RESUMEN
The acute-phase protein (APP) response to an infection caused by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized measuring serum concentrations of pig major acute-phase protein (pig MAP), haptoglobin (HPT), C-reactive protein (CRP) and apolipoprotein A-I (ApoA-I) in colostrum-deprived pigs. They were divided into six experimental groups: non-immunized control group (I); immunized with a non-commercial bacterin (II); with an OMP-vaccine (III); with a sublethal dose (IV); and with two commercial bacterins (V and VI). All groups were challenged intratracheally with 5 × 10(9)CFU of H. parasuis 37 days after immunisation. The highest levels of the positive APPs (pig MAP, HPT and CRP) and the lowest levels of the negative APPs (ApoA-I) were observed in the animals that died as a consequence of the infection, both those in the non-immunized and in the immunized groups. However, the surviving animals (all of them in groups II, V and VI, two pigs in group III, and three in group IV) showed a minor variation in APP response, mainly on day 1 post-challenge (p.c.), and then tended to recover the initial values. APP response was still less pronounced in the groups of pigs previously immunized with bacterins. In conclusion, APP response can reflect Glässer-disease ongoing, showing a correlation between the severity and duration of the clinical signs and lesions and the magnitude of changes in the APP levels.
Asunto(s)
Proteínas de Fase Aguda/análisis , Reacción de Fase Aguda , Infecciones por Haemophilus/veterinaria , Vacunas contra Haemophilus/inmunología , Haemophilus parasuis/inmunología , Enfermedades de los Porcinos/inmunología , Animales , Apolipoproteína A-I/sangre , Proteína C-Reactiva/análisis , Calostro , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/metabolismo , Haptoglobinas/análisis , Inmunización/veterinaria , Masculino , Porcinos , Enfermedades de los Porcinos/metabolismoRESUMEN
The objective of this study was to investigate, using a pulse-chase technique, the different incorporation of (1-(14)C) n -9, n -6 and n 3 fatty acids into hepatoma lipids and their secretion to the culture medium. Docosahexaenoic acid (DHA) accumulated preferentially into the triacylglycerol while arachidonic acid (AA) did into the phospholipid fraction. DHA was poorly secreted to the culture medium whereas AA was secreted to a large extent. The fatty acids were initially esterified mainly into phosphatidylcholine and phosphatidylethanolamine. During the 24 h chase, a general shift from phosphatidylcholine to phosphatidylethanolamine was observed. Linoleic acid was esterified in cardiolipin to a much greater extent than any other fatty acid and it was not converted to more polyunsaturated fatty acids. The supplementation of the culture medium with polyunsaturated fatty acids had no inhibitory effect on the growth of the hepatoma cells, in marked contrast to observations made in other tumoral cells. The reasons for the resistance of the hepatoma cells to polyunsaturated fatty acid toxicity, including the possible antioxidant effect of linoleic acid accumulation in cardiolipin, are also discussed.
Asunto(s)
Ácidos Grasos/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Albúminas/metabolismo , Animales , Radioisótopos de Carbono , Cardiolipinas/química , División Celular/efectos de los fármacos , Ácidos Grasos/farmacología , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Ácido Linoleico/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Ratas , Factores de Tiempo , Triglicéridos/química , Triglicéridos/metabolismo , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/metabolismoRESUMEN
The implication of oxidative damage and/or intact mitochondrial function in physiological Fas-based cytotoxicity has been tested using the cytolytic hybridoma d11S and the CD8(+) CTL clone KB5.C20, previously stimulated to express Fas ligand (FasL) on their surface, as effectors and U937 or U937-rho0 cells (depleted of mitochondrial DNA) as targets. Immobilized anti-Fas mAb, which induced death of U937 cells, inhibited the growth of U937-rho0 cells but without inducing cell death. By contrast, FasL-expressing effectors readily killed both targets, with induction of DNA fragmentation, in 20 h assays. These results demonstrate the lack of involvement of mitochondrial-derived free radicals and/or intact mitochondrial function in physiological Fas-based cytotoxicity. Supplementation of Fas-sensitive cells (Jurkat, U937, L1210Fas) with a polyunsaturated fatty acid, which induces cell death through the generation of lipid free radicals, resulted in the potentiation of Fas-based cytotoxicity. This potentiating effect, but not Fas-based cytotoxicity itself, was eliminated by the physiological antioxidant vitamin E. On the other hand, the IL-1beta-converting enzyme (ICE)-like protease tetrapeptide inhibitor Ac-YVAD-cmk partially inhibited Fas-based cytotoxicity, while the specific inhibitor of CPP32/Yama Ac-DEVD-CHO was a much more effective inhibitor of Fas-induced apoptosis. It was concluded that Fas-induced cytotoxicity was clearly dependent on ICE-like protease activation, and especially on that of CPP32 in Fas-sensitive cells, including mitochondrial DNA-depleted ones.
