Effective Blocking and Stabilizing Methods Using Synthetic Polymer on ELISA.

In ELISA, blocking reagents and stabilizers are important to improve the sensitivity and/or quantitative nature of the measurement system. Usually, biological substances such as bovine serum albumin and casein are used, but they still have problems such as lot-to-lot differences and biohazard. Here, we describe the methods using a chemically synthesized polymer, BIOLIPIDURE®, as a new blocking agent and stabilizer that can solve these problems.

Formation mechanisms of hollow manganese hexacyanoferrate particles and construction of a multiple-shell structure.

Formation mechanisms of hollow manganese hexacyanoferrate (Mn-HCF) particles have been investigated. Mn-HCF particles, which were precipitated by mixing an aqueous solution of K3[Fe(CN)6] with MnCl2 in the presence of sodium citrate, could be converted into a hollow structure just by washing with distilled water. The powder X-ray diffractometry suggested that the as-prepared particle has a core/shell morphology with different crystal structures: cubic-core and monoclinic-shell. The time evolutions of the particle size and shell thickness indicated that the core was rapidly (but not instantaneously) formed at the initial stage of the precipitation process, followed by a slower shell growth. In addition, the solubility of the cubic core was estimated to be about 2.5 times higher than that of the monoclinic shell, resulting in the preferential dissolution of the interior of the particle by the washing process. The formation procedure has been used to construct multiple-shell hollow Mn-HCF particles containing up to quadruple separated nesting shells by associating an additional growth technique.

Inhibition of Calcineurin and Glycogen Synthase Kinase-3ß by Ricinoleic Acid Derived from Castor Oil.

Ricinoleic acid (RA) is the main fatty acid component of castor oil and was found to inhibit Ca2+ -signal transduction pathway-mediated cell cycle regulation in a yeast-based drug screening assay. RA is expected to have antidiabetic, antiallergy, and/or anticancer properties but its target molecule is unknown. To identify a novel pharmacological effect of RA, we investigated its target molecule in the Ca2+ -signal transduction pathway. RA inhibition of calcineurin (CN) was examined in a yeast-based CN inhibitor screening assay using the rsp5A401E mutant and in a phosphatase assay using recombinant human CN. RA showed growth-restoration activity at 5 µg/spot in the CN inhibitor screening assay with the rsp5A401E yeast strain. Furthermore, it directly inhibited CN without immunophilins at Ki = 33.7 µM in a substrate-competitive manner. The effects of RA on CN in mammalian cells were further evaluated by measuring ß-hexosaminidase (ß-HEX) release in RBL-2H3 cells. RA at 50 µM suppressed the release of ß-HEX from RBL-2H3 cells. Moreover, this compound was found to inhibit glycogen synthase kinase-3ß (GSK-3ß), as determined by a kinase assay using recombinant human GSK-3ß. RA inhibited GSK-3ß at Ki = 1.43 µM in a peptide substrate-competitive manner. The inhibition of GSK-3ß by this molecule was further assessed in mammalian cells by measuring the inhibition of glucose production in H4IIE rat hepatoma cells. RA at 25 µM suppressed glucose production in these cells. These findings indicate that RA and/or castor oil could be a useful functional fatty acid to treat allergy or type 2 diabetes.

Synthesis of uniform Prussian blue nanoparticles by a polyol process using a polyethylene glycol aqueous solution.

A polyol process was applied to the synthesis of Prussian blue nanoparticles that have a narrow size distribution. Potassium hexacyanidoferrate(iii) and iron(iii) nitrate aqueous solutions were introduced into a 50% polyethylene glycol (PEG) aqueous solution under magnetic stirring at 50 °C and reacted for 48 h. The shape of the so-obtained particles was cubic with somewhat rounded edges and the mean size was 70 nm. In the formation process, nanoparticles of Prussian green, which is a partially oxidized state of Prussian blue, were firstly generated via reduction of the precursors by PEG molecules. The Prussian green nanoparticles were then reduced subsequently to Prussian blue nanoparticles. Rate constants for both the reduction steps have been estimated using the time evolution of absorbance.

Chiral and molecular recognition of monosaccharides by photoexcited tryptophan in cold gas-phase noncovalent complexes as a model for chemical evolution in interstellar molecular clouds.

