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Chikashi Nakamura

Publications and source records attributed to Chikashi Nakamura.

18 recordsLinked to original sources
PubMed2006

Simple and high-sensitivity detection of dioxin using dioxin-binding pentapeptide.

The purposes of this study are to construct a simple dioxin detection system using peptides that bind to dioxin, and to test the system on real environmental samples. In this method, dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) are competitively bound to the peptides synthesized on beads. The fluorescence intensity of the bead decreases with increasing dioxin concentration. The concentration of dioxin is determined by measuring the fluorescence intensity using a fluorescence microscope equipped with a CCD camera. The fluorescence microscope system was equipped with a motor-driven stage and could be used with 96-well microplates and analytical software that automatically measured the fluorescence intensity of the bead images in the wells. Dioxin detection conditions, reaction temperature, number of beads and concentration of the organic solvent were optimized. About 0.5 nM (150 pg mL(-1)) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could be detected under the optimized conditions. Environmental soil samples were subjected to the detection system using the peptide beads. Although the results obtained correlated poorly with the toxicity equivalency quantity (TEQ) concentration obtained by a GC/MS method, our method is robust enough as a prescreening method to detect at least 250 pg-TEQ g(-1), the survey level for soil as stipulated in the law concerning special measures against dioxins in Japan.

Dioxins↗
PubMed2006

Pyrroloquinoline quinone (PQQ) prevents fibril formation of alpha-synuclein.

Pyrroloquinoline quinone (PQQ) is a noncovalently bound cofactor in the bacterial oxidative metabolism of alcohols. PQQ also exists in plants and animals. Due to its inherent chemical feature, namely its free-radical scavenging properties, PQQ has been drawing attention from both the nutritional and the pharmacological viewpoint. alpha-Synuclein, a causative factor of Parkinson's disease (PD), has the propensity to oligomerize and form fibrils, and this tendency may play a crucial role in its toxicity. We show that PQQ prevents the amyloid fibril formation and aggregation of alpha-synuclein in vitro in a PQQ-concentration-dependent manner. Moreover, PQQ forms a conjugate with alpha-synuclein, and this PQQ-conjugated alpha-synuclein is also able to prevent alpha-synuclein amyloid fibril formation. This is the first study to demonstrate the characteristics of PQQ as an anti-amyloid fibril-forming reagent. Agents that prevent the formation of amyloid fibrils might allow a novel therapeutic approach to PD. Therefore, together with further pharmacological approaches, PQQ is a candidate for future anti-PD reagent compounds.

Amyloid↗
PubMed2005

Dioxin-binding pentapeptide for use in a high-sensitivity on-bead detection assay.

The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.

Dioxins↗
PubMed2005

Detection of polychlorinated biphenyls using an antibody column in tandem with a fluorescent liposome column. Effect of albumin on phospholipase A2-catalyzed membrane leakage.

Phospholipase A2 (PLA2)-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, 3,3-bis[N,N-di(carboxymethyl)aminomethyl]fluorescein (calcein). This enzymatic reaction was applied as signal amplification for biosensor detection of low concentrations of polychlorinated biphenyls (PCBs). In order to increase the fluorescent signal for improvement of PCBs detection, the effect of BSA on optimal lipid composition for PLA2-catalyzed membrane leakage from fluorescent liposomes has been investigated in this report. Various kinds of calcein-entrapped liposomes were immobilized in Sephacryl S1000 gel beads using avidin-biotin binding. In a contrast, free calcein was removed by size exclusion chromatography on Sephacryl S300 for free liposome suspensions. The PLA2-catalyzed membrane leakage was detected both in these gel-bead-immobilized liposomes and in free liposome suspensions. In both systems, the fluorescent release from the liposomes by PLA2 hydrolytic action significantly increased with increasing albumin concentration. The most rapid and greatest membrane leakage by PLA2 hydrolysis was found in anionic liposomes in the presence of albumin, both in free liposome suspensions and gel-bead-immobilized liposomes. Finally, the stabilities of various free liposomes and gel-bead-immobilized liposomes were monitored. Immobilized 1-palmitoy-2-oleoylphosphatidylcholine (POPC)/1-palmitoy-2-oleoylphosphatidylglycerol (POPG) liposome gel was chosen due to its excellent stability and large dye leakage by PLA2. A concentration of PCBs as low as 0.1 ng/mL was detectable using this tandem column system.

