Recombinant Superoxide Dismutase Research Articles

Using Recombinant Superoxide Dismutase to Control Oxidative Stress in the Gastrointestinal Tract of Cyclic Heat-Stressed Pigs.

Climate change is associated with an increased frequency and intensity of heat waves, posing a threat of heat stress to pig production. Heat stress compromises the efficiency of pig production partly due to causing oxidative stress, intestinal dysfunction, and inflammatory responses. Superoxide dismutase is an antioxidant enzyme reported to reduce oxidative stress and inflammation. Therefore, this experiment aimed to investigate whether recombinant superoxide dismutase (rSOD) could ameliorate oxidative stress and inflammatory responses in heat-stressed grower pigs. Sixty-four female pigs (Large White × Landrace, 27.8 ± 1.65 kg, mean ± SD) were randomly allocated to a control diet (standard grower feed, CON) or the control diet supplemented with 50 IU recombinant superoxide dismutase (rSOD) for 14 days. After acclimation to the diet, pigs were then housed under thermoneutral (TN, 20 °C, 35-50% relative humidity) or cyclic heat stress conditions (CHS, at 35 °C: 9 a.m. to 5 p.m. and 28 °C: 5 p.m. to 9 a.m., 35-50% relative humidity) for 3 days. Heat stress increased respiration rate (RR), skin and rectal temperature (RR and RT) (p < 0.001 for all), and reduced plasma thyroid hormone concentration (p < 0.001). The amount of oxidized glutathione (GSH:GSSG) was increased in the jejunum and ileum of CHS pigs. In the jejunum, rSOD also increased the amount of oxidized glutathione in both TN and CHS pigs, without any change in endogenous SOD activity. In the ileum, rSOD prevented increases in oxidized glutathione formation in the CHS pigs only. Taken together, this may reflect increased oxidative stress in both the jejunum and ileum in CHS pigs. Alternatively, rSOD increased the conversion of reduced to oxidized glutathione independently of CHS, possibly reflecting an increased overall SOD activity due to the addition of exogenous SOD. In conclusion, the use of in-feed SOD enzymes at a dose of 50 IU/kg may be a useful strategy for preventing oxidative stress in pigs.

Insertion of prpoD_rpoS fragment enhances expression of recombinant protein by dps auto-inducible promoter in Escherichia coli.

Nowadays, recombinant therapeutic proteins have been widely produced and consumed. For the safety and effectiveness of the protein production, an auto-inducible expression vector is required to replace inducer interference, which is uneconomic and could be harmful. In this research, an auto-inducible expression plasmid, pCAD2_sod (a pBR322 derivate plasmid), which was under dps (RpoS-dependent gene) promoter control, was modified to provide RpoS at earlier phase. Hence, accumulates more target protein and resulting a new plasmid, pCAD2+_sod. pCAD2_sod had been constructed to automatically induces the expression of recombinant superoxide dismutase (SOD) from Staphylococcus equorum (rMnSODSeq) in the stationary growth phase of Escherichia coli. This work aimed to obtain pCAD2+_sod and determine the expression level of rMnSODSeq on mRNA and protein level. A synthetic rpoS coding region under rpoD promoter control (prpoD_rpoS) was inserted to pCAD2_sod and generated pCAD2+_sod. The rMnSODSeq (24.3kDa) produced from pCAD2+_sod was ~ 1.5 fold higher at 37°C and more intense at 43°C compared to that from pCAD2_sod, likewise shifted to earlier phase (after 1h of incubation), as shown in the SDS-PAGE. The dismutase activity was also retained after zymography assay. The mRNA level from pCAD2+_sod was determined by qPCR and gave quantification cycle (Cq) values of cDNA lowest among others. It made the relative quantification (RQ) of the mRNA expression towards rho reference gene were high. The prpoD_rpoS insertion shifts and increases the rMnSODSeq production from stationary to exponential phase. The pCAD2+_sod plasmid is potential for further recombinant protein productions.

