Sensitive analysis of aconitine, hypaconitine, mesaconitine and jesaconitine in human body fluids and Aconitum tubers by LC/ESI-TOF-MS

The Aconitum species (Ranunculaceae) are widely distributed in northern Asia and North America. Their roots are popularly used in herbal medicines in China and Japan. Many cases of accidental, suicidal and homicidal intoxication with this plant have been reported; some of these were fatal because the toxicity of Aconitum is very high. It is thus important to detect and quantify Aconitum alkaloids in body fluids, with high sensitivity. We have developed a simple and sensitive method for measuring four kinds of Aconitum alkaloids (aconitine, hypaconitine, jesaconitine and mesaconitine) by LC/electrospray (ESI)-time-of-flight (TOF)-MS. For all of them, only molecular ions were observed at an orifice voltage of 75 V; at 135 V, base peaks corresponding to [M - 60 + H]+ ions were observed. These four compounds and methyllycaconitine (internal standard) in human plasma samples were purified by solid-phase extraction. The four extracted compounds were completely separated in mass chromatograms; the calibration curves showed good linearity in the range 10-300 ng/ml, and the detection limits were estimated to be 0.2-0.5 ng/ml. Using our method, we also determined the amounts of these compounds in tuber samples. The present method is applicable in clinical and forensic toxicology.     

Total synthesis of actinobolin from d-glucose by way of the stereoselective three-component coupling reaction

The total synthesis of (−)-actinobolin 3, an antipode of the natural product, starting from d-glucose is described. A three-component coupling reaction of functionalized cyclohexenone (+)-6 derived from d-glucose by way of Ferrier's carbocyclization reaction, with vinyl cuprate and 2-alkoxypropanal 7 effectively constructed the carbon framework of 3 in a highly stereoselective manner. In an aldol process of the three-component coupling reaction, stereochemical control (chelation and Felkin-Anh conditions) was achieved by the choice of the protecting groups of a hydroxy function in 2-hydroxypropanal and the reaction solvents. The formal synthesis of the natural enantiomer, (+)-actinobolin 1, starting from d-glucose was also accomplished.     

Thermodynamics of interactions of vancomycin and synthetic surrogates of bacterial cell wall

Glycopeptide antibiotics, including vancomycin, form complexes via a set of five hydrogen bonds with the acyl-l-Lys-d-Ala-d-Ala portion of the peptidyl stems of the bacterial cell wall peptidoglycan. This complexation deprives the organism from the ability to cross-link peptidyl stems of the peptidoglycan, leading to bacterial cell death. Four synthetic fragments as surrogates of the components of the bacterial cell wall have been prepared in our lab in multistep syntheses. These synthetic samples were used in investigations of the thermodynamics properties (DeltaG degrees , DeltaH degrees , and TDeltaS degrees ) for the complexation with vancomycin by isothermal titration calorimetry (ITC). Complexation with the glycopeptide analogues is largely enthalpy-driven (formation of five hydrogen bonds), and in the analogues with a single peptidyl stem, the complexation is 1:1. The complexation is more complicated with an approximately 2 kDa cell wall surrogate (compound 4), which possesses two peptidyl stems. The data were suggestive of interactions between the two vancomycin molecules, with an entropic penalty attributable to restriction of molecular movements within the complex due to restriction of motion of the highly mobile acyl-d-Ala-d-Ala moiety of the peptidyl stems. These data were reconciled with the recently determined NMR solution structure for the peptidoglycan fragment 4 and its implications for the larger cell wall.     

Synthesis and structure of new water-soluble and stable tantalum compound: ammonium tetralactatodiperoxo-mu-oxo-ditantalate(V)

The stable water-soluble tantalum complex with lactic acid (ammonium tetralactatodiperoxo-mu-oxo-ditantalate(V)), (NH4)4[Ta2(C3H4O3)4(O2)2O].3H2O, was prepared in the crystalline form. According to the single-crystal X-ray diffraction data, this compound forms a monoclinic cell with a = 13.85(2) A, b = 9.06(1) A, c = 12.32(2) A, and beta = 116.30 degrees , space group C2 (No. 2), and has Z = 2 molecules per unit cell. The solid-state 13C NMR data and low flack parameter are consistent with the determined structure. Appearance of the same vibration modes in Raman and IR spectra supports the choice of the space group without inversion symmetry. The solution of the tantalum complex was successfully applied for the synthesis of two photocatalytic materials, NaTaO3 and Sr2Ta2O7.     

One-Pot synthesis of gamma,delta-unsaturated carbonyl compounds from allyl alcohols and vinyl or isopropenyl acetates catalyzed by [IrCl(cod)]2

One-pot synthesis of gamma,delta-unsaturated carbonyl compounds from allyl alcohols and vinyl or isopropenyl acetates was achieved through in situ generation of allyl vinyl ethers by the action of the [IrCl(cod)]2 complex followed by Claisen rearrangement of the resulting ethers. For instance, the reaction of trans-2-methyl-3-phenyl-2-propen-1-ol with isopropenyl acetate in the presence of [IrCl(cod)]2 (1 mol %) and Cs2CO3 (5 mol %) at 100 degrees C for 3 h followed by 140 degrees C for 15 h afforded 5-methyl-4-phenyl-5-hexen-2-one in 72% yield. When vinyl acetate was employed in place of isopropenyl acetate, 4-methyl-3-phenyl-4-pentenal was obtained in 83% yield.     

