Specific binding of amyloid-beta-protein to IMR-32 neuroblastoma cell membrane.

In flow cytometry using two detecting methods, we have found that amyloid-beta-protein(1-40) [Abeta(1-40)] has high affinity to IMR-32 neuroblastoma cell membrane when it is aggregated to form beta-sheet conformation, whereas random coil small Abeta-species has low affinity. The difference in the binding ability to the cell membranes well accounts for the cytotoxicity of Abeta(1-40); namely, aggregated beta-sheet Abeta(1-40) gives cytotoxicity higher than random coil Abeta(1-40). Specific binding between Abeta(1-40) and ganglioside GM1 of the raft-like domain in lipid membrane is suggested from a surface plasmon resonance (SPR) experiment.

Practical assay and molecular mechanism of aggregation inhibitors of beta-amyloid.

Beta-Amyloid peptide (Abeta) is the main protein component of neuritic plaques in the brain of patients of Alzheimer's disease (AD), and its neurotoxicity would be exposed by the formation of aggregates. The aggregation inhibitors composed of an Abeta recognition element (KLVFF) and a hydrophilic moiety are evaluated by a novel fluorescence assay. These compounds inhibit growth of the model aggregates on the KLVFF immobilized surface. In addition, some compounds also possess disrupting activities of preformed aggregates. These compounds could be a key candidate for therapeutic drugs for AD by their novel molecular mechanisms.

Identification of the molecular interaction site of amyloid beta peptide by using a fluorescence assay.

Beta-amyloid peptides (Abeta) are the main protein components of neuritic plaques and are important in the pathogenesis of Alzheimer's disease. It is reported that Abeta itself is not toxic; however, it becomes toxic to neuronal cells once it has aggregated into amyloid fibrils by peptide-peptide interactions. In this study, to specify the molecular mechanism of aggregation, a novel fluorescence assay was designed. For this purpose, possible partial peptides (38 types of 5-mer) were synthesized on solid-phase. The molecular interactions were examined by a fluorescence probe possessing Lys-Leu-Val-Phe-Phe (KLVFF) as a molecular recognition site. KLVFF is known to be a minimum sequence for formation of the Abeta aggregate. A specific interaction was observed between labeled and immobilized KLVFF. It suggests that the aggregation of Abeta was controlled by the recognition of KLVFF itself by hydrophobic and electrostatic interactions.

Preparation of functional liposomes with peptide ligands and their binding to cell membranes.

Two novel lipopeptides, which have the peptide ligands [alpha-melanocyte stimulating hormone (alpha-MSH)] sequence and repeated [Gly-Arg-Gly-Asp-Se (GRGDS) sequence], are designed, synthesized by the solid-phase method, and introduced into liposome membranes by the freeze-thaw method. These liposomes bearing the peptide ligands on their surface are expected to bind to cell membranes. We have confirmed that the lipopeptides are introduced into liposome membranes almost quantitatively, while such a high degree of incorporation has not been accomplished in conventional methods. In this respect, the present method is superior to prepare surface-modified liposomes that are applicable to drug carriers and so on. We have also confirmed by using immunoelectron microscopy that the peptide ligands are actually located in an aqueous phase. It has been shown by flow cytometry that the liposome bearing alpha-MSH peptide ligand binds to B16 cells and the liposome bearing the repeated GRGDS sequence binds to NIH3T3 cells.

Interaction between a Solute Molecule and a Fluorocarbon Bonded Layer in Reversed-Phase High-Performance Liquid Chromatography.

Glassesand silica-gel packings modified with fluorocarbon and hydrocarbon silylation agents were prepared to investigate the separation mechanism of a fluorocarbon bonded layer in reversed-phase high-performance liquid chromatography (RP-HPLC). On the hydrocarbon layer the contact angles of benzene (Be) and hexafluorobenzene (FB) decreased with increasing carbon chain length of the modifier, and the mobile phase (MP) was independent of the modifier. The fluorocarbon layer gave a larger contact angle for each liquid than the hydrocarbon layer. The retention factor, k, of Be and FB on hydrocarbon packings increased with decreasing contact angle. It was demonstrated that the separation of Be and FB on hydrocarbon packings is caused by solvophobic interaction between solute molecules and the stationary layer. In contrast, k increased with increasing contact angle at the fluorocarbon layer. The retention of Be and FB on fluorocarbon packings may be attributed to the penetration of the solute molecules from the mobile phase into the fluorocarbon stationary layer. Copyright 1999 Academic Press.

