Stabilization of pericardial tissue by dye-mediated photooxidation

Bovine pericardial tissue was stabilized through a dye-mediated photooxidation reaction. Shrink temperature analysis of the stabilized tissue indicated a material with similar properties to untreated pericardial tissue and unlike identical tissue treated with glutaraldehyde. Photooxidized tissue was resistant to extraction when compared with untreated tissue or control tissues treated in the absence of light or dye. Photooxidized tissue was also resistant to enzymatic digestion by pepsin and to chemical digestion by cyanogen bromide (CNBr). In contrast, untreated or control treated tissues were readily digested by these reagents. Reduction of photooxidized tissue with beta-mercaptoethanol prior to CNBr digestion partially restored susceptibility of the tissue to CNBr digestion, indicating the photooxidation of methionine residues. Soluble collagen derived from bovine pericardium was used as a model compound for the photooxidation reaction. Polyacrylamide gel electrophoresis analysis indicated the photooxidative conversion of collagen into higher molecular weight aggregates consistent with intermolecular crosslink formation. Photooxidized tissue was stable to in vivo degradation when compared with control tissue. Results presented here indicate a crosslinked pericardial tissue produced by dye-mediated photooxidation possessing properties of chemical stability, enzymatic stability, in vivo stability, and biomechanical integrity suitable for use as a biomaterial.     

Isolation of anti-T cell receptor scFv mutants by yeast surface display

Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.