Impact of new Rexod® injection form on blood microcirculation in rat skin

Background. Free radical oxidation underlies many morbid processes in various organs and tissues, including skin. A major antioxidant preventing the free radical impact is superoxide dismutase (SOD). A particularly valued SOD-containing agent is recombinant human SOD, Rexod, possessing a wide spectrum of medical applications in form of lyophilisate. Marketing of a new Rexod® injection preparation in form of solution requires research into its properties, including the impact on blood microcirculation in skin, especially with the lack of relevant clinical trials.Objectives. Evaluation of anticipated positive effects of the new injection form of Rexod® on blood microcirculation in rat skin.Мethods. The new Rexod® preparation impact on blood microcirculation in skin was studied with laser Doppler fl uometry by recording the following non-oscillatory parameters of the basal blood fl ow: microcirculation (MC), mean squared deviation (MSD) and coeffi cient of variation (CV). Blood fl ow fl uctuations were measured in a wavelet analysis at different frequency bands: 0.0095–0.02, 0.02–0.046, 0.07–0.15, 0.15–0.4 and 0.8–0.16 Hz corresponding to endothelial (Ae), neurogenic (An), myogenic (Am), respiratory (Ar) and pulse (Ap) rhythm amplitudes, respectively.Results. The new Rexod® injection preparation at a dose of 8000 U/kg after 15 min of intraperitoneal administration in rats caused a statistically signifi cant (p &lt; 0.05) increase in the blood fl ow fl uctuation amplitudes Ae (43.1%), An (43.4%), Am (60.8%), Ar (58.3%) and Ap (32.0%) compared to the control group. Because the Ae fl uctuations coincide with nitric oxide (NO) emission episodes, such a growth indicates an elevated NO excretion by endothelial cells leading to endothelium-dependent vasodilation. The observed changes in blood microcirculation in skin are also associated with higher integral values of the basal blood fl ow, MC (33.4% increase), MSD (14.0%) and CV (27.6%), although only MC and CV values were statistically signifi cant (p &lt; 0.05). The results obtained suggest that the new injection form of Rexod® stimulates endothelial NO excretion, exerts adrenergic relaxation in smooth muscle cells of arteriolae and arteriovenular anastomoses, reduces precapillary sphincter and arteriolar contractility through Ca2+-dependent muscle relaxation, affects respiratory modulation of the venular blood microcirculatory compartment and vegetative cardiac support, intensifi es arterial blood fl ow by increasing the cardiac output.Conclusion. Application of the new Rexod® injection form improves blood microdynamics in rat skin via stimulating endothelium-dependent and independent vasodilatation and endothelial metabolism, decelerating adrenergic vasomotor activity and peripheral microvascular resistance. These processes in coupling improve blood fl ow to nutritive microvascular bed and normalise venular outfl ow. Our results provide further insights into pharmacodynamics of recombinant human SOD (Rexod).

Persistent Pulmonary Hypertension of the Newborn: Pathophysiological Mechanisms and Novel Therapeutic Approaches.

Persistent pulmonary hypertension of the newborn (PPHN) is one of the main causes of neonatal morbidity and mortality. It is characterized by sustained elevation of pulmonary vascular resistance (PVR), preventing an increase in pulmonary blood flow after birth. The affected neonates fail to establish blood oxygenation, precipitating severe respiratory distress, hypoxemia, and eventually death. Inhaled nitric oxide (iNO), the only approved pulmonary vasodilator for PPHN, constitutes, alongside supportive therapy, the basis of its treatment. However, nearly 40% of infants are iNO resistant. The cornerstones of increased PVR in PPHN are pulmonary vasoconstriction and vascular remodeling. A better understanding of PPHN pathophysiology may enlighten targeted and more effective therapies. Sildenafil, prostaglandins, milrinone, and bosentan, acting as vasodilators, besides glucocorticoids, playing a role on reducing inflammation, have all shown potential beneficial effects on newborns with PPHN. Furthermore, experimental evidence in PPHN animal models supports prospective use of emergent therapies, such as soluble guanylyl cyclase (sGC) activators/stimulators, l-citrulline, Rho-kinase inhibitors, peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists, recombinant superoxide dismutase (rhSOD), tetrahydrobiopterin (BH4) analogs, ω-3 long-chain polyunsaturated fatty acids (LC-PUFAs), 5-HT2A receptor antagonists, and recombinant human vascular endothelial growth factor (rhVEGF). This review focuses on current knowledge on alternative and novel pathways involved in PPHN pathogenesis, as well as recent progress regarding experimental and clinical evidence on potential therapeutic approaches for PPHN.

