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Ribosome is a bionanomachine that facilitates an orderly translation process during protein synthesis in living cells. Real-time monitoring of conformational changes in ribosomal subunits in aqueous solution is important to understand the regulatory mechanism of protein synthesis, because conformational changes in ribosome in E. coli have been predicted to operate the switch from translation initiation to an elongation process during translation. We performed an energy dissipation measurement by using a quartz-crystal microbalance-admittance (QCM-A) technique for in situ monitoring of conformational changes in pre-30S translation initiation complex in response to the binding of fMet-tRNA(fMet) in aqueous solution. The addition of fMet-tRNA(fMet) caused changes in the physical property (increased dehydration and elasticity) in 30S ribosomal subunit in the presence of mRNA and IF2/guanosine 5'-triphosphate (GTP) on the QCM plate. Furthermore, two sequential changes triggered by the addition of fMet-tRNA(fMet) were observed in 30S ribosomal subunit bound to mRNA in the presence of IF2/GTP and IF3. These observations suggest that the structural changes in 30S ribosomal subunit caused by the binding of fMet-tRNA(fMet) with IF2/GTP in the presence of IF3 could act as a switch to regulate the orderly processing in the construction of translation initiation complex, because the structural distinction can be a guidepost in the process for the relevant biomolecules.

Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet-tRNA(fMet) but did not lead to complete dissociation from the 30S-IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.

A quartz-crystal microbalance (QCM) technique was applied to analyze the effects of site-directed mutagenesis of soybean β-amylase (SBA) by using an amylose-immobilized 27-MHz QCM in buffer solution. We followed directly and quantitatively the formation and decomposition of an enzyme-substrate (ES) complex as frequency changes (mass changes) on the QCM plate. It has been predicted from X-ray crystallography of SBA that two carboxyl groups, Glu186 and Glu380, are the crucial residues for the catalytic hydrolysis of α-1,4-glycoside linkage, and the side chain Thr342 on the inner loop structure bends the glycoside at subsites −1 and +1 of the substrate, thereby accelerating the hydrolysis. When SBA wild-type (WT) was employed, the frequency increased (mass decreased) simply because of the hydrolysis of the substrate on the QCM plate. When T342S and T342A mutants having small side chains with no hydrogen bond on the inner loop were employed, the frequency change showed a sigmoidal curve pointing to an increase in ES complex formation and hydrolysis of the substrate. When E186A and E380A mutants were employed, the frequency decreases (mass increases) showed only enzyme binding to the substrate ES complex formation, but not hydrolysis. From curve fitting of these different patterns of frequency changes, we obtained all kinetic parameters for each step of the enzyme binding to the substrate ( k on ), the enzyme release from the ES complex ( k off ), and the catalytic rate constant ( k cat ) for WT and mutants. From the frequency change patterns of the substrate-immobilized QCM we evaluated how mutations of the enzyme affect k on , k off , and k cat values for ES complex formation and decomposition. We also evaluated the effects of accelerators and inhibitors on enzymes from the frequency change pattern.

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.

We monitored real-time DNA transcription by T7 RNAP using a 27-MHz DNA-immobilized quartz-crystal microbalance (QCM) in buffer solution to investigate the stepwise reaction of transcription. We designed a template double-stranded DNA that consisted of a T7 promoter, a stall position (15 bp downstream from the promoter), and a 73-bp transcription region. Based on the frequency (mass) changes of the template-immobilized QCM in response to the addition of T7 RNAP and monomers of NTP, we obtained the kinetic parameters of each step of the T7 RNAP reactions: the enzyme-binding rate ( k on ) to and the dissociation rate ( k off ) from the promoter, the proceeding rate ( k for ) from the promoter to the forward stall position, the polymerization rate ( k cat ) of RNA along DNA, and the release rate ( k r ) from the end of the template DNA. We found that k cat (120 s −1 ) was extremely large compared with k off (0.014 s −1 ), k for (0.062 s −1 ), and k r (0.014 s −1 ), revealing that the rate-limiting steps of T7 RNAP involve the binding to the promoter, the movement to the stall position, and the release from DNA. These kinetic parameters were compared with values for other DNA-binding enzymes.

