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Biomedical subjects

Mizuo Maeda

Publications and source records attributed to Mizuo Maeda.

44 records · Page 3Linked to original sources
PubMed2002

Gene mutation assay using a MutS protein-modified electrode.

A novel electrochemical biosensor for gene mutation detection was developed using a DNA mismatch recognizing protein MutS from E. coli. The MutS protein was immobilized onto an Au electrode by coordination of His-tag at its C-terminus to vacant sites of Ni(II)-nitrilotriacetato complex attached to the surface of electrode. When a target DNA duplex having a mismatch site was captured by the MutS protein on the electrode, the electrostatic repulsion arose between polyanionic DNA duplexes and negatively-charged ferrocyanide/ferricyanide 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. Using this assay, we could detect GT mismatch and deletion mutation in the double-stranded DNA.

Adenosine Triphosphatases↗
PubMed2002

Analysis of a biopolymer by capillary electrophoresis with a chemiluminescence detector using a polymer solution as the separation medium.

We developed capillary electrophoresis with a chemiluminescence detector using a polymer solution as the separation medium for the analysis of biopolymers, such as DNA and protein. A peroxyoxalate chemiluminescence reagent of bis(2,4,6-trichlorophenyl)oxalate was used together with fluorescein-labeling reagent. When a migration buffer solution containing carboxylmethylcellulose was used, the flow-type chemiluminescence detection cell was found to give a better resolution than the batch-type one. Fluorescein-labeled adenosine triphosphate of 1.0 x 10(-4) M was examined by means of capillary electrophoresis with absorption (260 nm), fluorescence (ex. 496 nm and em. 517 nm), and chemiluminescence detectors. The chemiluminescence detection showed the highest sensitivity among them; the S/N ratios obtained by absorption, fluorescence, and chemiluminescence detections were 4, 38, and 130, respectively. Fluorescein-labeled DNA was prepared through a polymerase chain reaction using fluorescein-labeled deoxyadenosine triphosphate. A mixture of the labeled DNA fragments (500, 600, 700, 800, 900, and 993 bp) was successfully separated and detected by the present system. A mixture of proteins (lysozyme, cytochrome C, and ribonuclease A) which were labeled with fluorescein isothiocyanate was also separated and detected.

Absorption↗
PubMed2002

Affinity precipitation separation of DNA binding protein using block conjugate composed of poly(N-isopropylacrylamide) grafted double-stranded DNA and double-stranded DNA containing a target sequence.

In this research, we synthesized a novel DNA-polymer conjugate and evaluated its application to an affinity precipitation separation of TATA-box binding protein (TBP), which is a representative general transcription factor. The conjugate was composed of two fractions. One was a double-stranded DNA modified by the grafting of poly(N-isopropylacrylamide) (PNIPAAm), which is known as a thermosensitive vinyl polymer. The other fraction is a native double-stranded DNA containing a specific base sequence (5'-TATAAA-3') called a TATA-box. These two fractions, which have EcoRI termini, were treated with T4 DNA ligase, and the block conjugate was obtained as a precipitate after two wash processes. When the resultant block conjugate was introduced into a sample solution containing TBP (0.26 microM) and bovine serum albumin (BSA) (0.39 microM), a rapid and selective precipitation separation of TBP under homogeneous conditions was achieved by controlling temperature. The purity of TBP in the precipitation fraction was estimated to be above 90%.

Acrylic Resins↗
PubMedDate not supplied

Intracellular signal-responsive artificial gene regulation for novel gene delivery.

We describe two types of artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) or caspase-3. These molecular systems use newly synthesized cationic polymers, PAK and PAC. The PAK polymer includes substrate oligopeptide for PKA, ARRASLG, as receptor of PKA signal, while the PAC polymer possesses oligopeptide that is comprised of a substrate sequence of caspase-3, DEVD, and a cationic oligolysine, KKKKKK. These polymers formed stable complexes with DNA to totally suppress the gene expression. However, PKA or caspase-3 signal disintegrates the PAK-DNA or the PAC-DNA complex, respectively. This liberates the DNA and activated the gene expression. These systems are the first concept of an intracellular signal-responsive gene-regulation system using artificial polymer. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy that is involved in our previously proposed new drug delivery concept, the drug delivery system based on responses to cellular signals.

Acrylic Resins↗
PubMedDate not supplied

Atomic force microscopic observation of in vitro polymerized poly[(R)-3-hydroxybutyrate]: insight into possible mechanism of granule formation.

Atomic force microscopy (AFM) was used to study the formation and growth of poly[(R)-3-hydroxybutyrate] (PHB) structures formed in the enzymatic polymerization of (R)-3-hydroxybutyryl coenzyme A [(R)-3-HBCoA] in vitro. Poly(3-hydroxyalkanoate) (PHA) synthase (PhaC(Re)) from Ralstonia eutropha, a class I synthase, was purified by one-step purification and then used for in vitro reactions. Before the reaction, PhaC(Re) molecules were deposited on highly oriented pyrolytic graphite (HOPG) and observed as spherical particles with an average height of 2.7 +/- 0.6 nm and apparent width of 24 +/- 3 nm. AFM analysis during the initial stage of the reaction, that is, after a small amount of (R)-3-HBCoA had been consumed, showed that the enzyme molecules polymerize (R)-3-HBCoA and form flexible 3HB polymer chains that extend from the enzyme particles, resulting in the formation of an enzyme-nascent PHB conjugate. When a sufficient amount of (R)-3-HBCoA was used as substrate, the reaction rapidly increased after the first minute followed by a slow increase in rate, and substrate was completely consumed after 4 min. After 4 min, spherical granules continued to grow in size to form clusters over 10 um in width, and in later stages of cluster formation, the cluster developed small projections with a size of approximately 100-250 nm, suggesting qualitative changes of the PHB clusters. Moreover, the high-resolution AFM images suggested that globular structures of approximately 20-30 nm apparent width, which corresponds to the size of PhaC(Re), were located on the surface of the small PHB granule particles.

Acyltransferases↗
PubMedDate not supplied

DNA-responsive hydrogels that can shrink or swell.

Molecule-responsive hydrogels are reputed to be smart materials because of their unique properties. We recently reported that hydrogels containing directly grafted single-stranded (ss) DNA or ssDNA-polyacrylamide conjugate in a semi-interpenetrating network (semi-IPN) manner that "only shrunk" by the addition of ssDNA samples. To date, however, no DNA-responsive hydrogels have been reported capable of "swelling" in response to specific DNAs. Smart materials capable of both shrinking and swelling in response to specific DNAs would be very useful in biochemical and biomedical applications. Here, we show a novel "shrinking or swelling" DNA-responsive mechanism. Novel hybrid hydrogels containing rationally designed ssDNA as the cross-linker were capable of shrinking or swelling in response to ssDNA samples and recognizing a single base difference in the samples. On the basis of the results presented in this paper, it is envisioned that these novel hybrid hydrogels could function and have potential in applications such as DNA-sensing devices and DNA-triggered actuators.

Acrylic Resins↗
PubMedDate not supplied

Photoinduced hydrogen production by direct electron transfer from photosystem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase.

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.

Bacterial Proteins↗