Molecular characterization, purification, and antioxidant activity of recombinant superoxide dismutase from the Pacific abalone Haliotis discus hannai Ino

Superoxide dismutase (SOD) is an acidic metalloenzyme that scavenges free radicals produced by endogenous and exogenous substances. In the present study, the tissue distribution of the superoxide dismutase HdhCu/Zn-SOD was investigated in Haliotis discus hannai Ino. The expression profile after lipopolysaccharide (LPS) challenge was determined using quantitative real-time polymerase chain reaction (qPCR). To study the antioxidant activity of a recombinant HdhCu/Zn-SOD protein, the HdhCu/Zn-SOD gene was cloned into the pPIC9K vector and transformed into the Pichia pastoris GS115 strain by electroporation. After induction by methanol, the recombinant product was purified using immobilized metal affinity chromatography and confirmed using mass spectrometry. The optimal expression conditions were determined to be incubation with 0.5% methanol at pH 6.0, resulting in a stable expressed product with the molecular weight of approximately 17 kDa and 21 kDa. The enzymatic activity of HdhCu/Zn-SOD consistently increased with increasing Cu2+ concentrations and showed good thermal stability. Recombinant HdhCu/Zn-SOD showed a strong ability to scavenge superoxide anions and hydroxyl radicals and protected L929 cells against the toxicity caused by H2O2 through its in vitro antioxidant activity. The heterologous expression of HdhCu/Zn-SOD in P. pastoris and the antioxidant activity of this enzyme are reported for the first time.Graphic abstract

Purification, biochemical characterization and DNA protection against oxidative damage of a novel recombinant superoxide dismutase from psychrophilic bacterium Halomonas sp. ANT108

A novel superoxide dismutase (referred hereafter to as HsSOD) from the psychrophilic bacterium Halomonas sp. ANT108 was purified and characterized. Escherichia coli (E. coli) was selected as the expression host. After recombinant HsSOD (rHsSOD) was purified, the specific activity was determined to be 213.47 U/mg with a purification ratio of approximately 3.61-fold. SDS-PAGE results demonstrated that rHsSOD has the molecular weight of 31.3 kDa, and type identification revealed that it belongs to Cu/Zn SOD. The optimum activity of rHsSOD was at 35 °C and 28% of its maximum activity remained at 0 °C. Further enzymatic assays indicated that rHsSOD exhibited thermal instability with a half-life of 20 min at 60 °C. Moreover, Cu2+ and Zn2+ significantly promoted rHsSOD activity. The values of Km and Vmax were 0.33 mM and 476.19 U/mg, respectively. Interestingly, rHsSOD could avoid DNA strand breakage formed by metal-catalyzed oxidation, demonstrating its antioxidant capacity. To summarize, the results suggested that rHsSOD has relatively high catalytic efficiency and oxidation resistance at low temperatures.

EXPERIENCE WITH THE USE OF RECOMBINANT SUPEROXIDE DISMUTASE IN THE COMPLEX TREATMENT OF PATIENTS WITH POSTHYPOXIC ENCEPHALOPATHY