Catalytic elongation by dextransucrase (DSase) was monitored directly on a dextran-acceptor- or DSase-immobilized 27 MHz quartz crystal microbalance (QCM). Kinetic parameters for the binding of the enzyme to the dextran acceptor (k(on), k(off), and K(d)) and enzymatic elongation in the presence of a sucrose monomer (K(m) for sucrose and k(cat)) were determined. The kinetic parameters obtained by both methods were consistent.

One-, two-, four-, and eight-branched globotriaosyl saccharides (Gb(3): Gal-alpha1,4-Gal-beta1,4-Glc), whose reducing ends were biotinylated, were prepared (1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, and 8Gb(3)-bio, respectively). They are dispersively immobilized as a glyco-array in the matrix of biotinylated maltotriose (Glc(3)-bio) on a streptavidin-covered 27 MHz quartz-crystal microbalance (QCM). The binding kinetics of the verotoxin B subunit (VTB) to various branched Gb(3)-bio ligands in the Glc(3)-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. VTB can recognize the Gb(3) unit but not the Glc(3) unit, where VTB is a pentamer having five binding sites for one Gb(3) unit per each B subunit (having a total of 15 binding sites for Gb(3)). By changing the Gb(3) multivalency, the Gb(3) packing density, and the Gb(3) cluster size in the Glc(3) matrix, association constants (K(a)), maximum amounts bound (Delta m(max)), and binding and dissociation rate constants (k(on) and k(off)) were obtained. When 15 sites of VTB were recognized by 16 Gb(3) units, K(a) was 100 times larger than that when 15 sites of VTB were recognized by only 2 Gb(3) units, with a 6-fold-larger k(on) and a 25-fold-smaller k(off). When the Gb(3) multivalency was changed by covering with two 1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, or 8Gb(3)-bio ligands on two pockets of one streptavidin, the K(a) values increased with increasing branch number from one to eight. When the Gb(3) cluster size was changed from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit in the matrix, the K(a) values increased but the Delta m(max) values decreased with increasing cluster size from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit. This is the first example of systematically obtaining all kinetic parameters of sugar-binding proteins to sugars on a glyco-array by changing the sugar multivalency, the sugar packing density, and the sugar cluster size in the matrix.

Utilizing the zwitterionic alpha-helix peptide bearing a cationic and anionic group at the N- and C-terminus, respectively, we first demonstrated that the vertically aligned multilayer film can be prepared by a simple cast and slow evaporation. The tilt angle of the peptide remained unchanged with ca. 30 degrees in the range between submicrometer and several micrometers in thickness. The key designs allowing simple vertical alignment of the helical peptide multilayer films were (i) monodispersity of the peptide, (ii) electrostatic interaction between anionic substrate and the cationic group bearing at the N-terminus of the peptide, and (iii) interlayer electrostatic interaction among terminal groups of the peptide.

Influenza is an infectious disease caused by the influenza virus, and each year many people suffer from this disease. Hemagglutinin (HA) in the membrane of type A influenza viruses recognizes sialylglycoconjugate receptors on the host cell surface at an initial step in the infection process; consequently, HA inhibitors are considered potential candidates for antiviral drugs. We identified peptides that bind to receptor-binding sites through a multiple serial selection from phage-displayed random peptide libraries. Using the HA of the H1 and H3 strains as target proteins, we obtained peptides that bind to both HAs. The binding affinities of peptides for these HAs were improved by secondary and tertiary selections from the corresponding sublibraries. A docking simulation suggested that, similar to sialic acid, the peptides are recognized by the receptor-binding site in HA, which indicates that these peptides mimic the sialic acid structure. N-stearoyl peptides inhibited infections by the A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains of influenza virus. Such HA-inhibitors are promising candidates for novel antiviral drugs.

