Adsorption behaviors of recombinant E-cadherin-IgG Fc fusion protein on polystyrene surface.

Adsorption behaviors of recombinant E-cadherin-IgG Fc (E-cad-Fc) fusion protein and mutated E-cad-Fcs on the polystyrene (PS) surface were investigated using a 27 MHz quartz-crystal microbalance (QCM) and ELISA. The amount of adsorbed E-cad-Fc on PS surface was increased with an increase of E-cad-Fc concentration as a Langmuir-type in a monolayer. Adsorbed E-cad-Fc on PS surface was stable even after washing if calcium ions are absent in the washing solution due to the calcium ion dependence in the adsorption. E-cadherin homophilic adhesion among E-cadherins during adsorption of E-cad-Fc was involved. Deglycosylation of the E-cad in the E-cad-Fc did not affect adsorption of E-cad-Fc on the PS surface although deglycosylation of the E-cad in the E-cad-Fc enhanced cell adhesion compared with E-cad-Fc.

Functionalization of multilayered DNA-coatings with bone morphogenetic protein 2.

The focus of the present study was to functionalize multilayered DNA-coatings with the osteoinductive factor bone morphogenetic protein 2 (BMP-2) using different loading modalities. The multilayered DNA-coatings were built up from either poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) and DNA using electrostatic self-assembly (ESA). The amounts of BMP-2 loaded into the multilayered DNA-coatings and its subsequent release characteristics were determined using radiolabeled BMP-2. Additionally, the effect of BMP-2 functionalized multilayered DNA-coatings on the in vitro behavior of bone marrow-derived osteoblast-like cells was evaluated in terms of proliferation, differentiation, mineralization, and cell morphology. The results demonstrate the feasibility of multilayered DNA-coatings to be functionalized by embedding BMP-2 according to three different loading modalities: superficial (s), deep (d), and double-layer (dl). BMP-2 was incorporated proportionally into the multilayered DNA-coatings as: s+(4*d)=dl. All differently loaded multilayered DNA-coatings showed an initial burst release followed by an incremental sustained release of the remaining BMP-2. In vitro experiments demonstrated that the loaded factor remained biologically active, as an accelerated calcium deposition was observed on s- and dl-loaded multilayered DNA-coatings, without affecting cell proliferation. In contrast, d-loaded multilayered DNA-coatings influenced osteoblast-like cell behavior by decreasing the deposition of calcium.

Antifungal activity of DNA-lipid complexes and DNA-lipid films against Candida species.

In this study amphiphilic lipids, DNA-lipid complexes, and DNA-lipid films were prepared, and their antifungal activity against Candida species was examined. The amphiphilic lipids were synthesized from a reaction of glycine or L-alanine with n-alkyl alcohol in the presence of p-toluene sulfonic acid. DNA-lipid complexes, which were prepared by the simple mixing of DNA and amphiphilic lipids, were insoluble in water. Self-standing, water-insoluble DNA-lipid films were prepared by casting the DNA-lipid complexes from a chloroform/ethanol solution. The antifungal activities of the lipids and DNA-lipid complexes against the Candida species were evaluated by minimum inhibitory concentrations (MICs); those of DNA-lipid films were evaluated by the disk diffusion method. The seven kinds of lipids, DNA-lipid complexes, and DNA-lipid films showed antifungal activity, and no differences were seen in the antifungal activities between glycine and L-alanine derivatives. The lipids, DNA-lipid complexes, and DNA-lipid films, which have shorter alkyl chain length in lipids, showed antifungal activity against all Candida species. However, the effect of antifungal activity against Candida species decreased with increased alkyl chain length in lipids. In this study, it was found that lipids, DNA-lipid complexes, and films with a decyl or dodecyl group exhibit more favorable antifungal activity.

Adsorption behaviors of poly(N-p-vinylbenzyl-4-o-beta-d-galactopyranosyl-[1-->4]-d-gluconamide) by quartz-crystal microbalance.

Adsorption behaviors of amphiphilic poly(N-p-vinylbenzyl-4-o-beta-d-galactopyranosyl-[1-->4]-d-gluconamide) (PVLA) on the polystyrene (PS) surface was studied using 27 MHz quartz-crystal microbalance (QCM). The amount of adsorbed PVLA on PS surface was increased with an increase of PVLA concentration as a Langmuir-type in a monolayer. The saturated mass change (DeltaM(max)) and association constant (K(a)) of PVLA on PS surface were 498.6 ng/cm(2) and 1.93 x 10(7)M(-1), respectively. The adsorbed PVLA on PS surface was specifically recognized by Allo A lectin due to specific interaction between galactose moieties in the PVLA and Allo A. The hydrophobic interaction between hydrophobic main chain of PVLA and hydrophobic surface of PS was reduced in the presence of urea and the diameter of PVLA aqueous solution was decreased with an increase of urea concentration.