Objective: to study the effect of recombinant superoxide dismutase, included in the complex intensive care, on the antioxidant properties of the blood of patients with acute posthypoxic encephalopathy syndrome. Material and methods. The study included 34 patients who were treated in the intensive care unit with a diagnosis of acute posthypoxic encephalopathy. Group 1 (n=21) was represented by patients who underwent standard intensive care. In group 2 (n=13), the treatment was supplemented with recombinant superoxide dismutase, administered intravenously with a drug dispenser at a dose of 12.8 million units within 12 hours. Consciousness was evaluated on a scale of Glasgow com and activation — sedation on a scale of Richmond. Spontaneous and induced biochemoluminescence (BCL) of whole venous blood was determined. Results: In patients of group 2 the phenomena of posthypoxic encephalopathy stopped faster than in group 1. In group 2, all patients were extubated 24 hours after the start of respiratory support, in group 1–57% of patients were extubated after 24 hours, the rest 48 hours after the start of treatment. By the end of the first day, in patients of group 1, spontaneous and induced venous blood BCL considerably increased, significantly exceeding the control values. In group 2, spontaneous venous blood BCL by the end of the first day of intensive care was, on average, lower than in patients of group 1 by 43, 85%, and induced by 52, 49% (p<0.05). By the end of the second day, spontaneous blood BCL in group 2, on average, was lower than in group 1 by 9.2%, and induced by 77.9% (p<0.05). Conclusion . Intravenous continuous infusion of recombinant superoxide dismutase, at a dose of 12.8 million units, included in the complex intensive care for patients with posthypoxic encephalopathy, reduces the free radical activity of whole venous blood. A decrease in the free radical activity of blood correlates with a decrease in the manifestations of acute posthypoxic encephalopathy.

A new recombinant MS-superoxide dismutase alleviates 5-fluorouracil-induced intestinal mucositis in mice.

Intestinal mucositis is a common side effect of anticancer regimens that exerts a negative impact on chemotherapy. Superoxide dismutase (SOD) is a potential therapy for mucositis but efficient product is not available because the enzyme is degraded following oral administration or induces an immune reaction after intravascular infusion. Multi-modified Stable Anti-Oxidant Enzymes® (MS-AOE®) is a new recombinant SOD with better resistance to pepsin and trypsin. We referred it as MS-SOD to distinguish from other SODs. In this study we investigated its potential to alleviate 5-FU-induced intestinal injury and the mechanisms. An intestinal mucositis model was established in C57/BL6 mice by 5-day administration of 5-FU (50 mg/kg every day, ip). MS-SOD (800 IU/10 g, ig) was given once daily for 9 days. 5-FU caused severe mucositis with intestinal morphological damage, bodyweight loss and diarrhea; MS-SOD significantly decreased the severity. 5-FU markedly increased reactive oxygen species (ROS) and inflammatory cytokines in the intestine which were ameliorated by MS-SOD. Furthermore, MS-SOD modified intestinal microbes, particularly reduced Verrucomicrobia, compared with the 5-FU group. In Caco2 cells, MS-SOD (250–1000 U/mL) dose-dependently decreased tBHP-induced ROS generation. In RAW264.7 cells, MS-SOD (500 U/mL) had no effect on LPS-induced inflammatory cytokines, but inhibited iNOS expression. These results demonstrate that MS-SOD can scavenge ROS at the initial stage of injury, thus play an indirect role in anti-inflammatory and barrier protein protection. In conclusion, MS-SOD attenuates 5-FU-induced intestinal mucositis by suppressing oxidative stress and inflammation, and influencing microbes. MS-SOD may exert beneficial effect in prevention of intestinal mucositis during chemotherapy in clinic.

Радиобиологические эффекты протонов

The article contains an analysis of literature data and the author’s own results on the radiobiological effects of protons at the cellular, systemic (intercellular) and organismic levels, as applied to the practical tasks of radiation therapy of oncological diseases and the protons effects on the astronauts’ organism.&#x0D; It is established that the proton RBE is a variable value, depending on the LET of the particles, the amount and dose rate, the presence or absence of oxygen. Proton RBE varies depending on the object of study, the type of tissue, proton energy and particle penetration depth, as well as the method for evaluating the biological efficiency of protons. which corresponds to general radiobiology.&#x0D; In particular, it has been shown that the RBE of protons adopted in radiation therapy at the level of 1.1 is conditional. A firmly established and repeatedly confirmed is an increase in RBE with a decrease in proton energy and, accordingly, an increase in LET.&#x0D; The use of elements of the physical protection of a spacecraft during exposure to protons with an energy of 170 MeV leads to an increase in LET and RBE of protons in terms of the cellularity of the bone marrow. &#x0D; Pharmacological agents effective in photon irradiation are also effective when exposed to a proton beam. It has been shown that natural melanin pigment and recombinant manganese superoxide dismutase helps to preserve and accelerate the resumption of blood formation in animals irradiated by protons. The Grippol vaccine increases radioresistance during proton irradiation. Neuropeptide Semax has a positive effect on the central nervous system and the strength of the forepaws of animals irradiated with protons at Bragg’s peak.