A weak laser irradiation (523 − 785 nm, 5 − 60 mW) onto an Au electrode surface of a 27-MHz quartz crystal microbalance (QCM) caused a frequency increase (a mass decrease) in the air phase. These frequency changes depended on the wavelength of the irradiated laser in the order of 523 nm > 636 nm > 785 nm, which corresponds to the light absorbance of the Au electrode of the QCM. The laser response increased linearly with increasing laser power (5 − 60 mW). In addition, the laser response showed a maximum at the incidence angle of 72 degrees when the P-polarized 636 nm laser was irradiated on the Au surface, due to the evanescent effect. These laser responses were also observed in the humid air of H2O, D2O, and in the vapors of various alcohols. Based on these findings, the observed frequency increase (mass decrease) can be explained by the photo-induced reversible desorption of water molecules from the Au electrode surface of the QCM due to the interfacial property changes.

A previous study revealed that DNA-chitosan complex prepared from the reaction between native DNA and chitosan in aqueous solution has suitable porosity for cell seeding, is nontoxic, and causes only a mild soft-tissue response. This simple and easy fabrication method for porous DNA-chitosan complex provides for a wide variety of applications as a scaffold material. The present study evaluated whether rinsing with PBS solution can fabricate DNA-chitosan complex in a mold and the histopathological responses of rat soft tissues to fabricated DNA-chitosan complexes. DNA-chitosan complex paste was prepared by mixing distilled water and freeze-dried water-rinsed DNA-chitosan complex powder. A DNA-chitosan complex disk could be fabricated by rinsing with PBS buffer and subsequently freeze-drying the DNA-chitosan complex paste in the mold. Thus, a wide range of applications of DNA-chitosan complex for tissue engineering can be anticipated using the present easy fabrication method. The porosity of the disk was 85%, and many pores were visible in the DNA-chitosan complex (before fabrication) and in the fabricated DNA-chitosan disk. The values of the complex disks gradually reduced in the tissues although 60% of disks remained in the tissues. In conclusion, an easy fabrication method for making porous DNA-chitosan complex disks was developed. It was found that the fabrication method can delay the biodegradation of the DNA-chitosan complex disk without serious tissue responses in vivo. DNA-chitosan complex is promising as a scaffold material, and a wide range of applications of DNA-chitosan complex for tissue engineering are anticipated.

The efficiency of protein synthesis is often regulated post-transcriptionally by sequences within the mRNA. To investigate the reactions of protein translation, we established a system that allowed real-time monitoring of protein synthesis using a cell-free translation mixture and a 27 MHz quartz-crystal microbalance (QCM). Using an mRNA that encoded a fusion polypeptide comprising the streptavidin-binding peptide (SBP) tag, a portion of Protein D as a spacer, and the SecM arrest sequence, we could follow the binding of the SBP tag, while it was displayed on the 70S ribosome, to a streptavidin-modified QCM over time. Thus, we could follow a single turnover of protein synthesis as a change in mass. This approach allowed us to evaluate the effects of different antibiotics and mRNA sequences on the different steps of translation. From the results of this study, we have determined that both the formation of the initiation complex from the 70S ribosome, mRNA, and fMet-tRNA(fMet) and the accommodation of the second aminoacyl-tRNA to the initiation complex are rate-limiting steps in protein synthesis.

Influenza virus hemagglutinin recognizes sialyloligosaccharides of glycoproteins and glycolipids as cell surface receptors in the initial stage of the infection process. We demonstrate that pentadecapeptides that bind to a sialylgalactose structure (Neu5Ac-Gal) inhibited the infection of cells by influenza virus. The pentadecapeptides were identified through affinity selection from a phage-displayed random peptide library using a monolayer of the ganglioside Neu5Acalpha2-3Galbeta1-4Glcbeta1-1'Cer (GM3). The peptides were found to have affinity for GM3, and alanine scanning showed seven amino acid residues that contribute to carbohydrate recognition. The binding of peptides to the cell surface was significantly inhibited in the presence of sialic acid or by the digestion of cell surface sialyl residues by neuraminidase. Plaque assays indicated that a molecular assembly of alkylated peptides inhibited the infection of Madin-Darby canine kidney cells by influenza virus. Carbohydrate-binding peptides that inhibit carbohydrate-virus interaction showed inhibitory activity. These results may lead to a new approach to the design of antiviral drugs.