Separation and characterization of octylphenol ethoxylate surfactants used by reversed-phase high-performance liquid chromatography on branched fluorinated silica gel columns.

The separation and characterization of octylphenol ethoxylate surfactants were carried out by reversed-phase high-performance liquid chromatography on branched fluorinated silica gel columns. For Triton X-100, simultaneous separation of octylphenol ethoxylate oligomers, positional isomers of octylphenyl group and butylphenol ethoxylate oligomers was achieved. These oligomers were completely separated and identified by means of MS spectra. Ethoxylated oligomers are eluted in the sequence from small to large oligomers. Fifty-five oligomers of Triton X-405 could be separated by using gradient elution. To separate octylphenol ethoxylate surfactant, non-end-capped branched fluorinated silica gel columns were superior to end-capped columns. The relationship between ln k' and methanol concentration was linear, indicating that branched fluorinated silica gel columns were operating in the reversed-phase mode. As Van 't Hoff plots of capacity factor for all oligomers gave straight lines, the equilibrium of conformation for the ethylene oxide chain might lay to one side of either zigzag or meander conformers.

Synthesis of a novel lipopeptide with alpha-melanocyte-stimulating hormone peptide ligand and its effect on liposome stability.

Introduction of liposomes into target cells is important for drug delivery systems. For this purpose, the surface of the liposome is equipped with ligand peptides, which may bind to specific receptors on the cell membrane. An artificial novel lipopeptide (MSH-C4A2) containing the alpha-melanocyte-stimulating hormone (alpha-MSH) sequence and two long alkyl chains was designed and synthesized, and the liposome, composed of egg phosphatidylcholine (EPC) and MSH-C4A2, was prepared. The stability of the liposome was estimated by measuring calcein leakage from the liposome inner phase. The stability of the liposome decreased upon addition of MSH-A4C2, which seemed to be attributable to the amphiphilic property of the peptide moiety (alpha-MSH) of MSH-A2C4. The stability was, however, recovered fairly well upon addition of cholesterol (Ch) or phosphatidylglycerol (PG). It was concluded therefore that the ternary system, MSH-C4A2/Ch/EPC or MSH-C4A2/PG/EPC, is suitable for preparing the functional liposome.

Adsorption Characteristics of Silica Gels Treated with Fluorinated Silylation Agents

Branched-chain polyfluorosilane (monochlorodimethyl[4,4-bis(trifluoromethyl)-5,5,6,6,7,7,7-heptafluoroheptyl)-bonded silica gels were prepared. The surface properties of the silica gels modified with various organic silanes were evaluated by the adsorption density of polar and nonpolar gases calculated from adsorption isotherms of nitrogen, carbon dioxide, and normal butane gases. The polyfluorocarbon-bonded silica gel surface was found to be both hydrophobic and oleophobic in nature, whereas the hydrocarbon-bonded silica gel surface was characterized only as hydrophobic. The trifunctional fluorinated silane provides the silica gel surface with a more hydrophilic nature than the monofunctional silane because of the formation of a denser polymeric coating layer with a larger number of silanols.

Synthesis of an N-methyl-N-nitrosourea linked to a methidium chloride analogue and its reactions with 32P-end-labeled DNA.

The synthesis and characterization of an N-methyl-N-nitrosourea (MNU) analogue that is covalently linked to a methidium nucleus is described. At 37 degrees C in pH 8.0 buffer 9 hydrolyzes via pseudo-first-order kinetics, with a calculated t1/2 = 77 min. By use of polyacrylamide sequencing gels the formation of piperidine-labile N7-methylguanine adducts from the reaction of 9 and MNU with 5'-32P-end-labeled DNA restriction fragments is reported. DNA methylation by 9 in 10 mM Tris buffer is enhanced with increasing ionic strength (50-200 mM NaCl), which contrasts to the inhibition of MNU-induced cleavage with increasing salt. In addition, 9 methylates all G sites equally, while MNU shows a clear preference for d(G)n (n greater than or equal to 3) runs and an asymmetrical methylation pattern within these G-rich regions. The results are discussed in terms of the delivery of the MNU moiety to the DNA target by a non-sequence-specific intercalation process and the subsequent hydrolytic generation of a nondiffusible alkylating intermediate.