A Study of the Activity of Recombinant Mn-Superoxide Dismutase in the Presence of Gold and Silver Nanoparticles

Superoxide dismutase (SOD) is one of the best characterized enzyme maintaining the redox state in the cell. A bacterial expression system was used to produce human recombinant manganese SOD with a His-tag on the C-end of the protein for better purification. In addition, gold and silver nanoparticles were chemically synthesized in a variety of sizes, and then mixed with the enzyme for immobilization. Analysis by dynamic light scattering and scanning transmission electron microscopy revealed no aggregates or agglomerates of the obtained colloids. After immobilization of the protein on AuNPs and AgNPs, the conjugates were analyzed by SDS-PAGE. It was determined that SOD was adsorbed only on the gold nanoparticles. Enzyme activity was analyzed in colloids of the gold and silver nanoparticles bearing SOD. The presence of a nanoparticle did not affect enzyme activity; however, the amount of protein and size of the gold nanoparticle did influence the enzymatic activity of the conjugate. Our findings confirm that active recombinant human superoxide dismutase can be produced using a bacterial expression system, and that the enzyme can be immobilized on metal nanoparticles. The interaction between enzymes and metal nanoparticles requires further investigation.

Separate Toxin-Antidote Stabilization System in Two Plasmids for Recombinant Protein Production.

Expression vector is an important component in the production of therapeutic recombinant proteins. Most of the commercialized expression vectors apply antibiotic-based selection system. Meanwhile, World Health Organization highly recommends for the alternative system due to its potentials to cause spreading of resistance gene and hypersensitivity to some people. In current work, we developed an expression system for Escherichia coli using the toxinantidote system in two separated plasmids. An antidote gene (ccdA) with its natural promoter and terminator was constructed in a plasmid (pDCSAsod) containing a DNA fragment encoding recombinant superoxide dismutase from Staphylococcus equorum (rMnSODSeq) as a model. The gene expression was directed under T7 promoter and regulated by lac operator. The toxin gene (ccdB) was located in a separate plasmid (pDCSB) under PBAD promoter. This study aimed to study the growth profile of the host in the presence of both plasmids, to determine plasmids stability, and the effect of the toxinantidote system on rMnSODSeq production and activity. The presence of both plasmids did not affect the growth profile of E. coli BL21(DE3), while the plasmid stability was 94% for pDCSAsod and 68% for pDCSB at the end of protein production time. The yield of purified rMnSODSeq was 3.2 mg/ml and the enzyme was shown to be active by a zymography assay. In conclusion, for the first time, we show that toxin-antidote system in two separated plasmids has the potential for the production of recombinant therapeutic proteins and is more flexible in choosing the E. coli strain compared to established chromosomally integrated toxin-antidote selection system.

Protective efficacy of a canine brucellosis vaccine candidate based on live attenuated Salmonella expressing recombinant Brucella BCSP31, Omp3b and SOD proteins in Beagles.

