Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a proteinconducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.

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The value of cultured cells and tissues is highly dependent on their degree of terminal differentiation. In order to obtain a tissue architecture similar to the in vivo situation, compatible cell supports and culture conditions must be optimised. This involves the development and evaluation of new thermoplastic elastomers, polyethylene (PE), polyurethane (PU) and polyethylene terephthalate (PET) for use in medical devices. A vascular implant currently under investigation incorporates human dermal microvascular endothelial cells seeded onto pretreated PET. Early indications are that it maintains differentiated functions in vitro. This represents a unique human angiogenesis model that permits the assessment of both potential stimulators and inhibitors of the angiogenic process. A novel cell culture rotary wall vessel (RWV) designed by NASA has been adopted for this study. This system allows the growth of differentiated 3-D cellular aggregates with and without polymer support. The system maintains a unique environment of low shear force, high mass transfer and microgravity which provides an ideal culture system for organotypic models. Our current objective is to identify multiple genes which show altered expression in this model using Restriction Fragment Differential Display-Polymerase Chain Reaction (RFDD-PCR), real time and conventional Reverse Transcription PCR (RT-PCR). Under investigation are the structural and inflammatory target components including connexins, cytokines, adhesive proteins and cytochrome P-450 isoforms. This technology allows one to investigate the exposure of long term differentiated 3D and monolayer cultures to a material with multiple end-point evaluation of the relationship between cells and their substratum.