Capillary electrophoretic study on pH dependence of enantioselective disopyramide binding to genetic variants of human alpha1-acid glycoprotein

A high-performance frontal analysis-capillary electrophoresis (HPFA-CE) method was applied to investigate the effect of pH on the drug binding properties of genetic variants of human alpha1-acid glycoprotein (AGP), A variant and a mixture of F1S variants. The unbound concentrations of a model basic drug, disopyramide (DP), in A variant solutions and in F1S variant solutions were measured by HPFA-CE to evaluate binding constants at pH 4.0, 5.0, 6.0 and 7.4. The binding between DP and A variant was gradually weakened by acidification of background buffer (from pH 7.4 to 4.0), while the binding between DP and FIS variants decreased at first (from pH 7.4 to 6.0), and then gained (from pH 6.0 to 4.0). Consequently, DP was more strongly bound to A variant than to FIS variants at pH 7.4, while at pH 4.0 DP was more strongly bound to F1S variants. At any pH (S)-DP was bound more strongly than (R)-DP, and the enantioselectivity of A variant was significantly higher than that of F1S variants. Electrophoretic mobilities of the AGP genetic variants decreased along with a decrease in pH. Fluorescent emission of these genetic variants indicated a distinct conformational change between pH 5.0 and 4.0. However, there was no significant difference in the electrophoretic mobility and the fluorescent emission spectrum between these variants at any pH. On the other hand, circular dichroism analyses revealed that beta-sheet content in FIS variants diminished as pH decreased, while that in A variant increased. These results suggest that the conformational change induced by acidification of background buffer differs between these genetic variants, and this causes the difference in DP bindability.     

Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay.

We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03 x 10(-20) and 2.05 x 10(-20) mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.     

Intensive optimization and evaluation of global DNA methylation quantification using LC-MS/MS

Analytical and Bioanalytical Chemistry - DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS...     

Allylation of alcohols and carboxylic acids with allyl acetate catalyzed by [Ir(cod)2](+)BF4- complex

A facile method for the synthesis of allyl alkyl ethers from alcohols with allyl acetate was developed by the use of [Ir(cod)(2)](+)BF(4)(-) complex. For instance, the reaction of allyl acetate with n-octyl alcohol in the presence of a catalytic amount of [Ir(cod)(2)](+)BF(4)(-) complex afforded allyl octyl ether in quantitative yield. Allyl carboxylates were also prepared by the exchange reaction between carboxylic acids and allyl acetate in good yields. The [Ir(cod)(2)](+)BF(4)(-) complex catalyzed the reaction of alkyl and aromatic amines with allyl acetate to lead to the corresponding allylamines in fair to good yields.     

Formation of multihollow structures in crosslinked composite polymer particles

Micron-size monodisperse polymer particles having multihollow structures were prepared as follows. First, micron-size monodisperse polystyrene/poly(styrene-divinylbenzene) (PS/P(S-DVB)) composite particles were produced by seeded copolymerization of S and DVB with 2,2-azobisisobutyronitrile as an initiator in the ethanol/water (76/24, weight ratio) medium in the presence of 2.37 m-size monodisperse PS seed particles produced by dispersion polymerization. The molar ratio of S/DVB was changed in the range of 1/110/1. The uncrosslinked polymer within the composite particles was extracted with toluene under reflux. For the highest DVB content (S/DVB=1/1, molar ratio), one large hollow was observed in a part of the composite particles after the extraction. For the middle DVB content (S/DVB=4/1, molar ratio), multihollow structure was observed in all the particles. For the lowest DVB content (S/DVB=10/1, molar ratio), fine multihollow structure was observed in all the particles.Part CXLIII of the series Studies on Suspension and Emulsion     

Multiple pickering emulsions stabilized by microbowls

Some researchers have focused on the adsorption of solid particles at fluid-fluid interfaces and prepared emulsions and foams called "Pickering emulsions/foams". However, while several reports exist on simple spherical emulsions, few reports are available on the formation of more complex structures. Here, we show that holes on particle surfaces are a key factor in establishing the variety and complexity of mesoscale structures. Microbowls, which are hollow particles with holes on their surfaces, form multiple emulsions (water-in-oil-in-water and oil-in-water-in-oil emulsions) by simply mixing them with water and oil. Furthermore, stable potato-like or coffee-bean-like emulsions are also obtained, although nonspherical emulsions are usually unstable because of their larger interfacial energies. These findings are useful in designing the building blocks of complex supracolloidal systems for pharmaceutical, food, and cosmetic products.     

COMPARISON OF FREE RADICALS PRODUCED BY LASER AND ULTRASOUND ABLATION OF CARDIOVASCULAR TISSUE

Abstract— We have used the method of spin trapping and EPR to study the nature of the free radicals produced by laser photoreaction of cardiovascular tissue. The results obtained with argon-ion (cw) and excimer (pulsed) lasers have been compared with radicals produced by ultrasonic disruption of the tissue samples. These comparative studies provide an understanding of the mechanism by which laser light energy effects photoablation of cardiovascular tissue in the pulsed vs cw mode of operation.     

Determination of pemoline in plasma, urine and tissues by reversed-phase high-performance liquid chromatography

A simple and selective high-performance liquid chromatographic method with ultraviolet detection at 215 nm for the determination for pemoline in rat plasma, urine and tissues is described. Pemoline in the samples was extracted with methylene chloride at pH 10 and the organic phase was evaporated after adding 5-methyl-5-phenylhydantoin used as an internal standard. Pemoline and the internal standard were separated on a Kaseisorb LC C8-60-5 reversed-phase column. The limits of determination of pemoline in 0.1-0.2 ml of plasma, urine and tissue homogenates were 2, 100 and 20 ng, respectively. The method should be useful for studies of the pharmacokinetics and distribution of pemoline in small animals.     

Preparation and biological activities of a bivalent poly(ethylene glycol) hybrid containing an active site and its synergistic site of fibronectin

A bivalent poly(ethylene glycol) or PEG hybrid of fibronectin-related peptides was prepared. An active site peptide (RGD) and its synergistic site peptide (PHSRN) of fibronectin were conjugated with an amino acid-type PEG (aaPEG) to form PHSRN-aaPEG-RGD. A moderate spatial array between RGD and PHSRN in fibronectin may be required for synergic activity. The bivalent hybrid exhibited potent cell spreading activity and exhibited potent anti-metastatic activity in a model of experimental metastasis with B16-BL6 cells in mice. PEG may serve as a spacer for maintaining the desired spatial array.