RESUMEN
Purpose: Hesperidin has anti-inflammatory and anti-oxidant stress effects, but its functions in chronic obstructive pulmonary disease (COPD) remains unknown. This study analyzed the role of hesperidin in COPD mice, aiming to provide a basis for the hesperidin application.Materials and methods: Mice were injected with cigarette smoke extract (CSE) to construct COPD models and then treated with budesonide or hesperidin. Hematoxylin-eosin (HE) and TUNEL assays were used to observe the pathological changes and cell death of lung tissue. The levels of interleukin (IL)-6, IL-8, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in bronchoalveolar lavage fluid (BLAF), as well as myeloperoxidase (MPO) content in lung tissues were confirmed. The expression levels of SIRT1, PGC-1α, and p65 proteins were measured by western blotting (WB) analysis.Results: CSE induced inflammatory cell infiltration and cell death in the lung tissues of mice, whereas budesonide and hesperidin effectively alleviated these pathological changes. The levels of IL-6, IL-8, and MDA in BLAF and pulmonary MPO content in the COPD mice were effectively increased, while the levels of SOD and CAT in BLAF were decreased, which could be reversed by budesonide and hesperidin. Moreover, the addition of budesonide or hesperidin reliably accelerated the expression levels of PGC-1α and SIRT1 but suppressed the phosphorylation of p65 in COPD mice. In general, high-dose hesperidin had a stronger regulatory effect on COPD mice.Conclusions: Hesperidin alleviated inflammation and oxidative stress responses in CES-induced COPD mice, associated with SIRT1/PGC-1α/NF-κB signaling axis, which might become a new direction for COPD treatment.
Asunto(s)
Hesperidina/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Sirtuina 1/genética , Animales , Líquido del Lavado Bronquioalveolar/química , Modelos Animales de Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/patología , Interleucina-6/química , Interleucina-8/química , Interleucina-8/aislamiento & purificación , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , FN-kappa B/genética , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/química , Peroxidasa/aislamiento & purificación , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Transducción de Señal/efectos de los fármacos , Humo/efectos adversos , Superóxido Dismutasa/química , Superóxido Dismutasa/aislamiento & purificación , Factor de Transcripción ReIA/genéticaRESUMEN
In the present study, superoxide dismutase (SOD) extracted from dry fruits; Juglans regia (Walnut; W) and Ribes nigrum (Munakka; M) was partially purified into 0%-40% and 40%-80% fractions based on ammonium sulfate saturation levels. The partially purified fractions (0%-40%) exhibited purification level of 3.09- (W) and 3.22- (M) fold with specific activity 79.32 Umg-1 (W) and 125.23 Umg-1 (M). SOD from both the sources was found to be thermally stable, that is, 80°C (W) and 70°C (M). Kinetic studies showed Km values to be 3.33 mM (W) and 2.86 mM (M), whereas the activation energy (Ea ) calculated as 24.52 KJ mol-1 (W) and 26.25 KJ mol-1 (M). Na+ , Mn2+ , and Ba2+ ions acted as potential inhibitors, whereas Fe2+ stimulated SOD from both the sources. Among these metal ions, Na+ exhibited uncompetitive inhibition in both cases; with Ki values of 0.7 mM (W) and 0.9 mM (M), suggesting the more prominent binding affinity and effectiveness. PRACTICAL APPLICATIONS: Awareness need to be created among people for multifactorial health benefits of nutraceuticals in day-to-day life. Nutritional consumption from fruits, nuts, and vegetables safeguard against various maladies like cardiovascular diseases, diabetes, and cancers. Superoxide dismutase (EC 1.15.1.1) is a standout among the most critical metal-containing enzymes that act as a main line of defense against oxidative stress. Antioxidant-based drugs and formulations have been developed in the recent years and research is emphasized on its impact on oxidative stress levels. In this study, Juglans regia (W) and Ribes nigrum (M) were found to have thermostable SOD enzyme with excellent antioxidant properties. Thermal stability of an enzyme improves its significance making it industry friendly with therapeutically vital products, alongside their utilization as supplement in numerous therapeutic formulations.
