Writing with DNA and protein using a nanopipet for controlled delivery

We present a new, general method for the controlled deposition of biological molecules on surfaces, based on a nanopipet operating in ionic solution. The potential applied to the pipet tip controls the flux of biological molecules from the pipet, allowing fine control of the delivery rate. We used the ion current to control the distance of the pipet from the surface of a glass slide and deposited the fluorescently labeled DNA or protein G at a defined location onto the surface. Features of 830 nm size were obtained by depositing the biotinylated DNA onto a streptavidin surface; 1.3 mum size spots were obtained by depositing protein G onto a positively charged glass surface.     

High-performance liquid chromatographic method for routine determination of vitamins A and E and beta-carotene in plasma.

A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the routine determination of vitamins A and E and beta-carotene in plasma (or serum) with wavelength-programmed ultraviolet-visible absorbance detection is described. A 200-microliters aliquot of serum or plasma sample, after deproteinization with ethanol, and containing tocopherol acetate as internal standard, was extracted with butanol-ethyl acetate. Sodium sulphate was added for dehydration. Analytes of extracted samples were found to be stable for at least four days. A 10-microliters aliquot of this organic extract was used for HPLC analysis. The mobile phase was methanol-butanol-water (89.5:5:5.5, v/v) and the flow-rate was set at 1.5 ml/min. The analytes of interest were well separated from other plasma constituents within 22 min at 45 degrees C. The lowest detection limits of vitamins A and E and beta-carotene were 0.02, 0.5 and 0.1 microgram/ml, respectively. The recovery and reproducibility of the present method were around 90%. The method is sensitive, specific and can be used for epidemiological studies and for routine determination of vitamin deficiency. Several important factors that may affect the analysis are also discussed in this paper.     

1-(Organosulfonyloxy)-3(1H)-1,2-benziodoxoles: Preparation and Reactions with Alkynyltrimethylsilanes

Organosulfonyloxy derivatives of 1,2-benziodoxol-3(1H)-one (3a-c) and 3,3-bis(trifluoromethyl)-3(1H)-1,2-benziodoxole (5a-c) can be prepared in high yield by the reaction of 1-hydroxybenziodoxoles 1 or 4 and the corresponding sulfonic acids or Me(3)SiOTf in the form of stable, but moderately hygroscopic, microcrystalline solids. Reaction of the triflate derivatives 3a and 5a with alkynyltrimethylsilanes affords either alkynyliodonium triflates 6, or (E)-beta-(trifluoromethanesulfonyloxy)alkenyliodonium triflates 7, while the same reaction in the presence of pyridine selectively gives the respective 1-alkynylbenziodoxoles 8 and 9 in 82-90% yield.     

Measuring trihalomethane concentrations in water using supported capillary membrane sampling-gas chromatography

The objective of this study was to develop and evaluate a supported capillary membrane sampling-gas chromatography method for the analysis of trihalomethanes (THMs) in drinking water. The effects of experimental parameters, such as flow rate of carrier gas, water temperature, ionic strengths of solutions and transfer line temperature on the system performance were investigated. The results of method detection limit and accuracy and precision studies are reported.     

A unified and quantitative receptor model for the microtubule binding of paclitaxel and epothilone

[formula: see text] Paclitaxel and epothilone represent the two major classes of antimicrotubule agents that promote tubulin polymerization and, presumably, mitotic arrest during cell division. A common minireceptor binding site model at beta-tubulin has been constructed for these structurally divergent compounds. Utilizing 20 amino acids identified in photoaffinity labeling experiments, the 3-D model correlates measured and predicted Ki's with r = 0.99 and rms(delta Gcalc-delta Gexp) = 0.2 kcal/mol. In addition, the model predicts the affinity of compounds not used in the training set and explains much of the SAR for the paclitaxel and epothilone families.     

A thermogravimetric analyzer for condensable gas adsorption under subatmospheric conditions

Potential alternative to petrochemical polymers, soy protein isolate (SPI), a plentily available, natural biopolymer is chemically modified with thiourea at 2.5, 5, 7.5, 10, 15 and 20 mass/mass% for better processing of plastic as a raw material. From the FTIR studies, it has been ascertained that there is no bonding reaction between SPI and thiourea and it acts as a modifier only. Thermogravimetric analysis of the modified material has been followed using a computer analysis method, LOTUS package, developed by us for assigning the degradation mechanism. A number of equations have been used to evaluate the kinetic parameters. The mechanism of degradation of the biopolymer is explained on the basis of the kinetic analysis.     

Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.     

Liquid chromatographic purification and detection of anabolic compounds.

The role of liquid chromatography within methods of analysis for steroids, related compounds and beta-agonists in biological samples is discussed. Special attention is given to the application of liquid chromatography in sample preparation and extract clean-up. Different forms of liquid chromatography, including immunoaffinity chromatography, are compared and evaluated. Methods for confirmation based on gas chromatography-mass spectrometry and cryotrapping Fourier transform infrared spectrometry are discussed.     

Spongistatin synthetic studies. An efficient, second-generation construction of an advanced ABCD intermediate

[reaction: see text] A short, efficient, and stereocontrolled synthesis of (-)-4, an advanced ABCD subunit of the spongistatins, has been achieved. Central to the synthetic strategy is the multicomponent linchpin union of silyl dithianes with epoxides to access both the AB and CD fragments. Fragment coupling was then achieved via an efficient stereoselective aldol reaction. The linear sequence required 22 steps and proceeded in 4.0% overall yield.     

Lateral interactions and multi-isotherms: nitrogen recombination from Rh(111)

Lateral adsorbate-adsorbate interactions result in variation of the desorption rate constants with coverage. This effect can be studied in great detail from the shape of a multi-isotherm. To produce the multi-isotherm, the temperature is increased in a (semi)stepwise fashion to some temperature, followed by maintaining this temperature for a prolonged time. Then, the temperature is stepped to a higher value and held constant at this new temperature. This cycle is continued until all of the adsorbates have desorbed. Using a detailed kinetic Monte Carlo model and an optimization algorithm based on Evolutionary Strategy, we are able to reproduce the shape of the experimentally measured multi-isotherm of nitrogen on Rh(111) and obtain the lateral interactions between the nitrogen atoms.