The study of proteoglycan metabolism in normal and diabetic tissues will be approached in a multidisciplined manner in order to uncover the mechanisms of the relationships of abnormalities of these large molecular weight, highly charged and strategically located compounds in diabetes mellitus to its complications. Four major avenues of endeavor will be undertaken: 1) in vivo and in vitro 35SO4-labelling studies to determine the turnover and species distribution of radiolabelled proteoglycan in normal and diabetic glomeruli and GBM, including analysis of the effects of glycoprotein synthesis inhibition with tunicamycin and correction of glucose homeostatic abnormalities with insulin; 2) preparation of heterologous and monoclonal antibodies to guanidine extracted, CsC1 gradient centrifugation and DEAE chromatographically purified proteoglycan isolated from various tissues, including normal and diabetic human aorta and kidney, Swarm rat chondrosarcoma, and bovine aorta and the use and development of techniques to assay antibody specificity including the ELISA and the transfer of antigen to nitrocellulose to assess antibody reactivity with electrophoretic components; 3) in vitro translation of mRNA isolated from tissue reactive with the above antibodies, such as kidney or liver or from tissue reactive with the above antibodies, such as kidney or liver, or from tissue producing large quantities of proteoglycan, such as chondrocytes and immunoprecipitation of labelled reticulocyte lysate translated products in addition to assessing the effects of diabetes on expression of proteoglycan genes by two-dimensional gel electrophoresis; and, 4) electron microscopic proteoglycan localization using cationic probes and the definition of the proteoglycan subspecies responsible for staining at a particular site using highly purified heparinase and other specific enzymes. Antibody will be localized in normal and disease tissue by immunofluorescent and immunoperoxidase (EM) techniques.
| Klein, D J; Brown, D M; Moran, A et al. (1989) Chondroitin sulfate proteoglycan synthesis and reutilization of beta-D-xyloside-initiated chondroitin/dermatan sulfate glycosaminoglycans in fetal kidney branching morphogenesis. Dev Biol 133:515-28 |
| Klein, D J; Oegema Jr, T R; Brown, D M (1989) Release of glomerular heparan-35SO4 proteoglycan by heparin from glomeruli of streptozocin-induced diabetic rats. Diabetes 38:130-9 |