Asunto(s)
Antioxidantes/farmacología , Suplementos Dietéticos , Juglans/enzimología , Ribes/enzimología , Superóxido Dismutasa/farmacología , Antioxidantes/aislamiento & purificación , Estabilidad de Enzimas , Frutas/enzimología , Calor , Estrés Oxidativo , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Superóxido Dismutasa/aislamiento & purificaciónRESUMEN
Superoxide dismutase (SOD) of the Tamarix aphylla leaves were detected at optimum conditions that collected in April, May and June. Results indicated the specific activity in the crude extract reaching to 36.76 unit/ mg protein. Crude SOD was purified by several techniques, precipitation with ammonium sulfate (50-75) %, Ion exchange chromatography using DEAE-cellulose and two steps of size exclusion chromatography on sephacryl S-200 column. The obtained specific activity (310 unit/mg protein) and purification fold 7.91. The purified enzyme revealed one band by SDS-polyacrylamide gel electrophoresis with molecular mass 85.703 kDa. while 89.125 kDa by Sephacryl S-200. The optimal pH and temperature for enzyme activity were 7.5, and 50ºC respectively. EDTA, SDS and NaN3 reduced activity, contrariwise of H2O2 and KCN, pointed to the studied SOD is MnSOD. Michalis constant Km and maximum velocity Vmax values were 0.016 mM and 55.86 mM/min, respectively by using Pyrogallol as substrate. According to the results, we conclude Tamarix aphylla produce MnSOD which can have purified by serial purification techniques for better activity and characterized for further studies.
Asunto(s)
Extractos Vegetales/química , Superóxido Dismutasa/química , Superóxido Dismutasa/aislamiento & purificación , Tamaricaceae/enzimología , Sulfato de Amonio/química , Quelantes/farmacología , Ácido Edético/farmacología , Inhibidores Enzimáticos/farmacología , Calor , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Hojas de la Planta/enzimología , Cianuro de Potasio/farmacología , Pirogalol/farmacología , Azida Sódica/farmacologíaRESUMEN
Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.
Asunto(s)
Caimanes y Cocodrilos/genética , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas/genética , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Etiquetas de Secuencia Expresada , Regulación Enzimológica de la Expresión Génica , Cinética , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Homología Estructural de Proteína , Superóxido Dismutasa/química , Superóxido Dismutasa/aislamiento & purificaciónRESUMEN
Medicinal plants have played a major role as a functional food and pharmacological source of active substances. Barley grass (BG) is young green barley leaves. It is the young grass of the common barley plant Hordeum vulgare L. of the family Poeaceae (Graminae). It is a type of green grasses, and the only vegetation on the earth that can supply sole nutritional support from birth to old age. It contains a wide spectrum of vitamins, minerals, as well as eight essential amino acids that we must get from our diets. BG possesses several pharmacological activities as anticancer activity, anti-oxidant activity and anti-inflammatory activity. It has been argued that BG helps blood flow, digestion and general detoxification of the body. The major pharmacologic interest of BG is its use in the treatment of chronic diseases. The beneficial effects observed in chronic disease may be related to bioactive compounds contained in BG such as superoxide dismutase (SOD) and bioflavonoids (lutonarin and saponarin). Thus, this paper is focused on the various studies that emphasize the therapeutic potential of BG in the prevention and treatment of chronic diseases.