Chiral and molecular recognition between amino acid and sugar molecules and their implications for chemical evolution were investigated using a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation of mass-selected and temperature-controlled gas-phase noncovalent complexes of protonated tryptophan (Trp) and monosaccharide enantiomers, such as aldohexose, aldopentose, and deoxyhexose, was examined as a model for chemical evolution in interstellar molecular clouds. Upon photoexcitation of noncovalent heterochiral H+(L-Trp)(D-aldohexose) complexes, NH2CHCOOH loss from protonated Trp via Cα-Cß bond cleavage occurred. Conversely, in homochiral H+(L-Trp)(L-aldohexose), the energy absorbed by Trp was released through the detachment of aldohexose, and dissociation of the amino acid was suppressed. In the photodissociation mass spectra of protonated Trp with aldopentose and deoxyhexose, which lacks the OH group of aldohexose, no dissociation of the molecules in the complexes or differences between enantiomers were observed. These results indicate that the OH groups in monosaccharides contribute to enantiomer-selective photodissociation in molecular clouds. The differences observed between enantiomers in the photodissociation mass spectra were applied to distinguishing and quantifying aldohexose enantiomers in solution using L-Trp as a chiral probe. The enantiomeric excesses of aldohexoses in solution could be determined from a single photodissociation mass spectrum by reference to the relative ion intensities for the NH2CHCOOH-elimination product and H+(L-Trp) formed via detachment of aldohexose. This analysis method could also distinguish and quantify two D-aldohexose mixtures, where L-Trp was employed as an isomer probe. Graphical abstract ᅟ.

A new eremophilane sesquiterpene from the fungus Xylaria sp. V-27 and inhibition activity against degranulation in RBL-2H3 cells.

A new eremophilane sesquiterpene, 13,13-dimethoxyintegric acid (1), together with known compound integric acid (2) have been isolated from a fungus, Xylaria sp. V-27, obtained from a dead branch. The structure of 1 was established by means of spectroscopic analyses. 1 and 2 promoted growth restoring activity against the mutant yeast strain (Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ)) and inhibited degranulation of rat basophilic leukemia RBL-2H3 cells stimulated by immnunoglobulin G + 2,4-dinitrophenylated-bovine serum albumin (IgE+DNP-BSA), thapsigargin and A23187.

Yeast Ca(2+)-signal transduction inhibitors isolated from Dominican amber prevent the degranulation of RBL-2H3 cells through the inhibition of Ca(2+)-influx.

A new norlabdane compound, named kujigamberol has previously been isolated from Kuji amber (but not from Baltic amber) by activity guided fractionation. However, there has been no study of biological compounds in Dominican amber. Biological activities were examined using the hypersensitive mutant yeast (zds1Δ erg3Δ pdr1Δ pdr3Δ) with respect to Ca(2+)-signal transduction, enzymes and rat basophilic leukemia (RBL)-2H3 cells. The structures were elucidated on the basis of spectral analysis including high resolution (HR)-EI-MS, 1D NMR and 2D NMR. Three diterpenoid compounds, 5(10)-halimen-15-oic acid (1), 3-cleroden-15-oic acid (2) and 8-labden-15-oic acid (3), which are different from the bioactive compounds in Kuji and Baltic ambers, were isolated from Dominican amber. They inhibited both calcineurin (CN) (IC50=40.0, 21.2 and 34.2µM) and glycogen synthase kinase-3ß (GSK-3ß) (IC50=48.9, 43.8 and 41.1µM) which are involved in the growth restored activity against the mutant yeast. The most abundant compound 2 showed inhibitory activity against both degranulation and Ca(2+)-influx in RBL-2H3 cells. The compounds having the growth restoring activity against the mutant yeast have potential as anti-allergic compounds.

Relationship between supersaturation ratio and supply rate of solute in the growth process of monodisperse colloidal particles and application to AgBr systems.

Supersaturation ratio, S, has been theoretically related to the supply rate of solute, Q, from growth rate and mass-balance equations in the quasi-steady state in the growth process of isotropic monodisperse particles. The derived equation, (S - 1) = (1/D + 1/kr)(Q/betaC(0)nr) + 2V(m)gamma/rRT, suggests a linear dependence of S on Q under constant n and r, where D is the diffusion coefficient, k is the rate constant for surface-reaction, C(0) is the solubility, n and r are the number and radius of growing particles, respectively, V(m) is the molar volume of particles, R is the gas constant, T is the absolute temperature, and beta is the shape factor defined by beta identical with (1/r(2)) dupsilon/dr, where upsilon is the volume of an individual particle. The equation was applied to the analysis of growth kinetics and determinations of critical supersaturation ratio in monodisperse AgBr particles in the controlled double-jet system with the assistance of a potentiometric supersaturation measurement. In both cubic and octahedral particles, growth rates were completely limited by diffusion and surface-reaction at pBr ( identical with -log[Br(-)]) 3.0 and 1.0, respectively, while the growths were intermediate of them at pBr 2.0 and 4.0. The growth parameters, DC(0) and kC(0), were experimentally determined. Also, critical supersaturation ratio was estimated as 1.28 as an average in the present study.