Papel de las actividades superóxido dismutasa y catalasa en la ...

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Journal of Fish Diseases 2006, 29, 355–364 PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida intervals by a spectrophotometer (Hitachi U-2000: Hitachi, Tokyo, Japan). The amount of enzyme resulting in 50% of maximum inhibition of NBT reduction was determined. Detection, characterization and quantification of catalase activity Catalase activity was visualized on non-denaturing acrylamide gels following the methodology of Woodbury, Spencer & Stahmann (1971). After electrophoresis, gels were washed three times in distilled water for 20 min and soaked in a solution of 0.015% H 2 O 2 (30%) (Merck, Darmstadt, Germany). Then, the activity was visualized by transferring the gels to a solution of 1% (w/v) ferric chloride (Panreac Quimica, Barcelona, Spain) and 1% (w/v) potassium ferricyanide (Sigma). Regions corresponding to catalase activity were identified as clear yellow bands on a dark green background. The metal cofactor of the catalase produced by P. damselae ssp. piscicida was determined by enzymatic inhibition studies according to Barnes, Bowden, Horne & Ellis (1999b). Lysates of P. damselae ssp. piscicida strains were incubated for 1 h with either 100 and 50 mm potassium cyanide (KCN; Sigma), 1 and 0.5 mm mercuric chloride (HgCl 2 ; Sigma), 25 and 12.5 mm sodium azide (NaN 3 ; Sigma) or 50 mm phosphate buffer as control. Equal volumes of treated extracts were electrophoresed and gels stained for catalase activity (Woodbury et al. 1971). Catalases with manganese as metal cofactor are resistant against sodium azide and potassium cyanide and sensitive to mercuric chloride (Kono & Fridovich 1983; Allgood & Perry 1986; Barnes et al. 1999b). Control wells inoculated with extracts of Escherichia coli (ATCC 13706) containing a ferric catalase retained the activity after treatment with mercuric chloride but not with sodium azide. Catalase activity was measured spectrophotometrically by monitoring the decrease in absorbance at 240 nm due to decomposition of hydrogen peroxide. One unit of catalase was defined as the activity causing the hydrolysis of 1 lmol of hydrogen peroxide per minute (Aebi 1984). Briefly, bacterial extracts were diluted (1:100) in 50 mm potassium phosphate buffer, pH 7.0 and the absorbance of the sample containing 660 lL of lysate and 340 lL of H 2 O 2 was measured against a blank with buffer. The decrease in absorbance at 240 nm (Hitachi U-2000) was monitored during a 10-min period. Bactericidal activity of sole phagocytes Monolayers of sole phagocytes were prepared following the methodology of Secombes (1990). Briefly, the kidneys of 100–300 g sole were dissected and pressed through a 100 lm nylon mesh with L-15 medium (Gibco, Gaithersburg, MD, USA) containing 2% fetal calf serum (FCS; Sigma), 1% penicillin/streptomycin (Sigma), 0.1% gentamicin sulphate (50 mg mL )1 distilled water; Sigma) and 10 U mL )1 sodium heparin. The resultant suspension was layered onto a 30–51% (v/v) Percoll (Amersham Pharmacia, Piscataway, NJ, USA) density gradient and the band of cells lying at the 30– 51% interface was collected. The cell suspension was washed and adjusted to 10 7 cells mL )1 in L-15 medium with antibiotics. The viability was determined by the exclusion test with trypan blue (Sigma) (0.5% in PBS). A volume of 100 lL per well was added to 96-well microtitre plates. Monolayers were maintained at 22 °C overnight until bactericidal assays were performed. Bacterial culture conditions to determine the ability to resist the bactericidal activity of phagocytes included growth until stationary phase, addition of two hydrogen peroxide pulses and growth in replete or reduced iron medium as previously described. The bacterial concentration was adjusted to 1 OD 600 , corresponding to 10 8 bacteria per mL. The methodology employed to test bacterial survival after contact with phagocytes was according to Secombes (1990). Phagocyte monolayers were washed twice with L-15 and the cells were then supplemented with 100 lL L-15, 5% FCS per well. Bacterial suspensions (20 lL) were added to triplicate wells containing macrophages. The microtitre plate was shaken and centrifuged at 150 g for 5 min to bring the bacteria into contact with cells and subsequently incubated at 22 °C for 0 and 5 h. At the end of the incubation period, the supernatants were removed and the killing stopped by lysing the phagocytes with 50 lL of cold sterile distilled water. Subsequently, 100 lL of TSBs was added to support the growth of the surviving bacteria for 18–20 h at 22 °C. The number of surviving bacteria was quantified colorimetrically following the methodology of Peck (1985) as modified by Graham, Jeffries & Secombes (1988). Briefly, 10 lL of 3 [4,5-di- Ó 2006 Blackwell Publishing Ltd 358