Asunto(s)
Enfermedad Crónica/tratamiento farmacológico , Enfermedad Crónica/prevención & control , Hordeum/química , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Aminoácidos Esenciales/análisis , Aminoácidos Esenciales/aislamiento & purificación , Antiinflamatorios , Antineoplásicos , Antioxidantes , Flavonoides/análisis , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Minerales/análisis , Minerales/aislamiento & purificación , Hojas de la Planta/química , Superóxido Dismutasa/análisis , Superóxido Dismutasa/aislamiento & purificación , Superóxido Dismutasa/farmacología , Vitaminas/análisis , Vitaminas/aislamiento & purificaciónRESUMEN
Superoxide dismutases (SODs) are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase (ecCuZnSOD) complementary DNA was cloned and characterized from the whitefly, Bemisia tabaci. Quantitative polymerase chain reaction analysis showed that the expression level of BtecCuZnSOD was more than 10-fold higher in the invasive Middle East Asia Minor 1 (MEAM1) than in the native Asia II 3 species of the B. tabaci species complex. After exposure to low temperature (4 °C), the expression of Bt-ecCuZnSOD gene was significantly up-regulated in MEAM1 but not in Asia II 3. Furthermore, the expression level of B. tabaci intracellular CuZnSOD (Bt-icCuZnSOD), Bt-ecCuZnSOD and mitochondrial MnSOD (Bt-mMnSOD) was compared after transferring MEAM1 and Asia II 3 whiteflies from favorable (cotton) to unfavorable host plants (tobacco). On cotton, both CuZnSOD genes were expressed at a higher level in MEAM1 compared with Asia II 3. Interestingly, after transferring onto tobacco, the expression of Bt-ecCuZnSOD was significantly induced in Asia II 3 but not in MEAM1. On the other hand, while Bt-mMnSOD was expressed equally in both species on cotton, Bt-mMnSOD messenger RNA was up-regulated in MEAM1 on tobacco. Consistently, enzymatic activity assays of CuZnSOD and MnSOD demonstrated that CuZnSOD might play an important protective role against oxidative stress in Asia II 3, whereas MnSOD activation was critical for MEAM1 whiteflies during host adaptation. Taken together, our results suggest that the successful invasion of MEAM1 is correlated with its constitutive high activity of CuZnSOD and inducible expression of MnSOD under stress conditions.
Asunto(s)
Hemípteros/enzimología , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario , Expresión Génica , Gossypium/parasitología , Hemípteros/genética , Interacciones Huésped-Parásitos , Especies Introducidas , Datos de Secuencia Molecular , Estrés Oxidativo , ARN Mensajero , Reacción en Cadena en Tiempo Real de la Polimerasa , Superóxido Dismutasa/aislamiento & purificación , Superóxido Dismutasa/metabolismo , Temperatura , Nicotiana/parasitologíaRESUMEN
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.
Asunto(s)
ADN Complementario/aislamiento & purificación , Fasciola/enzimología , Regulación Enzimológica de la Expresión Génica , Superóxido Dismutasa/aislamiento & purificación , Mataderos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Búfalos/parasitología , ADN Complementario/química , ADN de Helmintos/química , ADN de Helmintos/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Fasciola/genética , Fasciola/crecimiento & desarrollo , Fasciola hepatica/enzimología , Fasciola hepatica/genética , Fascioliasis/parasitología , Fascioliasis/veterinaria , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Estadios del Ciclo de Vida/genética , Nitroazul de Tetrazolio , ARN de Helminto/genética , ARN de Helminto/aislamiento & purificación , Conejos , Proteínas Recombinantes/química , Análisis de Secuencia de ADN , Superóxido Dismutasa/química , Superóxido Dismutasa/genéticaRESUMEN
BACKGROUND: The North American wood frog, Rana sylvatica, is able to overcome subzero conditions through overwintering in a frozen state. Freezing imposes ischemic and oxidative stress on cells as a result of cessation of blood flow. Superoxide dismutases (SODs) catalyze the redox reaction involving the dismutation of superoxide (O(2)(-)) to molecular oxygen and hydrogen peroxide. METHODS: The present study investigated the regulation of CuZnSOD and MnSOD kinetics as well as the transcript, protein and phosphorylation levels of purified enzyme from the muscle of control and frozen R. sylvatica. RESULTS: CuZnSOD from frozen muscle showed a significantly higher V(max) (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O(2)(-) (0.66 fold) in comparison to CuZnSOD from control frogs. MnSOD from frozen frogs showed higher phosphorylation of serine (2.36 fold) and tyrosine (1.27 fold) residues in comparison to MnSOD from control animals. Susceptibility to digestion via thermolysin after incubation with increasing amount of urea (C(m)) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53 M urea) and frozen (2.92 M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different. CONCLUSION: The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs. GENERAL SIGNIFICANCE: Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.