Antibacterial characteristics of newly developed amphiphilic lipids and DNA-lipid complexes against bacteria.

The purpose of this study was to investigate the antibacterial activity of newly developed amphiphilic lipids and DNA/lipid complexes against two types of oral bacteria and two types of hospital infection bacteria. Nine amphiphilic lipids were quantitatively prepared from the reaction of n-alkyl alcohol, alpha-amino acids, and p-toluenesulfonic acid. Nine DNA-lipid complexes were prepared by the simple mixing of DNA and amphiphilic lipids. The DNA-lipid complexes were insoluble in water. The antibacterial activity of lipids and DNA-lipid complexes against Porphyromonas gingivalis, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa were evaluated by the disk-diffusion method. Seven artificial lipids showed antibacterial behavior; in particular, the lipids prepared from n-decyl alcohol and glycine and from n-decyl alcohol and L-alanine showed antibacterial activity against the four bacterial strains used in this study. On the other hand, the lipids of glutamic acid derivatives did not show any antibacterial activity against the four bacteria strains except for the lipid with an n-octyl group. Five DNA-lipid complexes also had an antibacterial effect. The complex prepared from DNA and glycine decyl ester p-toluenesulfonic acid salt exhibited antibacterial activity against the four types of bacteria strains. In this study it was found that lipids and DNA-lipid complexes with a mono-decyl group or a mono-dodecyl group have more favorable antibacterial activity.

Effect of solvation on induce-fit molecular recognition in supercritical fluid to organic crystals immobilized on a quartz crystal microbalance.

The inclusion behavior of guest molecules to a solid apohost of an orthogonal anthracene-bis(resorcinol)tetraol (1) was investigated in supercritical carbon dioxide (scCO(2)) by using a 9 MHz quartz-crystal microbalance (QCM). Compound 1 forms crystals composed of molecular-sheet bound together by an extensive hydrogen-bonded network. The selective binding of gaseous ethyl acetate to the apohost-immobilized QCM in scCO(2) was observed, and the inclusion amount of ethyl acetate showed a drastic increase above a threshold concentration, [Guest](th) = 0.08 M, and the apparent Gibbs' free energy for the binding was DeltaG(app) = -1.3 kcal mol(-1). Similar selective bindings of ethyl acetate or ethanol had been observed in the gas phase and in water: [Guest](th) = 0.002 M with DeltaG(app) = -3.5 kcal mol(-1) and [Guest](th) = 0.5 M with DeltaG(app) = -0.41 kcal mol(-1), respectively. These values obtained in scCO(2) were intermediate between those in the gas and water phases. Since various physical properties (viscosity, density, polarity, diffusion constant, and solvation) of supercritical fluid are known to be intermediate between gas and liquid, these values clearly reflect the solvation behavior of guest molecules. Thus, the lower solvation of guest molecules indicates the lower threshold concentration and the larger binding energy in the following order: in air > in scCO(2) > in water.

Preparation of and tissue response to DNA-lipid films.

DNA-containing films have the potential to form complexes with antibiotics or cytokines by intercalation or groove-binding. This principle can be highly relevant for regenerative wound-healing around oral implants and in periodontology. In this study, we prepared DNA-lipid films and examined tissue responses to them as an indicator of their biological properties. The lipids were synthesized from the reaction of L-alanine, n-alkyl alcohol, and p-toluenesulfonic acid. We prepared the self-standing, water-insoluble DNA-lipid films by casting the DNA-lipid complex from chloroform/ethanol solution. The DNA-lipid complexes, which had 1:1 ratios of phosphate anions to cationic lipid, were found to have a double-helical B-form structure. The DNA-lipid films were almost dissolved 3 days after subcutaneous implantation in the backs of rats. There were no inflammatory reactions or inhibition of new tissue formation. We concluded that DNA-lipid films can be prepared by simple methods, and that they do not cause an unfavorable tissue response.

Direct monitoring kinetic studies of DNA polymerase reactions on a DNA-immobilized quartz-crystal microbalance.

