavenging of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In this study, a copper-zinc superoxide dismutase (Cu-ZnSOD) gene and a manganese superoxide dismutase (MnSOD) gene in aquatic midge, Chironomus riparius (CrSODs) was identified using an Expressed Sequence Tag (EST) database generated by 454 pyrosequencing. A multiple sequence alignment of C. riparius sequences revealed high homology with other insect sequences in terms of the amino acid level. Phylogenetic analysis of the CrSODs revealed that they were grouped with SODs of other organisms, such as Polypedilum vanderplanki, Drosophila melanogaster, Aedes aegypti, Anopheles gambiae, Culex quinquefasciatus and Bombyx mori. Expression of the corresponding CrSODs was analyzed during different developmental stages and following exposure to various environmental contaminants with different mode of actions i.e., paraquat, cadmium, benzo[a]pyrene, and chloropyrifos. CrSOD gene expression was significantly up or down regulated in response to exposure to the chemicals tested. The overall results suggested that SOD gene expression provided a platform for the understanding of oxidative stress responses caused by exposure to various environmental contaminants, and the SOD genes could be used as biomarkers for environmental disturbances such as oxidative stress initiated by xenobiotics.     

Mitochondrial manganese superoxide dismutase from the shrimp Litopenaeus vannamei: Molecular characterization and effect of high temperature,hypoxia and reoxygenation on expression and enzyme activity

Climate warming has been increasing ocean water temperature and decreasing oxygen concentrations, exposing aquatic organisms to environmental stress conditions. The shrimp Litopenaeus vannamei manages to survive these harsh environmental conditions by enhancing their antioxidant defenses, among other strategies. In this study, we report the mitochondrial manganese superoxide dismutase (mMnSOD) nucleotide and deduced amino acid sequences and its gene expression in L. vannamei tissues. The deduced protein has 220 amino acids with a signal peptide of 20 amino acids. Expression of mMnSOD was analyzed in hepatopancreas, gills and muscle, where gills had highest expression in normoxic conditions. In addition, shrimp were subjected to high temperature, hypoxia and reoxygenation to analyze the effect on the expression of mMnSOD and SOD activity in mitochondria. High temperature and hypoxia showed a synergistic effect in the up-regulation on expression of mMnSOD in gills and hepatopancreas. Moreover, induction in SOD activity was found in the mitochondrial fraction from gills of normoxia at high temperature, probably due to an overproduction of reactive oxygen species caused by an elevated metabolic rate due to the stress temperature. These results suggest that the combined stress conditions of hypoxia and high temperature trigger molecularly the antioxidant response in L. vannamei in a higher degree than only one stressor.     

Effect of temperature in multiple biomarkers of oxidative stress in coastal shrimp

Various studies in captivity and in the wild have pointed to the effect of season, and temperature in particular, in the levels of the oxidative stress biomarkers currently used for environmental quality assessment. However, knowledge on how temperature affects the oxidative stress response is unavailable for most species. This study investigated the effect of increasing temperature on lipid peroxidation, catalase activity, superoxide dismutase and glutathione-S-transferase in the shrimps, Palaemon elegans and Palaemon serratus. It was concluded that increasing temperatures significantly affect all the biomarkers tested in both species, with the exception of superoxide dismutase in P. serratus which was not affected by temperature. The oxidative stress response was more intense in P. elegans, than in P. serratus, producing higher peaks of all biomarkers at temperatures between 22 °C and 26 °C, followed by low levels at higher temperatures. It was concluded that monitoring of ecosystems using oxidative stress biomarkers should take into account the species and thermal history of the organisms. Sampling should be avoided during heat waves and immediately after heat waves.     

Transcriptional regulation of extracellular copper zinc superoxide dismutase from white shrimp Litopenaeus vannamei following Vibrio alginolyticus and WSSV infection

The cDNA encoding an extracellular copper zinc superoxide dismutase (LvECSOD) was cloned from the hepatopancreas of white shrimp Litopenaeus vannamei. It consisted of 915 bp nucleotides with an open reading frame corresponding to a deduced protein of 178 amino acids. The LvECSOD contains a putative signal peptide of 16 amino acids, two potential N-linked glycosylation sites (N(115)GTA and N(135)ITG) and a copper zinc superoxide dismutase family signature sequence (G(162)NAGaRvACctI(173)). It was found that four copper binding sites, four zinc binding sites and two cysteines involving in the formation of the disulfide bridge were conserved in the protein. LvECSOD shared 33-58% identity to ECSODs from other organisms. Expression analysis revealed that LvECSOD mRNA was widely distributed in all the tissues examined. When the shrimp challenged with Vibrio alginolyticus or white spot syndrome virus (WSSV), expression of LvECSOD mRNA in the hepatopancreas and hemocytes was mediated responsively. Our results suggested that LvECSOD was implicated in the immune response induced by V. alginolyticus and WSSV.     

