Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology

We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.     

Sulfide oxidation by gene expressions of sulfide-quinone oxidoreductase and ubiquinone-8 biosynthase in Escherichia coli

Sulfides (S2),SH-) such as hydrogen sulfide belong to a class of sulfur compounds with unpleasant odors. In order to confer sulfide-oxidizing ability on the intestine-inhabiting bacteria, the sulfide-quinone oxidoreductase gene (sqr) in Rhodobacter capsulatus DSM-155 and genes for quinone biosynthesis (ubiC, ubiA and ispB) in Escherichia coli XL1 Blue-MRF' were transduced into E. coli BL21(DE3). Plasmids pT7-7 and pSTV were used as vectors of sqr, and ubiCA and ispB, respectively. The recombinants sqr-BL21(DE3) and ubiCA,ispB-sqr-BL21(DE3) were successfully constructed. The maximal sulfide-removing activities of the whole cells and membrane fractions of sqr-BL21(DE3) attained at pH 8.0 and 7.8, were 267 nmol/mg cells (dry weight)/min and 1250 nmol/mg membrane fraction (protein)/min, respectively. The molecular ratio of sulfide (S2-) oxidized and oxygen (O2) consumed was 2:1. SQR activity in the recombinant cells was positively restricted under anaerobic conditions and also by the addition of electron transfer inhibitors. Ubiquinone-8 (UQ-8) biosynthesis in the cells of ubiCA,ispB-sqr-BL21(DE3) increased as much as 2.2-fold compared with that of (pSTV)-sqr-BL21(DE3) during the 12-16 h incubation period. The maximal sulfide removal in the quinone-raised E. coli was attained slightly earlier, however, SQR activities thereafter were lower than those in (pSTV)-sqr-BL21(DE3).     

Computational cavitation flows at inception and light stages on an axial-flow pump blade and in a cage-guided control valve

Cavitation flows induced around an axial-flow pump blade and inside a high pressure cage-type valve are simu-lated by a two-dimensional unsteady Navier-Stokes analysis with the simplest treatment of bubble dynamics.Thefluid is assumed as a continuum of homogeneous dispersed mixture of water and vapor nuclei.The analysis isaimed to capture transient stages with high amplitude pressure change during the birth and collapse of the bubbleespecially at the stage of cavitation inception.By the pump blade analysis,in which the field pressure is moderate,cavitation number of the inception and locations of developed cavitation are found to agree with experimental re-suits in a wide flow range between high incidence and negative incidence.In the valve flow analysis,in which thewater pressure of 5MPa is reduced to 2MPa,pressure change responding to the bubble collapse between the vaporpressure lower than 1 KPa and the extreme pressure of higher than 10~4 KPa is captured through a stable computa-tion.Location of the inception bubble and pressure force to the valve plug is found agree well with the respectiveexperimental features.     

Implementation of a decision support system using an interactive large-scale high-resolution display

In this research, we propose and evaluate a decision support system using an interactive large-scale high-resolution display. This decision support system supports the summarization and decision-making of a large amount of disaster information during the occurrence of a large-scale natural disaster. Municipal employees at the disaster control headquarters can display disaster information on the large-scale display with a touch or flick on a laptop or tablet. To evaluate the operability, readability, functionality, and necessity of the decision support system, we surveyed 23 municipal employees in the disaster prevention division using a questionnaire. The system received a great evaluation in all the evaluation items.     

A quick and simple method for the identification of meat species and meat products by PCR assay

The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw materials for products. By mixing seven primers in appropriate ratios, species-specific DNA fragments could be identified by only one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the six meats. The products showed species-specific DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken, cattle, sheep, pig and horse meats, respectively. Identification is possible by electrophoresis of PCR products. Cattle, pig, chicken, sheep and goat fragments were amplified from cooked meat heated at 100 or 120°C for 30 min, but horse DNA fragments could not be detected from the 120°C sample. Detection limits of the DNA samples were 0.25 ng for all meats.     

Micromachining of electroformed nickel mold using thick photoresist microstructure for imprint technology

In order to fabricate polymer-based microstructures with feature sizes on the order of micrometers, we have been developing a microimprint technology with a fine nickel (Ni) mold instead of a conventional photolithography technique. The Ni mold was successfully fabricated by electroforming using a positive thick photoresist microstructure patterned on a silicon substrate as a replication master. The photoresist microstructure with excellent edge quality can be obtained under irradiation with single wavelength (g line) selected from a high-pressure mercury lamp. In addition, its sidewall angle in the range of 65° to 84° can be controlled precisely by varying the distance between a photomask and a photoresist surface. On the structured photoresist master, Ni was electroplated up to a thickness of about 110 μm, and then removed from the master. In this process, two-step electroplating at different current densities was carried out in order to prevent deformation of the photoresist master due to stress generated in a Ni electrodeposit. With the Ni mold, fine patterns with a width of 10 or 30 μm and a depth of 24 μm were almost completely transferred to polymetric materials (PMMA). The geometrical dimensions of the fabricated PMMA microstructures were found to be only about 10% reduction against the Ni mold.     