During the immobilization process of proteins onto an Au-surface of a 27 MHz quartz crystal microbalance (QCM) in aqueous solutions, apparent large frequency changes (DeltaF(water)) were observed compared with those in the air phase (DeltaF(air)) due to the interaction with surrounding water of proteins. On the basis of an energy-transfer model for the QCM, the apparent added mass in the aqueous solution [(-DeltaF(water))/(-DeltaF(air)) - 1] could be explained by frictional forces at the interface of proteins with aqueous solutions. When [(-DeltaF(water))/(-DeltaF(air)) - 1] values for various proteins were plotted against values relating to the friction (antimobility), such as values of the molecular weight divided by the sedimentation coefficient (Mw/s), the inverse of the diffusion coefficient (1/D), and the volume divided by the surface area (volume/surface area = apparent radius) of proteins, there were good linear correlations. Thus, observations of the larger DeltaF(water) than DeltaF(air) for protein immobilizations on the QCM can be simply explained by the friction effect at the interface between proteins and the aqueous solution. Thus, QCM would be a mass sensor based on mechanical oscillation motion even in aqueous solutions.

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Deltam(max)), the dissociation constants (K(d)), the binding and dissociation rate constants (k(on) and k(off)), and the catalytic cleavage reaction rate constants (k(cat)) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.

pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex (k(on)), the decay of the ES complex (k(off)), and the formation of the product (k(cat)) could be analyzed by transient kinetics as mass changes on the QCM plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km calculated from (k(off) + k(cat))/k(on) due to k(cat)k(off). The rate-determining step was the binding step (k(on), and the catalytic rate k(cat) was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7, values of k(off) were independent of pH, and k(cat) increased gradually with decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of k(cat) at the lower pH correlated with the decrease of alpha-helix contents of the myoglobin substrate on the QCM.

Surface penetrated polymerization of tetrafluoroethylene (TFE) was carried out on a polycarbonate (PC) plate in supercritical fluoroform (scCHF3). Since the high diffusiveness is one of peculiar features of supercritical fluids, TFE monomers and initiators (perfluorinated benzoyl peroxide) could penetrate into the surface of polymer substrates and be photo-polymerized. After washing physisorbed homopolymers on the surface, polytetrafluoroethylene (PTFE) was found to penetrate into 50–800 nm depth from the surface and covered the PC surface in the proportion of 85%. The surface coverage density and the penetration depth could be controlled by adjusting of the pressure of scCHF3. The TFE-penetrated polymerization could be applied for various polymer plates such as polyethylene, polystyrene, polypropylene, poly(ethylene terephthalate), and polyimide. In addition to polymer plates, this technique could be applied to a cellulose paper, a nylon textile, and a porous PC membrane. The PTFE-penetrated nylon textile showed a high resistance for washing test with detergents, compared with the commercial fluoropolymer-sprayed nylon textile. The PTFE-penetrated porous PC membrane showed high oxygen permeability (P/P = 5.2), compared with that of the untreated PC membrane (P/P = 3.5) in gas permeation experiments of O2 and N2. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 1577–1585, 2008

