Studies on Interactions of Flectric Field and Biomembranes

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The main objects of this research project are (1) the elucidation of basic mechanisms of electroporation, diefectrophoresis and electrofusion, and (2) the development of standard application procedures for cell manipulation, such as gene transfer and membrane fusion. Investigations of electric field effects on various cellular phenomena, that were not yet studied, were also initiated.Major results obtained are as follows: (1) Pulse laser microscopy has been developed to observe directly electroporation on cellular membranes and liposomes. Time resolution of microseconds and spatial resolution of micrometers were obtained. Findings obtained shows that the electroporation takes place within a microsecond when membrane potential exceeds a threshold. The formation of unexpectedly large pores (micrometers in diameter) has been observed with liposomes. (2) High-performance power supply for electroporation and electrofusion was developed. (3) Diectrophoresis and high leictric field pulse were applied to induce membrane fusion in biomembrane vesicies as well as cells. (4) Intrinsic thiol proteases were found to take an important role in electrofusion of cells. (5) Millimolar Ca^<2+> and Mg^<2+> enhances the closing of pores formed by electric field. (6) A simple and efficient method of electroporation for gene transfer in mammalian cells was established. (7) Electrogenic ion pumps such as H^+- ATPase and Ca^<2+>-ATPase were incorporated into a planar lipid bilayer. This reconstituted membrane provides for ideal system for direct demonstration of the electrogenicity of ion pumps and the electric field effects, and for kinetic analysis of ion transport. (8) The effect of surface electric charge on membrane formation was examined.