RESUMEN
The sheep bot fly, Oestrus ovis, is a very common myiasis of nasal and sinus cavities of sheep and goats causing severe welfare and production implications. As the viability of O. ovis adult flies strictly depends on larval abilities to assimilate and to stock nutrients from the host, it was necessary to investigate proteolytic activities in larval excretory/secretory products (ESP). ESP of O. ovis larvae degrade mucosal and plasmatic components such as mucin, albumin or immunoglobulin G. A preliminary biochemical characterization, using substrate gel analysis and inhibitor sensitivity, demonstrated the presence of at least six major serine proteases (molecular weights from 20 to 100 kDa), mainly trypsin-like, secreted in the digestive tube of larvae. Their involvement in larval trophic activity and evasion from the host immune response is further discussed as O. ovis excretory/secretory serine proteases could represent potential vaccinal targets.
Asunto(s)
Dípteros/enzimología , Serina Endopeptidasas/análisis , Animales , Compuestos Azo/metabolismo , Benzoilarginina-Nitroanilida/metabolismo , Bovinos , Compuestos Cromogénicos/metabolismo , Colágeno/metabolismo , Electroforesis en Gel de Poliacrilamida/veterinaria , Concentración de Iones de Hidrógeno , Hidrólisis , Inmunoglobulina G/metabolismo , Larva/enzimología , Peso Molecular , Mucinas/metabolismo , Miasis/parasitología , Miasis/veterinaria , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/química , Serina Endopeptidasas/fisiología , Albúmina Sérica/metabolismo , Ovinos , Enfermedades de las Ovejas/parasitologíaRESUMEN
The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the effects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by caffeine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.
