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Biomedical subjects

Yoshio Okahata

Publications and source records attributed to Yoshio Okahata.

At least 19 recordsLinked to original sources
PubMed2007

Specific binding of GM1-binding peptides to high-density GM1 in lipid membranes.

The ganglioside Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1) is an important receptor. We have previously identified GM1-binding peptides based on affinity selection from a random peptide library. In the present study, we determined the amino acids essential for binding GM1 and investigated the specific interaction with GM1 in the lipid membrane. Arginines and aromatic amino acids in the consensus sequence (W/F)RxL(xP/Px)xFxx(Rx/xR)xP contributed to the ability of the peptides to bind GM1. The peptide p3, VWRLLAPPFSNRLLP, having the consensus sequence, showed high affinity for GM1 with a dissociation constant of 1.2 microM. Furthermore, the density-dependent binding of p3 was investigated using mixed monolayers of GM1 and Glcbeta1-1'Cer (GlcCer). p3 binds preferentially to high-density GM1, and its interaction with GM1 was found to be cooperative based on a Hill plot. These results indicated that a lateral assembly of GM1 molecules was required for the recognition of carbohydrates by p3. The GM1-binding peptide played a role as a unique anti-GM1 probe differing from the cholera toxin B subunit or antibodies.

Amino Acid Sequence↗
PubMed2007

Hydration and energy dissipation measurements of biomolecules on a piezoelectric quartz oscillator by admittance analyses.

By using a 27-MHz piezoelectric quartz oscillator connected with a vector network analyzer, we obtained resonance frequency decreases (-DeltaFwater) and energy dissipation increases (DeltaDwater) during binding of biotinylated bovine serum albumin, biotinylated ssDNA, biotinylated dsDNA, and biotinylated pullulan to a NeutrAvidin-immobilized 27-MHz quartz crystal microbalance (QCM) plate in aqueous solution, as well as in the wet air phase (98% humidity, -DeltaFwet and DeltaDwet) and in the dry air phase (-DeltaFair and DeltaDair). -DeltaFwater indicates the total mass of the molecule, bound water, and vibrated water in aqueous solutions. -DeltaFwet indicates the total mass of the molecule and bound water. -DeltaFair simply shows the real mass of the molecule on the QCM. In terms of results, (-DeltaFwet)/(-DeltaFair) values indicated the bound water ratios per unit biomolecular mass were on the order of pullulan (2.1-2.2) > DNAs = proteins (1.4-1.6) > polystyrene (1.0). The (-DeltaFwater)/(-DeltaFair) values indicated the hydrodynamic water (bound and vibrated water) ratios per unit biomolecular mass were on the order of dsDNA (6.5) > ssDNA = pullulan (3.5-4.4) > proteins (2.4-2.5) > polystyrene (1.0). Energy dissipation parameters per unit mass in water (DeltaDwater/(-DeltaFair)) were on the order of pullulan > dsDNA > ssDNA > proteins > polystyrene. Energy dissipation in the wet and dry air phases (DeltaDwet and DeltaDair) were negligibly small, which indicates even these biomolecules act as elastic membranes in the air phase (without aqueous solution). We obtained a good linear relationship between [(-DeltaFwater)/(-DeltaFair) - 1], which is indicative of hydration and DeltaDwater/(-DeltaFair) of proteins. The aforementioned values suggest that the energy dissipation of proteins was mainly caused by hydration and that proteins themselves are elastic molecules without energy dissipation in aqueous solutions. On the contrary, plots in cases of denatured proteins, DNAs, and pullulans were relatively deviant toward the large hydration and energy dissipation from the theoretical line as perfect elastic materials, meaning that the large energy dissipation occurs because of viscoelastic properties of denatured proteins, linear DNAs, and pullulans in the water phase, in addition to energy dissipation due to the hydration of molecules. These two parameters could characterize various biomolecules with structural properties in aqueous solutions.

Air↗
PubMed2006

Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance.