Albumins↗
PubMed2005

Gene expression using an ultrathin needle enabling accurate displacement and low invasiveness.

We have previously demonstrated a new cell manipulation technology by using an atomic force microscope (AFM) and ultrathin needles, named nanoneedles. The nanoneedle is an AFM tip etched by a focused ion beam (FIB) and is sharpened from 200 to 800 nm in diameter. In this study, we have evaluated the proper diameter of a needle required for insertion into human cells over a long period without causing cell death, and achieved highly efficient gene expression method for human cells using a nanoneedle and an AFM.

Cell Division↗
PubMed2005

Controllable growth of well-defined regular multiporphyrin array nanocrystals at the water-chloroform interface.

On the basis of the coordination geometry of metal ions, regular cubic, clubbed, and wirelike nanocrystals of Cd(2+)-/PtCl(6)(2-)-mediated, and Hg(2+)-/Ag(+)-/PtCl(4)(2-)-mediated multiporphyrin arrays have been grown at the water-chloroform interface. The nanocrystal growth process was monitored by the transmission electron microscopy (TEM), which revealed (1) an intrinsic rule for coordination polymers, that is, the geometries of metal ions (as connects for the coordination polymers) dominate the frameworks of the related polymeric nanocrystals, and (2) one kind of intuitive nanocrystal growth processes at the interfaces. Both electron diffraction and X-ray diffraction patterns indicated the formation of well-defined nanocrystals. It was found that single-/microcrystals were formed at first, and then they grew into polycrystals. The nanocrystal layer was transferred onto Si and quartz substrate surfaces by the Langmuir-Blodgett method, with its composition analyzed by X-ray photoelectron spectroscopy as well as the arrangement of porphyrin macrocycles in the nanocrystals by UV-vis absorption spectroscopy.

Journal Article↗
PubMed2005

A molecular delivery system by using AFM and nanoneedle.

We developed a new low invasive cell manipulation and gene or molecule transfer system in a single living cell by using an atomic force microscope (AFM) and ultra thin needle, a nanoneedle. DNA was immobilized on the surface of the nanoneedle by covalent bonding and avidin-biotin affinity binding. Immobilization of DNA on the nanoneedle was confirmed by measuring the unbinding force between avidin and biotin. The DNA-immobilized nanoneedle was successfully inserted into HEK293 cells. Though TO-PRO-3 iodide staining experiments using confocal microscopy, we observed the immobilized DNA on the surface of the nanoneedle, which was retained after 10 times insertions to and evacuations from a living cell.

Cell Line↗
PubMed2005

Mechanical sensing of the penetration of various nanoneedles into a living cell using atomic force microscopy.

Mechanical responses during insertion of a silicon nanoneedle into a living melanocyte were observed by using an atomic force microscope (AFM). In order to study the dependence of the mechanical response on the shape of the nanoneedle, we prepared various shapes of silicon AFM tips by focused-ion beam (FIB) etching. The force curves showed increases up to 0.65-1.9 nN after contact on the cell surface, and then the force dropped corresponding with the penetration of the needle through the cell membrane. The force required for penetration was significantly smaller than that using a normal pyramidal tip. The force curves with a cylindrical tip showed a shorter indenting distance before penetration than that with the cone-shaped tip. It is considered that the information about the geometry of penetrating material leads to the development of more suitable micro- and nano-materials to insert into a living cell for cell surgery.

Cells, Cultured↗
PubMed2005

Nanoscale operation of a living cell using an atomic force microscope with a nanoneedle.

We have developed a tool for performing surgical operations on living cells at nanoscale resolution using atomic force microscopy (AFM) and a modified AFM tip. The AFM tips are sharpened to ultrathin needles of 200-300 nm in diameter using focused ion beam etching. Force-distance curves obtained by AFM using the needles indicated that the needles penetrated the cell membrane following indentation to a depth of 1-2 microm. The force increase during the indentation process was found to be consistent with application of the Hertz model. A three-dimensional image generated by laser scanning confocal microscopy directly revealed that the needle penetrated both the cellular and nuclear membranes to reach the nucleus. This technique enables the extended application of AFM to analyses and surgery of living cells.

Cell Membrane↗
PubMed2004

Mechanical force analysis of peptide interactions using atomic force microscopy.

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.