Asunto(s)
Radicales Libres/metabolismo , Congelación , Ranidae/metabolismo , Superóxido Dismutasa/metabolismo , Adaptación Fisiológica , Secuencia de Aminoácidos , Animales , Western Blotting , ADN Complementario/química , ADN Complementario/genética , Estabilidad de Enzimas , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Masculino , Datos de Secuencia Molecular , Músculos/enzimología , América del Norte , Fosforilación , Ranidae/genética , Ranidae/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Superóxido Dismutasa/genética , Superóxido Dismutasa/aislamiento & purificaciónRESUMEN
Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5'- and 116 bp-3'- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.
Asunto(s)
Sepia/enzimología , Sepia/inmunología , Superóxido Dismutasa/aislamiento & purificación , Vibrio/fisiología , Animales , Cadmio/metabolismo , ADN Complementario , Hepatopáncreas , Datos de Secuencia Molecular , Filogenia , Sepia/microbiología , Superóxido Dismutasa/genética , Superóxido Dismutasa/inmunología , Superóxido Dismutasa/metabolismoAsunto(s)
Alérgenos/aislamiento & purificación , Phleum/enzimología , Poaceae/inmunología , Polen/inmunología , Superóxido Dismutasa/inmunología , Alérgenos/inmunología , Perfilación de la Expresión Génica , Humanos , Inmunoglobulina E/inmunología , Extractos Vegetales/inmunología , Proteómica , Análisis de Secuencia de Proteína , Superóxido Dismutasa/aislamiento & purificaciónRESUMEN
Superoxide dismutase (SOD) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species. We cloned cDNA encoding SOD activated with manganese (Mn-SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of Mn-SOD was 1,016 bp and had a 669 bp open reading frame encoding 222 amino acids. The deduced amino acid sequence of B. calyciflorus Mn-SOD showed 89.1, 71.3, and 62.1 % similarity with the Mn-SOD of the marine rotifer Brachionus plicatilis, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, respectively. The phylogenetic tree constructed based on the amino acid sequences of Mn-SODs from B. calyciflorus and other organisms revealed that this rotifer is closely related to nematodes. Analysis of the mRNA expression of Mn-SOD under different conditions revealed that expression was enhanced 5.6-fold (p < 0.001) at 30 °C after 2 h, however, low temperature (15 °C) promoted Mn SOD temporarily (2.5-fold, p < 0.001) and then decreased to normal level (p > 0.05). Moderate starvation promoted Mn-SOD mRNA expression (p 12 < 0.01, p 36 < 0.05), which reached a maximum value (15.3 times higher than control, p 24 < 0.01) at 24 h. SOD and CAT activities also elevated at the 12 h-starved group. These results indicate that induction of Mn-SOD expression by stressors likely plays an important role in aging of B. calyciflorus.
Asunto(s)
Regulación de la Expresión Génica , Rotíferos/genética , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , Datos de Secuencia Molecular , Filogenia , Rotíferos/fisiología , Inanición , Superóxido Dismutasa/aislamiento & purificación , TemperaturaRESUMEN
The total RNA was extracted from ginseng leaves of Panax ginseng. The Cu/Zn-SOD gene was amplified via RT-PCR and the pET-28(a)-Cu/Zn-SOD expression vector was constructed. The pET-28 (a)-Cu/Zn-SOD recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and was induced by IPTG in order to select optimal induction of expression conditions. The target protein was purified by the nickel ions (Ni ) affinity chromatography and the target protein enzyme activity was determinated by the xanthine oxidase method. The similarity of the Cu/Zn-SOD gene sequences and the Cu/Zn-SOD gene sequences of Korean ginseng in NCBI was 99. 00%. The target protein expression level was about 44.42%, and the molecular weight was 16.30 kDa after the pET-28(a)-Cu/Zn-SOD recombinants were induced by IPTG. The purified Cu/Zn-SOD protease activity reached 10,596.69 U x mg(-1). The P. ginseng pET-28(a)-Cu/Zn-SOD prokaryotic expression vector was built by the method of molecular biology, which provided the foundation for studying the Cu/Zn-SOD biology function.
