SELECTIN MEDIATED CELL ADHESION UNDER HYDRODYNAMIC SHEAR

选择流体力学剪切下介导的细胞粘附

• 批准号: 7330308
• 负责人: SRIRAM NEELAMEGHAM
• 金额: $ 22.21万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7330308
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; Acute; Address; Adhesions; Affect; Affinity; Anabolism; Area; Binding; Biochemical; Biochemistry; Biological Assay; Blood Vessels; Carbohydrates; Cardiovascular Diseases; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cell surface; Cells; Chemicals; Chimera organism; Complement; Complex; Condition; Data; Development; Disaccharides; Disease; Dose; Drug Design; Engineering; Epitopes; Family; Family member; Figs - dietary; Flow Cytometry; Fucose; Future; Glycolipids; Glycoproteins; HL60; Human; Immune response; Infiltration; Inflammation; Inflammatory; Kinetics; Lead; Lectin; Leukocytes; Ligand Binding; Ligands; Link; Liquid Chromatography; Measurement; Measures; Mediating; Membrane Glycoproteins; Metabolic; Metabolic Pathway; Methods; Modeling; Modification; Molecular Biology; Monosaccharides; Numbers; O-Glycans Biosynthesis Pathway; Oligosaccharides; P-Selectin; P-selectin ligand protein; Pathology; Pathway interactions; Peritonitis; Physiological; Polysaccharides; Process; Proteins; Range; Rate; Reaction; Reagent; Reporting; Research Personnel; Research Project Grants; Running; Selectins; Simulate; Source; Structure; Surface Plasmon Resonance; Systems Biology; Testing; Thioglycolates; Tissues; Trisaccharides; Ursidae Family; Vascular Diseases; Vascular Endothelial Cell; Western Blotting; Work; analog; base; carbohydrate binding protein; carbohydrate receptor; carbohydrate structure; chemical synthesis; concept; data modeling; design; feeding; fluid flow; glycosylation; glycosyltransferase; in vivo; inhibitor/antagonist; insight; interest; mathematical model; mouse model; novel; numb protein; prevent; programs; reaction rate; research study; small molecule; sulfated glycoprotein p50; tool

This research project examines the function of carbohydrate chains that are O-linked to leukocyte cell- surface glycoproteins. By acting as the natural ligands of the selectin family of adhesion molecules, these glycoproteins control the rates of leukocyte adhesion in the human vasculature during normal immune response, inflammatory diseases and certain types of cardiovascular disorders. It is widely believed that controlling the rate of leukocyte adhesion in vascular disorders can lead to new therapies to combat these ailments. Thus, in the current proposal, we evaluate two mechanisms for controlling selectin-ligand binding. In Aim 1, we develop and test the ability of unique molecules based on an unusual disaccharide carbohydrate structure (GalNAc(31,3GalNAca-O-Methyl) to competitively inhibit selectin binding interactions with its ligand. Our preliminary data suggests that this disaccharide alone can bind P- selectin. We also demonstrate that appropriate modification of this unit can dramatically enhance the binding affinity of the resulting carbohydrate for selectins, when compared with the prototypic selectin ligand sialyl Lewis-X. In Aim 2, we test an approach where small-molecule metabolic inhibitors are designed based on the structure of monosaccharides that compose natural selectin ligands. These modified monosaccharidesare fed to cells in order to interfere with the biosynthesis of specific carbohydrate epitopes on the glycoprotein ligands of selectins. More specifically, these molecules are directed to alter either the core or terminal residues of glycans expressed by an important leukocyte selectin-ligand called PSGL-1 (P-selectin glycoprotein ligand- 1). We evaluate the ability and mechanism by which these chemical inhibitors permeate cells, engage and modify glycan biosynthetic pathways and inhibit cell adhesion. In Aim 3, to complement the experimental work above, a Systems Biology based mathematical model is developed to simulate biochemical networks that regulate O-glycan biosynthesis in leukocytes. Many of the assumptions in this mathematical model are experimentally validated. Diverse experimental methods are applied to accomplish the above three aims. These include cell adhesion studies under controlled flow, in vivo experiments in a mouse model of acute inflammation, western blot analysis, molecular biology based approaches, flow cytometry, surface plasmon resonance and liquid chromatography. In the long run, we anticipate that small-molecule selectin-antagonists will be identified from this work that may aid future drug design. Mathematical models developed will enhance the application of metabolic engineering principles in the area of biological chemistry. Such analysis can also provide the rationale for the chemical synthesis of new inhibitors and for interpretation of experimental observations.

该研究项目检查了与白细胞O-连接的碳水化合物链的功能， 表面糖蛋白通过作为粘附分子的选择素家族的天然配体， 在正常免疫过程中，糖蛋白控制着人体血管中白细胞粘附的速率 反应、炎性疾病和某些类型的心血管疾病。外界普遍认为 控制血管疾病中白细胞粘附的速率可以导致对抗这些疾病的新疗法。 疾病因此，在目前的建议，我们评估两种机制控制选择素配体结合。 在目标1中，我们开发并测试了基于一种不寻常的二糖的独特分子的能力， 糖结构（GalNAc（31，3GalNH 4-O-甲基））竞争性抑制选择素结合相互作用 与配体我们的初步数据表明，这种二糖单独可以结合P-选择素。我们也 证明了对该单元的适当修饰可以显著增强该单元的结合亲和力。 当与原型选择素配体sialyl Lewis-X相比时，所产生的选择素的碳水化合物。在Aim中 2，我们测试了一种方法，其中小分子代谢抑制剂是基于以下结构设计的： 组成天然选择素配体的单糖。这些修饰过的单糖被喂入细胞， 为了干扰糖蛋白配体上特异性碳水化合物表位的生物合成， 选择素更具体地，这些分子针对改变蛋白质的核心或末端残基。 由称为PSGL-1的重要白细胞选择素配体表达的聚糖（P-选择素糖蛋白配体， 1）。我们评估了这些化学抑制剂渗透细胞，参与和 修饰聚糖生物合成途径并抑制细胞粘附。在目标3中，为了补充实验 在上述工作的基础上，建立了一个基于系统生物学的生化网络数学模型 调节白细胞中的O-聚糖生物合成。这个数学模型中的许多假设是 实验验证。为了实现上述三个目标，采用了多种实验方法。 这些研究包括在受控流下的细胞粘附研究、在急性炎症小鼠模型中的体内实验、在急性炎症小鼠模型中的体内实验、在急性炎症小鼠模型中的体内实验、在急性炎症小鼠模型中的体内实验、以及在急性炎症小鼠模型中的体内实验。 炎症，蛋白质印迹分析，基于分子生物学的方法，流式细胞术，表面等离子体 共振和液相色谱法。从长远来看，我们预计小分子选择素拮抗剂 将从这项工作中确定，可能有助于未来的药物设计。开发的数学模型将 加强代谢工程原理在生物化学领域的应用。这种分析 也可以为化学合成新的抑制剂和解释 实验观察。