Biomedical subjects
Publications and source records attributed to Y Okahata.
In vitro selection has been used as a method to determine the optimal binding site for DNA-binding proteins. We report here in vitro selection of dsDNA sequences that bind to mutated-GCN4-bZIP peptides. The GCN4-bZIP peptide mutated from alanine to histidine on a position-14 that contacts with DNA bound to different sequence from a binding site of wild type peptide.
Ganglioside Gal beta1 --> 3GalNAc beta1 --> 4(NeuAc alpha2 --> 3) Gal beta1 --> 4Glc beta1 -->1'Cer (GM1)-binding peptides were obtained from a phage-displayed pentadecapeptide library by an affinity selection. The selection processes were in situ-monitored by a quartz-crystal microbalance method, on which a ganglioside GM1 monolayer was transferred. After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected. The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP. Binding constants of these phage clones to the GM1 monolayer were 10(10) M(-1). Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC50 of 24, 13 and 1.0 microM, respectively. These peptides will be useful for searching functional roles of ganglioside GMI.
We investigated the interaction of GM3 lactone with influenza virus. The specific bindings of influenza virus and its hemagglutinin to GM3 lactone-containing mixed monolayers were studied by using a quartz-crystal microbalance. It has been known that gangliosides as receptors for influenza virus are also substrates for virus neuraminidase. GM3 lactone, however, was found to bind to influenza virus hemagglutinin, but not to be substrate for virus neuraminidase.
We report here kinetic analyses of DNA ligation by using a DNA-immobilized quartz-crystal microbalance (QCM), which enables in situ real-time monitoring of both the binding of ligase and ligation reaction on DNA strands, as mass changes.
A highly sensitive 27 MHz quartz-crystal microbalance (QCM) was applied as a device of dsDNA in vitro selection. When GCN4-bZIP peptides were immobilized on the small Au electrode of the QCM, dsDNAs having TGACTCA sequences could be mainly selected from DNA library having a 15-bp random region, which was consistent with the sequence observed by X-ray crystallography.
Specific protein-DNA interaction was studied quantitatively by using a highly sensitive 27-MHz quartz-crystal microbalance (QCM). Biotinylated DNA double strands (21 bp, having a CRE site of 5'ATGACGTCAT3') were immobilized on an avidin-bound QCM surface, and sequence-specific binding of bZIP 56-mer peptides (having both the basic region for binding and the leucine zipper region for dimerization) to the DNA strand on the QCM was observed. The binding amount (Deltam) at the nanogram level and kinetic parameters such as association constants (Ka) and binding and dissociation rate constants (k1 and k-1) could be obtained from time courses of QCM frequency decreases. A bZIP peptide as a dimer was observed to bind sequence-specifically to one DNA strand having a CRE site. Ka values of ss-bZIP, in which the leucine-zipper region of bZIP was substituted by a Cys-Cys linkage, were largely decreased, and the sequence selectivity also disappeared. Ka values obtained by the QCM method showed good agreement with those obtained from the conventional gel mobility shift assay or from circular dichroism spectrum changes. When the specific sequence of the CRE site of DNA strands was partly changed, Ka values decreased by about a half due to the increase of the dissociation rate constant (k-1) independent of the binding rate constant (k1).
A highly sensitive 27-MHz quartz-crystal microbalance, on which a 10-30-mer oligonucleotide was immobilized as a probe molecule, was employed to detect hybridization of complementary oligonucleotides in aqueous solution. From frequency decreases (mass increases due to the hybridization) with passage of time, kinetic parameters such as association constants (K(a)) and binding and dissociation rate constants (k(1) and k(-1)) could be obtained, as well as binding (hybridization) amount at the nanogram level (delta m). Kinetic studies were carried out by changing various parameters: (i) the immobilization method of a probe oligonucleotide on Au electrode, (ii) number of mismatching bases in sequences of target oligonucleotides, (iii) length of both probe and target oligonucleotides, (iv) hybridization temperature, and (v) ionic strength in solution. The obtained results were compared with those obtained by a surface plasmon resonance method using a BIAcore system.
