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Biomedical subjects

Norihiko Minoura

Publications and source records attributed to Norihiko Minoura.

11 recordsLinked to original sources
PubMed2006

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis.

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis (MIPGE), migration of the target dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This was confirmed by observation of deviations from the linear relationship between the migration distances of the DNA standard size markers in the polyacrylamide gel and those in the MIP gel. The migration distances of nontarget dsDNA maintained a linear relationship, however. In addition, the sequence selectivity of dsDNA in this method was investigated by using the Ha-ras gene and its point mutants. Except for A.T to T.A base pair substitution, mutant dsDNA (for example, substitution from A.T to C.G and from G.C to T.A) could be distinguished from the target (wild-type) dsDNA. Although some improvement in A.T (T.A) base pair distinction is still needed, this study is the first to demonstrate detection of a specific dsDNA sequence with MIPs and, as such, opens up a new realm for practical applications of MIPs.

DNA↗
PubMed2006

Detection of a specific DNA sequence by electrophoresis through a molecularly imprinted polymer.

To develop a simple and inexpensive DNA detection method, we prepared a molecularly imprinted polymer (MIP) gel for recognizing a specific double-stranded DNA (dsDNA) target sequence in MIP gel electrophoresis (MIPGE). During MIPGE, migration of the target sequence of dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This migration hindrance of target dsDNA was determined by plotting the relationship between the migration distance in the MIP gel and that in polyacrylamide gel, commonly used in gel electrophoresis. Using this plot, detection of a target dsDNA from a mixture of different-sized dsDNA fragments was achieved. Moreover, we found the detection method successfully distinguished between a target and its base-pair substitutes. These results suggest that MIPGE could be employed for detection of a target dsDNA sequence.

Base Sequence↗
PubMed2006

Self-cleavage of DNA in the presence of metal ions.

DNA is well known to be aggregated by metal ions including Mn ions, however, analysis of the aggregation process from a chemical aspect, which means identification of the product yielded during the process, has not been performed yet. On determination of what kinds of degraded materials were in the supernatant obtained on centrifugation of a DNA mixture aggregated under the conditions of 10 mM Mn ions ([Mn]/[P]=46.3) at 70 degrees C for 1 h, dAMP, dCMP, dGMP, and TMP produced through self-cleavage of DNA were found in the water-soluble part. These mononucleotides were purified by HPLC using TSKgel ODS-80Ts, and identified by LC-TOF/MS. The self-cleavage was effectively occurred under the conditions of more than 5 mM Mn ions, a reaction temperature of more than 70 degrees C, a reaction time of more than 30 min, and the use of DNA with a molecular weight of more than 140 bp. The self-cleavage was affected by the molecular size of the DNA.

Chromatography, High Pressure Liquid↗
PubMed2004

Imprinted polymer layer for recognizing double-stranded DNA.

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.

Acrylic Resins↗
PubMed2003

Synthetic potential of molluscan sulfatases for the library synthesis of regioselectively O-sulfonated D-galacto-sugars.

The substrate specificities of three molluscan sulfatases (E.C. 3.1.6.1; snail, abalone, and limpet origins) were investigated with assorted p-nitrophenyl (pNP) di-O-sulfonated beta-D-galactopyranosides and beta-lactosides [3,6-SO(3) Gal (1), 3',6'-SO(3) Lac (2), 4, 6SO(3) Gal (3), 2,6-SO(3) Gal (4), 3,4-SO(3) Gal (5), and 3,6-SO(3) GalNAc (6); Ac, acetyl; Gal, galactose; Lac, lactose] together with mono-O-sulfonated beta-D-galactopyranoside [pNP 3SO(3)-Gal (7)] and tri-O-sulfonated alpha-D-galactopyranoside [2,3,6-SO(3)-alpha-Gal (11)]. Some notable differences between the substrate specificity of the three sulfatases were disclosed; snail sulfatase hydrolyzed the 3O- and 2O-sulfo groups of 1 and 4, respectively, to afford 6SO(3) Gal (9) in high yields, while the abalone enzyme did not act on 4. Only the limpet enzyme could cleave the 3O-sulfo groups of 7 to give pNP beta-galactoside. In contrast, every enzyme could utilize 11 as a good substrate to afford a mixture of 6SO(3)-alpha-Gal (13) and 2,6-SO(3) alpha-Gal (12). None of the enzymes could cleave the O-sulfo groups of 5 and 6, which indicates that a primary 6O-sulfo group tends to promote the enzymatic hydrolysis of O-sulfo groups at the secondary positions.