Catalytic reactions of DNA polymerase I from E. coli (Klenow fragment, KF) were monitored directly with a template/primer (40/25- or 75/25-mer)-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 QCM electrode at the nanogram level. Three steps in polymerase reactions which include 1) binding of DNA polymerase to the primer on the QCM (mass increase); 2) elongation of complementary nucleotides along the template (mass increase); and 3) release of the enzyme from the completely polymerized DNA (mass decrease), could be monitored continuously from the time dependencies of QCM frequency changes. The binding constant (Ka) of KF to the template/primer DNA was 10(8)M(-1) (k(on) = 10(5)M(-1)s(-1) and k(off)= 10(-3)s(-1)), and decreased to 10(6)M(-1) (k'on = 10(4)M(-1)s(-1) and k'off = 10(-2)s(-1)) for completely polymerized DNA. This is due to the 10-fold decrease in binding rate constant (k(on)) and 10-fold increase in dissociation rate constant (k(off)) for completed DNA strands. Ka values depended slightly on the template and primer sequences. The kinetic parameters in the elongation process (k(cat) and Km) depended only slightly on the DNA sequences. The repair process during the elongation catalyzed by KF could also be monitored in real time as QCM frequency changes.

Mechanism of cell transfection with plasmid/chitosan complexes.

Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.

A lipid-coated lipase as an efficient hydrolytic catalyst in the two-phase aqueous-organic system.

A lipid-coated lipase has been known to be soluble in organic solvents and act as an efficient esterification catalyst in the dry organic solvent. It was also found to act as an efficient hydrolytic catalyst for lipophilic esters in the two-phase aqueous-organic system. Both the lipid-coated lipase and substrates are solubilized in the organic phase and the hydrolysis occurs with water molecules from the aqueous phase. Therefore, the reaction was 40-100 times faster than that of the native lipase, in which the enzyme and substrate exist separately in the aqueous and organic phase, respectively, and the reaction proceeded at the interface. The hydrolysis rates for the lipid-coated lipase were not affected by the aqueous pH and agitation speed of the two-phase. Enzymatic activity of the lipid-coated lipase was compared with that of the poly(ethylene glycol)-grafted lipase. Michaelis-Menten kinetics in the two-phase reactions was also studied.

In vitro gene delivery mediated by chitosan. effect of pH, serum, and molecular mass of chitosan on the transfection efficiency.

Aminopolysaccharides such as chitosan and polygalactosamine (pGalN) were used to transfer luciferase plasmid into tumor cells. Chitosan largely enhanced the transfection efficiency of luciferase plasmid (pGL3), while pGalN did not at all. Transfection efficiencies of the pGL3/chitosan complexes were dependent on pH of culture medium, stoicheometry of pGL3:chitosan, serum, and molecular mass of chitosan. Transfection efficiency at pH 6.9 was higher than that at pH7.6. Optimum charge ratio of the pGL3:chitosan was 1:5. Chitosan polymer of 15 and 52 kDa largely promoted luciferase activities. Transfection efficiency mediated by chitosan of > 100 kDa was less than that by chitosan of 15 and 52 kDa. Heptamer (1.3 kDa) did not show any gene expression. Cationic liposome (lipofectin)-associated gene expression was inhibited by serum, while chitosan showed resistance to serum.

Design and characterization of asparagine- and lysine-containing alanine-based helical peptides that bind selectively to A.T base pairs of oligonucleotides immobilized on a 27 mhz quartz crystal microbalance.

We have systematically designed and synthesized six kinds of 16-17 mer alanine-based peptides containing four to six lysine (K) and one to four asparagine (N) residues to achieve the selective binding to A.T base pairs of DNA duplexes. The position and number of K and N residues were changed in the helical structure according to common features of the DNA-binding proteins, in which K and N residues are expected to interact electrostatically with phosphate groups and to interact with A.T base pairs by hydrogen bonding, respectively. The time courses of binding of these peptides to dA(30).dT(30) and dG(30).dC(30) duplexes immobilized on a 27 MHz quartz crystal microbalance (QCM) were studied in 10 mM phosphate buffer (pH 7.5) and 40 mM NaCl at 10 degrees C. The maximum binding amounts (Deltam(max)) on a nanogram scale and binding constants (K(a)) could be obtained from the frequency decrease (mass increase) of the oligonucleotide-immobilized QCM. The conformation changes of the peptides upon binding to DNAs were monitored by circular dichroism (CD) spectroscopy. The four properly arranged N residues in the six-cationic K peptide, K6N4(d), resulted in a 5-fold higher affinity for A.T base pairs (K(a) = 5.9 x 10(5) M(-1)) than for G.C base pairs (K(a) = 1.2 x 10(5) M(-1)), and alpha-helices were clearly promoted by the binding to A.T base pairs from CD spectral changes.

