RESUMEN
AIMS To investigate the effects of sporidesmin on cells cultured from the epithelial surface of sheep gallbladder walls, and to examine cellular responses to sporidesmin using cultures prepared from the gallbladders of sheep from selection lines that differed in sensitivity to sporidesmin-induced liver damage. METHODS Gallbladders were obtained following slaughter of lambs that were selected for resistance or susceptibility to sporidesmin-induced liver damage, or were not selected (controls). Monolayer cell cultures were established after incubation of excised gallbladders with protease to detach the lining epithelial cells from the muscular and connective tissue of the gallbladder wall. Released cells were harvested and transferred to culture flasks or dishes, then incubated with 1â µg/mL sporidesmin and were examined at 5 minute intervals, up to 3 hours, using light microscopy to monitor loss of attachment of cultured cells. Immunofluorescence staining of cell cultures was used to identify cytokeratin 19 as a marker for biliary epithelial cells, and to characterise sporidesmin-induced change in the cellular distribution of actin microfilaments. Gallbladder size was also measured. RESULTS In cultures incubated with sporidesmin, cells at the margins of sheets of cells showed the first signs of change, becoming unanchored from the culture vessels while remaining attached to the cell mass. This was followed by progressive detachment of sheets of cells and clumps of rounded cells. Disruption of cytoplasmic actin microfilaments with accumulation of actin in the cytoplasm adjacent to the plasma membrane preceded major detachment of cells. Cells from susceptible line lambs were extensively rounded up within 1 hour with complete or almost complete detachment within 2 hours, whereas cultures from resistant line lambs generally only contained detaching rounded-up cells at the periphery of monolayers within 2 hours; detachment observed in cells from the control line lambs was intermediate. There was a trend for gallbladders to be smaller in male lambs from the resistant line compared to the control or susceptible lines. CONCLUSIONS Altered cell adhesion and disruption of microfilament actin in biliary cell cultures incubated with sporidesmin suggest that biliary tract pathology may be due to the effects of the toxin on cytoplasmic and cell surface protein networks that affect the integrity of the epithelial lining of the biliary tract. These effects can be interpreted in terms of the hepatobiliary pathology of facial eczema, including potential differences in sensitivity of biliary tract cells that may contribute to inherited resistance and susceptibility to sporidesmin and hence facial eczema.
Asunto(s)
Vesícula Biliar/efectos de los fármacos , Vesícula Biliar/patología , Esporidesminas/toxicidad , Mataderos , Análisis de Varianza , Animales , Sistema Biliar , Técnicas de Cultivo de Célula , Femenino , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Fotomicrografía , Ovinos , Enfermedades de las Ovejas , Esporidesminas/administración & dosificaciónRESUMEN
Plantlets were regenerated from protoplasts of in vitro shoot cultures and leaf-derived de novo shoots of the chrysanthemum Dendranthema zawadskii x D. grandiflora. Isolated protoplasts reformed cell walls and then began to divide within 24 hours of culture in streaky plate agarose lenses surrounded by liquid V-KM medium. Twenty one days after isolation, 1 mm diameter callus clumps were transferred to shoot regeneration medium. After a further 33 days leaves became visible. Elongated shoots were rooted on half strength hormone-free MS medium.
RESUMEN
Cows with histologically confirmed ocular squamous cell carcinoma were injected with autochthonous tumour brei in adjuvant. Lymphoproliferative responses of isolated peripheral blood lymphocytes exposed to phytomitogens and inhibition of cell migration indicated that afflicted animals were immunocompetent. Similar but lesser responses were evident when autochthonous tumour homogenates were used to stimulate lymphocytes.