Oxidative stress induced by amyloid beta-peptide (A beta) has been implicated in the neurodegeneration observed in Alzheimer's disease (AD) brain. However, the mechanism by which the predominant form of A beta found in AD brains, A beta(1--42), causes oxidative stress and neurotoxicity remains unknown. Numerous laboratories have used the smaller 11-amino acid fragment of the full-length peptide, A beta(25--35), as a convenient alternative in AD investigations since the smaller peptide mimics several of the toxicological and oxidative stress properties of the native full-length peptide. Our observation that the truncated peptide is more rapidly toxic and causes more oxidative damage than the parent A beta(1--42) led us to investigate the cause for this enhanced toxicity of A beta(25--35) in order to gain insight into the mechanism of action of these peptides. These studies reveal that two different mechanisms may be operative in the two peptides; however, the single methionine residue in the peptides appears to play a crucial role in both mechanisms. That methionine is C-terminal in A beta(25--35) seems to be the cause for its exaggerated effects. When the next amino acid in the sequence of A beta(1--42) (valine) is appended to A beta(25--35), the resultant peptide, A beta(25--36), in which methionine is no longer C-terminal, is neither toxic to cultured neurons nor does it cause oxidative damage. Additionally, oxidizing the sulfur of methionine to a sulfoxide abrogates the damaging effects of both A beta(25--35) and A beta(1--42). The putative mechanistic role of methionine in the observed properties of A beta peptides is discussed in the context of the obtained results as is the role of A beta(1--42)-induced oxidative stress in the neurodegeneration found in AD brain.

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Proteomic techniques were used to identify cardiac proteins from whole heart homogenate and heart mitochondria of Fisher 344/Brown Norway F1 rats, which suffer protein nitration as a consequence of biological aging. Soluble proteins from young (5 mo old) and old (26 mo old) animals were separated by one-and two-dimensional gel electrophoresis. One-and two-dimensional Western blots with an anti-nitrotyrosine antibody show an age-related increase in the immunoresponse of a few specific proteins, which were identified by nanoelectrospray ionization-tandem mass spectrometry (NSI-MS/MS). Complementary proteins were immunoprecipitated with an immobilized anti-nitrotyrosine antibody followed by NSI-MS/MS analysis. A total of 48 proteins were putatively identified. Among the identified proteins were ␣-enolase, ␣-aldolase, desmin, aconitate hydratase, methylmalonate semialdehyde dehydrogenase, 3-ketoacyl-CoA thiolase, acetyl-CoA acetyltransferase, GAPDH, malate dehydrogenase, creatine kinase, electron-transfer flavoprotein, manganese-superoxide dismutase, F1-ATPase, and the voltage-dependent anion channel. Some contaminating blood proteins including transferrin and fibrinogen ␤-chain precursor showed increased levels of nitration as well. MS/MS analysis located nitration at Y105 of the electron-transfer flavoprotein. Among the identified proteins, there are important enzymes responsible for energy production and metabolism as well as proteins involved in the structural integrity of the cells. Our results are consistent with agedependent increased oxidative stress and with free radical-dependent damage of proteins. Possibly the oxidative modifications of the identified proteins contribute to the age-dependent degeneration and functional decline of heart proteins.heart; mitochondria THERE IS INCREASING EVIDENCE for an age-dependent decline of cardiac performance (45). Several studies show this performance decline to be associated with oxidative stress (42, 43, 57, 58), i.e., elevated levels of reactive oxygen species. For example, various biomarkers of oxidative stress such as oxo-2-deoxyguanosine, H 2 O 2 , 3-nitrotyrosine (3-NT), N-⑀-methyllysine, malondialaldehyde, and advanced glycation end products (14,22,47,48,55,62) increase with the age in cardiac tissue. Moreover, dietary restriction significantly reduces the age-dependent accumulation of these oxidative markers in heart, which indicates the importance of oxidative stress in cardiac aging (46).A causal role of reactive oxygen species in the age-dependent decline of cardiac dysfunction can, however, only be defined if biological targets of these species are identified and the physiological impact of biomolecular modification is characterized. A first step to establish molecular mechanisms of age-dependent cardiac dysfunction is the proteomic identification of oxidatively modified proteins. Various studies have indicated the formation of reactive nitrogen species in acute myocardiac disorders such as heart failure (1, 28, 31). These reactive nitrogen species are met...

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