LAMININ ALTERATIONS AFTER NON-ENZYMATIC GLUCOSYLATION

在糖尿病中，高血糖扰乱了许多代谢途径， 每一种都可能导致糖尿病并发症。其中一个主要的 改变是蛋白质的非酶糖基化；它 分两步发生：第一步，可逆的一步(Amadori的形成 产品)和第二个不可逆转的(形成 最终产品)。因为它们的半衰期很长，地下室 膜大分子是这一过程的主要目标。在……里面 糖尿病肾病，其中最重要的变化是 肾小球增厚和通透性增加 基底膜。我们的长期目标是了解 在分子水平上，这些改变的发病机制。 层粘连蛋白是最重要的基底膜之一。 组件；它通过以下方式在其结构组织中发挥关键作用 与大多数其他基底膜大分子相互作用 和细胞表面。因此，我们建议调查这一影响 非酶糖基化对血管紧张素转换酶几种生物学功能的影响 层粘连蛋白与阿司匹林的作用(已知可抑制可逆的 步骤)和氨基胍(已知可以抑制不可逆的步骤) 在恢复受影响的功能方面。在我们的研究中，有两个体外实验 将使用模型系统：首先，从一个 产生基底膜的小鼠肿瘤(EHS)将 糖基化；第二，孤立的、完整的肾小球基底膜 大鼠的膜将被糖基化，然后层粘连蛋白将被 提取出来的。糖基化方案将包括添加阿司匹林 或者氨基胍。在这些治疗之后，将检测层粘连蛋白 对于它的结构外观和功能分析来检查 非酶糖基化对其性质的影响。这个 功能分析将包括：a)二聚体形成，b) 聚合，c)与IV型胶原结合，d)与 肝素，e)与硫酸肝素蛋白多糖结合。一种生物物理学 技术(浊度法)和生化技术(固相分析) 和一个形态的(旋转阴影的电子显微镜 样品)将用于这些检测。拟议的研究将 A)扩大我们在结构和功能方面的知识 糖尿病条件下基底膜的修饰， B)使我们能够测试对功能的影响和治疗 非酶修饰的每一阶段 糖基化，以及c)允许我们确定可能的副作用 看起来对预防癌症进展有希望的化合物 糖尿病并发症。

In diabetes, hyperglycemia disturbs many metabolic pathways and each may lead to diabetic complications. One of the major alterations is the non-enzymatic glucosylation of proteins; it occurs in two steps: a first, reversible one (formation of Amadori products) and a second, irreversible one (formation of endproducts). Because of their long half-life, basement membrane macromolecules are primary targets of this process. In diabetic kidney disease, among the most important alterations are the thickening and the increased permeability of the glomerular basement membrane. Our long term objective is to understand at the molecular level the pathogenesis of those alterations. Laminin is one of the most important basement membrane components; it plays a key role in their structural organization, by interacting with most other basement membrane macromolecules and cell surfaces. Therefore, we propose to investigate the effect of non-enzymatic glucosylation on several biological functions of laminin and the role of aspirin (known to inhibit the reversible step) and aminoguanidine (known to inhibit the irreversible step) in restoring affected functions. For our studies, two in vitro model systems will be used: first, laminin extracted from a basement membrane-producing murine tumor (EHS) will be glucosylated; second, isolated, intact glomerular basement membranes from rats will be glucosylated and then laminin will be extracted. Glucosylation protocols will include addition of aspirin or aminoguanidine. After these treatments laminin will be tested for its structural appearance and in functional assays to examine the effect of non-enzymatic glucosylation on its properties. The functional assays will include: a) dimer formation, b) polymerization, c) binding to type IV collagen, d) binding to heparin, e) binding to heparan sulfate proteoglycan. A biophysical technique (turbidometry) and biochemical one (solid-phase assays) and a morphological one (electron microscopy of rotary shadowed samples) will be used for these assays. The proposed studies will a) broaden our knowledge on structural and functional modifications of basement membranes under diabetic conditions, b) enable us to test for functional implications and treatment of each stage of the modifications due to non-enzymatic glucosylation, and c) allow us to determine possible side effects of compounds that appear promising for preventing progression of diabetic complications.