Enzymatic digestion with a type IIP restriction endonuclease EcoRV was investigated on a DNA-immobilized 27-MHz quartz crystal microbalance (QCM). Real-time observations of both the enzyme binding process and the DNA cleavage process of EcoRV were followed by frequency (mass) changes on the QCM, which were dependent on divalent cations such as Ca(2+) or Mg(2+). In the presence of Ca(2+), the site-specific binding of EcoRV to DNA could be observed, without the catalytic process. On the other hand, in the presence of Mg(2+), both the binding of the enzyme to the specific DNA (mass increase) and the site-specific cleavage reaction (mass decrease) could be observed continuously from QCM frequency changes. From time courses of frequency (mass) changes, each kinetic parameter, namely binding rate constants (k(on)), dissociation rate constants (k(off)), dissociation constants (K(d)) of EcoRV to DNA, and catalytic rate constant (k(cat)) of the cleavage reaction, could be determined. The binding kinetic parameters of EcoRV in the presence of Ca(2+) were consistent with those of the binding process followed by the cleavage process in the presence of Mg(2+). The k(cat) value obtained by the QCM method was also consistent with that obtained by other methods. This study is the first to simultaneously determine k(on), k(off), and k(cat) for a type IIP restriction endonuclease on one device.

Amino Acid Sequence↗
PubMed2006

The effect of natural extracellular matrix deposited on synthetic polymers on cultured primary hepatocytes.

It is well known that natural extracellular matrix (ECM) molecules are deposited on the surface of biomaterials during culture of cells and affect cellular behaviors. However, it has not been fully understood what kinds of ECM molecules are deposited on the surface of biomaterials although the cellular behaviors were affected by deposited ECM. In this study, to investigate the effect of deposited natural ECM on behaviors of hepatocytes cultured on biomaterials such as poly (N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-gluconamide) (PVLA) as a hepatocyte-specific matrix and poly (L-lysine) (PLL) as a non-specific one during the culture of hepatocytes in vitro, we investigated expression pattern of ECM genes and adsorption of ECM molecules onto PVLA- and PLL-coated surfaces. It was found that the expression levels of type I collagen and fibronectin genes in the hepatocytes cultured on PVLA-coated surface were different from them in the hepatocytes cultured on PLL-coated one. Also, the results showed that laminin was dominantly deposited on PVLA-coated surface whereas fibronectin was dominantly deposited on PLL-coated one. Hepatocytes maintained liver-specific functions on PVLA- and laminin-coated surfaces. It is thought that deposited laminin during the culture of hepatocytes affects the liver-specific functions of hepatocytes cultured on PVLA-coated surface.

Actins↗
PubMed2006

Buffer solution can control the porosity of DNA-chitosan complexes.

The current studies examine the pore properties and biological effects of DNA-chitosan complexes, which may be useful as scaffolds for tissue engineering. The porosity of the DNA-chitosan complexes was controlled by rinsing them with several different pH 7.2 buffer solutions, including phosphate-buffered saline (PBS), Tris-HCl, boric acid, and N-(2-hydroxyethyl)piperazine-N'-(2-ethanesufonic acid) (HEPES). Rinsing with PBS resulted in 84% porosity, whereas rinsing with Tris-HCl produced 94% porosity. It was further found that daunorubicin hydrochloride complex intercalated with and bound to the groove of the DNA-chitosan complexes, indicating that DNA in the complexes maintains its double-stranded helical structure. The DNA-chitosan complexes were not toxic to MG-63 osteoblast-like cells and caused only a mild tissue response when implanted subcutaneously in the backs of rats. These results suggest that buffer-rinsed DNA-chitosan complexes may be useful as a scaffold material in tissue engineering.

Buffers↗
PubMed2006

Effect of ultrasound on DNA polymerase reactions: monitoring on a 27-MHz quartz crystal microbalance.

Effects of ultrasound irradiation on DNA polymerase (Klenow fragment, KF) reactions were studied on the template/primer DNA-immobilized quartz crystal microbalance (QCM). Under ultrasound irradiation, binding of KF to the DNA was suppressed due to the decrease of the binding rate constant (k(1)) and the increase of the dissociation rate constant (k(-)(1)). The catalytic elongation rate (k(cat)) was increased, but the stability of the KF/DNA/monomer ternary complex (K(m)) was decreased by the ultrasound irradiation. Ultrasound effects are discussed in correlation with the conformation changes of domain structures in KF.

Base Sequence↗
PubMed2006

Kinetic analysis of ribosome binding process onto mRNA using a quartz-crystal microbalance.