Intramolecular electron conduction along DNA strands and their temperature dependency in a DNA-aligned cast film.

Electroconductivity along a long DNA strand (ca. 10 microns length) in a DNA-aligned cast film of DNA-lipid complex was measured on a comb-type electrode (5 microns distance), and it could be reversibly regulated by temperatures across 70 degrees C.

Adsorption to or desorption of beer components from a lipid membrane related to sensory evaluation.

The relationship between the adsorption to or desorption of beer from a lipid membrane and sensory evaluation was studied using a lipid-coated quartz crystal microbalance connected to a flow injection system. The adsorption and duration of adsorption of commercial beers showed a significant correlation with their body and smoothness in a sensory evaluation, respectively. Isohumulones, tartaric acid, NaCl, glutamic acid, and tannic acid were adsorbed onto the lipid membrane. Di- and trihydroxyoctadecenoic acids increased the duration of adsorption of the beer components onto the lipid membrane but not the extent of adsorption. They decreased the astringent duration of beer and the smoothness in the sensory evaluation but did not affect the intensity of bitterness or astringency or the body. It seems that this system, which modifies the electrostatic and hydrophobic interactions of the beer components with the tongue and throat surfaces, can mainly evaluate bitterness and/or astringency which significantly affect the body and smoothness of beer.

Kinetic analyses of ATP-dependent deoxyribonuclease (DNase) reactions on a quartz-crystal microbalance.

We report here kinetic analyses of the hydrolysis of DNA by the ATP-dependent DNase using a DNA-immobilized quartz-crystal microbalance (QCM), which enables in situ real-time monitoring both the binding of enzyme and the hydrolysis reaction on DNA strands, as mass changes.

Preparations of a RNA-lipid complex film and its physical properties.

We extracted RNA (aver. 70-80 bases) from yeasts and prepared a RNA-lipid complex by replacing sodium counter cations with cationic amphiphiles (2C12N+2C1). The RNA-lipid complex was soluble only in organic solvents and formed a self-standing film by casting from organic solutions. The RNA film was physically strong, although its molecular weight is small and the RNA molecules are oriented in one direction by stretching the film, in which RNAs form intermolecular hydrogen bonds and show the physical strength of the film.

Dynamic analyses on induced-fit gaseous guest binding to organic crystals with a quartz-crystal microbalance

The inclusion behavior of gaseous guest molecules in a solid apohost, an orthogonal anthracene-bis(resorcinol)tetraol (1), was investigated with a quartz-crystal microbalance (QCM). Compound 1 forms crystals composed of molecular sheets bound together by an extensive hydrogen-bonded network. An apohost of 1 was cast onto a QCM and the binding of gaseous guest molecules was followed as a function of time by observing the decrease in the oscillation frequency, which is directly related to the increase in mass. Ethyl acetate and methyl ethyl ketone were significantly included into the apohost, whereas benzene and cyclohexane were simply adsorbed onto the surface of the solid; all these guests have similar vapor pressures at 25 degrees C. On the other hand, a host analogue 2, a tetramethoxy derivative of 1, barely included these guest molecules. The inclusion amount and the rate of inclusion of ethyl acetate or methyl ethyl ketone showed a drastic increase above a threshold concentration of guests in the gas phase. Thus, the structure of the apohost changed cooperatively in order to bind guest molecules above the threshold guest concentration. This cooperativity of the binding behavior was kinetically analyzed.

In vitro selection and analysis of RNA aptamer recognize arginine-rich motif (ARM) model peptide on a QCM.

To study RNA-peptide interactions, we performed an in vitro selection of RNA on a 27 MHz quartz-crystal microbalance (QCM) on which a simple R5 helix peptide was immobilized as a model of N peptide from bacteriophade lambda. The consensus sequences including a GNRA tetraloop were obtained from a random RNA pool after the 7th cycle selection.

Quantitative detection of binding of PCNA protein to DNA strands on a 27 MHz quartz-crystal microbalance.

We report here binding kinetics of proliferating cell nuclear antigen (PCNA) to the dsDNA immobilized on a 27 MHz quartz-crystal microbalance. PCNA is a toroidal-shaped protein and encircles dsDNA sliding along the strand. The binding behavior of this sliding clamp protein was compared with those of other side binding proteins such as GCN4-bZIP and Zn-finger peptides.

In vitro selection by using mutated GCN4-bZIP peptides for analysis of peptide-DNA interactions.

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.