Fluorogenic Probing of Membrane Protein Trafficking.

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an efficient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag). Cell-surface FAST-tagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.

A comprehensive LC/MS analysis of novel cyclopentenedione library.

Cyclopentenediones (CPDs) are compounds with a variety of applications ranging from the preparation of functional polymers to the development of antimicrobial agents, suggesting the potential use of CPDs as novel bioactive compounds or drugs. For this reason, a detailed characterization of CPDs and the development of robust analytical methods for their trace analysis are being sought. Here we focused on the design and synthesis of a library of novelized benzylidene CPD derivatives that were consequently characterized by ultra-high performance liquid chromatography (UHPLC) on-line connected with tandem mass spectrometry (MS/MS). The library design was based on a 2-benzylidene-4-cyclopentene-1,3-dione skeleton substituted with a variety of hydroxy, methoxy, halogen, linear aliphatic, heterocyclic and saccharide moieties, primarily modulating the skeleton's hydrophobicity. The prepared CPDs were effectively ionized by positive/negative atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI). After careful optimization of the dopant composition and flow rate, positive-mode APPI proved to be more sensitive than APCI. In negative mode, both ionization techniques gave similar results. Further, a detailed MS fragmentation study was performed, confirming the structure of the compounds and enabling positional isomers of CPDs to be differentiated on the basis of their collision spectra analysis. Finally, an optimization of the composition of the mobile phase and reversed-phased separation mode were done, followed by a selection of the most suitable UHPLC stationary phases, i.e. C18, C8 and phenyl. The applicability of the method was evaluated by the inclusion of the other two substances in the study, i.e. monomeric and dimeric bioactive CPDs, compound TX-1123 and nostotrebin 6 with cytostatic and antimicrobial activities, respectively. The results presented here could be used in further investigations of the chromatographic retention and MS behavior of CPDs, which could be utilized for their isolation, detailed characterization and analysis in biological systems.

Prediction of liquid chromatographic retention behavior based on quantum chemical parameters using supervised self organizing maps.

Self organizing maps (SOMs) in a supervised mode were applied for prediction of liquid chromatographic retention behavior of chemical compounds based on their quantum chemical information. The proposed algorithm was simple and required only a small alteration of the standard SOM algorithm. The application was illustrated by the prediction of the retention indices of bifunctionally substituted N-benzylideneanilines (NBA) and the prediction of the retention factors of some pesticides. Although the predictive ability of the supervised SOM could not be significantly greater than that of some previously established neural network methods, such as a radial basis function (RBF) neural network and a back-propagation artificial neural network (ANN), the main advantage of the proposed method was the ability to reveal non-linear structure of the model. The complex relationships between samples could be visualized using U-matrix and the influence of each variable on the predictive model could be investigated using component planes-which can provide chemical insight.

Vibrational assignment of the normal modes of 2-phenyl-4-(4-methoxy benzylidene)-2-oxazolin-5-one via FTIR and Raman spectra, and ab inito calculations.

Raman and FTIR spectra of 2-phenyl-4-(4-methoxy benzylidene)-2-oxazolin-5-one were recorded in the regions, 100-3300 and 400-4000 cm(-1), respectively. Vibrational frequencies and intensities of the fundamental modes of this hetrocyclic organic molecule were computed using ab initio as well as AM1 semiempirical molecular orbital methods. Ab initio calculations were carried out with basis set up to RHF/6-311G. Conformational studies regarding the effect of moving the methoxy group in the 2-phenyl-4-(4-methoxy benzylidene)-2-oxazolin-5-one molecule to a different position on the ring was also carried out. Observed vibrational wavenumbers were found to be mostly consistent with ab initio values. The most intense mode of vibration observed at 1250 cm(-1) in Raman spectra, also observed as a strong band in FTIR, was assigned as C-O stretching vibration in the methoxy group. Asymmetric stretching vibrations between CC and CN bonds was predicted as most intense mode by our ab initio calculation.

Theoretical studies of Alzheimer's disease drug candidate 3-[(2,4-dimethoxy)benzylidene]-anabaseine (GTS-21) and its derivatives.

