This application is designed to continue studies aimed at a clarification of the intracellular formation of various collagens. Enough progress has been made recently in the techniques of radioautography and immunohistochemistry at the electron microscopic level to make it possible to combine these methods on the same sections. Thus, at various times after in injection of 3H-proline, radioautographic silver grains (which are spherical and about 50 nm wide) and immunogold particles (15 nm) can be counted separately over the same organelle (rER cisterna, Golgi saccule, secretory granule). The ratio of the two counts will provide the specific activity of collagen precursors in each organelle and its variation with time. The turnover rate of the precursors will when be calculated at each step along the intracellular pathway of collagen. Specific activity measurements will be applied to the study of the biogenesis of type IV collagen in active chondrocytes, type I collagen in odontoblasts, type IV collagen in endodermal cells and type III collagen in periodontal fibroblasts.
A second aim of the application is to continue the investigations of basement membrane and related structures. The major component of basement membrane is shown at high magnification of the electron microscope to be a tridimensional network of anastomosing strands, referred to as """"""""cords"""""""", whose average width is 4 nm. It is planned to combine purified type IV collagen with other basement membrane components in vitro in the hope of reproducibly obtaining 50-100 nm sheets made up of a network of 4-nm cords, that is, sheets similar to the lamina densa of authentic basement membranes. A minor component of some basement membrane, the basotuble - a 9 nm wide rod of indefinite length - has been shown to have the same dimension and ultrastructure as the microfibrils of connective tissues. The main structural element of both is a core consisting of stacked molecules of amyloid P component (a glycoprotein known to be present in normal serum and to accumulate in amyloid degeneration). Three substances, i.e. fibronectin, fibrillin and a proteoglycan, have been shown to be associated with microfibrils.
The aim of our work is to utilize immunolabeling for these substances using 5 nm gold particles to determine how they related to the amyloid P core of microfibrils and basotubules.
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