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Mizuo Maeda

Publications and source records attributed to Mizuo Maeda.

At least 19 recordsLinked to original sources
PubMed2007

Colorimetric biosensors based on DNA-nanoparticle conjugates.

In this review, we present an overview of the technologies in colorimetric biosensors based on DNA-nanoparticle conjugates. Two types of DNA-nanoparticles aggregation assays are summarized. One of the methods relies on cross-linking of the gold nanoparticle (GNP) by hybridization. The crosslinking system was used not only to detect target DNA sequences, but also to detect metal ions or small molecules which were recognized by DNAzymes. The other method is the GNP non-crosslinking system. This approach shows high performance in the detection of single nucleotide polymorphisms. These methods do not need special equipment and open up a new possibility of point-of-care diagnoses.

Biosensing Techniques↗
PubMed2006

Selective ultratrace detection of Al(III) and Ga(III) complexed with a calcein isomer by capillary zone electrophoresis with laser-induced fluorescence detection. Comparison of emissive polyaminocarboxylates as derivatizing ligands.

A highly sensitive and selective capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) for Al(3+) and Ga(3+) was developed using a calcein 4',5'-isomer as a pre-capillary complexing agent. Direct fluorescent detection without an agent added to the carrier buffer provided the notable detection limits of 3.9x10(-10) and 4.6x10(-10)moldm(-3) for Al and Ga, respectively. By applying this system successfully to a river water sample, its potential for practical applications was ascertained. A comparison of three aromatic polyaminocarboxylate ligands related to the coordination structure and the distance between the fluorophore and the chelating moiety, revealed some interesting factors regarding their selectivity.

Aluminum↗
PubMed2006

Detection of single-base mismatch at distal end of DNA duplex by electrochemical impedance spectroscopy.

Herein, we report an anomalous electrochemical behavior of surface-bound DNA duplex that has single-base mismatches at its distal end. Single-stranded 15-base DNA was immobilized at its 5'end onto gold electrode surfaces. After hybridization with complementary or mismatched DNA, electrochemical impedance spectra were obtained using [Fe(CN)(6)]3-/4- as redox marker ions. Hybridization with the complementary DNA reduced the charge-transfer resistance (R(CT)), whereas single-base mismatches at the distal end of the duplex largely increased the R(CT). This anomaly was found only with the distal end: the increase in R(CT) was not observed for mismatches at either the middle or the proximal end. These results indicate that electrochemical detection of single-base alterations at an end of sample DNA is exceptionally easy because of the diametrically opposite responses. This detection principle is promising for the typing of single-nucleotide polymorphisms in combination with the single-base primer extension protocol.

Base Pair Mismatch↗
PubMed2006

Effects of mutations in the substrate-binding domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 on PHB degradation.

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ(RpiT1)) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ(RpiT1) was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ(RpiT1). Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ(RpiT1) adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.

Amino Acid Sequence↗
PubMed2006

Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate.

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. The manner by which prefoldin interacts with a group II chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group II chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus alpha chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin.

Amino Acid Sequence↗
PubMed2006

Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K.

Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, gammaCys131-SO(2)H. When the SCNase alpha, beta and gamma subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (alphabetagamma)(4), like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase((+P15K))) possessed 0.86 Co atom/alphabetagamma trimer and exhibited 78% of the activity of native SCNase. SCNase((+P15K)) showed a UV-Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase((+P15K)) had the gammaCys131-SO(2)H modification. These results indicate that SCNase((+P15K)) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion.

Amino Acid Sequence↗
PubMed2006

Surface plasmon resonance imaging on a microchip for detection of DNA-modified gold nanoparticles deposited onto the surface in a non-cross-linking configuration.

Recently we reported that gold nanoparticles (GNPs) with fully matched duplexes on their surfaces are selectively deposited onto walls of poly(dimethylsiloxane) (PDMS) microchannels at high salt concentrations. In this study, the surface plasmon resonance (SPR) imaging technique was applied to monitor this phenomenon for improvement of detection sensitivity and elucidation of the phenomenon. The microchip was fabricated by bonding a surface-patterned PDMS plate and a gold thin film-deposited glass substrate. Probe oligonucleotide-modified GNPs were hybridized with target oligonucleotides to make fully matched or single-base-mismatched duplexes. The hybridized GNP solution was mixed with an NaCl solution in a Y-shaped microchannel. The deposition of the GNPs onto the gold sensor surface was detected by SPR imaging. Discrimination of the targets was possible with limit of detection of 32 nM (19 fmol) without temperature control in 5 min. Detailed analysis indicated that a seed layer of GNPs was initially adsorbed onto the sensor surface regardless of the target sequence. Therefore, in combination with a portable SPR device, the proposed method is promising for point-of-care testing of single-nucleotide polymorphsims.

DNA↗
PubMed2006

FRET-based monitoring of conformational change of the beta2 adrenergic receptor in living cells.