Theoretical and molecular modeling studies have been conducted for understanding the details of how 3-[(2,4-dimethoxy)benzylidene]-anabaseine dihydrochloride (GTS-21) and its metabolism derivatives bind with the receptor of alpha7 nicotinic acetylcholine dimer. Good accordance with experimental results has been achieved. It was found that the van der Waals repulsion makes the dominant contribution to the binding energy. GTS-21 and its metabolites are apparently too large for the binding sites of the alpha7 dimer. To improve the effectiveness of the drug, a possible approach is to reduce its volume while maintaining the presence of the active groups. Our studies, in combination with experimental studies, will lead to a promising basis for practical drug design against Alzheimer's disease.

Rapid determination of ascorbate by ESR spectroscopy.

Optimal conditions for the determination of ascorbate, based on the detection of its radical by ESR spectroscopy, were established. The optimal pH for the measurement of the ascorbate radical was 10, whereas that for the gallate and caffeate radical was much higher. This was due to the liability of the ascorbate radical at higher pH. When the radical intensity of ascorbate vs. concentration was plotted double logarithmically, a linear correlation was established between these two parameters within the ascorbate concentration of 0.03-10 mM. ESR analysis of the homogenates under alkaline condition demonstrated that ascorbate is distributed in various mouse organs and that the ascorbate radical is unstable at neutral pH in the brain and liver, suggesting the presence of metabolizing substances. The present study suggests the applicability of the present technique for the study of ascorbate metabolism and identification of physiological factors which regulate the radical intensity of ascorbate.

[Correlative study on the storage time and quality of rhizoma Dryopteris crassirhizomae].

The contents of dryocrassin in Rhizoma Dryopteris Crassirhizomae were determined by TLC-scanning in a period of two years of storage. Phloroglucinol content was calculated by area normalization method. The results show that in contrast with the fresh crude drugs of Rhizoma Dryopteris Crassirhizomae, the contents of dryocrassin in the crude drugs of one year and two years of storage have reduced by 22% and 27% respectively, and phloroglucinol contents have reduced by 2% and 8% respectively.

Resonance Raman spectra of chlorophylls dissolved in liquid crystal matrices. I. The interaction between chlorophylls and a liquid crystalline MBBA + EBBA mixture.

The resonance Raman (RR) spectra of chlorophyll (Chl) a, Chl b and pheophytin a dissolved in a liquid-crystalline MBBA + EBBA mixture were measured. The RR frequencies of chlorophylls in the liquid crystal (LC) are compared with those of solutions of various chlorophyll monomers and aggregates taken from the literature. It is concluded that Chl a and Chl b in LC exist largely as the solvated monomers, even at high concentration. The magnesium atoms in both Chl a and Chl b are pentacoordinated.

Analysis of sulindac and metabolites by combined isotope dilution-radioimmunoassay.

Sulindac, a new anti-inflammatory agent, and its sulfone and sulfide metabolites were conjugated to bovine serum albumin by the N-hydroxysuccinimide active ester procedure. Antiserum from rabbits immunized with each of these haptens exhibited extensive cross-reactivity, precluding differential analyses of the three species by displacement assay without prior separation. Therefore, an analytical method based on a combination of isotope dilution and radioimmunoassay was devised. A known mixture of the three chemical species, each labeled with tritium, was equilibrated with plasma or urine samples, reisolated chromatographically, and quantitated by binding to an appropriate immunoglobulin. The radiolabeled materials thus served as recovery standards as well as labeled antigens for each displacement assay. Sulindac and each of its metabolites in plasma or urine at concentrations as low as 500 ng/sample were differentially determined by this procedure. However, since an extraction is required, several milliliters of plasma can be used for each sample, thus increasing the actual sensitivity of the assay.

Determination of various drugs in rodent diet mixtures.

Methods employing solvent extraction, thin-layer chromatography, gas-liquid chromatography, and UV spectrophotometry are described for the quantitative determination of halofenate, cyclobenzaprine and sulindac in rodent diet mixtures. Halofenate was hydrolyzed to its free acid derivative and converted to a methyl ester prior to assay. The drugs were shown to be stable when stored in food mixtures at room temperature for seven days. Diet mixtures containing the three drugs were demonstrated to be uniformly mixed by the procedure employed.