Physiological responses and expression of metallothionein (MT) and superoxide dismutase (SOD) mRNAs in olive flounder, Paralichthys olivaceus exposed to benzo[a]pyrene

We cloned complementary DNA (cDNA) encoding metallothionein (MT) and superoxide dismutase (SOD) from the liver of olive flounder, Paralichthys olivaceus. The full-length MT cDNA consists of 183 base pairs (bp) and encodes a protein of 60 amino acids; partial SOD cDNA consists of 326 bp and encodes a protein of 109 amino acids. We investigated the dose- and time-related effects of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) on MT and SOD mRNA using quantitative polymerase chain reaction (QPCR). The expression levels of MT mRNA were highest at 24 h (about five times) in 10 microg/L BaP, and at 6 h (about twelve times) in 30 microg/L BaP. The expression levels of SOD mRNA were highest at 12 h (about three times) in 10 microg/L BaP, and at 6 h (about six times) in 30 microg/L BaP, and then decreased toward the end of the experiment. We also measured plasma glucose and cortisol, all of which increased with BaP exposure. These results suggest that MT and SOD play an important role in the detoxification of reactive oxygen species (ROS) caused by BaP exposure, and thus may be indicators of oxidative stress responses.     

Manganese-containing superoxide dismutase and its gene from Candida albicans

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.     

Changes in antioxidant defense system in gills of Capoeta umbla caught from Uzuncayir Dam Lake,Turkey

The aim of this work was to determine the seasonal changes in the activities of selected biomarkers in Capoeta umbla (Heckel, 1843) caught from Uzuncayir Dam Lake (Tunceli, Turkey) and to evaluate the effects of environmental factors on these activities. Fish were sampled on seasonal basis, and superoxide dismutase, catalase, glutathione peroxidase activities and levels of glutathione and malondialdehyde in gills were determined. Significant variations of oxidative stress biomarkers were observed between seasons and sites. The results of this study show that seasonal variations of oxidative stress responses and lipid peroxidation in gills of C. umbla are sensitive to the contaminants present in water of Uzuncayir Dam Lake and selected parameters are in valuable biomarkers for monitoring of water systems, since they give an early warning signal of effects of xenobiotics on aquatic organisms at molecular levels which help to prevent their effects at organismal level.     

C-terminal heat shock protein 90 inhibitor decreases hyperglycemia-induced oxidative stress and improves mitochondrial bioenergetics in sensory neurons

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in which hyperglycemia-induced mitochondrial dysfunction and enhanced oxidative stress contribute to sensory neuron pathology. KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple sensory deficits associated with the progression of DPN and protects unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically, KU-32 increased the expression of Hsp70, and this protein was critical for drug efficacy in reversing DPN. However, it remained unclear if KU-32 had a broader effect on chaperone induction and if its efficacy was linked to improving mitochondrial dysfunction. Using cultures of hyperglycemically stressed primary sensory neurons, the present study investigated whether KU-32 had an effect on the translational induction of other chaperones and improved mitochondrial oxidative stress and bioenergetics. A variation of stable isotope labeling with amino acids in cell culture called pulse SILAC (pSILAC) was used to unbiasedly assess changes in protein translation. Hyperglycemia decreased the translation of numerous mitochondrial proteins that affect superoxide levels and respiratory activity. Importantly, this correlated with a decrease in mitochondrial oxygen consumption and an increase in superoxide levels. KU-32 increased the translation of Mn superoxide dismutase and several cytosolic and mitochondrial chaperones. Consistent with these changes, KU-32 decreased mitochondrial superoxide levels and significantly enhanced respiratory activity. These data indicate that efficacy of modulating molecular chaperones in DPN may be due in part to improved neuronal mitochondrial bioenergetics and decreased oxidative stress.     

cDNA cloning and expression of ghrelin in giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. It plays an important role in stimulating growth hormone secretion, food intake, body weight gain and gastric motility. cDNA sequences coding for ghrelin precursor protein (prepro-ghrelin) were isolated from the stomach of a giant panda. Two different mRNA sequences of ghrelin were obtained. The long open reading frame of ghrelin (354 bp) encodes a precursor protein of 117 amino acids with a 23 amino acid signal peptide. The short one (351 bp) encodes a precursor protein of 116 amino acids with the same 23 amino acid signal peptide. The presumed giant panda mature ghrelin proteins also had two forms. Comparative analysis showed that the first and the fourth amino acids (Gly and Phe) were completely conserved and the third amino acid (Ser) was also highly conserved in the mature ghrelin. RT-PCR analysis of giant panda ghrelin mRNA in various tissues revealed high level of expression in stomach, relative lower levels of expression in small intestine, liver and kidney, and no expression in thymus, spleen and heart.     

