PEPTIDE EFFECTORS OF ENTERIC HOST DEFENSE

这项拟议的研究的目标是描述先天的机制 小肠的免疫力。隐形蛋白是潘氏细胞防御素， 持续释放到小分子体内的杀微生物肽 肠腔。这项建议的具体目标是有重点的 在定义密码的作用机制和它们的调节方面 综合。已经确定了六种老鼠密码的特征，它们是 对多种细菌具有选择性活性的强效抗菌剂 以微生物为目标。除了六个已知的多肽外，还有14个小说 基因证据已经预测了加密素的异构体。加密密码是 由不同的基因编码，某些基因是差异表达的 在出生后隐窝个体发育过程中，沿隐窝纵轴 成人小肠。大量数据支持潘氏细胞的假说 防御素是粘膜免疫的重要组成部分，而这个概念 将通过分析小鼠之间的生物相互作用来进行测试 并通过表征相应的基因。为了测试 关于Paneth细胞防御素作用机制的假说 密码素之间的协同相互作用将由 定量测定小鼠Small的区域密码蛋白亚型含量 肠道，评价修饰隐球菌素的杀菌作用 从肠腔中分离出来，并通过检测隐含蛋白，两者都 单独和组合反映它们的相对水平，对于 对一系列病原体的抗菌效力。复方丹参制剂 单独的和联合的Paneth细胞防御素将进行测试 对哺乳动物细胞系的潜在细胞毒作用，以及 细胞毒性将与髓系防御素进行比较。至 研究孔形成在密码素杀伤机制中的作用， 将对离子的动力学和化学计量进行测量- 隐球蛋白在脂类双层膜中形成通透性通道。为了测试 与密码素合成调控相关的假说，主要 从新生小鼠小肠中分离出的隐含蛋白的结构将是 确定，密码蛋白亚型的发育外观将 被建立起来。鼠标加密的相对位置和方向 同构基因将沿着物理图谱被建立。最后， 密码蛋白位置特异性的潜在顺式作用决定因素将 通过对构建了变量的转基因小鼠进行表征来进行研究 密码素-4基因的侧翼和内含子区域。这些研究将 阐明潘氏细胞防御素在先天免疫中的作用机制 通过阐明它们的行动模式、潜在的相互作用以及 它们在成体和发育小鼠体内的合成调节 肠子。

The goal of the proposed research is to characterize mechanisms of innate immunity in the small intestine. Cryptdins are Paneth cell defensins, microbicidal peptides that are released continually into the small intestinal lumen. The specific objectives of this proposal are focused at defining the mechanisms of cryptdin action and the regulation of their synthesis. Six mouse cryptdins have been characterized, and they are potent antimicrobial agents with selective activities against varied target microbes. In addition to the six known peptides, fourteen novel cryptdin isoforms have been predicted by genetic evidence. Cryptdins are coded by separate genes, and certain genes are expressed differentially during postnatal crypt ontogeny and along the longitudinal axis of the adult small bowel. Abundant data support the hypothesis that Paneth cell defensins are important components of mucosal immunity, and that concept will be tested by analyzing biological interactions between murine cryptdins and by characterizing the corresponding genes. To test hypotheses regarding mechanisms of Paneth cell defensin action, potential synergistic interactions between cryptdins will be investigated by quantitating the regional cryptdin isoform content of mouse small intestine, evaluating the microbicidal action of modified cryptdins isolated from the intestinal lumen, and by assaying cryptdins, both individually and in combinations that reflect their relative levels, for antimicrobial potency against a range of pathogens. Preparations of individual and combined Paneth cell defensins will be tested for potential cytotoxic effects against mammalian cell lines, and cytotoxicities will be compared with that of myeloid defensins. To investigate the role of pore-formation in the cryptdin killing mechanism, measurements will be made of the kinetics and stoichiometry of ion- permeable channel formation in lipid bilayers by cryptdins. To test hypotheses relevant to regulation of cryptdin synthesis, the primary structures of cryptdins isolated from newborn mouse small bowel will be determined, and the developmental appearance of cryptdin isoforms will be established. The relative placement and orientation of mouse cryptdin isoform genes will be established along the physical map. Finally, potential cis-acting determinants of cryptdin positional specificity will be studied by characterizing transgenic mice constructed with variable flanking and intronic regions of the cryptdin-4 gene. These studies will clarify mechanisms of Paneth cell defensin function in innate immunity by elucidating their mode of action, potential interactions, and the regulation of their synthesis in the adult and developing mouse small intestine.