Efficient method for visualization and isolation of proteins resolved in polyacrylamide gels

Polyacrylamide gel electrophoresis is a popular method used to purify proteins for reconstitution experiments, amino acid composition and sequence determinations. In this report we describe methods that will be of general use in the isolation and characterization of proteins and the benefits of substituting boric acid for glycine in the electrophoresis tray buffers. We also describe how proteins resolved in a variety of gel systems (including those containing sodium dodecyl sulfate) may be rapidly visualized with 8-anilino-1-naphthalene sulfonic acid and efficiently transferred to a second gel for two-dimensional gel analysis, or isolated by electroelution for subsequent characterization.     

DISCREPANCIES IN THE MEASUREMENT OF SPECTRAL SOURCES

Abstract— Comparison of spectroradiometric and meter measurements of a series of ultraviolet radiation sources indicates that a wide divergence between readings can occur. We found that with a xenon are filtered as a solar simulator producing UV-A (320–400 nm) and UV-B (290–320 nm) radiation, the meter can either over-or underestimate the emission of the source when different cutoff filters are used. The most severe discrepancy appears with the UV-B meter reading, although the UV-A reading can also be problematic. Meters should be calibrated against the specific sources they will be used to measure.     

DOSE-RESPONSE OF CHRONIC ULTRAVIOLET EXPOSURE ON EPIDERMAL FORWARD SCATTERING-ABSORPTION IN SK-1 HAIRLESS MOUSE SKIN

This work provides a dose-response model of UV-induced epidermal-stratum corneum thickening induced by irradiation at wavelength lambda. This model assumes that photobiochemical reaction(s) can give rise to hyperplasia in a manner which is predictable from a simple photochemical kinetic scheme. In this work, we derive an equation which predicts an approximately linear relationship between the logarithm of the increase in optical skin thickening measured at 320 nm (delta OD320) and total cumulative dose (DT) seen by the target cells in or near the basal layer. For each excitation wavelength lambda, the slope R(lambda) of the log delta OD320 vs DT plot is proportional to epsilon(lambda) phi rx, where epsilon(lambda) is the extinction coefficient for the target chromophore at excitation wavelength, and phi rx is the quantum yield for the photochemical reaction(s) leading to hyperplasia. Our data previously obtained from irradiation of SK-1 hairless mice with "monochromatic" UV wavebands at 280, 290, 300, 307 and 313 nm (Menter et al., 1988, Photochem. Photobiol. 47, 225-260.) and data from Sterenborg and van der Leun at 254 and 313 nm (1988, Photodermatology 5, 71-82) are in good agreement with this model, except for 254 and 280 nm excitation, which are greatly attenuated by epidermis-stratum corneum. For excitation at the latter wavelengths, "dark" regressive processes successfully compete with the "light" reaction(s) which lead to (pre)cancerous lesion. This difficulty notwithstanding, the "intrinsic" action spectrum for hyperplasia derived from these measurements indicates that the target chromophore preferentially absorbs in the UV-C region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chiral analytical method development of liquiritigenin with application to a pharmacokinetic study

Pharmacometric characterization studies of liquiritigenin have historically overlooked its chiral nature. To achieve complete characterization, an analytical method enabling the detection and quantification of the individual enantiomers of racemic (±) liquiritigenin is necessary. Resolution of the enantiomers of liquiritigenin was achieved using a simple high‐performance liquid chromatographic method. A Chiralpak® ADRH column was employed to perform baseline separation with UV detection at 210 nm.The standard curves were linear ranging from 0.5 to 100 µg/mL for each enantiomer. Limit of quantification was 0.5 µg/mL. The assay was applied successfully to stereoselective serum disposition of liquiritigenin enantiomers in rats. Liquiritigenin enantiomers were detected in serum as both aglycones and glucuronidated conjugates. Both unconjugated enantiomers had a serum half‐life of ~15 min in rats. The volume of distribution (V_d) for S‐ and R‐liquiritigenin was 1.49 and 2.21 L/kg, respectively. Total clearance (Cl_total) was 5.12 L/h/kg for S‐liquiritigenin and 4.79 L/h/kg for R‐liquiritigenin, and area under the curve (AUC_0‐inf) was 3.95 µg h/mL for S‐liquiritigenin and 4.23 µg h/mL for R‐liquiritigenin. The large volume of distribution coupled with the short serum half‐life suggests extensive distribution of liquiritigenin into tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

Chiral analytical method development and application to pre‐clinical pharmacokinetics of pinocembrin