The beta(2) adrenergic receptor (beta(2)AR) is a G protein-coupled receptor that is selective to epinephrine. We demonstrate herein monitoring of an agonist-induced conformational change of beta(2)AR in living cells. The monitoring method is based on fluorescence resonance energy transfer from a cyan fluorescent protein (CFP) to a biarsenical fluorophore, FlAsH, attached to the C-terminus, and the third intracellular loop (ICL3), respectively. Recombinant beta(2)ARs exhibited agonist-induced increases in the FlAsH/CFP emission ratio, indicating that the ICL3 approached the C-terminus upon activation. Since the emission ratio changes were on a time scale of seconds, the conformational change of beta(2)AR in living cells was more rapid than that of purified beta(2)AR measured in vitro. Interestingly, the direction of the emission ratio change of beta(2)AR was opposite to that of the norepinephrine-responsive alpha(2A) adrenergic receptor reported recently. It was suggested that this discrepancy corresponds directly to the diametric biological functions, i.e., the activation or inactivation of adenylyl cyclase.

Adrenergic beta-2 Receptor Agonists↗
PubMed2006

Sulfur K-edge XAS and DFT calculations on nitrile hydratase: geometric and electronic structure of the non-heme iron active site.

The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS(-))-, sulfenate (RSO(-))-, and sulfinate (RSO(2)(-))-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO(-) species changes upon protonation as the S-O bond is elongated (by approximately 0.1 A). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe(III) in the active site of NHase as CysS(-), CysSOH, and CysSO(2)(-) both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z(eff) of the Fe and reveals that the Fe in [FeNO](6) NHase species has a Z(eff) very similar to that of its photolyzed Fe(III) counterpart. DFT calculations reveal that this results from the strong pi back-bonding into the pi antibonding orbital of NO, which shifts significant charge from the formally t(2)(6) low-spin metal to the coordinated NO.

Binding Sites↗
PubMed2006

Mutational study on alphaGln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771.

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme having post-translationally modified cysteine ligands, alphaCys112-SO2H and alphaCys114-SOH. We replaced alphaGln90, which is conserved in all known NHases and involved in the hydrogen-bond network around the catalytic center, with glutamic acid or asparagine. The kcat of alphaQ90E and alphaQ90N mutants decreased to 24% and 5% that of wild type respectively, but the effect of mutations on Km was not very significant. In both mutants, the alphaCys114-SOH modification appeared to be responsible for the catalysis as in native NHase. We crystallized the nitrosylated alphaQ90N mutant and determined its structure at a resolution of 1.43 A. The structure was basically identical to that of native nitrosylated NHase except for the mutated site and its vicinity. The structural difference between native and alphaQ90N mutant NHases suggested the importance of the hydrogen bond networks between alphaGln90 and the iron center for the catalytic activity.

Crystallography, X-Ray↗
PubMed2006

A MutS protein-immobilized au electrode for detecting single-base mismatch of DNA.

A novel electrochemical biosensor was developed to detect gene mutation by using a DNA-mismatch binding protein: MutS from Escherichia coli. The MutS protein was immobilized onto an Au-electrode surface via complex formation between a histidine tag of the MutS protein and a thiol-modified nitrilotriacetic acid chemically adsorbed on the Au-electrode surface. When a target double-stranded DNA having a single-base mismatch was captured by the MutS protein on the electrode, some electrostatic repulsion arose between polyanionic DNA strands and anionic redox couple ions. Consequently, their redox peak currents on a cyclic voltammogram with the Au electrode drastically decreased, depending on the concentration of the target DNA, according to the redox couple-mediated artificial ion-channel principle. By using this assay, one can detect all types of single-base mismatch and single-base deletion.

Base Pair Mismatch↗
PubMed2005

Power-free sequential injection for microchip immunoassay toward point-of-care testing.

This paper presents a simple fluid handling technique for microchip immunoassay. Necessary solutions were sequentially injected into a microchannel by air-evacuated poly(dimethylsiloxane), and were passively regulated by capillary force at the inlet opening. For heterogeneous immunoassay, microchips are potentially useful for reduction of sample consumption and assay time. However, most of the previously reported microchips have limitations in their use because of the needs for external power sources for fluid handling. In this paper, an on-chip heterogeneous immunofluorescence assay without such an external power source is demonstrated. The microchip consisting of poly(dimethylsiloxane) (PDMS) and glass has a simple structure, and therefore is suitable for single-use applications. Necessary solutions were sequentially injected into a microchannel in an autonomous fashion with the power-free pumping technique, which exploits the high solubility and the rapid diffusion of air in PDMS. For deionized water, this method yielded flow rates of 3-5 nL s-1 with reproducibility of 4-10%. The inlet opening of the microchannel functioned as a passive valve to hold the solution when the flow was finished. Rabbit immunoglobulin G (rIgG) and human C-reactive protein (CRP) were detected using the microchannel walls as reaction sites. With the sample consumption of 1 microL and the assay time of approximately 20 min including the antibody immobilization step, the sandwich immunoassay methods for rIgG and CRP exhibited the limits of detection of 0.21 nM (0.21 fmol) and 0.42 nM (0.42 fmol), respectively.