Journal of Fish Diseases 2006, 29, 355–364 PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida methylthiazoyl-2-yl] 2,5-diphenyltetrazolium bromide (MTT, Sigma) (5 mg mL )1 distilled water) was added to the wells, plates were shaken and absorbance at 550 nm was read after 15-min incubation on a multiscan spectrophotometer (Microplate Reader 2001; Whittaker Bioproducts Inc., Walkersville, MD, USA). The percentage of surviving bacteria was calculated by dividing the absorbance obtained from the wells incubated with bacteria for 5 h by the values obtained from wells incubated with bacteria for 0 h. Statistical analysis Quantification of enzymatic activities was carried out in three independent experiments. Fish experiments were performed in triplicate, data corresponding to measurements were carried out with phagocytes from three different fish and three replicate wells for each fish. An anova test was performed to compare the results obtained. P < 0.05 was considered significant. Results (a) 1 2 3 4 5 6 7 8 9 10 (b) 1 2 3 4 5 6 7 8 9 10 Figure 1 Detection of superoxide dismutase (a) and catalase activity (b) in extracts of different strains of Photobacterium damselae subsp. piscicida grown until stationary phase. Lane 1: ATCC 17911; 2: B51; 3: B180; 4: DI-21S; 5: D 26/98 ; 6: Pp8H; 7: R45; 8: Lg h41/01 ; 9: MT 1415 and 10: MT 1379. All the extracts of the strains of P. damselae ssp. piscicida included in this study produced similar SOD and catalase activity bands (Fig. 1). Thus, a single band with identical mobility in native polyacrylamide gel electrophoresis gels was observed for all isolates and culture conditions assayed (Fig. 2). Similar protein concentrations were loaded in the gel lanes. However, differences in the intensity of the SOD and catalase bands were observed. Thus, SOD and catalase activity bands showed lower intensity in the extracts from cultures carried out under iron-limiting conditions, whilst increased intensity of SOD bands was observed in extracts from cultures under iron-supplemented conditions and in the presence of the cytoplasmic superoxide radical generator, methyl viologen (Fig. 2). Two isolates with different degrees of virulence for sole were selected for further characterization: one virulent, Lg h41/01 (LD 50 ¼ 2.8 · 10 4 cfu g )1 fish) and one non-virulent, EPOY-8803-II (LD 50 > 7 · 10 6 cfu g )1 fish). Cultures carried out until the early stationary phase of the non-virulent isolate contained significantly (P < 0.05) lower amounts of SOD than cultures of the virulent strain. However, when iron was added to the growth broth, EPOY-8803-II contained significantly higher amounts of SOD (Fig. 3). There was no significant hydrogen peroxide induction of SOD in any of the strains, and indeed a decrease in activity in strain Lg h41/01 was detected (Fig. 3). In contrast, cells of both strains cultured under iron limiting or replete conditions contained significantly different amounts of SOD activity. In all the cases, growth under iron-limiting conditions resulted in a significant decrease in SOD activity compared with iron replete conditions, this decrease being more important in the non-virulent strain than in the virulent strain. Unlike SOD, catalase activity in cultures of the non-virulent strain was lower than in the virulent strain (Fig. 4). Moreover, whilst no significant differences were observed in catalase contents of Lg h41/01 cultures grown until stationary phase and those pulsed with hydrogen peroxide, strain EPOY- 8803-II showed a considerably greater amount of catalase activity when cultures were pulsed with hydrogen peroxide. A significant decrease of activity was also observed for cultures of both strains carried out under iron-limiting conditions compared with iron-overloaded broths. Catalase activity could not be detected in the gels following exposure to 100 mm sodium azide and treatment with potassium cyanide resulted in a slight reduction of activity, suggesting that this bacterium contains an iron-cofactored enzyme. Ó 2006 Blackwell Publishing Ltd 359
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FACULTAD DE CIENCIAS DEPARTAMENTO D
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Los ensayos que constituyen esta Te
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- Díaz-Rosales, P, Arijo, S, Chabr
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Sabe esperar, aguarda que la marea
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ÍNDICE / INDEX Papel de las activi
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ÍNDICE / INDEX 2. Photobacterium d
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RESUMEN Photobacterium damselae sub
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INTRODUCCIÓN 1. LA ACUICULTURA. EL
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INTRODUCCIÓN saxatilis) (Hawke et
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INTRODUCCIÓN 2.2. SINTOMATOLOGÍA
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INTRODUCCIÓN las epidemias surgida
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INTRODUCCIÓN et al., 1997). En P.
