Summary Apoptosis is observed in the crypts of the small intestine of healthy animals and man (spontaneous apoptosis). The levels can be dramatically elevated 3-6 h following ionizing radiation exposure. Both the spontaneous and radiation-induced apoptosis in the small intestine crypts are most frequently observed at the positions in the crypt associated with stem cells (about four cell positions from the base of the crypt). The number of apoptotic deaths can be counted in routine histological preparations, but interpretation of the counts is complicated by numerous factors. However, recording the number of cells containing one or more apoptotic fragments in crypt sections provides a good estimate for the absolute number of cell deaths in crypts. Similarities are noted in the frequency and cell positional relationship of radiationinduced apoptosis in the small intestine of various strains of mice and one strain of rat. Apoptosis in the large intestine is generalty lower in frequency than in the small intestine and, for the mid-colonic and rectal regions, has a different cell positional frequency distribution, with the highest apoptotic yield at the crypt base. The caecal colon has a pattem of apoptotic distribution more similar to that in the small intestine. After exposure to 1 Gy ionizing radiation, the maximum apoptotic yield occurs over a period of 3-6 h in the small intestine. There is some unexplained variability in the values between groups of mice and between different mouse strains. After 8 Gy, the yield remains elevated for several days, however a similar maximum yield is still observed at the eariy times. In mouse large intestine and rat small intestine, the yield continues to rise until about 6 Gy in mouse large intestine and until at least 10 Gy in rat small intestine. Spontaneous apoptosis is interpreted as part of the homeostatic mechanism regulating stem cell numbers. About 1.6 cells per crypt are dying at any one time. Following irradiation, there is an apparent relationship between mitotic and apoptotic levels, suggesting that these processes are linked. The dose-response relationship suggests that there are about six apoptosis-susceptible cells in crypts of the small intestine, with about 2-4 of these occurring at cell positions in which there are other more resistant clonogenic cells. In the large intestine, the position of these apoptosis-susceptible cells varies with region, but the numbers are similar.

Emerging technologies use cell plasma membrane vesicles or "blebs" as an intermediate to form molecularly complete, planar cell surface mimetics that are compatible with a variety of characterization tools and microscopy methods. This approach enables direct incorporation of membrane proteins into supported lipid bilayers without using detergents and reconstitution and preserves native lipids and membrane species. Such a system can be advantageous as in vitro models of in vivo cell surfaces for study of the roles of membrane proteins as drug targets in drug delivery, host-pathogen interactions, tissue engineering, and many other bioanalytical and sensing applications. However, the impact of methods used to induce cell blebbing (vesiculation) on protein and membrane properties is still unknown. This study focuses on characterization of cell blebs created under various bleb-inducing conditions and the result on protein properties (orientation, mobility, activity, etc.) and lipid scrambling in this platform. The orientation of proteins in the cell blebs and planar bilayers is revealed using a protease cleavage assay. Lipid scrambling in both cell blebs and planar bilayers is indicated through an annexin V binding assay. To quantify protein confinement, immobility, etc., incorporation of GPI-linked yellow fluorescent protein (GPI-YFP) was used in conjunction with single-molecule tracking (SMT) microscopy. Finally, to investigate the impact of the bleb induction method on protein activity and expression level, cell blebs expressing human aminopeptidase N (hAPN) were analyzed by an enzyme activity assay and immunoblotting. This work enriches our understanding of cell plasma membrane bleb bilayers as a biomimetic platform, reveals conditions under which specific properties are met, and represents one of the few ways to make molecularly complete supported bilayers directly from cell plasma membranes.

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