LIPID AND PROTEIN EFFECTS ON MONOLAYER STABILITY

临床批准的肺表面活性物质治疗方法差异很大 由40-80%二棕榈酰磷脂酰胆碱(DPPC)组成的组合物， 不饱和脂类和阴离子脂类的变化也一样大。 表面活性物质中脂肪和蛋白质含量及组成的变化 灌洗与新生儿疾病的严重程度有关 成人呼吸窘迫综合征。然而，对 肺表面活性物质的脂类和蛋白质组分的作用仍不清楚 缺乏，很难将成分的变化与 最佳替代配方或特定生理学配方 各州。在这个修订的提案中，我们假设SP-B，in 与SP-C结合可增强表面活性剂的形成 在单层过程中保持附着在界面上的“储液层” 压缩。这一模型表明， SP-B和SP-C蛋白以及阴离子和不饱和脂类允许 这些脂类在高表面压力下保持在单层中 在压缩过程中形成(低表面张力)，同时促进 再膨胀时单层的再铺展。我们已经证明了SP- B的作用是将PG单层折叠成亚相，显然是通过增加 单层的柔性和弹性在某种程度上并不完全 明白了。SP-C是一种跨膜蛋白，可能会稳定这种情况 用折叠单层结构拼接的储集层结构 以形成更稳定的多层膜。为了检验这些假设，我们将使用 荧光、偏振荧光、布鲁斯特角和原子 根据第一项建议发展的力显微镜技术：(A) 确定不饱和和/或饱和卵磷脂是否可以 通过与SP-B的协同作用保留在单层中 和/或SP-C(B)确定增强 通过检测原生SP-B全长合成来生产褶皱 SP-B，1-25个氨基端肽，SP-B的不带电荷突变体，以及 KL4多肽。(C)将这些观察的结果与 以小牛肺表面活性物质为基准的形态特征 “天然”表面活性剂的形态和功能。如果有很大一部分 不饱和脂肪可以保留在单层中，很可能是 这些脂类是增强涂抹性能所必需的。为了测试 在这一假设下，我们计划(D)设计和建造一种沟道型单层 粘度计用于量化双分子层的流动性和重新铺展。一个系统的 单层粘度与蛋白质和蛋白质的关系 不饱和脂类浓度将提供一个很好的衡量 用于快速重新传播的最佳配方，并有助于解释 天然表面活性剂中含有大量不饱和脂类。

Clinically approved lung surfactant therapies vary widely in composition, from 40 - 80 percent dipalmitoylphosphatidylcholine (DPPC), with equally wide variations in unsaturated and anionic lipids. Variations in lipid and protein quantity and composition in surfactant taken by lavage are associated with severity of disease in neonatal and adult respiratory distress syndrome. However, an understanding of the roles of the lipid and protein components of lung surfactants is still lacking, and it is difficult to relate variations in composition to either optimal replacement formulations or to specific physiological states. In this revised proposal, we hypothesize that SP-B, in combination with SP-C, enhances the formation of a surfactant "reservoir" that remains attached to the interface during monolayer compression. This model suggests that synergistic interactions between the SP-B and SP-C proteins and the anionic and unsaturated lipids allows these lipids to be held in the monolayer at the high surface pressures developed during compression (low surface tensions), while facilitating re- spreading of the monolayers on re-expansion. We have shown that SP- B acts to fold PG monolayers into the subphase, apparently by increasing the flexibility and elasticity of the monolayer in a way not fully understood. SP-C, which is a transmembrane protein, may stabilize this reservoir structure by stitching together the folded monolayer structure to form more stable multilayers. To test these hypotheses, we will use the fluorescence, polarized fluorescence, Brewster angle and atomic force microscopy techniques developed under the first proposal to: (A) determine if unsaturated and/or saturated phosphatidylcholines can be retained in the monolayer through synergistic interactions with SP-B and/or SP-C (B) determine the protein features required to enhance the production of the folds by examining native SP-B, full length synthetic SP-B, the 1-25 amino terminal peptide, an uncharged mutant of SP-B, and the KL4 peptide. (C) Compare the results of these observations with the morphological characteristics of calf lung surfactant as a benchmark for "natural" surfactant morphology and function. If large fractions of unsaturated lipids can be retained in the monolayer, it is likely that these lipids are necessary to enhance the spreading properties. To test this hypothesis, we plan to (D) design and build a canal-type monolayer viscometer to quantify bilayer fluidity and respreading. A systematic investigation of monolayer viscosity as a function of protein and unsaturated lipid concentration will provide a good measure of the optimal formulation for rapid respreading and could help explain the large unsaturated lipid fraction in natural surfactant.