Animals↗
PubMed2005

Affinity capillary electrophoresis of DNA for detection of single-nucleotide polymorphisms and point mutations. Comprehensive study for optimization of the weak affinity.

A single-stranded DNA and its point mutant can be separated with affinity capillary electrophoresis (ACE) in which an appropriate ligand DNA is used as a pseudo-stationary affinity phase. In this paper, we systematically examine the effects of ligand sequence, capillary temperature, and cation concentration on the ACE separation quality, which is quantitatively evaluated in terms of resolution and peak height. For fine tuning of the affinity, control of MgCl(2) concentration and insertion of a spacer sequence into the ligand DNA are more effective than control of the capillary temperature. For design of the ligand DNA, a simple strategy is proposed, based on the prediction of melting temperature. This strategy was tested with eleven different sample sequences. All of them were separated under the same conditions (250 microM MgCl(2) and 25 degrees C), and nine cases of them had satisfactory separation qualities.

Cations↗
PubMed2005

Autonomous polymer loading and sample injection for microchip electrophoresis.

We have developed an extremely simple method for microchip electrophoresis. Loading of a sieving polymer solution and injection of a sample solution are autonomously executed by a microchip fabricated in poly(dimethylsiloxane) (PDMS). In advance, the energy for the fluid pumping is stored in bulk PDMS by evacuating air dissolved in PDMS, and the information for the sample plug regulation is coded into the microchannel design. Besides the simplicity, our method brings about an advantageous effect: sample compaction due to the discontinuous electrophoretic mobility at the sample/polymer interface. The sample compaction effect was moderate in ordinary size-dependent separation for double-stranded DNA and was extreme in affinity electrophoresis for single-stranded DNA (ssDNA). In the latter separation mode, ssDNA components were sequence-specifically separated by difference in affinity to a probe oligonucleotide immobilized to the sieving polymer matrix. We separated up to 60-mer ssDNA mixtures based on single-base substitutions. The separation processes included typically 100-fold sample compaction and were completed within 15-30 s. This technology provides easy, simple, and sensitive method for detection of gene point mutations and typing of single-nucleotide polymorphisms.

Base Sequence↗
PubMed2005

Non-cross-linking gold nanoparticle aggregation as a detection method for single-base substitutions.

Aggregation of DNA-modified gold nanoparticles in a non-cross-linking configuration has extraordinary selectivity against terminal mismatch of the surface-bound duplex. In this paper, we demonstrate the utility of this selectivity for detection of single-base substitutions. The samples were prepared through standard protocols: DNA extraction, PCR amplification and single-base primer extension. Oligonucleotide-modified nanoparticles correctly responded to the unpurified products from the primer extension: aggregation for the full match and dispersion for all the mismatches. Applicability of this method to genomic DNA was tested with five human tumor cell lines, and verified by conventional technologies: mass spectrometry and direct sequencing. Unlike the existing methods for single-base substitution analysis, this method does not need specialized equipments, and opens up a new possibility of point-of-care diagnosis for single-nucleotide polymorphisms.

Biosensing Techniques↗
PubMed2005

Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate.

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.

Acrylic Resins↗
PubMed2005

Assignment of hydrogen-bond structure in a ligand-nucleobase complex inside duplex DNA: combined use of quantum chemical calculations and 15N NMR experiments.

We present a combined experimental and theoretical approach, whereby a comparison of calculated and experimental 15N NMR chemical shifts allows the elucidation of hydrogen-bond structure in a ligand-nucleobase complex inside duplex DNA. In this work, we focus on the highly selective interaction of 2-amino-7-methyl-1,8-naphthyridine (AMND) to cytosine (C) base opposite the abasic site in DNA duplexes, despite the hydrogen-bond array of neutral AMND being fully complementary to guanine (G). Examination of the salt dependence of the binding constants reveals that the effective number of charges on the ligand is +1.0, indicating protonated AMND does bind to C. This is clearly supported by 15N NMR measurements, where the drastic changes in chemical shift are observed for the aromatic nitrogens on the ligand when binding to C. Furthermore, from the complexation-induced changes in chemical shift at 15N1 (83.1 ppm upfield), 15N8 (14.1 ppm upfield), and 15NH2 (18.3 ppm downfield) on AMND, the ligand is found to bind to C via three point hydrogen-bonds. The chemical shifts of the AMND-C complex, calculated by gauge-independent atomic orbital-DFT method, are in fair agreement with the experimental values. These results clearly explain the selective binding of AMND to C over G in abasic site-containing duplex DNA.

Cytosine↗