Biomechanical Phenomena↗
PubMed2003

Effect of lipid composition on phospholipase A2-catalyzed membrane leakage in immobilized liposomes: sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column.

Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.

Antibodies↗
PubMed2003

Rapid and specific detection of herbicides using a self-assembled photosynthetic reaction center from purple bacterium on an SPR chip.

In this study, a direct detection system for herbicides inhibiting photosynthetic electron transfer was developed using the photosynthetic reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The heavy-subunit-histidine-tagged RCs (HHisRCs) were immobilized on an SPR sensor chip via nickel chelation chemistry as a binder for one of the triazine herbicides, atrazine. Immediately after injection of atrazine solution on the HHisRCs-immobilized chip, the SPR responses increased and reached plateaus within 1 min. The SPR signals were proportional to the sample concentrations of atrazine in the range 1-100 microg/ml. To evaluate the binding specificity to atrazine, chlorinated aromatic herbicides, DCMU and MCPP, were investigated using the HHisRCs-immobilized chip. An RC inhibitor, DCMU, could also be detected with a higher detection limit of 20 microg/ml than atrazine (1 microg/ml). MCPP showed no signals because its inhibition mechanism against plants is different from that of atrazine and DCMU. These results indicated that the sensor chip immobilized RCs could be used for the specific detection of photosynthetic inhibitors.

2-Methyl-4-chlorophenoxyacetic Acid↗
PubMed2003

Conventional detection of 2,4-dinitrophenol using quartz crystal microbalance.

We present conventional detection of 2,4-dinitrophenol (DNP) for using the competitive reaction between DNP and DNP-conjugated albumin onto DNP antibody immobilized quartz crystal microbalance (QCM). This QCM method allows detection of DNP concentration in the range of 0.01 to 100 ng/ml; linear correlation obtains DNP concentration from 1 to 100 ng/ml.

2,4-Dinitrophenol↗
PubMed2002

Immobilized liposome chromatography to study drug-membrane interactions. Correlation with drug absorption in humans.

For rapid screening of drug-membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin-biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients (KLM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)-phosphatidylserine (PS)-cholesterol (chol) and EPC-PS-phosphatidylethanolamine (PE)-chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log KLM with the drug fraction absorbed in humans showed that the log KLM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin-biotin immobilization of the liposomes prolongs the lifetime of the columns.

Humans↗
PubMed2002

A hydrogen biosensor made of clay, poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode.

A hydrogen gas (H(2)) biosensor was developed in which hydrogenase (H(2)ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H(2)ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay-PBV-H(2)ase modified electrode. The clay-PBV-H(2)ase electrode catalyzed the oxidation of H(2) to protons (H(+)) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38+/-2 kJ/mol at pH 7. The catalytic current of the clay-PBV-H(2)ase electrode increased linearly when exposed to increasing concentrations of H(2) gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H(2) by the clay-PBV-H(2)ase electrode was from 7 to 10. Electron transfer process in the clay-PBV-H(2)ase electrode is discussed.

Aluminum Silicates↗
PubMed2002

Design and activity of antimicrobial peptides against sporogonic-stage parasites causing murine malarias.

Insects produce several types of peptides to combat a broad spectrum of invasive pathogenic microbes, including protozoans. However, despite this defense response, infections are often established. Our aim was to design novel peptides that produce high rates of mortality among protozoa of the genus Plasmodium, the malaria parasites. Using existing antimicrobial peptide sequences as templates, we designed and synthesized three short novel hybrids, designated Vida1 to Vida3. Each has a slightly different predicted secondary structure. The peptides were tested against sporogonic stages of the rodent malaria parasites Plasmodium berghei (in vitro and in vivo) and P. yoelii nigeriensis (in vitro). The level of activity varied for each peptide and according to the parasite stage targeted. Vida3 (which is predicted to have large numbers of beta sheets and coils but no alpha helices) showed the highest level of activity, killing the early sporogonic stages in culture and causing highly significant reductions in the prevalence and intensity of infection of P. berghei after oral administration or injection in Anopheles gambiae mosquitoes. The secondary structures of these peptides may play a crucial role in their ability to interact with and kill sporogonic forms of the malaria parasite.

Animals↗
PubMedDate not supplied

PCR-based ribosomal DNA detection technique for microalga (Heterosigma carterae) causing red tide and its application to a biosensor using labeled probe.

A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate-labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.

Base Sequence↗