Flexible membranes covered with cultured CECs, used as a new model that mimics in vivo conditions, minimized the mechanical damage caused by ultrasonic vibrations and turbulent currents, which destroy cells grown on hard surfaces. Phacoemulsification damage was not mediated by mechanical or shear forces but resulted from free-radical formation that apparently triggered cellular cascades, leading to apoptosis. Cell death was significantly reduced by the addition of ascorbic acid, probably via a free radical-scavenging mechanism.
Phosphotyrosine proteins involvement, particularly cortactin, was studied in cell-cell contacts of cultured bovine corneal endothelial (BCE) cells. These proteins, including alpha-catenin, vinculin and cortactin, are localized at cell-cell contacts separate from the cortical actin ring. Approximately 50% of cortactin isoforms p80 and p85 were associated with the Triton-insoluble fraction while phosphotyrosine proteins were in the soluble fraction. Disruption of cell-cell contacts by EDTA treatment was associated with a decrease in cortactin isoforms p80 (26%) and p85 (57%). Cortactin isoform p85 was phosphorylated at Y466, expressed in reattaching cells and associated with the Triton-soluble fraction, whereas cortactin isoform p80 was phosphorylated at Y421 and associated with the Triton-insoluble fraction. In sub-confluent cultures, pY421-cortactin was localized at the leading edge and pY466-cortactin at a perinuclear area. In confluent cultures both pY466- and pY421-cortactin isoforms were localized at the cell-cell contacts. In conclusion, in BCE cells, the most prominent appearance of cortactin was at the cell-cell contacts separate from the cortical actin ring. Isoform p80 was phosphorylated at Y421 and associated with the Triton-insoluble fraction and isoform p85 was phosphorylated at Y466 and associated with the Triton-insoluble fraction.
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