| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
1. The distributions of laminin-5 and laminin-α5 chain, were studied immunohistochemically in the developing submandibular glands of mice. The expression of laminin-5 was found in the basement membrane of developing duct but not epithelial end bud. In contrast, laminin-α5 chain was expressed in the entire epithelial basement membrane at the early embryonic stage, suggesting the role of laminin-α5 chain on the epithelial morphogenesis.
2. To elucidate roles of the laminin α5 chain on organogenesis, we tested 15 cell-adhesive synthetic peptides from a carboxy-terminal globular domain of laminin-α5-chain sequence in submandibular gland organ culture. We found a peptide sequence LVLFLNHGHFVA (residues 3307-3318 and designated A5G-77) strongly inhibits the organ development at 250μg/ml. Various amino- and carboxy-terminal truncated peptides of A5G-77 were synthesized and their activities were also tested in the same system. A carboxy-treminal deletion peptide A5G-77f (LVLFLNHGH) worked inhib
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itory, whereas an amino-treminal deletion peptide A5G-77c (FLNHGHFVA) failed to inhibit the organ development. The morphology of A5G-77-treated explants differed from those of AG-73-treated, indicating distinct role of individual laminin α chains for organ development.
3. We established a serum-free organ culture system of isolated vibrissal rudiments of embryonic day-13 mice. This system allowed us to test 22 laminin-1 peptides for their roles on the vibrissal growth and differentiation. We found that RKRLQVQLSIRT sequence, which is mapped at LG-4 subdomain of laminin-α1-chain carboxyl-terminal G-domain and designated AG-73, perturbed vibrissal growth in vitro. Other cell-adhesive laminin-1 peptides and a control scrambled-peptide, LQQRRSVLRTKI, failed to show any significant effects on vibrissal growth. An impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in AG-73-treated vibrissal epidermis, indicating that AG-73 signaling is involved in cytoskeletal organization. Less
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