Translation initiation is the most dynamic and important step along a series of protein synthesis processes. In bacteria, it is generally accepted that the 70S ribosome initially dissociates into the 30S and 50S subunit, and then, the 30S ribosomal subunit binds to the Shine-Dargalno (SD) sequence of mRNA. We analyzed binding kinetics of 70S, 50S and 30S ribosomes to the SD sequence by using a mRNA-immobilized 27 MHz quartz-crystal microbalance (QCM). The 70S ribosome was found to bind strongly to the SD sequence as a ratio of 1:1 without dissociation to each subunit from the lateral side, as well as the 30S subunit. The binding constant for 70S increased in the presence of the initiator tRNA, which suggests that the SD and initiator codon of AUG could be also recognized precisely with 70S.

Base Sequence↗
PubMed2005

Nonfibrous beta-structured aggregation of an Abeta model peptide (Ad-2alpha) on GM1/DPPC mixed monolayer surfaces.

Adsorption and aggregation of transformed peptides and proteins onto the cell membrane surface is commonly associated with forms of amyloidosis such as Alzheimer's disease and prion disease. To address dynamic features of these pathological phenomena molecularly, the in situ Ad-2alpha model peptide deposition on glycolipid-containing monolayers was studied by using a 9 MHz quartz-crystal microbalance (QCM). The Ad-2alpha peptide has two amphiphilic alpha-helix segments, each modified with a 1-adamantanecarbonyl group at the N-terminal as a hydrophobic defect. The peptide folds in a 2alpha-helix structure in the bulk solution. In the presence of mixed monolayers of glycolipids (GM1, asialo-GM1, GM3, or LacCer) and/or dipalmitoyl phosphatidylcholine (DPPC) laminated on the QCM plate, the peptide deposition and the conformational change to beta-structure on the monolayers were accelerated. The adsorption kinetics and the amount of Ad-2alpha were dependent on the sort and contents of the glycolipid in the DPPC matrix. Although the Ad-2alpha peptide adsorbs onto most of the glycolipid membranes as monolayer coverage, it adsorbed largely onto the GM1/DPPC (30/70 mol%) mixed monolayer with characteristic kinetic behaviors. The accumulation of beta-structured nonfibrous aggregations was confirmed by AFM and fluorescence microscopy with Thioflavin T (ThT).

Adsorption↗
PubMed2005

Fabrication, characterization, and biological assessment of multilayered DNA-coatings for biomaterial purposes.

This study describes the fabrication of two types of multilayered coatings onto titanium by electrostatic self-assembly (ESA), using deoxyribosenucleic acid (DNA) as the anionic polyelectrolyte and poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Both coatings were characterized using UV-vis spectrophotometry, atomic force microscopy (AFM), X-ray photospectroscopy (XPS), contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and for the amount of DNA immobilized. The mutagenicity of the constituents of the coatings was assessed. Titanium substrates with or without multilayered DNA-coatings were used in cell culture experiments to study cell proliferation, viability, and morphology. Results of UV-vis spectrophotometry, AFM, and contact angle measurements clearly indicated the progressive build-up of the multilayered coatings. Furthermore, AFM and XPS data showed a more uniform build-up and morphology of [PDL/DNA]-coatings compared to [PAH/DNA]-coatings. DNA-immobilization into both coatings was linear, and approximated 3microg/cm(2) into each double-layer. The surface morphology of both types of multilayered DNA-coatings showed elevations in the nanoscale range. No mutagenic effects of DNA, PDL, or PAH were detected, and cell viability and morphology were not affected by the presence of either type of multilayered DNA-coating. Still, the results of the proliferation assay revealed an increased proliferation of primary rat dermal fibroblasts on both types of multilayered DNA-coatings compared to non-coated controls. The biocompatibility and functionalization of the coatings produced here, will be assessed in subsequent cell culture and animal-implantation studies.

Animals↗
PubMed2005

Kinetic studies of site-directed mutational isomalto-dextranase-catalyzed hydrolytic reactions on a 27 MHz quartz-crystal microbalance.

