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 (a) (b) 1 2 3 4 5 6 1 2 3 4 5 6 Figure 2 Detection of superoxide dismutase (a) and catalase activity (b) in extracts of Photobacterium damselae subsp. piscicida (strain EPOY-8803-II) grown under different conditions. Lane 1: growth until exponential phase; 2: stationary growth phase; 3: exposure to hydrogen peroxide (20 lm H 2 O 2 mid-exponential phase and 2 mm H 2 O 2 early stationary phase); 4: addition of methyl viologen (0.2 mm) to the culture medium; 5: addition of 2,2¢-dipyridyl (100 lm) to the culture medium; 6: addition of FeCl 3 Æ6H 2 O (100 lm) to the culture medium. U SOD/mg prot. 20 15 10 5 0 EPOY-8803-II Lgh41/01 Figure 3 Superoxide dismutase activity (U mg )1 protein) of Photobacterium damselae subsp. piscicida strains grown under different culture conditions. ( ) Growth until stationary phase; ( ) exposure to hydrogen peroxide (20 lm mid-exponential phase and 2 mm early stationary phase); ( ) culture supplemented with FeCl 3 Æ6H 2 O 100 lm and (h) culture supplemented with the iron chelant 2,2¢-dipyridyl 100 lm. Data represent the mean (SD) of three independent determinations. U cat./mg prot. 140000 120 000 100000 80000 60000 40000 20 000 0 EPOY Lgh41/01 Figure 4 Catalase activity (U mg )1 protein) of Photobacterium damselae subsp. piscicida strains grown under different culture conditions. ( ) Growth until stationary phase; ( ) exposure to hydrogen peroxide (20 lm mid-exponential phase and 2 mm early stationary phase); ( ) culture supplemented with FeCl 3 Æ 6H 2 O 100 lm and (h) culture supplemented with the iron chelant 2,2¢-dipyridyl 100 lm. Data represent the mean (SD) of three independent determinations. In order to determine the influence of the levels of SOD and catalase activity on the resistance to the bactericidal activity of sole phagocytes, killing assays were carried out with a virulent and non-virulent strain of P. damselae ssp. piscicida. The percentages of surviving bacteria after 5 h contact with sole phagocytes are shown in Fig. 5. It can be observed that survival of the virulent strain in contact with phagocytes was significantly higher (P < 0.05) in all cases compared with the non-virulent strain. Despite this different survival rate, both strains showed a similar behaviour depending on the bacterial culture condition with highest rates corresponding in both cases to growth in ironreplete broths and lowest to growth under ironlimiting conditions. In addition, a significant increase in the survival percentages was observed %Survival 100 90 80 70 60 50 40 30 20 10 0 EPOY-8803-II Lgh41/01 Figure 5 Survival percentage of Photobacterium damselae subsp. piscicida after 5 h in contact with sole phagocytes. ( ) Growth until stationary phase; ( ) exposure to hydrogen peroxide (20 lm mid-exponential phase and 2 mm early stationary phase); ( ) culture supplemented with FeCl 3 Æ6H 2 O 100 lm and (h) culture supplemented with 2,2¢-dipyridyl 100 lm. Data represent the mean (SD) of nine wells containing phagocytes from three fish specimens. Ó 2006 Blackwell Publishing Ltd 360
Journal of Fish Diseases 2006, 29, 355–364 PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida in both strains pulsed with hydrogen peroxide compared with stationary phase cultures. Discussion Enzymes such as SOD and catalase, which neutralize ROS produced during aerobic metabolism or during respiratory burst in fish phagocytes are important virulence factors in many pathogens (Barnes et al. 1996, 1999b; Yesilkaya, Kadioglu, Gingles, Alexander, Mitchell & Andrew 2000; Vattanaviboon & Mongkolsuk 2001; Uzzau et al. 2002; Banin, Vassilakos, Orr, Martínez & Rosenberg 2003). In this study, all the strains of P. damselae ssp. piscicida assayed showed a single band of SOD activity with identical mobility on acrylamide gels. A unique band similar in all the strains was also observed on catalase activity gels. Similarly, Barnes, Balebona, Horne & Ellis (1999a), in a study that included a collection of P. damselae ssp. piscicida strains isolated from gilthead seabream, Sparus aurata (L.), reported only one SOD located in the periplasmic space and one cytoplasmic catalase. Several studies have reported that microorganisms contain different SOD and catalase isozymes inducible under certain growth conditions (Storz, Tartaglia, Farr & Ames 