Ultra-slim laminated capillary array for high-speed DNA separation

We developed a new kind of capillary array for electrophoresis by using the numerical-control (NC) wiring technique conventionally used to produce printed-circuit boards. Laminating two polyimide sheets after laying cylindrical capillaries between them according to designed geometries, we fabricated a 16-lane laminated capillary array (LCA) 9.9 cm long, 7.2 cm wide, and 0.5 mm thick in which the effective length of all capillaries was only 10.9 cm. This compact LCA thus had separation columns as short as those in capillary array electrophoresis chips fabricated by lithography techniques. Like conventional capillary arrays, it also enabled pipetting-less direct injection of analytes from sample preparation plates. Using the LCA with LIF detection and a replaceable fluid sieving matrix, we demonstrated high-speed ssDNA fragment separations. At an electric field strength of 316 V/cm, 15 fragments ranging from 50 to 500 bases were completely separated within 5.8 min in all lanes. The lane-to-lane CV of migration time was only 0.38%, and the fragment size for which the resolution per base was 0.59 was 258 +/- 15 bases (average +/-SD).     

Chemo-enzymatic production of fuel ethanol from cellulosic materials utilizing yeast expressing β-glucosidases

Ethanol was produced in a considerably high yield by fermenting hydrolyzates from cellulosic materials by means of a recombinant laboratory yeast expressing β-glucosidases. Tissue paper, cotton, and sawdust were hydrolyzed by two-step sulfuric acid hydrolysis to give mixtures containing glucose, cellobiose, and higher cello-oligosacc arides. After the cellulosic material was partially hydrolyzed with 80% sulfuric acid, the hydrolysis was continued with 5% sulfuric acid. Except for non-carbohydrate components, all constitutents in the hydrolyzates were fermented by the yeast that was preincubated in the medium that the plasmid encoded by the β-glucosidases gene was kept in the muliplicated yeast. A solution containing 4% hydrolyzates from paper was fermented to give as high as 1.9% maximum ethanol concentration and 70% ethanol conversion. Cotton also gave a similar result. Sawdust was converted into ethanol in approx 22% conversion. Accordingly, it was revealed that the β-glucosidases-expressing yeast can ferment the cello-oligosaccharides obtained by hydrolysis of cellulosic materials into ethanol. In addition, a hydrolyzate containing a high glucose proportion gave a high ethanol concentration in a short time.     

Evaluation of left ventricular function with cardiac magnetic resonance imaging using Fourier fitting

The aims of this study were to investigate the applicability of Fourier fitting in the magnetic resonance (MR) evaluation of left ventricular (LV) function and to determine the optimal number of harmonics for fitting. Cine cardiac MR imaging was performed in 10 subjects, and an LV time–volume curve was generated. Fourier fitting was applied to the original curve using 1–10 harmonics, and the qualities of the time–volume curve and first-derivative curve were evaluated. LV functional parameters were calculated from curves generated with and without fitting. The quality of the original time–volume curve was good, and Fourier fitting had no substantial effect on functional parameters obtained directly from the time–volume curve such as ejection fraction. The first-derivative curve generated without fitting showed substantial artificial fluctuation. The application of Fourier fitting depressed the fluctuation and tended to decrease estimates of peak ejection rate and peak filling rate. Five or six harmonics appeared to be appropriate for obtaining a high-quality first-derivative curve. In conclusion, Fourier fitting was indicated to aid in reducing the artificial fluctuation of the first-derivative curve generated from cine cardiac MR imaging and to contribute to the evaluation of functional parameters derived from the first-derivative curve.     

Holographic femtosecond laser processing with multiplexed phase Fresnel lenses

Holographic femtosecond laser processing with multiplexed phase Fresnel lenses for high-speed parallel fabrication of microstructures is proposed. Use of a spatial light modulator (SLM) allows independent tunability of the diffraction peaks, three-dimensional parallelism, and arbitrary, variable features. The diffraction peaks are made uniform by changing the center phase and size of each phase Fresnel lens while taking account of the intensity distribution of the irradiated laser pulse and the spatial frequency response of the SLM.     

Sarcospan: ultrastructural localization and its relation to the sarcoglycan subcomplex

Sarcospan is a 25 kDa transmembrane component of dystrophin-associated glycoprotein. We generated a rabbit polyclonal antibody against synthetic peptide of the N-terminal domain of human sarcospan. Using this antibody we investigated the localization of sarcospan and its spacial relation to the components of sarcoglycan subcomplex in normal human skeletal myofibers by immunofluorescent microscopy and immunogold electron microscopy. In immunofluorescence the reaction was observed continuously at the myofiber surface. Ultrastructurally the gold signals of rabbit anti sarcospan antibody were present along the muscle plasma membrane, mainly at its inside surface. The triple immunogold labeled muscle samples showed that the signals of rabbit or sheep polyclonal anti alpha-, beta-, gamma- and delta-sarcoglycan antibodies and/or mouse monoclonal anti beta-, gamma- and delta-sarcoglycan antibodies were located along the muscle plasma membrane, and the cluster formation of different two or three sarcoglycan molecules was observed. The triple immunogold labeling also revealed that the signal of sarcospan molecules are present frequently in doublets and/or triplets with the components of sarcoglycan subcomplex, resulting in the cluster formation of signals of sarcoglycan and sarcospan molecules. The result of this study showed that sarcospan was expressed at the myofiber surface and that sarcospan was present in close association with alpha-, beta-, gamma- and delta-sarcoglycans and formed a functional unit with sarcoglycan subcomplex.