Monosialogangliosides (GM1, GM2, GM3 and GM4) were reconstituted in lipid monolayers at the air-water interface. The binding amounts and the initial binding rates of wheat germ agglutinin (WGA) to the monosialoganglioside monolayers were quantitatively studied by use of a quartz-crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase, and the binding behavior (mass increase) was followed by the frequency decrease of the QCM. WGA binding affinities for the ganglioside monolayers were influenced by hydrophilic head groups of lipid matrices, densities of gangliosides, and sequences of oligosaccharide in gangliosides. Binding of WGA to the gangliosides reconstituted in a phosphatidylcholine (sphingomyelin and distearoylphosphatidylcholine) matrix was strongly suppressed, but not in a neutral glycolipids (GlcCer, GalCer, and LacCer), dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine matrix. WGA showed high affinity for monolayers containing 20 mol% gangliosides, but only low affinity for 100% ganglioside monolayers. WGA preferably binds to gangliosides in the following sequence: GM3 > GM4 >> GM2 = GM1. No affinities of WGA for GM2 and GM1 were observed. The combined techniques of monolayer and QCM have the advantages of investigating recognition properties of gangliosides.
The effects of trivalent metal ions such as lanthanoid (La3+, Ce3+, Nd3+, Sm3+, Gd3+, Er3+, Yb3+, Lu3+) and Al3+ ions on the morphological change and proliferation of B16 melanoma cells in culture are discussed. These metal ions induced morphological transformations and decreased growth rates at doses of 1 mM. B16 melanoma cells treated with La3+, Ce3+, Nd3+, Sm3+, and Gd3+ showed polyhedrical spreading. Elongation of axones was dependent on the metal ions. B16 melanoma cells treated with Er3+, Yb3+, Lu3+, and Al3+ showed a long slender shape. Growth rates of melanoma cells in the presence of 1 mM of metal ions (La3+, Ce3+, Nd3+, Sm3+, Gd3+, Yb3+, Al3+) were significantly lower than that of control cells. Measurements of cell cycle indicated that the metal ions arrested the transitions from G0/G1 to S state.
A lipid-coated beta-D-galactosidase was prepared in which the enzyme surface is covered with a lipid monolayer and two long alkyl lipophilic tails serve to solubilize the enzyme in organic solvents. In a two-phase aqueous-organic system, a lipid-coated enzyme exists in the organic (2-propyl ether) phase and acts as an efficient transgalactosylation catalyst for various hydrophobic alcohols with lactose in the aqueous buffer solution. When a native beta-D-galactosidase was employed in the two-phase system, neither the transgalactosylation nor the hydrolysis reaction proceeded due to denaturation of the enzyme at the interface. Effects of coating lipid molecules, origins of enzymes, reaction in organic solvents, and chemical structures of acceptor alcohols on the transgalactosylation catalyzed by the lipid-coated enzyme were studied. This system could also be applied in a large-scale synthesis on the 0.1-1 g scale.
Lipoglutamides with tetraethylene glycol tails were synthesized. Physicochemical features of the DNA/lipoglutamide complexes were investigated by light scattering method, phase transition, and CD-spectrum. Aggregation of the DNA/lipoglutamide complex was significantly depressed compared with DNA complexes without ethylene glycol tails, and the solution showed no turbidity. The DNA/lipoglutamide complex showed a high resistance to nuclease, and an efficient internalization into tumor cells, compared with those of DNA alone. Furthermore, the DNA complex was found by confocal laser scanning fluorescence microscopy to distribute in the cytoplasmic region.
We synthesized a lipoglutamide having tetraethylene glycol tails (PEG lipid), and prepared a DNA/PEG lipid complex. Observation by a confocal laser scanning fluorescence microscope showed that the oligonucleotide/PEG lipid complex distributed in cytoplasmic region. On the other hand, a oligonucleotide/lipofectin complex distributed in nucleus. Interactions of the oligonucleotide complexes with isolated nucleus were also investigated, and mechanisms of the subcellular distributions were discussed.
