Genomic/proteomic analysis of human salivary glands

子项目3的总体目标是比较蛋白质和转录本在正常人腮腺和来自特发性口干个体的腮腺组织中的表达。总体假设是特发性口干是由关键调节或效应蛋白表达的变化引起的。为了鉴定人类腮腺特异性蛋白，在特定目标1中：我们将创建人类腮腺的表达序列标签[EST]数据库。为了实现这一目标，我们将从正常成人腮腺构建定向cDNA文库。为了实现这一目标，我们将从正常成人腮腺组织构建定向cDNA文库，并将随机选择的克隆的5'端测序。然后将序列与可用数据库进行比较。我们假设特发性口干是由于关键调节或效应蛋白表达的变化导致液体产生失调。为了验证这一假设，在具体目标2：我们将比较正常腺体中的转录本的表达与那些来自个体的特发性口干使用微阵列和PCR扩增的转录本的直接测序。作为对特定目标1中概述的EST数据库的补充，我们在特定目标3中提出，绘制正常人腮腺和下颌下/舌下腺分泌的蛋白质谱。将使用二维凝胶电泳分离蛋白质，并将使用免疫印迹、Edman降解和基质辅助激光解吸/电离飞行时间（MALDI/TOF）质谱法的组合来鉴定蛋白质。我们假设，这种“正常”的配置文件和使用唾液收集的特发性口干，谁提出了看似“正常”的流速之间的差异将被观察到。在其表达水平上显示差异的蛋白质将是参与维持口腔处于润滑和水合状态的功能作用的候选物。总之，这些方法将使我们能够确定导致口腔干燥的关键调节或效应蛋白的潜在缺陷。

The overall goal of Subproject 3 is to compare the expression of proteins and transcripts in normal human parotid gland with parotid tissue derived from individuals with idiopathic dry mouth. The overall hypothesis is that idiopathic dry mouth is caused by a change in the expression of key regulatory or effector proteins. To identify human parotid gland-specific proteins, in Specific Aim 1: we will create an expressed-sequence-tag [EST] data base of the human parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland tissue and will sequence the 5' end of randomly selected clones. Sequences will then be compared against available databases. We hypothesize that idiopathic dry mouth results from a change in the expression of key regulatory or effector proteins leading to dysregulation of fluid production. To test this hypothesis, in Specific Aim 2: we will compare the expression of transcripts in normal glands with those derived from individuals with idiopathic dry mouth using microarrays and direct sequencing of PCR amplified transcripts. As a complement to the EST database outlined in Specific Aim 1, we propose in Specific Aim 3, to map the spectrum of proteins which are secreted by the normal human parotid and submandibular/sublingual glands. Two dimensional gel electrophoresis will be used to separate proteins and combinations of immunoblotting, Edman degradation and matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) mass spectrometry will be used to identify the proteins. We hypothesize that differences will be observed between this "normal" profile and one obtained using saliva collected from subjects with idiopathic dry mouth, who present with seemingly "normal" flow rates. Proteins displaying variance in their level for expression will be candidates for functional roles involved in maintaining the oral cavity in a lubricated and hydrated state. Taken together, these approaches will allow us to identify underlying defects in key regulatory or effector proteins which lead to oral dryness.