INTESTINAL BRUSH BORDER MEMBRANES

这项提案的长期目标是研究 钴胺素（Cbl;维生素B12）的运输缺陷， 肠上皮细胞和各种外周组织中， 转钴胺素II（TC II）缺乏，这是一种Cbl转运蛋白。 将讨论的具体问题包括分析 在mRNA水平上调节TC II基因表达的因素 吸收性肠上皮细胞及对Cbl转胞吞作用的影响 杂交体阻止TC II mRNA翻译。 这些研究中的许多将使用 自发分化的人结肠腺癌（Caco-2）细胞， 和大鼠（胎儿和成年）。 使用[32 P]-的核糖核酸酶保护测定 将使用从TC II cDNA的Nde I消化物转录的cRNA（350 bp） 为了测量上皮细胞分化期间TC II mRNA水平， 肠发育，沿着水平（回肠）和 垂直轴（隐窝到绒毛）。 此外，混合的效果 用反义寡核苷酸阻断TC Ⅱ mRNA的翻译 产生TC II cDNA上不同区域的内因子 [57 Co]Cbl介导的转胞吞作用将使用极化的Caco- 2个细胞在培养插入物上生长。 这些研究将提供重要的 TC II在正常人中的表达调节和作用的线索 Cbl跨肠细胞的转胞吞作用，这反过来可能有助于 了解Cbl肠吸收不良的病理生理学 在TC II缺乏的患者中观察到。 使用体外诱变 TC II cDNA是有助于或重要的结构元件 在TC II的结合反应及其分泌将被检查 使用从突变的TC II转录的cRNA， 网织红细胞裂解物或非洲爪蟾卵母细胞表达系统。 的 TC II多态性的结构基础，包括 将使用皮肤成纤维细胞在RNA/DNA水平对患者进行检查 从这些病人身上。 通过PCR扩增DNA后 将对TC II中分子缺陷的性质和位置进行检查 在DNA水平使用限制性片段长度多态性和/或 变性梯度凝胶电泳。 为了开始研究 人体肠道组织和区域的变化规律 TC II基因的表达，我们将首先分离全长基因 并绘制出内含子和外显子。 结构分析 第二个技术合作委员会将提供关于第二个技术合作委员会在以下方面的作用的重要见解： 维持Cbl穿过细胞膜的通量。 这种研究 帮助我们更好地了解Cbl吸收不良的病理生理学， 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.