The binding of influenza A virus to GM3-containing monolayers at an air/water interface was quantitatively investigated by use of a quartz crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase and the binding behavior of influenza virus was followed by the frequency changes of the QCM. GM3 was reconstituted in the momolayer of sphingomyelin (SM) or glucosylceramide (GlcCer). When the mole fraction of GM3 was below 30 mol%, the binding rate of the influenza A virus to the GM3/GlcCer membrane was significantly faster than that to GM3/SM membranes. When the mole fraction of GM3 in SM was below 20 mol%, specific binding of influenza virus was not observed at all. Binding of the virus to the GM3/GlcCer mixed membrane was inhibited by the addition of sialyllactose (Neu5Ac alpha 2-3Gal beta 1-4Glc). The virus binding was also visually observed by scanning electron microscopy. Viruses selectively bound to GM3/GlcCer (20:80, by mol%) membrane, but not to GM3/SM (20:80, by mol%) membrane. Furthermore, it was suggested that specific binding of influenza virus to the GM3/GlcCer membrane induced the changes in morphology of virus. It was clearly demonstrated that binding of influenza virus to GM3 was influenced by matrix lipids surrounding GM3.
Recently, development of delivery system for oligonucleotides and genes into mammalian cells has been remarkably improved. Basic requirements for the therapeutic use of nucleotides are both of increase in their stability and efficient cell uptake. In this paper, we newly prepared DNA complexes with cationic lipoglutamates such as dibutyl glutamate (2C4N+), dihexyl glutamate (2C6N+) and dioctyl glutamate (2C8N+). Formations of the DNA/lipoglutamate complexes were confirmed by gel chromatography, elemental analysis, CD spectra, and light scattering measurement. Compaction of DNA by binding with the cationic lipoglutamate was revealed by means of multi-angle light scattering. The DNA/lipoglutamate complexes showed the increase in the stability against enzymatic hydrolysis by DNase I. The DNA/lipoglutamate complexes showed increase in cell uptake and transfection efficiency (CAT assay). The transfection efficiency of DNA/2C8N+ complex was comparable with that of commercially available lipofectin. Furthermore, a DNA complex with lipoglutamide (2EO4C10N+) having tetraethyleneglycol tails was prepared. This DNA/2EO4C10N+ complex showed high water solubility. And, the complexes of DNA or antisense (S-oligo) with 2EO4C10N+ showed efficient uptake by Hera cells, and showed intracellular distribution to the cytoplasmic reagion.
Adhesion and spreading of tumor cells to the films of a galactose-, glucose-, or phosphatidylcholine-bearing lipid was studied. Human adenocarcinoma Hela cells, B16 mouse melanoma cells, and HuH-7 human hepatoma cells selectively adhered and spread on galactose-bearing lipid in serum-containing medium, but not in serum-free medium. The spreading of the tumor cells in serum-containing medium was inhibited in the presence of lactose, but not in the presence of maltose. Cell spreading also occured on the galactose-bearing glycolipid film pre-treated with serum. From quantitative analysis for the the adsorption of serum components by a quartz-crystal microbalance, the surfaces of the lipid films were found to be entirely covered with serum components. These results suggested that serum components pre-adsorbed on the galactose-bearing lipid influence the cell spreading.
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Specific binding of Wheat Germ Agglutinin (WGA) to GM3-containing mixed monolayer at the air-water interface was studied by using a quartz crystal microbalance to know the binding amount and initial binding rate of WGA to GM3. GM3 in the glucosylceramide matrix membrane showed the high recognizability compared to GM3 in sphingomyeline matrix membrane. It is demonstrated that matrix lipids surrounding GM3 regulated the recognition of GM3.
Antifluorescyl IgG antibody and Fab binding to two fluorescein-conjugated lipids was measured using the quartz crystal microbalance methodology. By use of the Langmuir-Blodgett technique, the fluorescein lipids, which were diluted to 5% in a L-alpha-dipalmitoyl phosphatidylethanolamine (DPPE) matrix, were deposited directly onto one gold electrode of the quartz crystal. Binding to films containing the fluorescein hapten was significantly enhanced compared to films of the pure DPPE matrix lipid, indicating that binding occurred primarily through a specific interaction. Association constants were 40-300 times less than for binding to haptens free in solution. Binding of IgG to the lipid in which the hydrocarbon chains and the fluorescein hapten were linked via a hydrophilic spacer was approximately 7 times as great as to the lipid containing no spacer. IgG binding to the lipid containing the spacer was increased 1.5-4.4 times compared to Fab binding for the same lipid. Equilibrium binding curves and kinetic measurements are analyzed quantitatively and compared.