Miscibility and crystallization behavior of poly(ether ether ketone ketone)/poly(ether imide) blends

The miscibility and crystallization behavior of poly(ether ether ketone ketone) (PEEKK)/poly(ether imide) (PEI) blends prepared by melt‐mixing were investigated by differential scanning calorimetry. The blends showed a single glass transition temperature, which increased with increasing PEI content, indicating that PEEKK and PEI are completely miscible in the amorphous phase over the studied composition range (weight ratio: 90/10–60/40). The cold crystallization of PEEKK blended with PEI was retarded by the presence of PEI, as is apparent from the increase of the cold crystallization temperature and decrease of the normalized crystallinity for the samples anealed at 300°C with increasing PEI content. Although the depression of the apparent melting temperature of PEEKK blended with PEI was observed, there was no evidence of depression in the equilibrium melting temperature. The analysis of the isothermal crystallization at 313–321°C from the melt of PEEKK/PEI (100/0, 90/10, and 80/20) blends suggested that the retardation of crystallization of PEEKK is caused by the increase of the crystal surface free energy in addition to the decrease of the mobility by blending PEI with a high glass transition temperature. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 769–775, 2001     

Bactericidal effect of cationic hydrogels prepared from hydrophilic polymers

This study investigated whether hydrogels comprising hydrophilic cationic polymers have similar bactericidal effects. Bacteria were seeded on hydrogels and agar and their viability was assessed with time. Cationic hydrogels displayed bactericidal effects upon long-term bacterial contact. Furthermore, we assessed the areal density of cationic monomer unit of the cationic hydrogels, water content, and the initial elastic modulus. We examined correlations between each factor and bacterial death ratios; consequently, the bacterial death ratios were strongly correlated with the areal density of cationic hydrogel monomers. Elastic energy (W_el) generated at the cytomembrane ion-binding region and the cationic hydrogel and the cytomembrane interfacial energy (W_f) were estimated; consequently, W_el exceeded W_f at higher contact areas. The cationic hydrogel may extract cytomembranes with a reasonable adsorption area. Therefore, cationic hydrogels may be used as probes for ultrasonic echo to sterilize medical equipment.     

Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches

ALG-2 is a penta-EF-hand Ca(2+)-binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, PXYPXnYP (X, variable; n = 4), in ALIX and PLSCR3; type 2, PXPGF, in Sec31A and PLSCR3. The previously solved X-ray crystal structure of the complex between ALG-2 and an ALIX peptide containing type 1 motif showed that the peptide binds to Pocket 1 and Pocket 2. Co-crystallization of ALG-2 and type 2 motif-containing peptides has not been successful. To gain insights into the molecular basis of type 2 motif recognition, we searched for a new hydrophobic cavity by computational algorithms using MetaPocket 2.0 based on 3D structures of ALG-2. The predicted hydrophobic pocket designated Pocket 3 fits with N-acetyl-ProAlaProGlyPhe-amide, a virtual penta-peptide derived from one of the two types of ALG-2-binding sites in PLSCR3 (type 2 motif), using the molecular docking software AutoDock Vina. We investigated effects of amino acid substitutions of the predicted binding sites on binding abilities by pulldown assays using glutathione-S-transferase -fused ALG-2 of wild-type and mutant proteins and lysates of cells expressing green fluorescent protein -fused PLSCR3 of wild-type and mutants. Substitution of either L52 with Ala or F148 with Ser of ALG-2 caused loss of binding abilities to PLSCR3 lacking type 1 motif but retained those to PLSCR3 lacking type 2 motif, strongly supporting the hypothesis that Pocket 3 is the binding site for type 2 motif.     

Formation mechanism for hexagonal-structured self-assemblies of nanocrystalline titania templated by cetyltrimethylammonium bromide

Hexagonal-structured self-assemblies of nanocrystalline (anatase) titania templated by cetyltrimethylammonium bromide (C(16)H(33)N(CH(3))(3)Br; CTAB) (Hex-ncTiO(2)/CTAB Nanoskeleton) were formed after mixing of aqueous solutions containing CTAB spherical micelles and titanium oxysulfate acid hydrate (TiOSO(4).xH(2)SO(4).xH(2)O) as a titania precursor in the absence of any other additives. Formation mechanism of the Hex-ncTiO(2)/CTAB Nanoskeleton was examined in terms of the reaction temperature, titania precursor/CTAB mixing ratio, surfactant type, electrostatic interaction, micelle formation and molecular component. We found that crystal growth of crystalline (anatase) titania (polymorphic crystallization) was promoted with higher temperature and lower titania precursor content in aqueous solutions. In addition, we revealed that the crystalline (anatase) titania was formed in polycation, poly(allylamine hydrochloride ([CH(2)CH(CH(2)NH(2))HCl](n); PAH), and formamide (HCONH(2)) solutions. On the other hand, no titania formation was observed in anionic systems such as sodium dodecyl sulfate (CH(3)(CH(2))(11)OSO(3)Na; SDS) and poly(sodium 4-styrenesulfonate ([C(8)H(7)SO(3)Na](n); PSSS). This indicates that hydrolysis reaction of the titania precursor is initiated by not only cations but also nitrogen atoms in molecules and polymers. Hexagonally structure was formed in only cationic surfactant micellar solutions but not in polycation solutions and formamide.