L-Asparaginyl and L-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing L-isoaspartyl and D-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77). The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal L-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues. To define the physiological relevance of this protein-repair pathway and to determine whether deficient protein repair would cause central nervous system dysfunction, we used gene targeting in mouse embryonic stem cells to generate protein L-isoaspartate(D-aspartate) O-methyltransferase-deficient mice. Analyses of tissues from methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain, heart, liver, and erythrocytes. The knockout mice showed significant growth retardation and succumbed to fatal seizures at an average of 42 days after birth. These results suggest that the ability of mice to repair L-isoaspartyl-and D-aspartylcontaining proteins is essential for normal growth and for normal central nervous system function.The success of an aging organism depends on its ability to maintain the integrity of its unstable macromolecular machinery over time (1, 2). We have been particularly interested in the consequences of protein damage on aging. Proteins are subject to a variety of spontaneous degradation processes, including oxidation, glycation, deamidation, isomerization, and racemization (1, 3-9). These nonenzymatic modifications can produce functionally damaged species that reflect the action of aging at the molecular level (5, 10).L-Asparagine and L-aspartate are among the most unstable residues in proteins, being particularly susceptible to linked deamidation, isomerization, and racemization reactions (6, 11-13). The initial event in these nonenzymatic reactions at neutral pH is generally the nucleophilic attack of the side-chain carbonyl carbon by the peptide-bond nitrogen to yield an unstable five-membered L-succinimidyl ring (Fig. 1). The spontaneous hydrolysis of the succinimide produces either an L-isoaspartyl residue (in which the peptide backbone is redirected through the ␤-carboxyl group) or a normal L-aspartyl residue. In addition, the ␣-carbon of the succinimidyl residue is racemization-prone, and D-aspartyl and D-isoaspartyl residues can also form, although in lower yields (6,14). Under physiological conditions, succinimide-linked deamidation of asparagine can occur with half-times as short a...

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8

Reporter genes (eg ␤-galactosidase, chloramphenicol-aceimage reporter gene expression following somatic gene tyltransferase, green fluorescent protein, luciferase) play transfer.