Asunto(s)
Escherichia coli/genética , Ingeniería Genética/métodos , Vectores Genéticos/genética , Panax/enzimología , Panax/genética , Superóxido Dismutasa/genética , Clonación Molecular , Expresión Génica , Análisis de Secuencia , Superóxido Dismutasa/aislamiento & purificación , Superóxido Dismutasa/metabolismoRESUMEN
Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383 bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6 h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83 % after 10 min incubation at 10-50 °C, and more than 87 % after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.
Asunto(s)
Bivalvos/enzimología , Bivalvos/genética , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica , Espacio Intracelular/enzimología , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Estabilidad de Enzimas , Perfilación de la Expresión Génica , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Superóxido Dismutasa/química , Superóxido Dismutasa/aislamiento & purificación , Temperatura , Factores de TiempoRESUMEN
A novel iron superoxide dismutase (Fe-SOD) gene from Jatropha curcas was cloned and expressed in Escherichia coli BL21 (DE3). This recombinant enzyme was isolated by a combined procedure involving immobilized metal-ion affinity chromatography and ion-exchange chromatography. The apparent molecular mass of this purified enzyme (designated as JcFe-SOD) was 35 kDa on SDS-PAGE. The full-length complementary DNA sequence of JcFe-SOD contained a 918-bp open reading frame encoding a 305-amino-acid precursor of 34.589 kDa. The result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed that the purified enzyme may own two forms: a dimer and a monomer. The enzyme was relatively stable and showed 54% activity when incubated in 70°C for 60 Min. JcFe-SOD was found to have good pH stability in the pH range of 5.5-9.5 at 25°C over 1 H incubation. The activity of this enzyme was gradually inhibited by increasing concentration of H2O2, 2-mercaptoethanol, and ethylenediaminetetraacetic acid. An assay of the atomic absorption spectrum showed the presence of 0.41 atom of Fe in each SOD subunit.
Asunto(s)
Jatropha/enzimología , Superóxido Dismutasa/genética , Superóxido Dismutasa/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Estabilidad de Enzimas , Expresión Génica , Concentración de Iones de Hidrógeno , Jatropha/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , TemperaturaRESUMEN
A superoxide dismutase (SOD) with the molecular weight of 31,079 has been purified as a homodimer from Panax ginseng by employing neutral pH buffer extraction, ammonium sulfate precipitation, isoelectric point precipitation and ion exchange methods. The enzyme's specific activity determined by an improved Marklund method was 9480.43 U/mg. Metal analysis showed that the SOD contained iron with the stoichiometry of 0.9 ± 0.3 Fe/subunit and exhibited high thermal stability (70 °C) over the pH range from 4.0 to 9.0. Its maximum absorption wavelength was 278 nm and it was sensitive to hydrogen peroxide, trichloromethane-ethanol and urea. These results indicate that the enzyme is an iron SOD.
