Application of an integrated microchip system with capillary array electrophoresis to optimization of enzymatic reactions

In this work, a combination of complementary metal-oxide semiconductor (CMOS) microchip system with capillary array electrophoresis (CAE) is demonstrated as a system for optimizing conditions for enzymatic reaction. Dimethylacridinone (DDAO)-phosphate substrate and alkaline phosphatase conjugate were selected for the enzymatic reaction, which was applicable to the enzyme-linked immunosorbent assay (ELISA) technique. Laser-induced fluorometry with a miniature semiconductor laser was used to detect the enzymatic products. The speed of the enzymatic reaction between the DDAO-phosphate and the alkaline phosphatase conjugate was investigated as a function of reaction time. The microchip-CAE detection system could determine the pH condition and the concentration of enzyme that are suitable for rapid and low-cost analysis. This result shows the feasibility of using the microchip-CAE system for application to miniaturized screening systems.     

Electrospray ionization tandem mass spectrometric study of the aconitines in the roots of aconite

Fragmentation pathways of aconitine-type alkaloids were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry. Low-energy collision-induced dissociation of protonated aconitines follows a dominant first step, the elimination of the C(8)-substituent as acetic acid or fatty acid in MS(2) spectra. Successive losses of 1-4 CH(3)OH molecules, 1-3 H(2)O, CO, benzoic acid, and CH(3) or C(2)H(5) (N-substituents) are all fragmentation pathways observed in MS(3) and MS(4) spectra. By applying knowledge of these fragmentation pathways to the aconitines in the ethanolic extract of aconite roots, all the known aconitines were detected and also 23 unknown aconitine-type alkaloids, in which the lipo-alkaloids containing residues of 15C, 17C and 19C saturated or unsaturated fatty acids were characterized. These odd-carbon-number fatty acid substituents have not been reported previously.     

Trajectory studies of S(N)2 nucleophilic substitution. 8. Central barrier dynamics for gas phase Cl(-) + CH(3)Cl

Quasiclassical direct dynamics trajectories, calculated at the MP2/6-31G level of theory, are used to study the central barrier dynamics for the C1(-) + CH(3)Cl S(N)2 reaction. Extensive recrossings of the central barrier are observed in the trajectories. The dynamics of the Cl(-)-CH(3)Cl complex is non-RRKM and transition state theory (TST) is predicted to be an inaccurate model for calculating the Cl(-) + CH(3)Cl S(N)2 rate constant. Direct dynamics trajectories also show that Cl(-) + CH(3)Cl trajectories, which collide backside along the S(N)2 reaction path, do not form the Cl(-)-CH(3)Cl complex. This arises from weak coupling between the Cl(-)-CH(3)Cl intermolecular and CH(3)Cl intramolecular modes. The trajectory results are very similar to those of a previous trajectory study, based on a HF/6-31G* analytic potential energy function, which gives a less accurate representation of the central barrier region of the Cl(-) + CH(3)Cl reaction than does the MP2/6-31G* level of theory used here. Experiments are suggested for investigating the non-RRKM and non-TST dynamics predicted by the trajectories.     

Clinical significance of molecular genetic changes in sporadic invasive pituitary adenomas

Several molecular and genetic changes have been found in pituitary adenomas. We looked for correlations between these changes and the degree of invasiveness of the tumors. The invasiveness of 11 pituitary adenomas was graded by Hardy classification. We examined the retinoblastoma gene (RB1.20 on chromosome 13q) and the region around the MEN1 locus (chromosome 11q13.1-5) for loss of heterozygosity. Also examined are p53 mutations using single strain conformation polymorphism, p53 protein overexpression using immuno cytochemistry, homozygous deletions of p15 and p16 by polymerase chain reaction, and cellular proliferative activity using MIB-1 antibody. Six tumors (54.5%) had an LOH at either RB1.20 or the MEN1 locus. LOHs were found more frequently in Grade 4 and stage E tumors (72% and 67%) than in Grade 3 and stage D tumors (25% and 40%). However, no mutation or overexpression of p53 was found. No homozygous deletions of p15 or p16 were identified. The cell proliferative index ranged from 0 to 3%. LOH at 11q13 and 13q may be valuable in predicting the invasiveness of pituitary adenomas.     

