INTESTINAL BRUSH BORDER MEMBRANES

这项提议的长期目标是研究分子基础 钴胺(Cb1；维生素B12)在体内的转运有缺陷 肠上皮细胞和各种外周组织 Cbl转运蛋白转钴胺II(TC II)缺乏。 将解决的具体问题包括对 在基因水平上调节TC II基因表达的因素 肠吸收细胞及其对慢性淋巴细胞性白血病穿细胞性的影响 TC II基因的杂交抑制翻译。这些研究中的许多将使用 自发分化人结肠腺癌(Caco-2)细胞， 和大鼠(胎儿和成年)。[32P]-核糖核酸酶保护分析 将使用从TC II基因的Nde I酶切转录的cRNA(350bp) 为了检测上皮细胞分化过程中TC II基因的表达水平， 肠道发育，沿水平方向(十二指肠-回肠)和 垂直(隐窝到绒毛)轴。此外，杂交的效果 反义寡核苷酸抑制TC II基因的翻译 产生于TC II基因不同区域的内源性因子 [57Co]Cbl的介导性转胞作用将用极化CaCO- 2个细胞生长在培养片上。这些研究应该提供重要的 TC II在正常人群中表达和作用的调控线索 Cbl在肠道细胞中的跨细胞转运，这反过来可能有助于 对慢性阻塞性肺疾病肠道吸收不良的病理生理认识 在TC II缺乏的患者中注意到。利用体外诱变技术对沙门氏菌进行研究 TC II基因：构成或起重要作用的结构元件 在与TC II的结合反应中将检测其分泌情况 使用从突变的TC II转录而来的cRNA在 网织红细胞裂解物或非洲爪哇卵母细胞表达系统。这个 TC II多态的结构基础，包括TC II缺乏 患者将使用皮肤成纤维细胞在RNA/DNA水平上进行检查 从这些病人那里得到的。用聚合酶链式反应扩增DNA 我们将研究TC II中分子缺陷的性质和位置。 在DNA水平上使用限制片段长度多态性和/或 变性梯度凝胶电泳法。为了开始研究…… 人体组织与肠道调控的区域变异 在TC II基因的表达中，我们将首先分离出一个全长基因 并绘制出内含子和外显子。对其结构的分析 TC II将为TC II在以下方面的作用提供重要见解 维持Cbl跨细胞膜的通量。这样的研究应该 帮助我们更好地了解Cbl吸收不良的病理生理学和 先天性TC II缺乏患者的Cbl转运

The long term goal of this proposal is to examine the molecular basis for the defective transport of Cobalamin (Cbl; Vitamin B12) out of the enterocytes and into various peripheral tissues in patients who exhibit deficiency of transcobalamin II (TC II), a Cbl transport protein. Specific questions that will be addressed include an analysis of the factors that regulate the expression of TC II gene at the mRNA level in the absorptive enterocytes and the effect on Cbl transcytosis following hybrid arrest translation of TC II mRNA. Many of these studies, will use spontaneously differentiating human colon adenocarcinoma (Caco-2) cells, and rats (fetal and adult). Ribonuclease protection assay using [32P]- cRNA (350 bp) transcribed from Nde I digest of TC II cDNA will be used to measure TC II mRNA levels during epithelial cell differentiation, intestinal development, and along the horizontal (duodenum-ileum) and vertical (crypt to villus) axis. Furthermore, the effect of hybrid arrest translation of TC II mRNA by using anti-sense oligonucleotides generated to different regions of TC II cDNA on the intrinsic factor mediated transcytosis of [57Co]Cbl will be examined using polarized Caco- 2 cells grown on culture inserts. These studies should provide important clues on the regulation of expression and role of TC II in the normal transcytosis of Cbl across the enterocytes, which in turn may help in understanding the pathophysiology of intestinal malabsorption of Cbl noted in patients with TC II deficiency. Using in vitro mutagenesis of TC II cDNA the structural elements that contribute to or are important in the binding reactions of TC II and its secretion will be examined using the cRNA transcribed from mutagenized TC II expressed in either reticulocyte lysate or Xenopus laevis oocyte expression systems. The structural basis for TC II polymorphism, including TC II deficiency in patients will be examined at the RNA/DNA levels using skin fibroblasts obtained from these patients. Following amplification of the DNA by PCR the nature and locus of the molecular defect in TC II will be examined at the DNA level using restriction fragment length polymorphism and/or denaturing gradient gel electrophoresis. In order to begin studies on the human tissue and regional variation in the intestine of regulation of the TC II gene expression, we will first isolate a full length gene for TC II and map out the introns and exons. Analysis of structure of TC II will provide important insights into the role of TC II in maintaining flux of Cbl across cellular membranes. Such studies should help us to better understand the pathophysiology of Cbl malabsorption and transport of Cbl in patients with congenital lack of TC II.