ENAMEL WITHOUT ENAMELIN

描述(申请人提供)：不含釉质的釉质：我们的长期目标是了解正常和疾病牙釉质形成的分子机制。人类釉质基因突变导致常染色体显性显性釉质发生不全。在本研究中，我们研究了釉质蛋白在成釉过程中的作用。 这两个具体目标建议通过以下方式研究釉质蛋白的功能： SA 1：在没有小鼠釉质蛋白表达的情况下表征釉质的形成。 SA 2：表征釉蛋白的结构和功能特性。 在特定的目标1中，我们产生了一种敲入小鼠，它用细菌13-半乳糖苷酶的编码区与小鼠核定位信号(NLS-LacZ)融合来取代釉蛋白基因。然后，我们将在没有釉质蛋白表达的情况下形成的釉质与在野生型小鼠中形成的釉质进行比较。釉蛋白-NLS-LacZ报告基因的表达将通过冰冻切片和整体支架的X-Gal染色来表征。生化分析将包括SDS-PAGE、Western blotting和氨基酸分析。矿物分析将包括光学透射显微镜(LTM)、扫描电子显微镜(SEM)用于寻找点蚀、碎裂或其他表面缺陷、透射电子显微镜(TEM)用于确定晶体形态、X射线显微分析用于确定元素组成、选区电子衍射(SAED)用于确定矿物类型、双能X射线吸收(DEXA)用于测量矿物密度、组织形态测量和Faxitron放射学用于高分辨率X射线成像以确定牙釉质厚度，以及微型计算机断层扫描用于矿物面积和体积的三维重建。 在特定目的中，从猪牙釉质基质中分离出2种釉质蛋白水解性切割产物，并对其进行表征以揭示其翻译后修饰。通过pH值恒定和羟基磷灰石形成的恒定组成分析、种子生长抑制羟基磷灰石生长的实验和X射线衍射仪分析羟基磷灰石的生长习性，分析了这些釉质蛋白对体外晶体生长的影响。 这项研究将确定釉质是否催化釉质晶体成核和晶体伸长，或控制晶体习性。这些发现将为建立关于釉质蛋白控制釉质生物矿化的分子机制的未来假说奠定适当的知识基础。

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.