1990; Privalle & Fridovich 1992; Barnes et al. 1996; Yesilkaya et al. 2000; Geslin, Llanos, Prieur & Jeanthon 2001; Vattanaviboon & Mongkolsuk 2001). However, culture conditions assayed in this work have not induced new SOD or catalase isozymes in P. damselae ssp. piscicida. Mn-SOD activity has been reported to be modulated by oxidative stress and iron-limiting conditions (Privalle & Fridovich 1992; Barnes et al. 1999b) but in the case of P. damselae ssp. piscicida neither production of intracellular superoxide by methyl viologen nor culture under ironrestricted conditions induced the production of a different type of SOD. Although further studies are necessary, this lack of induction of a new SOD could be due to the presence of only one sod gene, i.e. sod B encoding Fe-SOD (Lynch & Kuramitsu 2000). In contrast, differences in the intensity of the bands were observed in extracts obtained under different culture conditions for both SOD and catalase activities. As the amount of protein loaded in the electrophoretic lanes was similar in all cases, the different intensities suggest variations in the levels of activity in the extracts depending on the culture condition. These results are in agreement with those obtained by Barnes, Balebona, Horne & Ellis (1999a), who also detected differences in cultures carried out under iron replete and depleted conditions and high- and low-aerated broths. The quantification of both SOD and catalase activities carried out in this study corroborated that different band intensities corresponded to variations in the levels of activity. The lowest levels of SOD activity were detected when bacteria were grown under iron-restricted conditions. The ferric nature of P. damselae ssp. piscicida SOD described by Barnes et al. (1999a) could explain this lower activity in the presence of an iron chelant. Iron also influenced the levels of catalase activity in P. damselae ssp. piscicida. The role of iron as cofactor in this enzyme has been demonstrated with inhibition studies. Thus, catalase activity could not be detected in the gels following exposure to sodium azide and it was slightly reduced after treatment with potassium cyanide. These results suggest that the enzyme is an iron cofactored catalase, as Mn-containing catalases retain activity after treatment with azide and cyanide and are inhibited by mercuric chloride (Kono & Fridovich 1983; Allgood & Perry 1986; Barnes et al. 1999b). This ferric nature of the catalase may explain the lower catalase activity observed in cultures with added iron chelant and lower survival with H 2 O 2 observed by Díaz-Rosales, Chabrillón, Moriñigo & Balebona (2003). Lower survival of P. damselae ssp. piscicida in sole phagocytes has been observed for strain EPOY- 8803-II compared with the virulent strain. Contradictory results have been reported on the ability of P. damselae ssp. piscicida to survive inside macrophages from several fish species. In a study using macrophages from sea bass, gilthead sea bream and rainbow trout, Skarmeta et al. (1995) concluded that head kidney macrophages from these fish species were able to kill the pathogen. However, Noya et al. (1995b) reported that whilst bacteria within granulocytes and macrophages from large gilthead sea bream were morphologically altered, bacteria inside small fish remained unaffected. In addition, data on the ability of P. damselae to survive inside fish macrophages have been reported by several authors who observed that bacteria can multiply inside fish macrophages (Kubota et al. 1970; Hawke, Plakas, Minton, McPherson, Zinder & Guarino 1987; Noya et al. 1995a; Elkamel, Hawke, Henk & Thune 2003). Ó 2006 Blackwell Publishing Ltd 361
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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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Page 131 and 132: CONCLUSIONS Studies carried out on
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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 171: Journal of Fish Diseases 2006, 29,
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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
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Page 221 and 222: 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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