PANETH CELL AND GASTROINTESTINAL HOST DEFENSE

这些研究的目的是确定 粘膜宿主防御通过研究内源性 Paneth细胞的抗菌肽。 潘氏细胞防御素，或 已经暗示穴状蛋白是粘膜屏障的组分， 因为它们是丰富的颗粒组分，具有不同的初级 结构，释放到管腔中，和有效的杀微生物近端 8号染色体的区域，具有高度保守的两个外显子结构。 尽管成熟cryptdin肽的一级结构不同，但它们的 前原编码区和原片段几乎相同，这表明， 潘氏细胞防御素前体中的某些残基在 在成年人中，Cryptdin基因沿着近端-远端轴包装， 发展小肠，调查潜在的前体 翻译后加工的重要性。 第一个目标是 确定个体隐窝是否在特定的 cryptdin基因;扩增的cDNA从分离的隐窝沿promixmal- 通过以下方法测定远端轴的穴状蛋白同种型序列 与肽特异性寡核苷酸杂交。 同样， 将测定隐蛋白同种型mRNA出现的动力学， 新生小鼠的肠道发育，以测试Cryptdin基因是否 都是有选择性的或者是一致的。 第二个目标是测试 穴蛋白中氨基酸的变化影响或调节生物学特性 活性;重组的生物化学和抗菌特性 对应于天然存在的和诱变的穴状蛋白的肽 将被定性。 第三个目标是测试密码蛋白 前体包含将肠防御素靶向帕内特的决定因素 细胞颗粒;天然和突变的cryptdin-1前体对 嵌合报告基因靶向的分布将在 从小鼠分离的隐窝中分离Paneth细胞并与包装进行比较 巨噬细胞系，以确定是否防御素贩运到 颗粒通过保守的或细胞特异性的机制发生。 这些研究 应该表征肠防御素的分子分布， 定义生物活性的功能决定因素，并提供 了解导致其释放到管腔中的事件。

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.