PANETH CELL AND GASTROINTESTINAL HOST DEFENSE

这些研究的目的是定义 通过研究内源性激素的合成来防御粘膜宿主 潘氏细胞的抗菌肽。Paneth细胞防御素，或 隐形蛋白被认为是粘膜屏障的组成部分， 因为它们是丰富的颗粒成分，原生物质多样 结构，释放到管腔内，并有效地杀灭近端的微生物 8号染色体上具有高度保守的双外显子结构的区域。 尽管成熟的隐蛋白多肽具有不同的一级结构，但其 预编码区和蛋白质几乎完全相同，这表明 Paneth细胞防御素前体中的某些残基在 成人和成人隐形蛋白基因的近远轴包装 发展中的小肠，研究潜在的前体 在翻译后处理中的重要性。第一个目标是 确定单个隐窝在特定基因的表达上是否存在差异 密码素基因；从分离的隐窝中扩增出的cDNA- 将通过以下方法检测远端轴上的加密蛋白亚型序列 与多肽特异的寡核苷酸杂交。类似地， 密码素异构体mRNA出现的动力学将在 检测新生小鼠肠道发育中是否存在隐形蛋白基因 是选择性地或一致地诱导的。第二个目标是测试 隐球蛋白中的氨基酸变化影响或调节生物 重组蛋白的生物活性、生化和抑菌性能 与自然产生和诱变的隐含蛋白相对应的多肽 将被描述为。第三个目标将测试加密 前体含有将肠道防御素靶向Paneth的决定因素 天然和突变的隐蛋白-1蛋白对细胞颗粒的影响 嵌合报告基因打靶的分布将在 小鼠隐窝分离培养Paneth细胞及其与包装方法的比较 巨噬细胞系确定防御素是否运输到 颗粒通过保守的或细胞特有的机制发生。这些研究 应该描述肠道防御素的分子分布， 定义生物活动的功能决定因素，并提供 了解导致它们释放到管腔内的事件。

The objective of these studies is to define the molecular basis of mucosal host defense by investigating the synthesis of endogenous antimicrobial peptides by Paneth cells. Paneth cell defensins, or cryptdins, have been implicated as components of the mucosal barrier, because they are abundant granule components with diverse primary structures, release into the lumen, and potent microbicidal proximal region of chromosome 8 that have highly-conserved, two-exons structures. Despite diverse primary structures of mature cryptdin peptides, their prepro-coding regions and propieces are almost identical, suggesting that certain residues within Paneth cell defensin precursors have roles in the packaging of cryptdin genes along the proximal-distal axis in adult and developing small intestine, to investigate precursors of potential importance in posttranslational processing. The first aim is to determine whether individual crypts differ in the expression of specific cryptdin genes; amplified cDNAs from isolated crypts along the promixmal- distal axis will be assayed for cryptdin isoform sequences by hybridization to peptide-specific oligonucleotides. Similarly, the kinetics of cryptdin isoform mRNA appearance will be determined during intestinal development in neonatal mice to test whether cryptdin genes are induced selectively or in concert. The second aim is to test whether amino acid changes in cryptdins influence or modulate biological activity; the biochemical and antimicrobial properties of recombinant peptides corresponding to naturally-occurring and mutagenized cryptdins will be characterized. The third aim will test whether cryptdin precursors contain determinants in targeting enteric defensins to Paneth cell granules; the effect of natural and mutated cryptdin-1 propieces on the distribution of chimeric reporter gene targeting will be measured in Paneth cells from isolated crypts and compared with packaging in mouse macrophage cell lines to establish whether defensin trafficking to granules occurs by conserved or cell-specific mechanisms. These studies should characterize the molecular distribution of enteric defensins, define functional determinants for biological activity, and provide knowledge of events leading to their release into the lumen.