Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence. Analysis of A549 cells

Detection of benzo[a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6 x 10(5) and 1 x 10(6) plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-10) M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells.     

Evaluation of a high-performance thin-layer chromatographic technique for the determination of salbutamol serum levels in clinical trials.

Salbutamol concentrations were determined by high-performance thin-layer chromatography in the sera of two sets of ten volunteers at hourly intervals for 6 h after taking one 8-mg slow-release tablet. The influence of time lapse in processing of serum samples, i.e. centrifugation, extraction and chromatography, was studied. A statistical significant instability of salbutamol in the sera of patients was found which was not present in standard drug-free serum samples spiked with salbutamol and used for construction of standard curves.     

The role of β-cyclodextrin and hydroxypropyl β-cyclodextrin in the secondary chemical equilibria associated to the separation of β-carbolines by HPLC

The use of cyclodextrins (CDs) in HPLC as mobile phase additives provides a flexible alternative for the separation of chemically related compounds because these separations can be performed on conventional columns and are economically advantageous over the use of chiral stationary phases. The present paper describes the influence of the presence of β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HPβ-CD) on the separation of the β-carboline alkaloids norharmane, harmane and harmine. The nature of the stationary phase (reverse phases C₁ and C₁₈) affects the chromatographic separations and also the stability of the inclusion complexes that are developed. The changes in the proportion of the organic solvents at constant concentration of CDs (3 mM for β-CD and 15 mM for HPβ-CD) modify the retention factors (k′) for all alkaloids studied. The nature of the organic solvent in the mobile phase also changes the chromatographic parameters. The logarithm of the capacity factor (k′) is linearly increased with the proportion of water in the hydro-organic mobile phase (ethanolic or methanolic) but the slopes obtained vary depending on the CD added to the mobile phase. The role of competitive equilibria, i.e., chromatographic distribution and inclusion complexes formation is discussed. This paper was presented at XIIIth International Cyclodextrin Symposium. Torino, Italy, May, 14–17, 2006     

Synthesis of (+)-1,3:2,5-dianhydroviburnitol ((+)-(1R,2R,3S,5R,6S)-4,7-dioxatricyclo[3.2.1.03,6]octan-2-ol)

Epoxidation of (?)-(1R,2R,4R)-2-endo-cyano-7-oxabicyclo[2.2.1]hept-5-en-2-exo-yl acetate ((?)-5) followed by saponification afforded (+)-(1R,4R,5R,6R)-5,6-exo-epoxy-7-oxabicyclo[2.2.1]heptan-2-one ((+)-7). Reduction of (+)-7 with diisobutylaluminium hydride (DIBAH) gave (+)-1,3:2,5-dianhydroviburnitol ( = (+)-(1R,2R,3S,4R,6S)-4,7-dioxatricyclo[3.2.1.0^3,6]octan-2-ol; (+)-3). Hydride reductions of (±)-7 were less exo-face selective than reductions of bicyclo[2.2.1]heptan-2-one and its derivatives with NaBH₄, AlH₃, and LiAlH₄ probably because of smaller steric hindrance to endo-face hydride attack when C(5) and C(6) of the bicyclo-[2.2.1]heptan-2-one are part of an exo oxirane ring.     

An application of coupled reference interaction site model/molecular dynamics to the conformational analysis of the alanine dipeptide

We present an application of our recently proposed coupled reference interaction site model (RISM) molecular dynamics (MD) solvation free energy methodology [Freedman and Truong, Chem. Phys. Lett. 381, 362 (2003); J. Chem. Phys. 121, 2187 (2004)] to study the conformational stability of alanine dipeptide in aqueous solution. In this methodology, radial distribution functions obtained from a single MD simulation are substituted into a RISM expression for solvation free energy. Consequently, iterative solution of the RISM equation is not needed. The relative solvation free energies of seven different conformations of the alanine dipeptide in aqueous solution are calculated. Results from the coupled RISM/MD methodology are in good agreement with those from earlier simulations using the accurate free energy perturbation approach, showing that the alphaR conformation is most stabilized by solution. This study establishes a framework for applying this coupled RISM/MD method to larger biological systems.     

Decoding ‘Maximum Entropy’ Deconvolution

For over five decades, the mathematical procedure termed “maximum entropy” (M-E) has been used to deconvolve structure in spectra, optical and otherwise, although quantitative measures of performance remain unknown. Here, we examine this procedure analytically for the lowest two orders for a Lorentzian feature, obtaining expressions for the amount of sharpening and identifying how spurious structures appear. Illustrative examples are provided. These results enhance the utility of this widely used deconvolution approach to spectral analysis.     

Lentinus edodes Polysaccharides Alleviate Acute Lung Injury by Inhibiting Oxidative Stress and Inflammation

Acute lung injury (ALI) is a kind of lung disease with acute dyspnea, pulmonary inflammation, respiratory distress, and non-cardiogenic pulmonary edema, accompanied by the mid- and end-stage characteristics of COVID-19, clinically. It is imperative to find non-toxic natural substances on preventing ALI and its complications. The animal experiments demonstrated that Lentinus edodes polysaccharides (PLE) had a potential role in alleviating ALI by inhibiting oxidative stress and inflammation, which was manifested by reducing the levels of serum lung injury indicators (C3, hs-CRP, and GGT), reducing the levels of inflammatory factors (TNF-α, IL-1β, and IL-6), and increasing the activities of antioxidant enzymes (SOD and CAT) in the lung. Furthermore, PLE had the typical characteristics of pyran-type linked by β-type glycosidic linkages. The conclusions indicated that PLE could be used as functional foods and natural drugs in preventing ALI.     

A Modified Vancomycin Molecule Confers Potent Inhibitory Efficacy against Resistant Bacteria Mediated by Metallo-β-Lactamases

Multidrug-resistant bacterial infections mediated by metallo-β-lactamases (MβLs) have grown into an emergent health threat, and development of novel antimicrobials is an ideal strategy to combat the infections. Herein, a novel vancomycin derivative V_b was constructed by conjugation of triazolylthioacetamide and vancomycin molecules, characterized by reverse-phase high performance liquid chromatography (HPLC) and confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The biological assays revealed that V_b effectively inhibited S. aureus and methicillin-resistant S. aureus (MRSA), gradually increased the antimicrobial effect of β-lactam antibiotics (cefazolin, meropenem and penicillin G) and exhibited a dose-dependent synergistic antibacterial effect against eight resistant strains tested, which was confirmed by the time-kill curves determination. Most importantly, V_b increased the antimicrobial effect of meropenem against the clinical isolates EC08 and EC10 and E. coli producing ImiS and CcrA, resulting in a 4- and 8-fold reduction in MIC values, respectively, at a dose up to 32 μg/mL. This work offers a promising scaffold for the development of MβLs inhibitors, specifically antimicrobials for clinically drug-resistant isolates.