Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the destabilization of preformed fAbeta in the central nervous system, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAbeta formation from Abeta(1-40) and Abeta(1-42) and destabilize preformed fAbeta(1-40) and fAbeta(1-42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of curcumin (Cur) and rosmarinic acid (RA) on the formation, extension, and destabilization of fAbeta(1-40) and fAbeta(1-42) at pH 7.5 at 37 degrees C in vitro. We next compared the anti-amyloidogenic activities of Cur and RA with NDGA. Cur and RA dose-dependently inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42), as well as their extension. In addition, they dose-dependently destabilized preformed fAbetas. The overall activities of Cur, RA, and NDGA were similar. The effective concentrations (EC(50)) of Cur, RA, and NDGA for the formation, extension, and destabilization of fAbetas were in the order of 0.1-1 microM. Although the mechanism by which Cur and RA inhibit fAbeta formation from Abeta and destabilize preformed fAbeta in vitro remains unclear, they could be a key molecule for the development of therapeutics for AD.

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Cerebral deposition of amyloid b-peptide (Ab) in the brain is an invariant feature of Alzheimer's disease (AD). A consistent protective effect of wine consumption on AD has been documented by epidemiological studies. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of wine-related polyphenols (myricetin, morin, quercetin, kaempferol (+)-catechin and (-)-epicatechin) on the formation, extension, and destabilization of b-amyloid fibrils (fAb) at pH 7.5 at 37°C in vitro. All examined polyphenols dose-dependently inhibited formation of fAb from fresh Ab(1-40) and Ab(1-42), as well as their extension. Moreover, these polyphenols dose-dependently destabilized preformed fAbs. The overall activity of the molecules examined was in the order of: myricetin ¼ morin ¼The effective concentrations (EC 50 ) of myricetin, morin and quercetin for the formation, extension and destabilization of fAbs were in the order of 0.1-1 lM. In cell culture experiments, myricetin-treated fAb were suggested to be less toxic than intact fAb, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which these polyphenols inhibit fAb formation from Ab, and destabilize pre-formed fAb in vitro are still unclear, polyphenols could be a key molecule for the development of preventives and therapeutics for AD.

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.

Background-Osteoprotegerin (OPG) is a secretory glycoprotein that belongs to the tumor necrosis factor receptor family.OPG-deficient mice develop severe osteoporosis and medial arterial calcification of the aorta and renal arteries. OPG immunoreactivity was demonstrated in the normal blood vessels and in early atherosclerotic lesions. A recent clinical study suggests that there is a significant correlation between elevated serum OPG levels and cardiovascular mortality. We examined whether serum OPG levels are associated with the progression of coronary artery disease (CAD). Methods and Results-Serum OPG levels were examined in 201 patients who underwent coronary angiography because of stable chest pain. The number of diseased vessels was used to represent the severity of CAD. Serum OPG levels were measured by ELISA and were significantly greater in patients with significant stenosis of the coronary arteries than in those without stenosis. As the severity of CAD increased, there was a significant increase in serum OPG levels. Serum OPG levels were 0.94Ϯ0. 1 Recently, it has been demonstrated that OPG is produced by a variety of tissues, including the cardiovascular system (heart, arteries, veins), lung, kidney, and immune tissues, as well as bone, 1,2 and that the expression and production of OPG are regulated by various cytokines and hormones. 3 It has been shown that OPGdeficient mice develop severe osteoporosis and medial arterial calcification of the aorta and renal arteries, 4 and that the development of osteoporosis and arterial calcification was completely prevented by restoration of the gene. 5 OPG is also expressed in vascular cells such as coronary smooth muscle cells and endothelial cells in vitro. 6 In endothelial cells, OPG has been demonstrated to act as an anti-apoptotic factor. 7 Moreover, OPG immunoreactivity was demonstrated not only in the nondiseased vessel wall, but also in early atherosclerotic lesions in human tissues. 8 These findings suggest that OPG may play an important role in the development of vascular disease. A recent clinical study reported that there is a significant correlation between elevated serum OPG levels and cardiovascular mortality, 9 suggesting that OPG may contribute to the progression of coronary artery disease (CAD). In this study, we assessed the severity of CAD by coronary angiography and examined whether serum OPG levels are associated with the progression of CAD. Methods PatientsThe present study involved 201 patients who underwent coronary angiography. All patients fulfilled the criteria of stable chest pain and/or signs of myocardial ischemia on exercise electrocardiography for clinical indication for cardiac catheterization. Patients with an acute coronary syndrome were excluded. At the time of a physical examination, blood pressure, body mass index (BMI), and a hematological and biochemical profile were determined. Age and history of cigarette use were assessed through an interview preceding the physical examination. Ninety-six subjects were receiving antian...

The protective role of Sirt1 in renal damage was investigated. Sirt1 in proximal tubules (PT) was downregulated before albuminuria occurred in streptozotocin-induced or obese-type (db/db) diabetic mice. PT-specific Sirt1 transgenic (TG) and knockout (KO) mice showed prevention and aggravation of the glomerular changes occurring in diabetes, respectively, and non-diabetic KO mice exhibited albuminuria, suggesting that Sirt1 in PT affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by Sirt1-mediated epigenetic regulation in podocytes contributed to albuminuria. These phenomena were not observed in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PT to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the auto-fluorescence of photoactivatable NMN, and injection of fluorescence-labeled NMN. In human subjects with diabetes, Sirt1 and Claudin-1 levels were correlated with proteinuria level. Sirt1 in PT protects against albuminuria in diabetes through maintaining NMN concentrations around glomeruli and controlling podocyte function.

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a beta-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into beta-sheet-rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble beta-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic beta-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.

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