ENAMEL WITHOUT ENAMELIN

描述（由申请人提供）：无釉质的釉质：我们的长期目标是了解正常和患病牙釉质形成的分子机制。人釉蛋白基因突变导致常染色体显性遗传性釉质发生障碍。本研究旨在探讨釉蛋白在牙釉质形成过程中的作用。 这两个特定目标建议通过以下方式研究釉蛋白功能： SA 1：在小鼠釉蛋白表达不存在的情况下表征釉形成。 SA 2：表征釉蛋白的结构和功能特性。 在特定目标1中，我们产生了一个敲入小鼠，该小鼠用融合到小鼠核定位信号（NLS-lacZ）的细菌13-半乳糖苷酶的编码区替换釉蛋白基因。然后，我们比较了在没有釉蛋白表达的情况下形成的釉质与野生型小鼠形成的釉质。将通过冷冻切片和完整封片中的X-gal染色表征釉蛋白-NLS-lacZ报告基因的表达。生化分析将包括SDS-PAGE、蛋白质印迹和氨基酸分析。矿物分析将包括光透射显微镜（LTM）、扫描电子显微镜（SEM）以寻找点蚀、碎屑或其他表面缺陷，透射电子显微镜（TEM）以确定晶体形态，X射线显微分析以确定元素组成，选区电子衍射（SAED）以确定矿物类型，双能X射线吸收（DEXA）以测量矿物密度，组织形态测量和Faxitron放射学用于高分辨率X射线成像以确定釉质厚度，以及微计算机断层扫描用于矿物质面积和体积的3-D重建。 在具体目标2釉蛋白水解裂解产物分离的猪釉质基质和其特征在于揭示其翻译后修饰。这些釉蛋白如何影响晶体生长在体外进行了分析，使用pH稳态和恒定组成的羟基磷灰石形成的测定，抑制羟基磷灰石生长的种子生长测定，并通过X射线衍射法分析羟基磷灰石生长习性 这项研究将确定釉蛋白是否催化釉质晶体成核和晶体伸长，或控制晶体习性。这些发现将为构建未来关于釉蛋白控制釉质生物矿化的分子机制的假设建立一个适当的知识基础。

DESCRIPTION (provided by applicant): Enamel Without Enamelin: Our long-term objective is to understand the molecular mechanisms of normal and diseased dental enamel formation. Mutations in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. In this investigation, we study enamelin's functions during amelogenesis. The two Specific Aims propose to investigate enamelin function by: SA 1: characterizing enamel formation in the absence of mouse enamelin expression. SA 2: characterizing enamelin protein structural and functional properties. In Specific Aim 1 we generate a knock-in mouse that replaces the enamelin gene with the coding region for bacterial 13-galactosidase fused to a mouse nuclear localization signal (NLS-lacZ). We then compare enamel formed in the absence of enamelin expression to enamel formed in the wild-type mice. Expression of the enamelin-NLS-lacZ reporter gene will be characterized by X-gal staining in frozen sections and whole mounts. Biochemical analyses will include SDS-PAGE, Western blotting, and amino acid analysis. Mineral analyses will include Light Transmission Microscopy (LTM), Scanning Electron Microscopy (SEM) to look for pitting, chipping or other surface defects, Transmission Electron Microscopy (TEM) to determine crystal morphology, X-ray microanalysis to determine elemental composition, Selected-Area Electron Diffraction (SAED) to determine mineral type, Dual-Energy X-ray Absorption (DEXA) to measure mineral density, histomorphometric measurement and Faxitron radiology for high-resolution X-ray imaging to determine enamel thickness, and Micro-computed tomography for 3-D reconstruction for mineral area and volume. In Specific Aim 2 enamelin proteolytic cleavage products are isolated from the pig enamel matrix and characterized to reveal their posttranslational modifications. How these enamelins influence crystal growth in vitro is analyzed using pH stat and constant composition assays of hydroxyapatite formation, seeded growth assays for inhibition of hydroxyapatite growth, and by analysis of hydroxyapatite growth habit by X-ray diffractometry This study will determine if enamelin catalyzes enamel crystal nucleation and crystal elongation, or controls crystal habit. The findings will establish an appropriate knowledge base for framing future hypotheses concerning the molecular mechanisms by which enamelin controls enamel biomineralization.