Cell permeabilization technique is widely used for the purpose of inserting membrane-impermeable molecules across cell membranes. However, cellular lipid metabolism could be affected, since cell permeabilization can be a drastic disturbance for the lipid bilayer structure of cell membranes. In this study, the changes of cellular lipid compositions as a result of cell permeabilization were investigated utilizing the lipid analysis by HPLC and GC. In mouse leukemic lymphocyte L1210 cells, a broad alteration of lipid composition was observed upon cell permeabilization by digitonin, a typical pore-forming agent. Major changes observed were the increase of free fatty acids (5.0 fold) and phosphatidylethanol (phospholipase D product in the presence of ethanol) and the decrease of phosphatidylcholine. Small but significant increases were also detected in the levels of diacylglycerol, phosphatidic acid, lysophosphatidylethanolamine, and lysophosphatidylcholine. The lipid changes by digitonin were almost identical with those by ultrasonic disruption of cells, suggesting that digitonin-permeabilization affects the lipid metabolism as severely as the membrane disruption. Similar pattern of lipid changes upon cell permeabilization was observed in various cell lines including HL60, P388D1, VER076, NIH3T3, S49.1, and PC12 cells. These results thus imply that the alteration of lipid composition of mammalian cells during cell permeabilization seems to be a common phenomenon. Additionally the lipid changes of non-specific cell permeabilization were compared with those of specific cell pore-forming processes such as P2z receptor opening by ATP and calcium transport by calcium ionophore A231 87 [Supported by KOSEF and BK21 Project].Desmocollins and desmogleins are members of the superfamily of cell adhesion molecules, the cadherins, which constitute the desmosome type of cell-cell junction. Like the classical cadherins such as E-cadherin, they are single-pass transmembrane proteins characterised by five extracellular tandem repeats of -110 amino acids with conserved calcium binding motifs and a cytoplasmic domain which is specialised to interact via a plaque with the cytoskeIeta1 filaments. This link between cadherins and the cytoskeleton allows lateral clustering of cadherins which is thought to play a role in their adhesive function. We have used the purified recombinant first two N-terminal domains of desmocollin 2 (Dsc2) and desmoglein 2 (Dsg2) to carry out quantitative studies of protein-protein interactions undergone by these molecules. The use of the technique of BIAcore has shown that these proteins preferentially undergo calcium-dependent heterotypic interactions in contrast to the classical cadherins which exhibit homotypic interactions. These findings are consistent with the results of analysis of these molecules by analytical ultracentrifugation. Further evidence for hetrodimerisdation is being sought by using a combination of chemical cross-linking and immunoprecipitation. Additionally, circular dichroism spectroscopy ...
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