Background/Aims: TFF3, a member of the TFF (trefoil factor family) peptides, and epidermal growth factor (EGF) actively support the repair of mucosal barriers, particularly during restitution. The aim of this study was to compare the motogenic effects of TFF3 and EGF. Methods: The influence of recombinant human TFF3 (dimeric form) and EGF on the migration of IEC-18 cells was characterized in an in vitro restitution model (scratch wound assay) with the help of time-lapse video microscopy, morphometry, and immunocytochemistry including confocal laser scanning microscopy. Results: TFF3- and EGF-treated cells re-populated the wounded area via different migration patterns; TFF3 treatment resulted in the formation of continuous sheets of migrating cells with only a few gaps. In contrast, EGF-treated cells formed a network of migrating cells (often with a fibroblast-like morphology) with numerous gaps and only punctual contacts. TFF3 and EGF treatment also changed the localization of E-cadherin indicating endocytotic recycling and/or degradation of E-cadherin. Conclusion: TFF3, in contrast to EGF, enhanced a collective cell migration ensuring a precise coverage of the re-populated area avoiding gaps.

Coagulation factor XIII (FXIII) is activated by thrombin and catalyses crosslinking between fibrin monomers thereby providing mechanical strength to the fibrin network. V34L is a common FXIII-A polymorphism found in the activation peptide. FXIII-A V34L is activated faster by thrombin and provides formation of a tighter clot at fibrinogen concentrations in the low end of the physiological range. FXIII-A variants with potentially increased activation rates were generated. Introduction of an optimal thrombin cleavage site, V34L+V35T, increased the activation rate 7.6-fold and facilitated the formation of a fibrin network more resistant to fibrinolysis than obtained with wt FXIII-A. In contrast, introduction of fragments of fibrinopeptide A into the activation peptide resulted in severely impaired activation rates.

Backbone cyclic insulin was designed and prepared by reverse proteolysis in partial organic solvent of a single-chain precursor expressed in yeast. The precursor contains two loops to bridge the two chains of native insulin. The cyclisation method uses Achromobacter lyticus protease and should be generally applicable to proteins with C-terminal lysine and proximal N-terminal. The presence of the ring-closing bond and the native insulin disulfide patterns were documented by LC-MS peptide maps. The cyclic insulin was shown to be inert towards degradation by CPY, but was somewhat labile towards chymotrypsin. Intravenous administration of the cyclic insulin to Wistar rats showed the compounds to be equipotent to HI despite much lower insulin receptor affinity.

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