Deposition of aggregated amyloid -protein (A), a proteolytic cleavage product of the amyloid precursor protein (1), is a critical step in the development of Alzheimer's disease (2). However, we are far from understanding the molecular mechanisms underlying the initiation of A polymerization in vivo. Here, we report that a seeding A, which catalyzes the fibrillogenesis of soluble A, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this A depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel A and our recent study (3), it is suggested that a conformationally altered form of A, which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains. A1 is physiologically secreted into the extracellular space; however, why and how soluble A aggregates and forms amyloid fibrils remains to be elucidated. A great deal of effort has been made to clarify this issue, using mainly in vitro systems. In most such experiments, it has been found that A at much higher concentrations than those prevailing in biological fluids is needed for A aggregation. Thus, it has been hypothesized that aggregation of soluble A involves seeded polymerization (4, 5), although this assumption has not yet been proved in vivo.We have recently reported the detection of a novel A in the apical compartment of cultures of MDCK cells that had been stably transfected with APP cDNA (⌬C MDCK cell) with a truncated cytoplasmic domain (⌬C APP) (3). This A species possesses unique molecular characteristics including its appearance as a smear on immunoblots and altered immunoreactivity. Significantly, these molecular characteristics disappeared dramatically following treatment of the cells with compactin or filipin, an inhibitor of de novo cholesterol synthesis and a cholesterol-binding drug, respectively. Based on previously reported evidence for ⌬C APP being missorted to the apical surface (6) and the cholesterol concentrations of the apical plasma membrane and apical transport vesicles being higher than those in other cellular membranes (7), we concluded that a novel A is generated from apically missorted APP in a cholesterol-dependent manner.Regarding the involvement of cellular cholesterol in the generation of the pathogenic protein, it must be noted that cholesterol-rich lipid microdomains within cells, called caveolae-like domains, have been reported to be the likely sites of the conversion of the normal cellular form of prion protein (PrP c ) to its pathogenic form (PrP sc ) (8 -11). Thus, it would be of great interest to investigate whether the novel A detected in our recent study (3), the generation of which is exclusively dependent on the presence of cholesterol in the cell, has the potential to act as a seed for fibrillogenesis of soluble A. EXPERIMENTAL PROCEDURESMDCK Cell Culture-Dulbecco's modified Eagle's medium (Life Technologies, Inc...
From the rhizomes of Ligularia dentata Hara two new compounds, a phenolic norsesquiterpene ligudentatol (2), and a eudesmanoid ligucyperonol (6), have been isolated in addition to the known caryophillene (1) and additional three phenolic norsesquiterpenes (3, 4, and 5). The structures and stereochemistries of 2 and 6 have been established by a combination of spectroscopic and chemical methods, and by correlation with known ligujapone (4) and (+)-α-cyperone (11), respectively.
Phthalocyaninatozinc(II) complexes bearing dendritic substituents have been prepared. It was found that their generation number and positions on the phthalocyanine ring caused unusual absorption and emission spectral features.
Dinuclear and tetranuclear copper(II) complexes with a ligand bearing phthalocyanine and Schiff-base coordination sites were prepared and characterized. The dinuclear complex has two copper(II) phthalocyanine units linked by 2,6-diiminomethyl-4-methylphenol. The imino nitrogen (Schiff-base) coordinates to two more copper(II) ions to give a tetranuclear copper(II) complex composed of a hydroxo- and phenoxo-bridged copper(II) dinuclear unit and two copper(II) phthalocyanine units. Magnetic, spectroscopic, and electrochemical studies showed that there is no significant interaction among the two copper(II) phthalocyanine units and the hydroxo- and phenoxo-bridged dinuclear unit.
Conotoxin GS, a y-carboxyglutamic acid(Gla)-containing neurotoxic peptide composed of 34 amino acid residues with'one Gla residue and three intramolecular disulfide bonds, was synthesized in solution by the Boc strategy, using the cyclohexyl group to protect the y,y-dicarboxyl functional side chain of the Gla residue. All of the protecting groups were removed by the HF procedure. During the synthesis, the Gla residue was completely stable and no decarboxylated product was observed. The free peptide was subjected to the oxidative folding reaction. The reaction proceeded almost quantitatively in the presence of reduced and oxidized glutathione; however, no product was formed in the absence of redox reagents concomitant with the formation of disulfide isomers or intermediates. The final product was confirmed to be identical to natural conotoxin GS on reversed phase-and ion exchange-HPLC as well as capillary zone electrophoresis. The disulfide structure of synthetic conotoxin GS was determined by gas-phase sequencing and mass spectrometry of its proteolytic fragments and was found to be identical to those of other o~-conotoxins. The major disulfide isomer obtained during the oxidative folding reaction without redox reagents was determined in the same manner. To clarify the role of the Gla residue and the disulfide structure in the conotoxin GS molecule, decarboxylated conotoxin GS and its disulfide isomer were also synthesized, and the neurotoxic activities and circular dichroism spectra of these peptides were compared with those of conotoxin GS and its disulfide isomer. The results showed that the correct disulfide structure was necessary for expression of the toxicity; however, the presence of the Gla residue was not a prerequisite for both the activity and the calciumdependent conformational transition.
Experimental Peptides synthesis and determination of their concentrationPeptides were synthesized by Fmoc peptide chemistry using a Shimadzu PSSM-8 automated peptide synthesizer (Shimadzu, Kyoto, Japan), and purified by reverse-phase HPLC on a C18 column. The identity and purity of the peptides were confirmed by matrix-assisted laser desorption/ionization (MALDI)-TOF MS using a MicroflexAI mass spectrometer (Bruker Daltonics, Yokohama, Japan) and α-cyano-4-hydroxycinnamic acid as a matrix. Peptides with one disulfide bond were prepared by air oxidation of reduced peptides without disulfide bonds (SH type). For air oxidation, 10 -7 M peptides were suspended in 50 mM ammonium acetate buffer (pH 8.2), stirred and left at room temperature for one or two days. After oxidation, 0.4% 2012 © The Japan Society for Analytical Chemistry † To whom correspondence should be addressed. In order to elucidate the role of desorption/ionization efficiency of peptides in MALDI-MS, we focused on peptides with disulfide bonds, which form a rigid tertiary structure. We synthesized seven sets of peptides with one disulfide bond (oxytocin, somatostatin, [Arg 8 ]-vasopressin, [Arg 8 ]-vasotocin, cortistatin, melanin-concentrating hormone, urotensin II-related peptide) and five sets of peptides with two disulfide bonds (tertiapin, α-conotoxin GI, α-conotoxin ImI, α-conotoxin MI and α-conotoxin SI). Each peptide set consisted of three peptides: the oxidized form (S-S type), the reduced form (SH type), and an internal standard peptide in which all cysteine residues were substituted with alanine residues. In the case of urotensin II-related peptide, tertiapin, α-conotoxin ImI and α-conotoxin MI, the reduced form showed higher desorption/ionization efficiency than the oxidized form. In contrast, the other peptides revealed higher desorption/ionization efficiency in the oxidized form relative to the reduced form. These results imply that a rigid structure of peptides formed by disulfide bonds does not correlate with desorption/ionization efficiency in MALDI-MS.
BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.