Asunto(s)
Panax/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Superóxido Dismutasa/química , Superóxido Dismutasa/aislamiento & purificación , Precipitación Química , Cromatografía por Intercambio Iónico , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Peso Molecular , Panax/químicaRESUMEN
* Author for correspondence and reprint requests Z. Naturforsch. 65c, 141-147 (2010); received June 17/July 21, 2009 The superoxide radical (O2-), hydrogen peroxide (H2O2) and nitric oxide (NO) are pleiotropic inflammatory mediators which play an important role in inflammatory joint diseases. They are overproduced during rheumatoid arthritis and its experimental model - adjuvant-induced arthritis in rodents--and may be detected both systemically and intra-articularly. Their secretion is up-regulated by proinflammatory cytokines such as IFN-gamma, IL-12, IL-6 and TNF-alpha, and they are responsible for the destruction of joint tissue. In this work, the effect of superoxide dismutase (SOD) from a thermotolerant yeast strain, Kluyveromyces marxianus, on the production of proinflammatory cytokines, reactive oxygen and nitrogen species was studied. Mice received three intraperitoneal injections of yeast SOD at a dose of 10 mg/ kg body weight (30,000 U/kg) on consecutive days starting on the day after arthritic induction. On days 3, 8 and 14 post induction peritoneal macrophages were isolated and both spontaneous and stimulated production of reactive oxygen and nitrogen metabolites were measured. Early in arthritic development yeast SOD treatment did not influence the O2- production, but on day 14 both spontaneous and PMA-induced secretion were dramatically reduced. Spontaneous H2O2 release was inhibited on day 14, while PMA-stimulated production was decreased from the beginning of the arthritic development. Yeast SOD treatment effectively suppressed the spontaneous and recombinant mouse IFN-gamma + LPS induced release of NO as well. Serum levels of proinflammatory cytokines, IL-12, IFN-gamma, IL-6 and TNF-alpha, were also significantly reduced. The obtained results show some of the mechanisms of action of SOD in reducing the severity of arthritic inflammation. Besides direct inhibition of joint tissue destruction exogenous SOD substantially limits the existing positive feedback between secretion of reactive oxygen species and inflammatory cytokine production.
Asunto(s)
Artritis Experimental/fisiopatología , Inflamación/tratamiento farmacológico , Inflamación/etiología , Kluyveromyces/enzimología , Superóxido Dismutasa/uso terapéutico , Animales , Artritis Experimental/sangre , Artritis Experimental/tratamiento farmacológico , Citocinas/sangre , Edema/etiología , Inflamación/sangre , Inyecciones , Ratones , Ratones Endogámicos ICR , Superóxido Dismutasa/administración & dosificación , Superóxido Dismutasa/aislamiento & purificación , Superóxidos/metabolismoRESUMEN
INTRODUCTION: Rheumatoid arthritis is an autoimmune inflammatory disease of unknown etiology, free radicals have been implicated in the genesis and perpetuation of damage in this pathology. OBJECTIVE: To evaluate the anti-inflammatory effect of Cu,Zn-superoxide dismutase (SOD) obtained from two different sources (bovine erythrocytes, Be-SOD, and Debaryomyces hansenii, Dh-SOD) with Type II Collagen-induced Arthritis model in rats. MATERIAL AND METHODS: Arthritis was induced by repeated injection of a porcine type II collagen-incomplete Freund adjuvant suspension on the back of Dark Augui (DA) rats. Arthritis was clinically evaluated throughout the study. Body weight was determined at three different times. Two different doses for each treatment (Be-SOD, Dh-SOD) were tested: 100 and 1,000 U/kg. At the end of the trial (day 28), histological analyses of the most inflamed ankle joint, as well as serum anti-collagen antibodies, were determined. RESULTS: Both sources of SOD decreased, although to a different extent, the incidence and severity of the disease. Arthritis score was lower in all treatments, except for the low dose of Be-SOD. Groups receiving either source of SOD showed a significant weight increase compared to the placebo group. Histological damage was similar in all groups. Only the group that received the highest dose of Dh-SOD showed a significant lower antibody titer; nevertheless, no correlation appears to derive from arthritis score and antibody titer. CONCLUSION: Our findings suggest that, although unable to counteract the arthritis syndrome, SOD may still be beneficial due to its anti-inflammatory activity. In the case of Dh-SOD, the best effect was observed at the highest dose tested.