Asunto(s)
Cisteína Endopeptidasas/fisiología , Citotoxicidad Inmunológica/efectos de los fármacos , Estrés Oxidativo/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Receptor fas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Caspasa 1 , Cisteína Endopeptidasas/efectos de los fármacos , Ácidos Grasos Insaturados/farmacología , Humanos , Células L , Activación de Linfocitos/efectos de los fármacos , Ratones , Oxidación-Reducción , Inhibidores de Proteasas/farmacología , Células Tumorales Cultivadas , Receptor fas/efectos de los fármacosRESUMEN
It has been proposed that synthesis of docosahexaenoic acid (22:6(n-3) in rat hepatocytes occurs by a route independent of delta 4-desaturase, which involves delta 6-desaturation and retroconversion (Voss A., Reinhart M., Sankarappa S. and Sprecher H. (1991) J. Biol. Chem. 266, 19995-20000). However, most cells exhibit these enzymatic activities and nevertheless synthesize low to undectectable amounts of 22:6(n-3). Moreover, there are few data on the occurrence of this pathway in human cells. In the present work, we have analysed the biosynthetic pathway of 22:6(n-3) in human Y-79 retinoblastoma and Jurkat T-cells. Y-79 cells were supplemented with 18:3(n-3) and 20:5(n-3) or incubated with [1-14C]18:3(n-3) and [1-14C]20:5(n-3) and lipids analysed by argentation TLC, reverse-phase TLC and GLC-mass spectrometry. Pulse-chase experiments revealed that synthesis of 22:6(n-3) from 20:5(n-3) in Y-79 cells occurred through two successive elongations, followed by a delta 6-desaturation of 24:5(n-3) to 24:6(n-3) and retroconversion to 22:6(n-3). Incubation of Y-79 cells with [1-14C]18:3(n-3) in medium containing 50 microM trans-9,12-18:2, a potent inhibitor of delta 6-desaturase, caused a reduction of 22:6(n-3) synthesis mainly by interfering with the desaturation of 18:3(n-3). However, when [1-14C]20:5(n-3) was used as precursor, synthesis of 22:6(n-3) was depressed to a lesser extent and mainly by reduction of 24:6(n-3) retroconversion. Neuronal differentiation of Y-79 cells caused a great increase in delta 6-desaturase activity on 18:3(n-3), though the amount of 22:6(n-3) synthesized did not change or diminish, suggesting the existence of a particular delta 6-desaturase involved in the synthesis of 22:6(n-3). The existence of a distinctive delta 6-desaturase activity could also explain why Jurkat cells growing in serum-free medium showed a near 3-fold increase in the synthesis of pentaenes from 18:3(n-3) and, at the same time, a large decrease in the synthesis of 22:6(n-3). The verification of the involvement of two delta 6-desaturase activities in 22:6(n-3) synthesis would have important implications for the formulation of the nutritional requirements of this fatty acid during development.
Asunto(s)
Ácidos Docosahexaenoicos/metabolismo , Ácido Graso Desaturasas/metabolismo , Bucladesina/farmacología , Diferenciación Celular , Medio de Cultivo Libre de Suero , Neoplasias del Ojo/metabolismo , Ácidos Grasos/metabolismo , Humanos , Cinética , Leucemia/metabolismo , Linoleoil-CoA Desaturasa , Retinoblastoma/metabolismo , Células Tumorales CultivadasRESUMEN
Unsaturated fatty acids are essential for the proliferation of many haematopoietic cells, but little is known about their biosynthetic pathways in these cells. We have studied the activity of the main desaturation-elongation enzymes in human B-(Reh-6, Raji, Ramos) and T-(CEM, Jurkat) lymphocytic, promonocytic (U937), promyelocytic (HL-60) and pluripotent myeloid (K562) cell lineages, as well as the changes induced by cell differentiation. Cells were incubated with 14C-labelled 18:0, 18:2(n - 6) and 18:3(n - 3) or supplemented with the corresponding unlabelled fatty acid and synthesis of polyunsaturated fatty acids (PUFA) was evaluated by argentation-TLC and GLC. The main activity present in most cells was delta 9-desaturase (range between 200-1000 pmol/24 h per 10(6) cells) that was regulated by the type of free fatty acids in culture media. A great variability in the activities of delta 6- and delta 5-desaturase was observed. They were virtually absent in B-cells and only one (Jurkat) T-cell line synthesized significant amounts of (n - 6) and (n - 3) PUFA. The main PUFA formed by Jurkat cells were 20:3 and 20:4(n - 6) (30 and 40%, respectively, of cell lipid radioactivity) and 20:5, 22:5 and 22:6(n - 3) (60, 20 and 10%, respectively, of cell radioactivity). Cell differentiation caused complex changes in desaturase activities. The activity of delta 9-desaturase increased with the degree of differentiation of B-cells. Differentiation of U937 cells to macrophages with PMA caused a 2-3-fold increase in the activity of (delta 6 + delta 5)- and delta 9-desaturases and no changes and a 2-fold decrease, respectively, if the inducer was DMSO. Differentiation of HL-60 cells to granulocytes with DMSO virtually abolished delta 9-desaturase activity and greatly reduced that of delta 6- and delta 5-desaturases. delta 9-Desaturase activity increased (2.5-fold) in myeloid K562 cells differentiated to erythroblasts with hemin. No induction of delta 6-desaturase, absent in K562 cells, occurred after differentiation to erythroblasts or megakaryoblasts and they synthesized alternative PUFA through sequential elongation and delta 5-desaturation of 18:2(n - 6) and 18:3(n - 3). The activities of delta 6- and delta 5-desaturase in HL-60 and U937 cells increased when differentiation also stimulated the synthesis of eicosanoids and extracellular release of PUFA.