| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
We developed a novel culture system in which mouse bone marrow-derived, immature, mast cells (BMMC) coclultured with Swiss 3T3 fibroblasts in the presence of stem cell factor quickly underwent morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. Thus, cocultured BMMC contained more histamine, heparin proteoglycan and serine protease MMCP-4, produced more PGD_2 in response to Fc_εRI crosslinking, and importantly, reponded to substance P and compound 48/80, well-known polycationic secretagogues that elicit IgE-independent, G protein-dependent activation of CTMC phenotype only.
The cDNA subtraction strategy was conducted to identify a series of genes whose expression levels were dramatically changed during this in vitro mast cell maturation process. Of approximately 100 sequenced clones, nearly 50% were chromosome 14-associated serine proteases, including MMCP-4 and apoptosis-inducing protease granzyme B.Approximately 14% encoded Ndr-1, a 43-k
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Da cytosolic protein that has been reported to be induced in several cell types during differentiation or stress responses. Induction of Ndr-1 in BMMC preceded the maturation into CTMC-like cells and was accompanied by enhancement of the IgE-dependent and -independent exocytotic responses. Forcible introduction of Ndr-1 into rat mastocytoma RBL-2H3 cells led to augmentation of IgE-dependent and -independent degranulation. Deletion of three hydrophilic tandem repeats near the C-terminus in Ndr-1 abrogated the degranulation-enhancing function. These results suggest that Ndr-1, a strongly inducible gene during in vitro maturation of mast cells, is a novel component that may play a pivotal role in the regulation of stimulus-induced degranulation.
Another novel gene, AVRL-2, encoded a 100-kDa protein, the C-terminal half of which showed a weak homology with angiotensin/vasopression receptor-like (AVRL). AVRL-2 contained several motifs that are presumed to participate in protein-protein interaction, such as helix-loop-helix and leucine zipper, and unique C-terminal three tandem repeats. At least three alternative splicing variants were identified, each of which lacked one or more of the tandem repeats. Full-length AVRL-2 and its splicing variants showed distinct induction profiles in BMMC during coculture. Less
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