The aim of this study was to establish a proof-of-concept of protective efficacy of Salmonella-based B. abortus vaccine candidate in Beagles. Group A Beagles (n=10) were subcutaneously (SC) inoculated with S. Typhimurium delivery strain containing pMMP65 (vector to deliver antigens) only as vector control. Group B Beagles (n=10) were SC vaccinated with the mixture of the three Salmonella delivery strains expressing the recombinant B. abortus BCSP31, Outer membrane protein 3b (Omp3b), and superoxide dismutase (SOD) proteins, respectively. No Salmonella delivery strains were isolated from all tissues tested. Serum IgG, interleukin-4, tumor necrosis factor-alpha, and interferon-gamma concentrations were significantly higher in group B than in group A. Following intraconjunctival challenge with B. abortus 544, among 5 group B Beagles, the challenge strain was isolated from mandibular, and retropharyngeal lymph nodes of three Beagles, and no isolates were observed from all tissues of two Beagle. However, the challenge strains were detected from spleen, uterus (except two Beagles), and mandibular, prescapular, retropharyngeal, and superficial inguinal lymph nodes of all group A Beagles. These results suggest that the mixture of three S. Typhimurium delivery strains be a good vaccine candidate against brucellosis by B. abortus in dogs. Further investigations are needed to improve the protective efficacy of the Salmonella-based B. abortus vaccine candidate and explore its practical application in dogs.

Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation

Thermophilic or hyperthermophilic SODs (superoxide dismutase) usually offer substantial biotechnological advantages over mesophilic SODs. Previously a 244-amino acid N-terminal domain (NTD) from a heatresistant SOD of Geobacillus thermodenitrificans NG80-2 was discovered and demonstrated to be able to confer thermostability to homologous mesophilic SODs, which revealed a new type of heat resistance mechanism. To further improve the heat resistance and stress tolerance of thermophilic cambialistic superoxide dismutase (Fe/Mn- SOD Ap ) from Aeropyrum pernix K1 through metal incorporation and fusion with the newly found peptide NTD for broadening its industrial application, the wildtype SOD Ap and NTD-fused ntdSOD Ap were expressed in E. coli BL21 and incorporated with metal cofactors by two ways. Recombinant fusion SOD obtained by in vitro reconstitution (Mn-rec ntdSOD Ap ) exhibited improved optimum temperature at 70°C and dramatically enhanced thermostability especially at 110°C with enhanced pH stability from 4 to 10 and higher tolerance for denaturants and organic media than Mn-rec SOD Ap . To the best of our knowledge, Mn-rec ntdSOD Ap could be the most heat resistant SOD. In addition, metal incorporation of SOD Ap and ntdSOD Ap via in vivo modification have been developed and proved to be more practical for industrial use. These results indicate that fusion with NTD along with metal incorporation can generate superimposed effect and be applied to enhance the stability of cambialistic thermophilic SODs, thus providing a universal and convenient bioengineering method for generating extremely stable SODs.

Statistical optimization for the production of recombinant cold-adapted superoxide dismutase in E. coli using response surface methodology

ABSTRACTCold-adapted superoxide dismutase (SOD) with higher catalytic activity at lower temperature has great amount of applications in many aspects as an industrial enzyme. The application of recombinant enzyme in gene engineering and microbial fermentation technology is an effective way to obtain high-yield product. In this study, to obtain the recombinant SOD in E. coli (rPsSOD) with the highest activity, the Box-Behnken design was first applied to optimize the important parameters (lactose, tryptone and Tween-80) affecting the activity of rPsSOD. The results showed that the optimal fermentation conditions were Tween-80 (0.047%), tryptone (6.16 g/L), lactose (11.38 g/L). The activity of rPsSOD was 71.86 U/mg (1.54 times) as compared with non-optimized conditions. Such an improved production will facilitate the application of the cold-adapted rPsSOD.

Overexpression of Mn-superoxide dismutase in Oxya chinensis mediates increased malathion tolerance

