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MOLECULAR & BIOCHEMICAL ANALYSIS OF MUTANT VWF RECEPTORS

分子

基本信息

批准号：
2771134
负责人：
CLARA N FINCH CRUZ
金额：
$ 11.59万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-01 至 2000-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2771134
关键词：
CHO cells chemical binding genetic recombination glycoproteins molecular genetics molecular pathology mutant phenotype phenylalanine polymerase chain reaction protein sequence receptor expression ristocetin site directed mutagenesis thrombasthenia transfection valine von Willebrand factor

项目摘要

The P.I. has been focused upon developing a career in academic medicine since her early years as a part-time research student in her native Puerto Rico. For the past few years, the P.I. has done research with the sponsor, Dr. Jonathan Miller. The P.I. has studied patients with defective platelet glycoprotein (GP) Ib/IX receptor complexes for von Willebrand factor (vWF). This receptor consists of GPIb-alpha, GPIb-beta, and GPIX. In the course of her research training, the P.I. has learned numerous molecular biology techniques, including: isolation and purification of DNA and RNA, Southern and Northern blot hybridization, RNA dot blot, reverse transcription and PCR techniques, competitive oligonucleotide priming, DNA cloning and sequencing, site-directed mutagenesis, and in vitro expression of human glycoproteins in insect cells. The P.I. will apply these and other newer techniques to accomplish the proposed research work. One of the goals of the proposed work is to express mutant GPIb-alpha on the surface membrane of stable mammalian cell lines expressing GPIb-beta and GPIX. Initially, the work will be focused in the expression of receptor complexes containing the mutant valine GPIb-alpha or the mutant phenylalanine GPIb-beta described in our patients with platelet-type von Willebrand disease and variant Bernard-Soulier disease, respectively. The P.I.'s research approach will be to express homozygous (containing mutant GPIb-beta alone) or heterozygous (containing mutant GPIb-alpha and wild-type GPIb-alpha) mutant vWF receptors. The P.I. will then study the functional activity of each complex. To accomplish this goal, the P.I. will study the binding of vWF to these mutant receptors in the presence of ristocetin or of other modulators. It is anticipated that these studies will reveal the roles of particular amino acid sequences within the mature GPIb-alpha that are important in modulating vWF binding. Finally, it is the P.I.'s intent to identify the molecular abnormality responsible for the classic Bernard-Soulier disease phenotype in patients with normal GPIb-alpha genes. This work should yield relevant information about the interaction among the components of the GPIb/IX receptor, as well as between this receptor and its ligand, vWF. To accomplish these goals, the institution, through the P.I.'s chairman, is committed to providing adequate laboratory space and the necessary resources to create a productive research environment for the P.I.

私家侦探一直致力于发展学术事业，医学，因为她早年作为一个兼职研究生，她家乡波多黎各。在过去的几年里，P.I.做了与赞助商乔纳森米勒博士进行研究。私家侦探具有研究了血小板糖蛋白（GP）Ib/IX缺陷患者血管性血友病因子（vWF）受体复合物。该受体由GPIb-α、GPIb-β和GPIX组成。在她的研究训练私家侦探学过许多分子生物学技术，包括：DNA和RNA的分离和纯化， Southern和北方印迹杂交，RNA斑点杂交，反向转录和PCR技术，竞争性寡核苷酸引发、DNA克隆和测序、定点诱变，以及人糖蛋白在昆虫细胞中的体外表达。的 P.I.将应用这些和其他更新的技术来实现建议的研究工作。拟议工作的目标之一是在稳定的细胞膜表面表达突变型GPIb-α，表达GPIb-β和GPIX的哺乳动物细胞系。最初工作将集中在受体复合物的表达含有突变的缬氨酸GPIb-α或突变的苯丙氨酸 GPIb-β在我们的血小板型血管性血友病患者中的描述 Willebrand病和变体Bernard-Soulier病，分别私家侦探的研究方法将是表达纯合子（仅含突变GPIb-β）或杂合子（含有突变型GPIb-α和野生型GPIb-α）突变型vWF 受体。私家侦探然后将研究每一个细胞的功能活动复杂.为了实现这一目标，P.I.将研究 vWF与这些突变型受体的结合，其他调制器。预计这些研究将揭示了特定氨基酸序列的作用，成熟GPIb-α在调节vWF结合中是重要的。最后是私家侦探。的意图是确定分子一种导致典型Bernard-Soulier病的异常 GPIb-α基因正常的患者的表型。这项工作应该产生有关相互作用的相关信息， GPIb/IX受体的组成部分，以及受体及其配体vWF。为了实现这些目标，通过P.I.的董事长，致力于提供足够的实验室空间和必要的资源，以建立一个为私家侦探创造一个高效的研究环境