Synthesis of methacryloyloxyethyl phosphate copolymers and in vitro calcification capacity.

Methacryloyloxyethyl phosphate is a methacrylic monomer used to modify different substrates by copolymerisation, in order to enhance hydroxyapatite deposition onto their surfaces. We report the synthesis of two copolymers series using increasing concentrations of methacryloyloxyethyl phosphate with (diethylamino) ethyl methacrylate and 1-vinyl-2-pyrrolidinone. Reactivity ratios were evaluated for the two copolymer systems. The influence of phosphate content and distribution on the capacity to form a calcium-rich layer was evaluated after immersion for 15 days in a synthetic body fluid. Corresponding homopolymers were synthesised as controls. Calcium-phosphorus globules were developed only on samples containing (diethylamino) ethyl methacrylate, and presenting a low density of phosphate groups. The amounts of calcium increased when higher concentrations of methacryloyloxyethyl phosphate were used. The use of 1-vinyl-2-pyrrolidinone was associated with greater calcium amounts, (compared to (diethylamino) ethyl methacrylate). The amine groups may favour the attraction of phosphorus, thus creating another way for the nucleation of calcium/phosphate crystals.

A conserved N-terminal sequence targets human DAP3 to mitochondria.

Human DAP3 (death-associated protein-3) has been identified as an essential positive mediator of programmed cell death. Structure-function studies have shown previously the N-terminal extremity of the protein to be required in apoptosis induction. Analysis of human DAP3 gene structure predicted 13 exons and subsequent targeting prediction by two software packages (MITOPROT and TargetP) gave a high probability for mitochondrial targeting. The predicted N-terminal targeting structure was also found in the mouse, Drosophila, and C. elegans orthologues with a strong sequence homology between mouse and human. Secondary structure analyses identified alpha-helical structures typical of mitochondrial target peptides. To confirm experimentally this targeting we constructed a fusion protein with N-terminal human DAP3 upstream of enhanced green fluorescent protein (EGFP). Confocal analysis of transfected human fibroblasts clearly demonstrated EGFP localization exclusive to mitochondria. The positioning of this key apoptotic factor at the heart of the mitochondrial pathway provides exciting insight into its role in programmed cell death.