RESUMEN
Previously it was reported that supplementation of insect cell culture with Lonomia obliqua hemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58-63). In this work the anti-apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf-9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti-apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf-9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliqua hemolymph is due to the presence of potent anti-apoptotic factors.
Asunto(s)
Apoptosis/efectos de los fármacos , Hemolinfa/química , Mariposas Nocturnas/química , Proteínas , Animales , Apoptosis/fisiología , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Medios de Cultivo/farmacología , ADN/química , Dactinomicina/farmacología , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/farmacología , Spodoptera/citología , Spodoptera/metabolismo , Factores de TiempoRESUMEN
Eighteen out of 27 lots of fuenzalida & palacios type antirabies vaccine for human use produced at the Butanta, were filteres through two different Millipore systems, i.e., couple of cartridges (pre-filter & cart ridge of 0.55 aem porosity) or sandwich of membranes (pre-filter & membranes of 5.0 & 1.2 aem porosity). Afterwards, a study on the stability of the lots carried out during 18 months of storage at 2-8§C has demonstrated that filtration: a) reduced the protein contents of the vaccine, b) decreased the number of ampoules discarded due to the formation of clumps, c) didn`t interfere with the stability of the vaccine. It was also shown that combination of membranes was more suitable than cartridges for the filtration of vaccine lots and did not turn production costlier