The overall specific aim of this research is to study the role of altered forms of amino acids, particularly D-aspartate (D-Asp) and N- methyl-D-aspartate (NMDA), in normal and dysfunctional physiologic systems; namely Alzheimer's disease (AD), Down's syndrome (DS), and osteoarthritis (OA). It is hypothesized that these post-translationally modified amino acids may result in unique stereochemical alterations with important implications for altered structure-function relationships for the systems involved, e.g. AD and DS brain and OA cartilage. The research proposed here will investigate this hypothesis by concentrating efforts on the following specific areas: 1. D-Aspartate and N-Methyl-D-Aspartate in Brain. It has been shown that D-Asp is present at elevated levels in Alzheimer brain proteins (tangles and plaques) and cerebrospinal fluid, whereas free D-Asp is decreased in some regions of AD brain where the D-Asp may have been converted to NMDA, a potent neuroexcitatory agent for which there are receptors in brain. It is hypothesized that NMDA may be present at increased levels in AD and DS brains, having arisen from D-Asp. Using protocols developed here by Drs. Fisher and D'Aniello to isolate and characterize D-Asp and NMDA in human nervous tissue, these altered forms of aspartate in human brains will be measured in order to determine if there is a correlation of increased levels of NMDA with decreased levels of D-Asp and to assess their relationship to AD and DS. In addition, in collaboration with Dr. Antimo D'Aniello, Naples, Italy, the possibility that free D-Asp might be the precursor for the formation of NMDA and that D-Asp induces brain pathology will be investigated by in vivo experiments in which rats are injected with D-Asp. These studies will give a clearer picture of the location and role of D-Asp and NMDA in brain and their correlation with neurological disorders. Finding NMDA in degenerated nervous tissue of vertebrates and it synthesis from D-Asp would also be very significant discoveries. 2. D-Asp in Osteoarthritic Cartilage. It is hypothesized that osteoarthritic damaged cartilage may be correlated with racemized D-Asp due to aging, lowered pH and hyperthermia associated with joint inflammation. Therefore, using techniques developed in the past by Dr. Fisher et al. to isolate and quantify D-amino acids, the extent of D-Asp in degenerated cartilage from osteoarthritic knee joints will be investigated compared to normal cartilage. These studies will further elucidate whether this altered amino acid (D-Asp) has a role in osteoarthritic dysfunctional systems. 3. Effect of DMSO on Aspartate Racemization. A long-range goal of this research is to find ways of preventing or reversing the formation of D- Asp. Preliminary experiments indicate that dimethyl sulfoxide (DMSO) catalyzes the net conversion of racemized D-Asp to L-Asp within chiral environments of other L-amino acids in peptides. The synthetic peptides containing D- and L-Asp as models to investigate the possibility of repairing damage peptides/proteins using DMSO to convert abnormal D-Asp to normal L-Asp.
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