Miniature biochip system for detection of Escherichia coli O157:H7 based on antibody-immobilized capillary reactors and enzyme-linked immunosorbent assay

In this work, we report Escherichia coli O157:H7 detection using antibody-immobilized capillary reactors, enzyme-linked immunosorbent assay (ELISA), and a biochip system. ELISA selective immunological method to detect pathogenic bacteria. ELISA is also directly adaptable to a miniature biochip system that utilizes conventional sample platforms such as polymer membranes and glass. The antibody-immobilized capillary reactor is a very attractive sample platform for ELISA because of its low cost, compactness, reuse, and ease of regeneration. Moreover, an array of capillary reactors can provide high-throughput ELISA. In this report, we describe the use of an array of antibody-immobilized capillary reactors for multiplex detection of E. coli O157:H7 in our miniature biochip system. Side-entry laser beam irradiation to an array of capillary reactors contributes significantly to miniaturized optical configuration for this biochip system. The detection limits of E. coli O157:H7 using the ELISA and Cy5 label-based immunoassays were determined to be 3 and 230 cells, respectively. This system shows capability to simultaneously monitor multifunctional immunoassay and high sensitive detection of E. coli O157:H7.     

Hetero-calix[4]pyrroles: incorporation of furans, thiophenes, thiazoles or fluorenes as a part of the macrocycle

Furans, thiazoles, fluorene or thiophene incorporated calix[4]pyrrole analogues were synthesized and characterized. The synthesis was achieved by utilization of various building blocks such as 7, 13, 14, 18 and 21. Acid catalyzed condensation of those building blocks with acetone or meso-disubstituted dipyrromethanes afforded desired macrocycles.     

Molecular variations in Th1-specific cell surface gene Tim-3

The family of T-cell immunoglobulin domain and mucin domain (TIM) proteins is identified to be expressed on T cells. A member of Tim family, Tim-3 (T cell immunoglobulin mucin 3) is selectively expressed on the surface of differentiated Th1 cells. Tim-3 might have an important role in the induction of autoimmune diseases by regulating macrophage activation and interacts with Tim-3 ligand to regulate Th1 responses. To determine the variation sites in the coding and promoter region of human Tim-3 gene, we performed variation scanning by direct sequencing using the genomic DNA isolated from the patients with asthma or allergic rhinitis and healthy controls without asthma and allergic rhinitis. We identified four single nucleotide polymorphisms (SNPs) including one novel SNPs (-1541C>T) and two variation sites (-1292_-1289delTAAA and -1282_-1278dupTAAAA) in the coding and promoter region of human Tim-3 gene in both the patients and healthy groups.     

Effect of water in the precursor solution on the catalytic property of LaMnAl11O19 high temperature combustion catalyst

Manganese-substituted lanthanum hexaaluminates were prepared by the sol-gel method. The effect of water content on the catalyst preparation was examined. Large water content caused a rapid decrease in the surface area. When the R (R is the mole ratio of H₂O/ Al(OC₃H₇)₃) is equal to 1.5, the catalyst retains a specific surface area of 13.5 m² g⁻¹ and shows higher activity in CH₄ combustion.     

NMR and X-ray crystallography, complementary tools in structural proteomics of small proteins

NMR spectroscopy and X-ray crystallography, the two primary experimental methods for protein structure determination at high resolution, have different advantages and disadvantages in terms of sample preparation and data collection and analysis. It is therefore of interest to assess their complementarity when applied to small proteins. Structural genomics/proteomics projects provide an ideal opportunity to make such comparisons as they generate data in a systematic manner for large enough numbers of proteins to allow firm conclusions to be drawn. Here we report a comparison for 263 unique proteins screened by both NMR spectroscopy and X-ray crystallography in our structural proteomics pipeline. Only 21 targets (8%) were deemed amenable to both methods based on an initial 2D 15N-HSQC NMR spectrum and optimized crystallization trials. However, the use of both methods in the pipeline increased the total number of targets amenable to structure determination to 107, with 43 amenable to NMR only and 43 amenable to X-ray crystallographic methods only. We did not observe a correlation between 15N-HSQC spectral quality and the success of the same protein in crystallization screens. Similar results were found for an independent set of 159 proteins as reported in the accompanying paper by Snyder et al. Thus, we conclude that both methods are highly complementary, and in order to increase the number of proteins suited for structure determination, we suggest that both methods be used in parallel in screening of all small proteins for structure determination.     

高效离子排斥色谱法测定饮料中的糖精钠

提出了一种用高效离子排斥色谱法测定饮料中糖精钠的新方法。以ＩｏｎＰａｃ ＩＣＥ－ＡＳ ６柱为分离柱，０．１０ ｍｍｏｌ／ＬＨ２ＳＯ４＋甲醇（９＋１）为淋洗液，２０２ｎｍ波长下紫外检测。在１－１００ｍｇ／Ｌ范围内，糖精钠的含量与峰面积呈 良好线性关系。检出限为０．６０ｎｇ，灵敏度优于以往采用离子交换色谱－电导检测法的结果。在实验条件下，饮料 中常见有机酸以及其它人工合成甜味剂等均不产生干扰。方法用于饮料中糖精钠的测定，加标回收率为９８％～ １０５％。此外还比较了电导检测、紫外检测和经化学抑制系统后紫外检测等三种检测方式对测定灵敏度的影响。