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2.4. Isolation of head kidney phagocytes Five specimens of soles of 200 g mean weight were sacrificed by an overdose of clave oil and head kidney phagocytes to test respiratory burst activity were isolated following the technique described by Secombes [32]. Briefly, the kidney was removed aseptically and pushed through a 100 μm nylon mesh with Leibovitz medium (L-15) containing 2% foetal bovine serum (FBS, Sigma), 1% penicillin-streptomycin (Sigma), 0.1% (5 mg ml -1 ) gentamicine sulfate (Sigma) (P/S/G) and 10 U heparine ml -1 . This cell suspension was layered on a 30 to 51% Percoll (Amersham) gradient and centrifuged at 600 xg for 30 min, without brake. Then, the bands separated at the interface were collected, centrifuged for 15 min at 500 xg and resuspended in L-15 medium supplemented with P/S/G. The viable cell concentration was determined after staining with trypan blue and microscope counting. Aliquots of 100 μl containing 1x10 7 cells ml - 1 in L-15 medium supplemented with P/S/G were added to 96-well microtitre plates. After 3 h incubation at 22ºC, non-adherent cells were removed and medium was substituted by L-15 and P/S/G supplemented with 2%FBS. Monolayers were incubated overnight at 22ºC, before use. 2.5. Respiratory burst activity The generation of intracellular superoxide radicals by sole phagocytes, in response to in vitro contact with algal polysaccharide, was determined by the reduction of nitro-blue tetrazolium (NBT) according to the technique described by Secombes [32] and Boesen et al. [33]. Volumes of 20 μl containing 1, 2, 5 and 10 mg ml -1 of the lyophilized polysaccharidic fraction from P. cruentum were added to the wells containing phagocyte monolayers obtained as described above. The response of sole phagocytes to β-glucan from Euglena gracilis was also evaluated and 20 μl of serial dilutions containing 1, 2, 5 and 10 mg ml -1 to 12 wells with sole phagocyte monolayers. Response of sole phagocytes to the infection with P. damselae subsp. piscicida was determined after inoculation of algal polysaccharide or β-glucan treated monolayers with 20μl of bacterial suspensions containing 10 8 bacteria ml -1 . Phorbol myristate acetate (PMA, Sigma) (1 μg ml -1 ) was used as a positive control to stimulate the respiratory burst activity of sole phagocytes (data not shown). The specifity of the 6
eaction was tested by adding superoxide dismutase (SOD) (300 I.U. per well) to some wells containing PMA-stimulated phagocytes (data not shown). The effect of inoculation of polysaccharidic fraction to unvaccinated and vaccinated soles against P. damselae subsp. piscicida on the production of superoxide anions by sole phagocytes was also determined. In this case, phagocytes from control fish, fish inoculated with algal polysaccharide, and fish inoculated first with the algal polysaccharide and with the bacterin were isolated and monolayers prepared. In all the cases, NBT (100 μl) dissolved at 1 mg ml -1 in HBSS was added to the wells and phagocytes incubated at 22 ºC for 30 min. After incubation, cells were fixed in 70% methanol and the reduced formazan within phagocytes was solubilised by adding 120 μl 2M KOH and 140 μl dimethyl sulfoxide (DMSO, Sigma). Finally, absorbance was read at 630 nm in a multiscan spectrophotometer (UV-1601 Spectrophotometer, Whitakker Bioproducts). 2.6. Statistical analysis Results are expressed as phagocyte stimulation index, calculated by dividing each sample value by the mean control value. Values above 1 reflect an increase and under 1 a decrease in each parameter. Data were statistically analysed by one-way analysis of variance (ANOVA) and Tukey’s comparison of means using SPSS for Windows. Differences were considered statistically significant when P< 0.05. 3. Results Results obtained in vitro after incubation of phagocytes with algal extracts are shown in Figure 1a. The data are expressed as a phagocytic stimulation index obtained by dividing superoxide anion produced by phagocytes incubated with algal polysaccharide or with algal polysaccharide plus bacteria by the values of phagocytes incubated without the algal polysaccharidic fraction. Data obtained after incubation with β-glucan instead of algal polysaccharide are expressed in Figure 1b. Results obtained show that only at the commercial β-glucan significantly enhanced the respiratory burst activity of sole phagocytes at the highest concentration (10 mg ml -1 ), compared to non 7
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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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RESULTS AND DISCUSSION resist 100 m
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RESULTS AND DISCUSSION present work
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RESULTS AND DISCUSSION reason, once
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RESULTS AND DISCUSSION great number
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RESULTS AND DISCUSSION dominant spe
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CONCLUSIONS Studies carried out on
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R R E F E R E N C I A S E F E R E N
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REFERENCIAS / REFEREN CES Aoki, T,
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REFERENCIAS / REFEREN CES Bell, MV,
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REFERENCIAS / REFEREN CES Dalmo, RA
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REFERENCIAS / REFEREN CES Hamaguchi
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REFERENCIAS / REFEREN CES Kono, Y &
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REFERENCIAS / REFEREN CES Magariño
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REFERENCIAS / REFEREN CES Ortuño,
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REFERENCIAS / REFEREN CES Salinas,
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REFERENCIAS / REFEREN CES Thompson,
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S E C C I Ó N D E A R T Í C U L O
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A RTÍCULO 1.1. A RTICLE 1.1.
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Journal of Fish Diseases 2003, 26,
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Journal of Fish Diseases 2003, 26,
Page 167 and 168: 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: On the other hand, β-1,3-glucan fr
Page 221 and 222: Intraperitoneal inoculation of the
Page 223 and 224: defense mechanisms and protective i
Page 225: phagocytes from sole (Solea senegal
Page 229: Figure 1a Stimulation index 3 2.5 2
Page 233: Figure 2 Stimulation index 1.4 1.2
Page 237 and 238: Effect of dietary administration of
Page 239 and 240: 1. Introduction Senegalese sole cul
Page 241 and 242: 2.2. Fish and experimental design S
Page 243 and 244: 2.5. Challenge with Photobacterium
Page 245 and 246: the densitometric curves of the pro
Page 247 and 248: most efficient probiotics for aquac
Page 249 and 250: acterial diversity only the dominan
Page 251 and 252: [9] Verschuere, L, Rombaut, G, Sorg
Page 253 and 254: [28] Burr, G, Gathin, D and Ricke,
Page 255 and 256: WB, editors. Techniques in Fish Imm
Page 257: and host-specific communities of ac
Page 261: Table 1 Primer Sequence (5’-3’)
Page 265: 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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