MINERAL-MATRIX RELATIONS IN CALCIFYING TISSUES

描述：(改编自调查人员摘要)本提案 打算获得有关脊椎动物骨骼的新信息 骨骼、软骨和肌腱中的钙化。正常的小鸡，老鼠，大鼠， 和火鸡；骨和软骨培养；以及异常的成骨 一个不完美的老鼠突变体将被用作模型。在这些方面的研究 示例将检查中的某些结构和化学作用 它们各自的细胞外基质、细胞内基质和 细胞内和细胞外接口。工作将有系统地进行， 定义蛋白质分泌途径，细胞基质黏附关系，以及 矿物基质协会。超微结构，生化， 将应用免疫化学和生物物理方法来扩大 申请人以前的结果，描述其存在、性质、位置、 脊椎动物中有机基质与矿物成分的相互作用 钙化。需要检验的具体假设是：有机的 脊椎动物钙化组织的基质对 矿物沉积在晶体大小、形状、位置、 方向和分布。具体目标，以解决结构问题- 在原子、分子和大分子水平上的功能关系 层级，是为了：(1)识别胶原蛋白的存在和位置 和参与矿化的非胶原蛋白，以及 细胞基质中选择的整合素和细胞骨架元素 界面，利用常规和高压电子显微镜， 三维(3D)图像重建和视频技术，以及2D 和3D免疫化学；(2)通过以下方法确定矿物沉积位置 类似的手段，还有电子衍射，x射线探针微分析， 和新颖的傅里叶变换红外光谱；(3)表征 细胞外基质蛋白与矿物质的相互作用 上述方法与原子力显微镜和 计算机模拟(分子动力学)；以及(4)评估 骨细胞培养对基质-矿物质相互作用的影响 蛋白质与抑制物莫能菌素一起运输。有人建议， 结果数据将有助于了解组织的结构和功能 细胞、细胞-基质界面、细胞外基质和事件 脊椎动物钙化的基础。

DESCRIPTION: (Adapted from investigator's abstract) This proposal intends to obtain new information concerning vertebrate skeletal calcification in bone, cartilage, and tendon. Normal chicks, mice, rats, and turkeys; bone and cartilage cultures; and an abnormal osteogenesis imperfecta mouse mutant will be used as models. Studies in these examples will examine certain structural and chemical interactions in their respective extracellular matrices, intracellular matrices, and intra-and extracellular interfaces. Work will be done systematically, defining protein secretory pathways, cell matrix adhesion relations, and mineral-matrix associations. Ultrastructural, biochemical, immunochemical, and biophysical methods will be applied to extend the applicants' previous result describing the presence, nature, location, and interaction of organic matrix and mineral components in vertebrate calcification. The specific hypothesis to be examined is: the organic matrix of vertebrate calcifying tissues critically influences the deposition of mineral in terms of crystal size, shape, location, orientation, and distribution. Specific Aims, to address structure- function relations at atomic, molecular, and macromolecular levels of hierarchy, are to: (1) identify the presence and location of collagenous and non-collagenous proteins involved in mineralization, as well as selected integrins and cytoskeletal elements at the cell-matrix interface, utilizing conventional and high voltage electron microscopy, three-dimensional (3D) image reconstruction and video techniques, and 2D and 3D immunochemistry; (2) determine the sites of mineral deposition by similar means and also electron diffraction, x-ray probe microanalysis, and novel Fourier transform infrared spectroscopy; (3) characterize interaction between extracellular matrix proteins and mineral through correlation of the methods above and by atomic force microscopy and computer modeling (molecular dynamics); and (4) assess the effects in bone cell culture on matrix-mineral interaction following modulation of protein transport with the inhibitor, monensin. It is suggested that outcome data will contribute to knowledge of structure and function of cells, the cell-matrix interface, and extracellular matrices and events fundamental to vertebrate calcification.