Sub-lethal effect of synthetic pyrethroid pesticide on metabolic enzymes and protein profile of non-target Zebra fish,Danio rerio

Extensive application of pesticide in agricultural field affects the enzymatic activity of non-target animals, including fishes. In this study, the impact of sublethal concentration of fenvalerate on marker enzymes of freshwater Zebra fish was evaluated. Pesticide-induced stress can specifically affect non target fishes, through elevated level of reactive oxygen species which is responsible for biochemical, cell metabolism and physiological activities. The oxidative stress mediated by fenvalerate at sub lethal concentrations after 28 days of exposure of Zebra fish. Following 28 days of exposure of pesticide, catalase, superoxide dismutase, aspartate amino transferases, alanine amino transferase, alkaline phosphatase and acid phosphatase were assessed. Results revealed reduction of superoxide dismutase activity after 28 days of exposure in sub lethal concentration of fenvalerate in liver and gills. In liver, catalase activity was found to be less in fenvalerate exposed fish than control fish. In liver, increase of 75.75% aspartate amino transferase and 38% increase in alanine amino transferase in gills. SGPT activity was relatively higher than SGOT suggests more contribution of phyruvalate than oxaloacetate formation. Fenvalerate induced changes in acid phosphatase and alkaline phosphatase activity in the liver and gills of Zebra fish after four weeks of exposure. Fenvalerate induced expression of various stress proteins in gill, liver, followed by muscle. Some proteins lost its intensity due to fenvalerate toxicity. Result revealed that enzyme assays and SDS-PAGE analysis for protein subunits determination is relevant tool to monitor stress in freshwater ecosystem. The findings suggest that in monitoring fenvalerate toxicity programme, enzyme activities can be potent diagnostic tool for fenvalerate induced toxicity.     

Oxidative stress is a consequence, not a cause, of aluminum toxicity in the forage legume Lotus corniculatus

? Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. ? Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. ? The exposure of plants to 10 μM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 μM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. ? We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 μM Al.     

Cloning and expression of the Mycobacterium bovis BCG gene for extracellular alpha antigen.

The gene for the extracellular alpha antigen of Mycobacterium bovis BCG was cloned by using a single probe restricted to G or C in the third position. This technique should have great potential for the isolation of mycobacterial antigen genes. The gene analysis revealed that the alpha antigen gene encoded 323 amino acid residues, including 40 amino acids for signal peptide followed by 283 amino acids for mature protein. This is the first report on the structure of the mycobacterial signal peptide. The promoter-like sequence and ribosome-binding site were observed upstream of the open reading frame. In the coding region, the third position of the codon showed high G + C content (86%). The gene was expressed as an unfused protein in Escherichia coli by using an E. coli expression vector. This protein, which reacted with polyclonal antibody raised against alpha antigen from Mycobacterium tuberculosis, would be applicable to the immunodiagnosis of tuberculosis.     

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.     

Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

Characterization of two genes expressed in the salivary glands of the Hessian fly, Mayetiola destructor (Say)

Two genes, SSGP-11A1 and SSGP-12A1, have been isolated that encodes proteins with a secretion signal peptide at theN-terminals from the Hessian fly (Mayetiola destructor (Say)). The SSGP-11A1 gene contains one small intron (89 bp) and encodes a putative protein with 79 amino acids. The first 18 amino acids constitute a putative secretion signal peptide. The SSGP-12A1 gene contains three small introns and encodes a putative protein with 234 amino acids. The first 19 amino acids constitute a putative secretion signal peptide. Northern blot analysis revealed that both of the genes are primarily expressed in the salivary glands of Hessian fly larvae, the feeding stage of the insect. These observations are consistent with the possibility that the proteins encoded by them are secreted into host plants during feeding. Even though both genes are exclusively expressed in Hessian fly larvae, the expression profiles between them were quite different in insects at different instars. The SSGP-11A1 gene was expressed in all instars of larvae while the SSGP-12A1 gene was almost exclusively expressed in the first instar larvae. The differential expression suggests that the proteins encoded by these two genes may perform different functions. In situ hybridization revealed that the SSGP-11A1 gene is located on the short arm of chromosome A1 while SSGP-12A1 gene is on the long arm of chromosome A2.     

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions). In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system. Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR). Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions.