Coupling stimuli-responsive magnetic nanoparticles with antibody-antigen detection in immunoassays

Because current homogeneous immunoassays show some limitations, particularly low sensitivity, we developed a new immunoassay to overcome these limitations. The approach was based on magnetic nanoparticles with a thermoresponsive polymer layer, a negatively charged polymer, and streptavidin-biotin-based antibody-antigen detection and yielded higher sensitivity than commonly used heterogeneous immunoassays. Because no special equipment is needed, it can be applied to currently available absorbance-based systems for high-throughput assays.     

Determination of pindone in animal products, fishery products, and honey by LC-MS/MS

A sensitive and selective analytical method for the determination of the rodenticide pindone in animal products, fishery products, and honey by LC-MS/MS was developed. Pindone was extracted with acidified acetone, and the crude extract was purified by liquid-liquid partitioning, followed by silica gel and ODS column chromatography. LC separation was performed on an ODS column with methanol/water containing ammonium acetate as the mobile phase, and detection was carried out using tandem mass spectrometry (MS/MS) with electrospray ionization (ESI) in the negative mode. The average recoveries from fortified bovine muscle, bovine liver, bovine fat, chicken muscle, salmon, eel, freshwater clam, egg, milk, and honey spiked at 0.001 mg/kg were in the range of 76-92%, and the relative standard deviations were 4-8%. The limit of quantitation (S/N≥10) of the developed method was 0.001 mg/kg for all the tested foods.     

STUDIES ON FROZEN BEER PRECIPITATES I. FORMATION AND GENERAL CHARACTERS

When beers were kept frozen at ?8° to ?10°C. and then thawed at room temperature, white flake-like precipitates were formed in most of the commercial beers tested. The frozen beer precipitates consisted mainly of non-starchy polysaccharides. Quantitative analysis of acid hydrolysates showed that the precipitates contained 83% glucose and 6% pentoses (xylose and arabinose). Using a partially purified preparation, various characters such as susceptibility to hydrolytic enzymes, infra-red absorption spectra, and optical rotation were examined, and the results suggested that the main component of the frozen beer precipitate is a β-linked glucose polymer, β-glucan. Effects of temperature and duration of freezing on the formation of the precipitate were examined, and the mechanism of formation of the frozen beer precipitate is discussed.     

The processing of high-molecular-weight xylanase (XynE, 110 kDa) from Aeromonas caviae ME-1 to 60-kDa xylanase (XynE60) in Escherichia coli and purification and characterization of XynE60

A xylanase gene (xynE) encoding XynE (110 kDa) was cloned from a lambda phage genomic library of Aeromonas caviae ME-1 which is a multiple-xylanase-producing bacterium. Upon nucleotide sequence analysis, we found that xynE comprises 2823 by and encodes a protein of 941 amino acid residues (104,153 Da), which was similar to endo-beta-1,4-xylanases which are categorized to glycosyl hydrolase family 10. An Escherichia coli transformant that harbored pXED30 carrying xynE produced 110-, 84-, 72-, and 66-kDa xylanases in the cell-free extract, and 72- and 66-kDa xylanases in the culture supernatant. We purified the 66-kDa xylanase to electrophoretic homogeneity from a culture supernatant by a series of column chromatographies. The calculated molecular mass of the purified xylanase determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was 60,154.50 Da, and the xylanase was designated XynE60. Analysis of the N-terminal 10 amino acid residues and the determined molecular mass of XynE60 revealed that XynE60 is a product processed at the Gly26-Gly27, and Thr565-Ala566 sites of XynE by proteolytic cleavage. XynE60 showed optimal activity at 55 degrees C and pH 8.0, and was stable below 45 degrees C and at pH 7.0-8.5. The K(m) and V(max) of XynE60 were calculated to be 8.1 mg/ml and 6897 nkat/mg, respectively.     

Molecular identification of Sphingomonas sp. A1 alginate lyase (A1-IV') as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution

Sphingomonas sp. A1 (strain A1) produces three endotypes (A1-I [65 kDa], A1-II [25 kDa], and A1-III [40 kDa]) and an exotype (A1-IV [86 kDa]) alginate lyases in cytoplasm. These four enzymes cooperatively depolymerize alginate into constituent monosaccharides. In addition to the genes for these lyases, novel genes encoding hypothetical proteins homologous with A1-IV were found in the genomes of many bacteria including strain A1. One such protein, A1-IV' (90 kDa) of strain A1, was overexpressed in Escherichia coli cells, purified, and characterized. A1-IV' catalyzed the cleavage of glycosidic bonds in alginate through a beta-elimination reaction and released unsaturated di- and trisaccharides as main products, thus indicating that the enzyme is an endotype alginate lyase. A1-IV', which differed from A1-IV in some enzymatic properties, was not expressed in strain A1, suggesting that A1-IV' has no significant role in alginate metabolism. A1-IV' and other A1-IV homologs facilitate the creation of novel polysaccharide lyase family 15 based on their primary structures, implying the evolution route of alginate lyases in family PL-15.     

Preparation and evaluation of liquid-crystal formulations with skin-permeation-enhancing abilities for entrapped drugs

The usefulness of liquid crystals (LC) in topical formulations for application to skin was evaluated by measuring the in vitro permeation profile of a model compound, calcein, entrapped in a LC formulation, through excised hairless rat skin and a three-dimensional cultured human-skin model; the viability was determined using the MTT assay. Two physically stable LCs were prepared from a mixture of mono-, di-, and tri-esters 1, and monoesters 2, composed of erythritol and phytanylacetic acid. Cryo-transmission electron microscopy (cryo-TEM), electron diffraction patterns, and small-angle X-ray diffraction (SAXS) observations of the LC nanodispersions showed that the structures of the LCs were reverse hexagonal (LC-A) and cubic (LC-B). The skin-permeation properties of calcein were enhanced by entrapping in the LCs as a result of the increase in calcein partition from the LC dispersion solution into the skin; the properties were analyzed using a skin-permeation-time profile. Drug partitioning could also be modified by the LC structure. No skin damage was caused by the LC formulation in these experiments.The present study suggests that LC dispersions are potential additives in topical drug formulations and cosmetic formulations.     

Larval Feeding Stimulants for a Rutaceae-Feeding Swallowtail Butterfly, Papilio xuthus L. in Citrus unshiu Leaves

Larvae of a swallowtail butterfly, Papilio xuthus L., feed exclusively on plants of the family Rutaceae, including various Citrus crops. Larvae were strongly stimulated to feed on paper strips impregnated with ethanolic extracts of host-plant leaves. Stimulation of feeding on extracts of Citrus unshiu leaves required a mixture of chemicals including sugars (d-glucose, d-fructose, and d-sucrose), a betaine [(−)-stachydrine], a cyclic peptide (citrusin I), a polymethoxyflavone (isosinensetin), and the lipids 1-linolenoylglycerol, 1-linoleoylglycerol, 1-octadecenoylglycerol, 1-stearoylglycerol, and 1,2-dilinolenoyl-3-galactosyl-sn-glycerol. When these compounds were assayed individually, few larvae consumed test strips. However, larvae readily chewed the test strips treated with a mixture of all compounds, indicating that host recognition by P. xuthus larvae is mediated by a specific combination of both primary and secondary substances. Comparison of 11 stimulant components with 10 compounds from C. unshiu leaves previously reported as stimulant components for oviposition by P. xuthus adult females revealed only one compound, stachydrine, as an ingredient in common. While the larval feeding-stimulant mixture is dominated by nutrients and other compounds of general significance for primary metabolism, the component oviposition stimulants are mostly secondary substances, including flavonoid glycosides, protoalkaloids, a cyclitol, and a betaine, that have restricted distributions in plants. Reliance by adult females on unique profiles of secondary compounds presumably reflects the need to locate and recognize specific host-plant species within a diverse flora. Since the initial host choice for the larvae is made typically by the ovipositing female, however, unique secondary compounds may be less important for larval feeding than are compounds useful for indicating food and microhabitat quality once on the host plant.     

Application of flamelet model to large‐eddy simulation of turbulent reacting liquid flows

Large‐eddy simulations using the flamelet models are applied to turbulent reacting liquid flows and validated by comparing with the experiments. The computations are performed for two reaction conditions, namely a rapid reaction and a moderately fast reaction in a grid‐generated turbulent flow. For the flamelet models, both the steady flamelet model and the unsteady Lagrangian flamelet model are tested. A second‐order, irreversible, and isothermal reaction is considered. The results show that the flamelet models inherently developed for turbulent combustion are applicable to turbulent reacting liquid flows, provided that the model coefficient in evaluating the subgrid scale variance of mixture fraction in the scale‐similarity model is set to be 5.0. The rapid reaction can be adequately predicted by both the steady and unsteady Lagrangian flamelet models, whereas the moderately fast reaction can be predicted only by the unsteady Lagrangian flamelet model which is capable to take slow chemical processes into account. © 2010 American Institute of Chemical Engineers AIChE J,, 2011     

Study of Superconductor Recovery Time Characteristics and High‐Speed Reclosing of Electromagnetic Repulsion Switch

Using a high‐temperature superconductor, we constructed and tested a model superconducting fault current limiter (SFCL). The superconductor and a vacuum interrupter serving as the commutation switch were connected in parallel using a bypass coil. When the fault current flows in this equipment, the superconductor is quenched and the current is then transferred to the parallel coil due to the voltage drop in the superconductor. This large current in the parallel coil actuates the magnetic repulsion mechanism of the vacuum interrupter and the current in the superconductor is interrupted. Using this equipment, the current flow time in the superconductor can easily be minimized. On the other hand, the fault current is also easily limited by the large reactance of the parallel coil. This system has many advantages. Thus, we introduced an electromagnetic repulsion switch. High‐speed reclosing after interrupting the fault current in the electrical power system is essential. Thus, the SFCL should recover to the superconducting state before high‐speed reclosing. But the superconductor generates heat at the time of quenching, and it takes time to recover to the superconducting state. Therefore, the recovery time is an issue. In this paper, we study the superconductor recovery time. We also propose an electromagnetic repulsion switch with a reclosing system. © 2011 Wiley Periodicals, Inc. Electr Eng Jpn, 175(3): 12–19, 2011; Published online in Wiley Online Library ( wileyonlinelibrary.com ). DOI 10.1002/eej.21072