| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
CD40 antigen, a member of the TNF/NGF receptor family, is a 45-50 KDa glycoprotein expressed by B cells, dendritic cells, macrophages and thymic epithelial cells. CD40-mediated signals play essential roles in B cell growth, activation, class switching of Ig genes and memory B cell formation. CD40 also regulates cell death, rescuing immature B cells from anti-Ig induced apoptosis and inducing Fas expression and Fas susceptibility in mature B cells. Little is known, however, about the molecular events that govern such diverse functions from the same CD40 receptor. As a first step toward understanding the molecular mechanism of CD40 function, we have employed a modified subtractive cloning strategy (PCR select) to identify novel genes that are induced or suppressed by CD40-mediated signals. These novel genes, termed Clasts 1-6 (for CD40 ligand-activated specific transcript), have unique characteristics in their expression pattern in cells of the immune system. For example, Clast1 expressi
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on is selectively induced by CD40 ligation but down regulated by B cell antigen receptor signaling. The predicted amino acid sequence indicates that Clast 1 is a tetraspaning membrane protein structurally related to the high affinity Fcε receptor β chain and the pan-B cell antigen CD20. In contrast to FcεRβ chain which contains ITAM motifs in its cytoplamic region that transmit activation signals, Clast1 protein contains an ITIM motif in its N-terminal cytoplamic region which may function to inhibit signal transduction. Northern blot analysis of various cell lines demonstrated that Clast1 expression is mainly restricted to mature B cells and macrophages. RT-PCR analysis revealed that Clast1 in an immature B cell line, WEHI231, enhanced the anti-IgM indued apoptosis, suggesting a role for Clast1 in the regulation of B cell death. We have generated polyclonal antibodies against Clast1 extracellular domain. We also generated 6 lines of transgenic mice overexpressing Clast1 in B lineage cells. Expriments are currently in progress to reveal the role of Clast1 in the regulation of B lymphocyte differentiation, activation and apoptosis. Less
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