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INTRODUCCIÓN 3.1. ESTALLIDO RESPIR
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INTRODUCCIÓN radicales generados e
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INTRODUCCIÓN Tabla 2 Ejemplos de m
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INTRODUCCIÓN de parasitismo en el
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INTRODUCCIÓN razón de la búsqued
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INTRODUCCIÓN araquidónico, están
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INTRODUCCIÓN simplificar la admini
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INTRODUCCIÓN sino también cuando
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INTRODUCCIÓN La necesidad de mejor
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OBJETIVOS El trabajo planteado en e
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MATERIA L Y MÉTODOS La metodologí
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RESULTADO S Y DISCUSIÓN El primer
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RESULTADO S Y DISCUSIÓN 1996; Lync
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RESULTADO S Y DISCUSIÓN Una vez de
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RESULTADO S Y DISCUSIÓN inmunoesti
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RESULTADO S Y DISCUSIÓN cruentum s
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RESULTADO S Y DISCUSIÓN permiten c
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RESULTADO S Y DISCUSIÓN harían fa
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CONCLUSIONES Tras los estudios real
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ABSTRACT Photobacterium damselae su
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INTRODUCTION 1. AQUACULTURE. THE CU
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INTRODUCTION With regard to Solea s
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INTRODUCTION 3.1. RESPIRATORY BURST
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INTRODUCTION Some catalases have al
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INTRODUCTION pathogens, study of th
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INTRODUCTION increase their bacteri
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INTRODUCTION 4.3.1. Porphyridium cr
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INTRODUCTION Different mechanisms h
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A I M S
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M A T E R I A L S A N D M E T H O D
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R E S U L T S A N D D I S C U S S I
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Page 122 and 123: RESULTS AND DISCUSSION present work
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Page 131 and 132: CONCLUSIONS Studies carried out on
Page 133: R R E F E R E N C I A S E F E R E N
Page 136 and 137: REFERENCIAS / REFEREN CES Aoki, T,
Page 138 and 139: REFERENCIAS / REFEREN CES Bell, MV,
Page 140 and 141: REFERENCIAS / REFEREN CES Dalmo, RA
Page 142 and 143: REFERENCIAS / REFEREN CES Hamaguchi
Page 144 and 145: REFERENCIAS / REFEREN CES Kono, Y &
Page 146 and 147: REFERENCIAS / REFEREN CES Magariño
Page 148 and 149: REFERENCIAS / REFEREN CES Ortuño,
Page 150 and 151: REFERENCIAS / REFEREN CES Salinas,
Page 152 and 153: REFERENCIAS / REFEREN CES Thompson,
Page 155: S E C C I Ó N D E A R T Í C U L O
Page 159: A RTÍCULO 1.1. A RTICLE 1.1.
Page 162 and 163: Journal of Fish Diseases 2003, 26,
Page 169: Journal of Fish Diseases 2006, 29,
Page 177: A RTÍCULO 2.1. A RTICLE 2.1.
Page 180 and 181: Abstract The stimulatory effect of
Page 182 and 183: alga Porphyridium cruentum. For thi
Page 184 and 185: Feeding assays were carried out wit
Page 186 and 187: (10 8 bacterias ml -1 ) and used to
Page 188 and 189: are in agreement with the data repo
Page 190 and 191: unvaccinated fish. However, the ser
Page 192 and 193: immune response of gilthead seabrea
Page 194 and 195: [27] Duncan, PL and Klesius, PH. Ef
Page 196 and 197: (Macrogard) and alginic acid (Ergos
Page 198 and 199: [66] Salinas, I, Díaz-Rosales, P,
Page 201: Figure 1a 3 2.5 Stimulation index 2
Page 205: Figure 1c Stimulation index 3 2.5 2
Page 209: Figure 2b Stimulation index 3 2.5 2
Page 213 and 214: Effect of the extracellular polysac
Page 215 and 216: 1. Introduction Solea senegalensis
Page 217 and 218: On the other hand, β-1,3-glucan fr
Page 219 and 220: eaction was tested by adding supero
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Intraperitoneal inoculation of the
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defense mechanisms and protective i
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phagocytes from sole (Solea senegal
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Figure 1a Stimulation index 3 2.5 2
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Figure 2 Stimulation index 1.4 1.2
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Effect of dietary administration of
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1. Introduction Senegalese sole cul
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2.2. Fish and experimental design S
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2.5. Challenge with Photobacterium
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the densitometric curves of the pro
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most efficient probiotics for aquac
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acterial diversity only the dominan
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[9] Verschuere, L, Rombaut, G, Sorg
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[28] Burr, G, Gathin, D and Ricke,
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WB, editors. Techniques in Fish Imm
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and host-specific communities of ac
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Table 1 Primer Sequence (5’-3’)
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Figure 1b Stimulation index 2.5 2 1
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Table 2 Primer Control Pdp11 Pdp13
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Figure 3 Pearson correlation [0.0%-
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que perdieras parte de tu tiempo en
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