ABSTRACT
Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3...
Subject(s)
Animals , Antibodies , Apoptosis , Oncorhynchus mykiss , Blotting, Western , Enzyme-Linked Immunosorbent Assay , ImmunohistochemistryABSTRACT
De un total de 7.370 nacidos vivos durante 1983 en un hospital metropolitano de Santiago, 730 fueron referidos para estudio en la Unidad de Cardiología pediátrica, encontrando 101 pacientes con cardiopatías congénitas, lo que representa una incidencia de 13,70 por mil nacidos vivos. La mayoría de ellas (74%) fueron pesquisadas durante el primer mes de vida, el resto, en edades mayores, a lo largo de un período de seguimiento de 20 a 32 semanas. La presencia de soplos anorgánicos se detectó en 68 de 730 niños referidos a cardiología, desapareciendo en 30 de ellos (45%) en los controles sucesivos. De los 22 síndromes genéticos detectados en la población estudiada, 14 correspondieron a síndrome de Down y 11 de ellos (79%) tenían cardiopatías congénitas. Se encontraron malformaciones asociadas en 15 de los 101 (14,8%) pacientes con cardiopatías congénitas. Durante el primer año de vida, fallecieron 17 de los 101 pacientes: letalidad 168 por mil. La tasa descrita para cardiopatías congénitas en este estudio es la primera que se comunica en nuestro medio, y es mayor que la descrita en el extranjero. La distribución porcentual relativa concuerda en general con la descrita por otros autores