Hydrophilic polysaccharides such as dextran and hyaluronan were immobilized on a hydrophobic polystyrene (PSt) plate by a nanometer-scale surface penetration method in the emulsion of aqueous solutions in supercritical fluoroform (scCHF3). Since a supercritical fluid has high diffusiveness, water emulsions of polysaccharides can penetrate into the polymer surface. Dextran was surface-penetrated by two different methods: (1) the penetration of sucrose as a glucose donor and then the enzymatic polymerization to dextran near the surface catalyzed by dextransucrase, and (2) the direct penetration of dextran polymer into the PSt plate. The contact angle for water of the dextran-penetrated PSt plate was decreased to 78 degrees from 95 degrees of the untreated plate. The surface coverage and the penetration depth of polysaccharides could be obtained to be 10-30% and 10-20 nm, respectively, by X-ray photoelectron spectroscopy. These values could be controlled by the pressure of scCHF3. The transparency of the PSt dish did not change after the dextran penetration. Dextrans on the PSt plate could be elongated enzymatically by dextransucrase in the presence of sucrose as a glucose donor, and be detected by the enzyme-linked biotin-avidin assay. When anionic hyaluronan was surface-penetrated on the PSt plate instead of the neutral dextran, the plate showed the specific adhesion for human T-cells having hyaluronan receptors.

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.

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (kon, koff, and Ka, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA30·dT30 and dG30·dC30) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA30·dT30 and dG30·dC30, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG30·dC30 with high Ka and small koff values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low kon and koff values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high Ka values owing to the high kon values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.

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 pullulandsDNAssDNAproteinspolystyrene. 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.

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.

Protamine is an FDA-approved compound with a documented safety profile that facilitates efficient plasmid condensation for gene delivery by various types of cationic liposomes. It also improves adenoviral vector-mediated gene transfer as a transfection accelerator. However, there is no consensus as to the mechanism of protamine on gene delivery into cells. To analyze the uptake and subcellular distribution, plasmid and protamine were labeled with FITC and Texas-Red, respectively. Although the uptake of FITC-labeled plasmid/protamine complexes into the cells was the same as that of free FITC-labeled plasmid in HeLa, SOJ and A549 cells, they improved the transfection efficiency by several orders of magnitude. Moreover, we found that protamine derived from different sources (salmon, herring and trout sperm) had different transfection efficiencies; however, the gene transfer efficiency with protamine was lower than with optimized poly(L-lysine) and DEAE-Dextran. There were likely two main reasons: firstly, the uptake of plasmid mediated by protamine was complete within the first 10min because the particle size increased as time passed, and secondly, the plasmid/protamine complexes were not released from endosomal membrane. These results indicate that as a transfection accelerator from an appropriate protamine source, with controlled particle size and facile release from endosomes would lead to successful gene delivery with protamine.

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.

Contains fulltext : 35831.pdf (Publisher’s version ) (Closed access) 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.

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.

Homogeneous polymerizations of tetrafluoroethylene (TFE) were executed in supercritical fluoroform (scCHF3), and the resulting poly(tetrafluoroethylene) (PTFE) was uniformly dispersed and twined fibers (ca. 100 nm in width and 10 μm in length). The number-average molecular weight and the conversion of PTFE can be controlled by pressures of scCHF3 from Mn = 104 to 106 and 60−80%, respectively. Heterogeneous detergent-free or stabilizer-free emulsion polymerization could be performed in the narrow condition of the scCHF3/water (5:3 in v/v) system at 17 MPa of the pressure with the 20−40% conversion, and the uniform spherical submicron particles (ca. 200 nm) of PTFE were obtained, in comparison with twined fibers from the homogeneous polymerization.

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.

Objective evaluation systems for beer tastes were developed using a lipid-coated quartz-crystal microbalance (QCM). The adsorption and desorption on the lipid membrane in a water flow system showed...

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.

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.

We performed the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS–S,S–S embedded in supported lipid bilayers immobilized on a 27-MHz quartz-crystal microbalance, under vario...

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.

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.

We have developed a quartz-crystal microbalance (QCM), in which fundamental frequency decreases with increasing mass on the electrode in aqueous solution. When a 27 MHz QCM is employed, a mass of 0.62 ng/cm2 deposited on the electrode decreases the frequency by 1 Hz. Thus, when the host molecule is immobilized on the QCM electrode, the binding behavior of guest molecules could be observed in nanogram level in aqueous solution. In this review, we report that the biomolecular recognitions and reactions at the water-substrate interface by using the QCM technique, such as 1) protein binding to a sugar lipid monolayer at the air-water interface, and 2) oligo nucleotides biding to a nucleolipids monolayer at the air-water interface.

