| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
In order to determine the roles of extracellular matrix tenascin-X (TNX) in collagen fibrillogenesis, we first developed an easy and fast method to isolate TNX from various tissues in mice. We purified approximately 350-kDa cellular interstitial TNX from spleen, liver and kidney. Next, we investigated the effects of TNX on type I collagen fibril formation. Consequently, we found that TNX from these tissues increased the rate and the final quantity of fibril formation. Furthermore, glycobiochemical properties of TNX were characterized by lectin blot analysis. Lectin blots revealed the presence of N-glycan in TNX. In addition, the TNX from liver and kidney also possessed O-glycan based on the reaction to PNA. The binding to AAL indicated that TNX from the three tissues possesses fucose linked a1,6 to a pentasaccharide core. The reaction to SSA but not to MAM suggested the presence of sialic acid linked a2,6 to galactose in TNX. During the purification of cellular TNX from various organs, we identified the serum form of TNX (sTNX) with a molecular mass of 200 kDa in mouse. The 200-kDa sTNX contains 15 fibronectin type III-like (FNIII) repeats and a fibrinogen domain identical to the C-terminal portion of cellular TNX. sTNX was generated by proteolytic cleavage of cellular TNX by spleen homogenate.
Subsequently, L, MCF7 or H1299 cells were tested for adhesion to TNX. L cells did not adhere to TNX, whereas MCF7 or H1299 cells adhered to it just a few, but most of the adhered cells remained rounded or spread minimally without forming significant lamellae. Furthermore, MCF7 cells were plated on tissue culture plastic coated on fibronectin (FN) with TNX. TNX was found to have mild inhibitory effects on the cell attachment and spreading promoted by FN. These results indicated that TNX has its ability to modulate cell-extracellular matrix interactions.
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