An analytical method enabling the detection and quantification of the individual enantiomers of racemic (±) pinocembrin is required to fully characterize its pharmacokinetic disposition. Direct resolution of the enantiomers of pinocembrin was achieved using a novel and simple reversed‐phase high‐performance liquid chromatography method with electrospray ionization and detection by mass spectrometry in rat serum. A Chiralcel® AD‐RH column was employed to perform baseline separation with electrospray positive‐mode ionization with selected ion monitoring detection. The standard curves were linear from 0.5 to 100 µg/mL for each enantiomer. The limit of quantification was 0.5 µg/mL. The assay was applied successfully to stereoselective serum disposition of pinocembrin enantiomers in rats. Pinocembrin enantiomers were detected in serum. Both enantiomers had a serum half‐life of ~15 min in rats. Similar values of volume of distribution between the enantiomers were also observed: 1.76 L/kg for S‐pinocembrin and 1.79 L/kg for R‐pinocembrin. Total clearance was 5.527 L//h/kg for S‐pinocembrin and 5.535 L/h/kg for R‐pinocembrin, and the area under the curve was 1.821 µg h/mL for S‐pinocembrin and 1.876 µg h/mL for R‐pinocembrin. The large volume of distribution coupled with the short serum half‐life suggests extensive distribution of pinocembrin into the tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

Copper(II)-mediated autoxidation of tert-butylresorcinols

Although copper(II)-mediated oxidation of phenols results in oxidative coupling rather than in oxygenation, it was recently reported that naturally occurring 5-alkylresorcinols undergo oxygenation in the presence of copper(II). To explore the generality of this reaction, the copper(II)-mediated autoxidation of 4-tert-butylresorcinol and 4,6-di-tert-butylresorcinol was investigated and was found to result in direct oxygenation at open activated positions and, at the tert-butyl-substituted positions, in oxygenation with competing loss of (as isobutylene) and 1,2-rearrangement of the tert-butyl group. 5-tert-Butyl-2-hydroxy-1,4-benzoquinone is the major product from both starting materials, and the final product mixture reflects, in part, coupling of metastable initially formed electrophilic and nucleophilic side products. Mechanisms that are consistent with the observed products and control reactions are proposed. The key step appears to be equilibration of a copper(II)-resorcinolate with a charge-transfer radical form that reacts regioselectively with O(2) as prescribed by resonance.     

On the rank-dependence of coal tar secondary reactions

The secondary reactions of volatile compounds, including coal tar and light gases, accounts for a great portion of soot formation and the subsequent heat release and pollutant emissions in the combustion zone. While coal primary pyrolysis has been extensively studied over the last few decades and several network pyrolysis models has been developed to describe this process, coal secondary pyrolysis is still not well understood. The Babcock and Wilcox Company has been investigating coal secondary pyrolysis in order to develop a comprehensive mechanism for inclusion in predictive computational fluid dynamics and coal combustion models. Supportive experiments were carried out in an entrained-flow reactor. Tar was extracted from the pyrolysis byproducts of seven coals of widely-distributed rank at temperatures ranging from 923 to 1473 K, and analyzed by ¹³C NMR. Tars formed from higher rank coals generally demonstrated higher sooting propensities. This rank-dependent sooting propensity is associated with tar’s chemical structure properties. With increased heat treatment severity, tar molecules lose a substantial amount of aliphatic attachments, and the average size of substitution per cluster decreases. Compared to tars formed from high-rank bituminous coals, those formed from low-rank sub-bituminous coals have a larger attachment portion, higher averaged substitution, and higher oxygen-containing functional groups. These differences contribute to the higher cracking propensity observed for low-rank coal tars.     

An alternative total synthesis of pentosidine

Pentosidine, a fluorescent advanced glycation endproduct that serves as a biomarker of diabetic complications, kidney dysfunction, oxidative stress, and aging and age‐related diseases, was synthesized from 2,3‐diaminopyridine and benzyloxycarbonyl (Cbz) protected chiral amino acids N^α‐Cbz‐lysine and N^δ‐Cbz‐ornithine. Regioselective alkylation of 2‐(methylthio)imidazo[4,5‐b]pyridine, chlorination of methylthio group, and amination of 2‐chloro‐imidazo[4,5‐b]pyridine are the key steps. Hydantoin protection of amino acids was used and the deprotection under acidic condition was achieved in the presence of glycine. J. Heterocyclic Chem., (2011).