[CITATION][C] Evidence for a 24R, 25 (OH) 2-Vitamin D3Receptor/Binding Protein in a Membrane Fraction Isolated from a Chick Tibial Fracture-Healing Callus

EG Seo, A Kato, AW Norman - Biochemical and biophysical research …, 1996 - Elsevier
EG Seo, A Kato, AW Norman
Biochemical and biophysical research communications, 1996Elsevier
MATERIALS AND METHODS Animals. Male White Leghorn chicks were obtained at
hatching (Hyline International, Lakeview, CA) and were fed a vitamin D3-replete diet (2000
IU vitamin D3/kg diet) containing 0.6% calcium and 0.4% phosphate. When chicks were
three weeks old, a tibial fracture surgery (approved by University of California Chancellor's
Committee on Laboratory Animal Care: A-9412056-1) was performed on the right tibia: the
chick was anesthetized with Metofane (methoxyflurane: Pitman-Moore, IL) inhalation and a …
MATERIALS AND METHODS
Animals. Male White Leghorn chicks were obtained at hatching (Hyline International, Lakeview, CA) and were fed a vitamin D3-replete diet (2000 IU vitamin D3/kg diet) containing 0.6% calcium and 0.4% phosphate. When chicks were three weeks old, a tibial fracture surgery (approved by University of California Chancellor’s Committee on Laboratory Animal Care: A-9412056-1) was performed on the right tibia: the chick was anesthetized with Metofane (methoxyflurane: Pitman-Moore, IL) inhalation and a skin incision (Ç1 cm) was made over the tibia. After the separation of overlying muscle tissues, a hole (Ç2 mm in diameter) was drilled with a dental burr in the diaphysis. The skin was then closed, sutured, and a complete fracture was made by applying light manual pressure. No form of fixation of the fracture was made. Ten days after fracture treatment, the birds were euthanized and the callus tissue was dissected and utilized for the various studies.
Tissue preparation. Immediately after the dissection of the callus, 10% homogenates in TED (10 mM Tris, 1.5 nM EDTA, 1 mM dithiothreitol at pH 7.4) were prepared using a Polytron (Brinkmann Instruments, Westburg, NY) briefly. The homogenate was centrifuged at 500 1 g for 10 min (4 C) and the post nuclear supernatant was ultracentrifuged in a 60 Ti rotor (Beckmann Instruments) at 203,900 1 g for 30 minutes. The resulting pellet was washed twice with TED by homogenizing (Potter-Elvehjem homogenizer) and centrifuging at 203,900 1 g for 30 min. After the washes, the final post nuclear pellet was resuspended in TED and used for saturation analysis and steroid competition assay and described as follows.
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