Complete sequencing and characterization of equine aggrecan.

OBJECTIVES: To fully sequence and characterize equine aggrecan and confirm conservation of major aggrecanase, calpain and matrix metalloproteinase (MMP) cleavage sites. METHODS: Reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends were used to generate clones that encompassed the complete equine aggrecan sequence. Clones were sequenced and compared with the equine genome database to determine intron-exon boundaries. RESULTS: The aggrecan gene spans over 61 kb on chromosome 1 and is encoded by 17 exons. Two major variants of aggrecan were cloned; one containing 8187 bp (2728 amino acids) and a second sequence of 8061 nucleotides (2686 amino acids). The variation was due to a CS1 domain polymorphism. Both sequences are substantially larger than predicted by the genomic database; 11 CS1 repeat elements are absent in the database sequence. The equine amino acid sequence was compared with human, bovine and murine sequences. Globular domains 1, 2 and 3 are highly conserved (overall identity over 80%). Equine CS1 is considerably larger than in other species and, therefore, is the least conserved domain (an overall amino acid identity of 22%). Previously defined aggrecanase, calpain and MMP cleavage sites were identified. Western blotting of chondrocyte culture samples showed complex post-secretion processing. CLINICAL SIGNIFICANCE: The complete equine aggrecan sequence will support more in-depth research on aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.

Effect of fibrin glue derived from snake venom on the viability of autogenous split-thickness skin graft

The aim of this study was to analyze the effect of snake venom derived from fibrin glue on the viability of split-thickness skin graft. Nine crossbreed dogs were used. Full-thickness skin segments measuring 4X4 cm were bilaterally excised from the proximal radial area on each dog. A split-thickness skin graft was harvested from left lateral thoracic area using a freehand graft knife, and was secured to the left recipient bed using several simple interrupted sutures of 3-o nylon (sutured graft). A split-thickness skin graft was harvested from the right lateral thoracic area using a graft knife. Fibrin glue derived from snake venom was applied to the recipient bed, and 8 equidistant simple interrupted sutures of 3-0 nylon were used to secure the skin graft (glued graft). Viable and nonviable areas were traced on a transparent sheet and measured using a Nikon Photomicroscope connected to a KS-300 image analysis system. The skin graft and recipient bed were collected from three dogs at day 7,15, and 30 postoperative. The glued grafs had statistically higher graft viability than sutured grafts. Histological examination showed that the tissue repair process in the glued grafts was more accentuated than sutured grafts. It was possible to conclude that fibrin glue derived from snake venom increased survival of autogenous split-thickness skin graft.