Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.