A quartz-crystal microbalance (QCM) technique was applied to analyze effects of site-directed mutagenesis of a glycosidase (isomalto-dextranase) on the hydrolysis mechanism of the substrate binding (k(on), k(off), and K(d)) and the catalytic process (k(cat)), separately, by using a dextran-immobilized QCM in buffer solution. D266N, D198N, and D313N mutants, which are predicted as critical residues of the isomalto-dextranase hydrolytic activity, dramatically decreased the apparent enzyme activity. The D266N mutant, however, did not change the substrate binding ability (K(d)), and the D198N and D313N mutants largely increased K(d) values due to the increase of k(off) and/or the decrease of k(on) values, as well as the negatively small k(cat) values. From these results, we estimate the reaction mechanism, in which Asp266 acts as only a general acid in the catalytic process, Asp198 acts as both nucleophile in the catalytic process and binding the substrate, and Asp313 acts as only the substrate binding.

Amino Acid Sequence↗
PubMed2005

Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles.

The nuclear membrane is a tight barrier against the delivery of therapeutic genes into non-dividing tissue cells. Overcoming this barrier with the aid of peptidic nuclear localization signals (NLS) is crucial for improving the performance of synthetic gene-delivery vehicles. In this article, we examine the nuclear transport of lambda phage particles displaying various peptides containing the minimum NLS of SV40 T antigen on their surface. As the minimum NLS (PKKKRKV) is a binding domain to importin alpha, recombinant proteins and molecular conjugates containing this peptide accumulate into the nucleus efficiently. However, we find that the C-terminal and N-terminal structures besides the minimum NLS profoundly affect the efficiency of the nuclear transport of the phage particles as well as their binding capacity to importin alpha: either truncation of a few amino acid residues from the C-terminus or the replacement of the N-terminus with a FLAG- or c-myc-tag abolish both of these biological activities. The structure of the optimized NLS is unpredictable from conventional protein transport assay and from the structural analysis in silico. Our results reveal that the objects with 50 nm in diameter can pass through the nuclear pore complex when the optimized NLS is displayed at a sufficient density on their surface.

Active Transport, Cell Nucleus↗
PubMed2005

Kinetic studies of DNA cleavage reactions catalyzed by an ATP-dependent deoxyribonuclease on a 27-MHz quartz-crystal microbalance.

Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.

Adenosine Diphosphate↗
PubMed2004

Kinetic study of phosphorylation-dependent complex formation between the kinase-inducible domain (KID) of CREB and the KIX domain of CBP on a quartz crystal microbalance.

We report quantitative analysis of peptide-peptide interactions on a 27 MHz quartz crystal microbalance (QCM) in aqueous solution. The KID (kinase-inducible domain) of transcription factor CREB (cyclic AMP response element binding protein) is known to interact with the KIX domain of coactivator CBP (CREB binding protein), facilitated by phosphorylation at Ser-133 of the KID. The KIX domain peptide (86 aa) was immobilized on the QCM gold electrode surface by means of a poly(ethylene glycol) spacer. Binding of the KID peptide (46 aa) to the KIX peptide was detected by frequency decreases (mass increases) of the QCM. Both maximum binding amount (Deltammax) and association constants (Ka) obtained from the QCM measurements increased as a result of phosphorylation of Ser-133 of the KID peptide. The Ka values for KIX peptide to the phosphorylated (pKID) and unphosphorylated KID peptides were (93+/-2) x 10(3) and (5+/-1) x 10(3) M(-1), respectively. This difference was explained by the dissociation rate constant (k(-1)) of the pKID being 20 times smaller than that of the KID, while association rate constants (k1) were independent of phosphorylation.

Amino Acid Sequence↗
PubMed2004

Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance.

Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.

Adenosine Monophosphate↗
PubMed2004

Intercalation behavior and tensile strength of DNA-lipid films for the dental application.

In this study, we prepared DNA-lipid films and examined their intercalation behavior and tensile strength as an indicator for usefulness as a dental material. The lipids were synthesized from the reaction of glycine, l-alanine, or l-glutamic acid with n-alkyl alcohol in the presence of p-toluenesulfonic acid. The self-standing, water-insoluble DNA-lipid films were prepared by casting the DNA-lipid complex from chloroform/ethanol solution. The DNA-lipid films formed intercalation complexes with ethidium bromide. This indicates that DNA-lipid films maintain a double helical structure. The tensile strengths of DNA films were 0.8-2.4MPa and were compatible with a commercially available material (Membrane) for guided tissue regeneration in dental use. We conclude that DNA-lipid films have potential for use as a material for the surface treatment of implanted materials or as a bone-guiding scaffold for dental application.

DNA↗