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FATTY ACID AND PEPTIDE AMIDATION--A SHARED MECHANSIM

脂肪酸和肽酰胺化——共享机制

基本信息

批准号：
6639546
负责人：
GREGORY P MUELLER
金额：
$ 22.28万
依托单位：
HENRY M. JACKSON FDN FOR THE ADV MIL/MED
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-04-01 至 2006-03-31
项目状态：
已结题

项目摘要

DESCRIPTION: (Adapted from Applicant's Abstract ) Sleep is ubiquitous among mammals and essential for life. More than seventy types of sleep disorders chronically affect millions of Americans in all age groups. Recently, it was shown that the primary fatty-acid amide, oleamide, is a mediator of sleep and may thus contribute to the genesis of sleep disorders. Oleamide accumulates in cerebral spinal fluid during sleep deprivation and induces profound motor quiescence and a sleep-like state upon administration. Primary fatty-acid amides represent a new class of receptor-active signaling molecules whose biogenesis is unknown. Understanding the pathway involved will offer targets for the therapeutic interventions for treating sleep disorders. Recently, we established in vitro that peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is able to catalyze the formation of oleamide. PAM is known for its role as the rate-limiting enzyme in the production of peptide messengers and may thus regulate the production of primary fatty-acid amides as well. The objective of this research is to elucidate the mechanisms that govern oleamide production in brain. Three specific aims are proposed for testing the hypothesis that PAM is the physiologic mediator of oleamide biosynthesis. Aim 1. Establish in a cell-free system that oleamide biosynthesis is dependent upon the actions of PAM. Cell-free models permit the direct investigation of fundamental reaction components. Subcellular fractions of N18TG2 mouse neuroblastoma cells, a line which expresses PAM and synthesizes oleamide, will be used to determine the effects of PAM inhibition on the biosynthesis of oleamide. Aim 2. Establish in cell culture that the induction of PAM during neuronal differentiation mediates an increase in oleamide production. Antisense RNA inhibition of PAM expression will be used to directly establish PAM as an integral component of the oleamide biosynthesis pathway in whole cells. Aim 3. Establish that the production of oleamide in vivo is dependent upon the action of PAM. Experiments conducted in rats will take into account the full biologic complexity of the mammalian organism. Direct measures of oleamide in brain and assessments of tissue activity for oleamide biosynthetic labeling will be used to demonstrate the extent to which inhibition of PAM in vivo impairs oleamide biosynthesis. These studies will define the physiologic role of PAM in mediating the biosynthesis of oleamide. Demonstrating that oleamide biosynthesis proceeds through PAM, or via an alternative pathway, will provide a basis for improving the diagnosis and treatment of sleep disorders.

描述：(根据申请者摘要改编)睡眠普遍存在于哺乳动物和生命的必需品。70多种睡眠障碍长期影响着所有年龄段的数百万美国人。最近，它是研究表明，主要的脂肪酸酰胺，油酰胺，是睡眠和可能因此导致睡眠障碍的发生。油酰胺在体内积聚睡眠剥夺时的脑脊液和深部运动服药后处于静止状态和睡眠状态。初级脂肪酸酰胺类代表了一类新的受体活性信号分子，其生物发生尚不清楚。了解所涉及的途径将提供靶点用于治疗睡眠障碍的治疗干预。最近，我们在体外建立了肽甘氨酸-α-酰胺化单加氧酶(PAM； EC 1.14.17.3)能够催化油酰胺的形成。帕姆以它在多肽信使生产中作为限速酶的作用并因此也可以调节初级脂肪酸酰胺的产生。这个本研究的目的是阐明油酰胺的调控机制。大脑中的生产。提出了三个具体目标来测试假设PAM是油酰胺生物合成的生理介体。目标 1.在无细胞体系中建立油酰胺生物合成依赖于 PAM的作用。无细胞模型允许直接调查基本反应组件。N18TG2小鼠亚细胞组分的研究神经母细胞瘤细胞，一种表达PAM和合成油酰胺的细胞，将用来测定PAM抑制对生物合成的影响油酰胺。目的2.在细胞培养中建立PAM的诱导神经元分化介导了油酰胺产量的增加。反义 RNA抑制PAM的表达将用于直接建立PAM作为一种整个细胞中油酰胺生物合成途径的组成部分。目标3. 证实体内油酰胺的产生依赖于这种作用 PAM。在老鼠身上进行的实验将考虑到完整的生物学哺乳动物有机体的复杂性。脑组织和脑组织中油酰胺的直接测量将使用油酰胺生物合成标记的组织活性评估为了证明体内PAM抑制对油酰胺的损害程度生物合成。这些研究将确定PAM在体内的生理作用调节油酰胺的生物合成。证明油酰胺生物合成通过PAM进行，或通过替代途径，将提供为提高睡眠障碍的诊断和治疗水平奠定了基础。