1. Continued studies of a mutant mouse which stores cystine in lysosomes as do tissues from cystinotic and I-cell patients. Investigation of effects of various cystine lowering drugs on levels of stored cystine in the mouse organs since there has not previously been an animal model for cystinosis. 2. Continued studies of anomalies in cholesterol metabolism transport in Niemann-Pick C (NPC), the mutant mouse and cystinotic fibroblasts. Investigation of cholesterol movement from the lysosomal compartment (where it is stored in NPC disease and in the mutant mouse) to the microsomes where cholesterol can be esterified and packaged for storage in lipid vesicles. Use of hydrophobic amines and progesterone to produce NPC disease in normal fibroblasts. 3. Characterization of a cystinotic cell protein associated with cystine and present in a 2-fold excess in cystinotic versus normal fibroblasts. Amino acid analysis of this protein, N-terminal sequencing, a search for similar proteins and investigation of their cystine binding characteristics. Analysis of a cystine binding protein (CBP) of E. Coli used for quantitative analysis of the cystine content of tissues. This investigation has included purification by HPLC, amino acid analysis, N- terminal sequencing of two purified components and comparison to known proteins in data banks. One protein was similar to a histidine binding protein of S. Typhimurium and bound histidine. Another protein was not similar to any known protein and bound cystine. Further work involves antibody production to the N-terminal peptide of the purified CBP, a search for similar proteins in eukaryotic cells and characterization of the actual cystine binding site. 4. Investigation of alternative ways of depleting cystine and cholesterol levels in mutant mouse tissues and cystinotic fibroblasts, using single and combined drugs that influence intracellular accumulation. 5. Investigation of uptake and metabolism of ascorbic acid in normal and cystinotic fibroblasts, and the effects of ascorbic acid uptake on the production of type I collagen.
