A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of bisoprolol in human plasma using multiplexing technique

A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of bisoprolol in human plasma using multiplexing technique (two HPLC units connected to one MS). Bisoprolol was extracted from human plasma using solid-phase extraction technique using metoprolol as internal standard. A Betabasic 8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 326.2-->116.1 for bisoprolol and m/z 268.2-->191.0 for metoprolol. The method involves a simple multiplexing, rapid solid-phase extraction, simple isocratic chromatography conditions and mass spectrometric detection which enable detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.5-70.0 ng/mL with correlation coefficient > or =0.9991. The precision and accuracy were within 10% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for bisoprolol and metoprolol were 93.89% and 77.65%, respectively. Total MS run time was 0.90 min only. The developed method was applied for the determination of pharmacokinetic parameters of bisoprolol following a single oral administration of a 10mg bisoprolol tablet in 18 healthy male volunteers.     

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture.     

Chemical biology approaches in plant stress research

In addition to their importance as essential agrochemicals and life-saving drugs, small molecules serve as powerful research tools to address questions at all levels of biological complexity from protein function to plant biotic interactions. In certain contexts, chemical tools are complementary or even preferred to genetic analysis, since not all experimental systems are amenable for genetic dissection. For example, mutants impaired in oxygen sensing cannot easily be recovered. Pharmacological and chemical genetics approaches have come to the rescue of biologists in unraveling such genetically intractable systems. In this review, I have discussed my own efforts to analyze oxygen deprivation signaling in plants to illustrate the validity of small molecular approaches in elucidating an essential pathway such as oxygen sensing. Chemical biology is also a potent approach to tease out genetically redundant biological processes. The recent breakthrough in identifying the elusive abscisic acid receptors has clearly demonstrated the power of chemical tools in dissecting redundant pathways and led to the blossoming of this area as a distinct discipline of plant biology research. I present a summary of this work and conclude the review with potential challenges in using chemical tools.     

Association of lysolecithin acyltransferase with the high density lipoproteins and its activation by the low density lipoproteins in normal human plasma.

The lysolecithin acyltransferase of human plasma is shown to be associated with the high-density lipoprotein fraction. Although the low density lipoproteins do not have intrinsic enzyme activity, their presence activated the enzyme 3--7-fold. This activation is not affected by heat-treatment of the low density lipoproteins, but is abolished by the addition of heparin.     

Effect of solvents on photophysical properties and quenching of 2‐{[3‐(1H‐benzimidazole‐2‐yl) phenyl] carbonoimidoyl}phenol

The effect of solvents of varying polarity on the absorption and fluorescence emission of the Schiff base, 2‐{[3‐(1H‐benzimidazole‐2‐yl) phenyl]carbonoimidoyl}phenol, was studied using Lippert‐Mataga bulk polarity function, Reichardt's microscopic solvent polarity parameter and Kamlet's multiple linear regression approach. The spectral properties follow Reichardt's microscopic solvent polarity parameter better than Lippert‐Mataga bulk polarity parameter, indicating the presence of both general solute–solvent interactions and specific interactions. Catalan's multiple linear regression approach indicates the major role of solvent polarizability/dipolarity influence compared with solvent acidity or basicity. The solvatochromic effect was utilized to calculate the dipole moments of ground and excited states of the Schiff base using different methods. Bathochromic shift in the emission spectrum and the increase in dipole moment in the excited state signifies the intramolecular charge transfer character in the emitting singlet state. Fluorescence quenching by aniline was also studied in 1,4‐dioxane and n‐butanol, and the results were analyzed using sphere of action static quenching and finite sink approximation models. Copyright © 2014 John Wiley & Sons, Ltd.     

Incorporation profiles of conjugated linoleic acid isomers in cell membranes and their positional distribution in phospholipids

Although the conjugated linoleic acids (CLA) have several isomer-specific biological effects including anti-carcinogenic and anti-adipogenic effects, their mechanisms of action remain unclear. To determine their potential effects on membrane structure and function, we studied the incorporation profiles of four CLA isomers (trans-10 cis-12 (A), trans-9 trans-11 (B), cis-9 trans-11 (C), and cis-9 cis-11 (D)) in CHO and HepG2 cells. All four isomers were incorporated into cellular lipids as efficiently as linoleic acid (LA), with the majority of the incorporated CLA present in membrane rafts. Of the four isomers, only CLA-A increased the cholesterol content of the raft fraction. Over 50% of the incorporated CLAs were recovered in phosphatidylcholine of CHO cells, but in HepG2 the neutral lipids contained the majority of CLA. The desaturation index (18:1/18:0 and 16:1/16:0) was reduced by CLA-A, but increased by CLA-B, the effects being apparent mostly in raft lipids. The Δ? desaturase activity was inhibited by CLAs A and C. Unlike LA, which was mostly found in the sn-2 position of phospholipids, most CLAs were also incorporated significantly into the sn-1 position in both cell types. These studies show that the incorporation profiles of CLA isomers differ significantly from that of LA, and this could lead to alterations in membrane function, especially in the raft-associated proteins.     

Molecular dynamic simulation study of cholesterol and conjugated double bonds in lipid bilayers

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is believed to have anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be associated with anti-obesity effects. In this paper we extend earlier molecular dynamics (MD) simulations of pure CLA–phosphatidylcholine bilayers to investigate the comparative effects of cholesterol on bilayers composed of the two respective isomers. Simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed in which, for each isomer, the simulated bilayers contained 10% and 30% cholesterol (Chol). From MD trajectories we calculate and compare structural properties of the bilayers, including areas per molecule, thickness of bilayers, tilt angle of cholesterols, order parameter profiles, and one and two-dimensional radial distribution function (RDF), as functions of Chol concentration. While the structural effect of cholesterol is approximately the same for both isomers, we find differences at an atomistic level in order parameter profiles and in two-dimensional radial distribution functions.