Animals↗
PubMed2003

Chemoenzymatic synthesis of the sialyl-alpha-(2-->3')-lactosamine trisaccharide with a 3-aminopropyl group as a spacer at the reducing end.

The trisaccharide, 3-aminopropyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid-(2-->3)-beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside has been synthesized chemoenzymatically for the first time. First, the acceptor, 3-aminopropyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized in a conventional chemical manner, and then it was coupled with CMP-sialic acid using alpha-(2-->3)-(N)-sialyltransferase to afford the desired trisaccharide by an enzymatically stereocontrolled manner.

Carbohydrate Conformation↗
PubMed2003

Enzymatic degradation behavior of porous silk fibroin sheets.

We investigated the degradation behavior of porous silk fibroin sheets by in vitro enzymatic experiments with alpha-chymotrypsin, collagenase IA, and protease XIV. With 1.0 U/ml protease XIV, 70% of a silk fibroin sheet was degraded within 15 days at 37 degrees C. When the fibroin sheet was exposed to collagenase IA, the amount of Silk II crystalline structure in the sheets decreased slightly, and a small amount of Silk I crystalline structure was formed. When protease XIV was used, almost all Silk II disappeared, but the crystallinity increased overall because the amount of Silk I increased. During digestion with protease XIV, the pore size of the fibroin sheets increased with increasing degradation time, until the sheets finally collapsed and became totally shapeless. The average molecular weight of the products after degradation with the three enzymes followed the order protease XIV < collagenase IA < alpha-chymotrypsin. More than 50% of the products resulting from degradation with protease XIV were free amino acids.

Amino Acids↗
PubMed2002

Structure and properties of silk fibroin-poly(vinyl alcohol) gel.

A series of porous silk fibroin-poly(vinyl alcohol) blend gels were prepared from corresponding aqueous mixtures by freeze- or air-drying, and their structure and mechanical properties were examined. The air-dried gels had higher crystallinity and much greater strengths than the freeze-dried gels, and therefore are suitable for mechanical uses. The freeze-dried gels had characteristic porous structure potentially useful as cell culture substrate. Its structure could be systematically varied by changing freezing temperature and freeze-thaw pretreatments before drying.

Fibroins↗
PubMed2000

Passive Transport of Ionic Drugs through Membranes with pH-Dependent Fixed Charges.

We have studied both theoretically and experimentally the passive transport of ionic drugs through membranes with pH-dependent fixed charge. The system considered constitutes a simplified model for pH-controlled drug delivery through membranes of biochemical and pharmaceutical interest. The theoretical approach employed is based on the Nernst-Planck flux equations and all of the species present in the system (the neutral or ionic drug and the hydrogen and hydroxide ions) have been taken into account together with a Langmuir-type isotherm for the adsorption of the ionic drug onto the membrane surface. The membrane permeabilities of cationic, anionic, and neutral drugs through porous membranes with graft-polymerized weak polyelectrolytes have been measured as a function of the external pH. According to the nature of the grafted polyelectrolyte, the ionized membrane fixed groups can be negative or positive. For the amphoteric membrane, both fixed charge groups are present in the grafted chains. In all cases, the ionization state of the weak polyelectrolyte fixed groups changes with the local pH within the membrane. A comparison of the theoretical results with the experimental data allows one to explain qualitatively the changes of the membrane flux with the external pH and gives new physical insights into the transport problem. Copyright 2000 Academic Press.

Journal Article↗
PubMedDate not supplied

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand.

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.

Binding, Competitive↗