Bertil Björklund

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis

A neo‐epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo‐epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30‐positive cells appear in the ‘apoptotic’ subG1 peak. Tests with synthetic peptides define positions 387–396 of CK18, with a liberated C‐terminus at the caspase cleavage site DALD‐S, as the ten‐residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo‐epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin‐fixed material. Copyright

The cytostat, an apparatus for automatically controlled continuous propagation of suspended cells.

Summary The cytostat and its operation have been described. It would seem to be a useful aid to further in vitro studies of cell populations.

Curtain electrophoretic separation of phenyl acetate hydrolysing esterases of human serum. Demonstration of an EDTA-dependent enzyme.

Summary By repeated continuous flow curtain electrophoresis of human serum, 2 distinct phenyl acetate hydrolyzing esterases were purified 120 and 850 times. One esterase was heat-labile and inactivated by EDTA, while the other was heat-stable and required EDTA for activity. EDTA-inactivation could be prevented by manganese. The methods for purification and assay are described and the results discussed.