Proteomic analysis of equine amniotic membrane: characterization of proteins.

INTRODUCTION: Human amniotic membrane (AM) has been used as a biomaterial for surgical wound skin and ocular surface reconstruction for several years. Currently, equine AM has been used for corneal reconstruction in several animal species, and appears to have the same properties as human AM. Despite the observed positive healing abilities of this tissue in horses with ulcerative keratitis the proteins of equine AM have not been described. OBJECTIVE: To identify proteins known to be associated with corneal healing from frozen equine AM. PROCEDURES: Placentas were acquired from healthy live foal births from a local Thoroughbred breeding farm. The amnion was removed from the chorion by blunt dissection, washed with phosphate-buffered saline (PBS), and treated with 0.05% trypsin and 0.02% ethylene diaminetetraacetic acid in PBS. Amnion was attached to nitrocellulose paper (epithelial side up), and cut into 4 × 4 cm pieces. The sheets were frozen at -80 °C. The protein samples were solubilized, and analyzed by 2D gel electrophoresis and shotgun proteomics. RESULTS: A reference identification map of the equine AM proteins was produced and 149 different proteins were identified. From gel-based proteomics, 49 spots were excised and 43 proteins identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Shotgun proteomics identified 116 proteins with an overlap of 10 proteins in both analyses. CONCLUSIONS: We have described a reference map for equine AM proteins that may provide a background to explain the positive results found in horses with ulcerative keratopathies using this biomaterial.

Cytogenetics and embryology of Eupatorium laevigatum (Compositae)

Embryological studies indicate Eupatorium laevigatum to have Antennaria type diplospory with precocious embryony. The embryo sac is of the Polygonum type and the polar nuclei fuse before anthesis (maturation of the stamens). Endosperm development is autonomous and the central cell divides only after the initial stages of embryo formation. It is estimated that about 10 percent of the florets in anthesis contain an undivided egg which can be used for sexual reproduction. The study of microsporogenesis revealed abnormalities in chromosome pairing which result in the formation of univalents, bivalents, trivalents and higher polyvalents, with the consequent production of lagging chromosomes, unbalanced nuclei, micronuclei and sterile pollen. We found that, as represented by the material studied, E. laevigatum is an autohexaploid (2n = 6x = 60) in which each chromosome of a basic set of ten chromosomes is repeated six times and that E. laevigatum is an essentialy obligate apomictic.