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Debaryomyces/enzimología , Proteínas Fúngicas/uso terapéutico , Superóxido Dismutasa/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/aislamiento & purificación , Antirreumáticos/administración & dosificación , Antirreumáticos/aislamiento & purificación , Artritis Experimental/sangre , Artritis Experimental/patología , Artritis Experimental/prevención & control , Artritis Reumatoide , Autoanticuerpos/sangre , Bovinos , Colágeno Tipo II/toxicidad , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Eritrocitos/enzimología , Femenino , Fibrosis , Proteínas Fúngicas/administración & dosificación , Proteínas Fúngicas/aislamiento & purificación , Hiperplasia , Inyecciones Intraperitoneales , Ratas , Especificidad de la Especie , Superóxido Dismutasa/administración & dosificación , Superóxido Dismutasa/aislamiento & purificaciónRESUMEN
Muskmelons (Cucumis melo L.) are well-known as excellent sources of several vitamins, minerals and non-enzymatic antioxidant phytochemicals such as vitamin C and pro-vitamin A. Less well-studied is their potential role as sources of enzymatic antioxidants such as superoxide dismutase (SOD), which have been associated with enhanced reactive oxygen species scavenging capacity in some muskmelon fruits. In this study, we investigated the variability in SOD activities among diverse advanced breeding lines and commercial muskmelon cultivars grown in two different soil types-clay or sandy loam. Specific and total SOD activities varied significantly among the genotypes (P Asunto(s)
Antioxidantes/metabolismo
, Cucumis melo/enzimología
, Frutas/enzimología
, Extractos Vegetales/metabolismo
, Superóxido Dismutasa/metabolismo
, Antioxidantes/aislamiento & purificación
, Cucumis melo/genética
, Variación Genética
, Genotipo
, Extractos Vegetales/química
, Suelo
, Superóxido Dismutasa/aislamiento & purificación
RESUMEN
Reactive oxygen species are implicated in cancer development and antioxidants in general and superoxide dismutases and superoxide dismutase mimetic in particular, and they inhibit malignant transformation. We examinated the effects of an isolated manganese superoxide dismutase from a medicinal plant Allium sativum. The protein was prepared by a serial of chromatographic techniques: gel filtration and diethylaminoethyl ions exchanger. The enzyme has a specific activity equal to 55 U/mg. Two tumoral cell lines, porcine endothelial cells and mouse melanoma cells were exposed to garlic superoxide dismutase. The exogenous manganese superoxide dismutase is able to modify the intracellular level of reactive oxygen species by eliminating superoxide anion and producing hydrogen peroxide. The cell viability of the two lines was not significantly affected but the cell multiplication was arrested. This effect obtained in the presence of manganese superoxide dismutase correlates with the activation and modulation of phospho-extracellular signal-regulated kinases proteins, implicated in the control of several biological processes including cell proliferation.
Asunto(s)
Ajo/enzimología , Neoplasias/tratamiento farmacológico , Superóxido Dismutasa/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/aislamiento & purificación , Superóxidos/metabolismoRESUMEN
The stalk, a characteristic structure of the large ribosomal subunit, is directly involved in the interaction with the soluble factors during translation. In the Mediterranean mussel Mytilus galloprovincialis, the stalk consists of one 32 kDa protein, MgP0, and two smaller, 12 kDa acidic proteins, MgP1 and MgP2, of pI 3.0 and 4.0, respectively, as revealed by analysis of purified ribosomes with electrophoresis and Western blot with a specific monoclonal antibody. Treatment of the ribosomes with alkaline phosphatase showed movement of the bands corresponding to the acidic MgP1 and MgP2 proteins to more basic pH after isoelectrofocusing, implying phosphorylation. The cDNA molecules of M. galloprovincialis ribosomal proteins MgP0, MgP1 and MgP2 and superoxide dismutase (MgSOD) were isolated from a cDNA library or constructed by RT-PCR, cloned in expression vectors and expressed in Escherichia coli. The recombinant proteins were purified with immobilized metal ion affinity chromatography (IMAC) and identified with immunoblotting. Exposure of mussels at cadmium and sorbitol and analysis of gill tissue extracts showed over expression of MgP0 protein.