Two methods for measuring astringency were developed using a lipid-coated and gelatin-immobilized quartz-crystal microbalance (QCM). It was found that the level of adsorption of astringent tannins ...

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.

An objective evaluation method for the bitter characteristics of beverages was developed with a lipid-coated quartz-crystal micro-balance connected with a flow injection system. Observation of the adsorption and desorption of beer and coffee on the lipid membrane in the buffer system specifically noted the hydrophobic interactions between the beer and coffee components and the lipid membrane, which could simulate the bitter reception reactions on the tongue. The adsorption or duration of beer or coffee on the lipid membrane in the acetate buffer successfully agreed with the bitter intensity or duration in the sensory evaluation, respectively.

The relationship between the adsorption or desorption of beer on a lipid membrane and the beer's body or smoothness in a sensory evaluation was studied using a lipid-coated quartz crystal microbala...

We have already reported that poly(2-methoxyethylacrylate) (PMEA) showed excellent blood compatibility featured by the significantly low adsorption of plasma protein and the low platelet adhesion. In this study, we tried to analyze the adsorption behavior of the plasma proteins (albumin and fibrinogen) on PMEA surface in terms of kinetics in the early stage of the adsorption reaction by using dynamic quartz crystal microbalance (QCM) method. In addition, the conformational changes of the proteins on the polymer surfaces were investigated. It was concluded from the results that the QCM method could be applied to the analysis of the kinetics in such a polymer–protein system. The characteristic of PMEA is that its detachment rate constant k−1, was higher than those from poly(2-hydroxyethylmethacrylate) (PHEMA) and polypropylene (PP) which were used as references. The degree of the conformational changes of the proteins decreases in the following order; PP>PHEMA>>PMEA. This was strongly related to the difference of the detachment rate constant k−1.

stringent reception mechanism of tannins, which has not been fully elucidated, was studied using a lipid-coated quartz-crystal microbalance. We found that the adsorption of astringent tannins on the lipid membrane significantly increased in the presence of peptides, while NaCl, tartaric acid, quinine-sulfite, sucrose, and glutamic acid had no effect. The adsorption of tannin-peptide complexes showed a linear relationship with tannin concentration and had an optimum concentration of peptides. The adsorption of red wines on the lipid membrane in the presence of bovine serum albumin agreed with astringent intensity in sensory evaluation. It seems that adsorption of tannin-peptide complexes on the oral lipid membrane could be important to the astringent sensation.

Despite high theoretical sensitivity, low-cost manufacture, and compactness potentially amenable to lab-on-a-chip use, practical hurdles have stymied the application of the quartz crystal microbalance (QCM) for aqueous applications such as detection of biomolecular interactions. The chief difficulty lies in achieving a sufficiently stable resonance signal in the presence of even minute fluctuations in hydrostatic pressure. In this work, we present a novel versatile planar sensor chip design (QCM chip) for a microliter-scale on-line biosensor. By sealing the quartz resonator along its edges to a flat, solid support, we provide uniform support for the crystal face not exposed to solvent, greatly decreasing deformation of the crystal resonator under hydrostatic pressure. Furthermore, this cassette design obviates the need for direct handling when exchanging the delicate quartz crystal in the flow cell. A prototype 27-MHz sensor signal exhibited very low noise over a range of flow rates up to 100 microL/min. In contrast, signals obtained from a conventional QCM sensor employing an O-ring-based holder were less stable and deteriorated even further with increasing flow rate. Additional control designs with intermediate amounts of unsupported undersurface yielded intermediate levels of stability, consistent with the interpretation that deformation of the crystal resonator under fluctuating hydraulic pressure is the chief source of noise. As a practical demonstration of the design's high effective sensitivity, we readily detected interaction between myoglobin and surface-bound antibody.