| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
(1)Differentiation capacity of TBR 31-2 cells
To study the differentiation capacity of TBR 31-2 cells, the m-RNA expression of differentiation factor, osteoblast core binding factor 1 and differentiation phenotypes such as alkaline phosphatase (ALP), type 1 collagen, osteocalcin and bone Gla-protein were examined by reverse transcription-polymerase chain reaction. Expression of these proteins was only observed in the stage of cell differentiation. In addition, the increase of activity of ALP and accumulation of Ca^<2+> level were also observed. These observations suggest that TBR 31-2 cells show the property of differentiating toward osteoblasts.
(2)Purification and identification of GPI-anchored proteins in TBR 31-2 cells
After biotinylation of peripheral proteins present on the plasma membranes of TBR 31-2 cells, GPI-anchored proteins were released by the treatment of PI-PLC. Solubilized GPI-anchored proteins were separated by SDS-PAGE, blotted to nitrocellulose membrane, and then analyzed chemiluminescence using peroxidase-conjugated streptoavidin. Six proteins having molecular weights of 120, 112, 96, 60, 55 and 41 kDa were specifically solubilized by the action of PI-PLC.
(3)Purification and identification of GPI-anchored proteins in HeLa 229 cells
By the treatment of sodium butyrate, two proteins having molecular weights of 60 and 38 kDa were specifically increased and solubilized by the action of PI-PLC. It is highly possible that the 60 kDa protein is the typical GPI-anchored protein, ALP, since the antibody against human placental ALP was found to crossreact to this protein. The 38 kDa protein was identified as folate receptor using the time of flight mass spectrometer.
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