The aim of the project is to define the genetically induced group of storage diseases of children known as the ceroid-lipofuscinoses in biochemical terms using as an experimental tool the ovine model. The long term objective would be to use this information as a basis to prenatal diagnosis, heterozygote detection and the development of therapeutic and control strategies. Analyses of isolated storage lipopigment indicate that this disease could reflect a proteinosis as the dominant chemical species stored are polypeptides. These could accumulate either as a result of a specific protease deficiency or as a consequence of disordered protein processing or recycling. The origin of the specific polypeptides will be sought by electron microscopic immunocytochemistry. Experiments will also purify and characterize these enriched polypeptides in order to compare them for common features that might help elicit the underlying biochemical anomaly. Methods will include amino acid analyses, determination of C and N terminals, peptide mapping by HPLC and two dimensional electrophoretic/TLC methods, with amino acid sequencing of peptides of primary interest. Differences in lysosomal and tissue proteases between affected and control animals will be looked for by a variety of methods. The chemical and flurophor content of lipopigment will be compared with that of other lipopigments including age pigment and pigment of brown atrophy in cattle to find those features of lipopigments which are common to lipopigments and hence probably secondary phenomena. Pathophysiological experiments will involve electronmicroscopy of retinal pigment epithelium, other tissues, and of platinum plated freeze fractured preparations of isolated lipopigment. Prenatal diagnosis will be attempted by SDS-PAGE of amniotic fluid. Evidence of a secondary auto-oxidation of lipopigment will be sought by histochemistry of brain and TLC of lipids from isolated lipopigment. Collaborative studies include brain metabolism studies by topical-magnetic-resonance spectrometry and development of an early diagnostic technique by measurement of native retinal fluorescence in the live individual.
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