Superoxide dismutase (SOD) is the first line of defense against oxidative damage. Malathion is an organophosphate insecticide and can induce the production of reactive oxygen species (ROS) and cause the intracellular oxidative stress. The present study was undertaken to examine the effects of malathion on SODs activity and their transcriptional levels in Oxya chinensis (Thunberg) (Orthoptera: Acrididae). The results showed that total SOD and MnSOD activities increased as a dose-dependent manner while CuZnSOD activity has no significant changes after malathion treatments. Total SOD and MnSOD activities were the highest at the concentration of 0.8 μg μL−1 malathion treatment and increased significantly about 1.81- and 2.48-fold compared with the control, respectively. Increased mRNA expression of MnSOD, ecCuZnSOD1, and ecCuZnSOD2 were observed after malathion treatments. Moreover, the alteration of MnSOD transcript was similar to the profiles of MnSOD activity. These results suggested that the up-regulation expression of MnSOD transcript led to the increase of MnSOD activity in order to eliminate the excessive ROS caused by malathion. In addition, we evaluated the role of individual SOD gene in malathion stress by using RNAi and recombinant SOD proteins. The results showed that ROS contents increased significantly after the silencing of MnSOD and ecCuZnSOD1 genes. The OD values of the E. coli cells transformed with pET-28a-OcMnSOD plasmid were 1.13–1.31-fold and 1.08–1.33-fold higher than those of cells with pET-28a plasmids under 0.4 and 0.8 μg μL−1 malathion treatments, respectively. These findings indicated that MnSOD exerted an important role in defense oxidative stress caused by malathion.

L-аргінін та рексод покращують стан печінки при експериментальному перитоніті

&lt;p&gt;РЕЗЮМЕ. Мета роботи – встановлення впливу попередника синтезу оксиду азоту – L-аргініну та рекомбінантної супероксиддисмутази (рексоду) на стан печінки при гострому експериментальному перитоніті. L-аргінін (“Sigma”, США, вводили внутрішьоочеревинно по 25 мг/кг маси, чотириразово – за 30 хв до і через 12, 24 та 36 год після моделювання перитоніту) сприяв покращенню стану печінки тварин, що проявлялось пригніченням процесів ліпопероксидації, нормалізацією активності антиоксидантної системи та ферментів мітохондрій, супроводжувалось зростанням синтезу оксиду азоту у печінці. Препарат супероксиддисмутази – рексод (SODrec, РФ, вводили внутрішьоочеревинно по 0,05 мг/кг, чотириразово на тлі перитоніту за тією ж схемою) сприяв зменшенню вмісту у піддослідних тварин показників пероксидного окиснення ліпідів, зниженню рівня ендогенної інтоксикації, активував антиоксидантну систему та активність ферментів мітохондрій.&lt;/p&gt;&lt;p&gt;КЛЮЧОВІ СЛОВА:&lt;strong&gt; &lt;/strong&gt;гострий перитоніт, печінка, L-аргінін, рексод.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;

Cloning, expression and biochemical characterization of recombinant superoxide dismutase from Antarctic psychrophilic bacterium Pseudoalteromonas sp. ANT506.

In this study, a superoxide dismutase gene (PsSOD) from Pseudoalteromonas sp. ANT506 was cloned and over expressed in Escherichia coli. The PsSOD has an open reading frame of 582 bp with a putative product of 193 amino acid residue and an estimated molecular size of 21.4 kDa. His-tagged PsSOD was subsequently purified 12.6-fold by Ni-affinity chromatography and the yield of 22.9%. The characterization of the purified rPsSOD exhibited maximum activity at 30 °C and pH 8.0. The enzyme exhibited 13.9% activity at 0 °C and had high-thermo lability at higher than 50 °C. rPsSOD exhibited well capability to 2.5 M NaCl (62.4%). These results indicated that rPsSOD exhibited special catalytic properties.

Biochemical and Molecular Characterization of a Novel Cu/Zn Superoxide Dismutase from Amaranthus hypochondriacus L.: an Intrinsically Disordered Protein.

A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50% of catalytic activity after incubation at 100°C for 30min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability.

Isolation and characterization of Cu/Zn–superoxide dismutase in Fasciola gigantica

A full-length complementary DNA (cDNA) encoding Cu/Zn–superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn–superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn–SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)–polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn–SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0–10.0. Native Cu/Zn–superoxide dismutase protein was detected in the somatic extract and excretory–secretory products of the adult F. gigantica by Western blotting. NBT–PAGE showed a single Cu/Zn–SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.

Expression and characterization of a recombinant psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica E. Desv. [Poaceae

We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5-8 and a temperature interval of 0-40 °C. Furthermore, DaSOD was still active at -20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products.