Biomedical subjects

James G Boyd

Publications and source records attributed to James G Boyd.

5 recordsLinked to original sources

PubMed2006

Nanoscale electrostatic actuators in liquid electrolytes.

Equilibrium and energy analyses were performed for an electrostatic actuator consisting of two plane parallel electrodes, operated using DC voltages, separated by a liquid electrolyte. One electrode is fixed, and the other electrode is connected to a spring and is free to move. The mechanical equilibrium includes the spring force, the van der Waals force, and the electrochemical force as given by the solution of the linearized Poisson-Boltzmann equation. The electrode separation is determined as a function of the applied potential, the natural (i.e., zeta) potential, the Debeye length, the initial electrode separation, the spring constant, and the Hamaker constant. The actuator exhibits the classical "pull-in" instability. The natural potential increases the critical applied potential but does not significantly affect the critical separation. For zero natural potential, the spring constant does not affect the critical separation. Ratios of the maximum spring energy, the maximum van der Waals energy, and the maximum electrochemical energy were plotted as functions of the Hamaker constant and the initial electrode separation.

Journal Article↗

PubMed2006

A semiquantitative method for the determination of reactive metabolite conjugate levels in vitro utilizing liquid chromatography-tandem mass spectrometry and novel quaternary ammonium glutathione analogues.

An in vitro semiquantitative reactive metabolite detection assay is described that incorporates NADPH-supplemented human liver microsomes, a novel quaternary ammonium glutathione analogue conjugating agent (QA-GSH), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection. The assay was developed to have high sample capacity and the potential for high sample throughput. MS/MS detection is selective and sensitive for the QA-GSH conjugating agent and semiquantitation of QA-GSH-reactive metabolite conjugates is performed using QA-GSH standards added to samples prior to analysis [i.e., internal standards (ISs)]. The reactive metabolite trapping capability of the free thiol group in QA-GSH was assessed using model drugs acetaminophen, clozapine, and flutamide, which are bioactivated to afford reactive metabolites. MS signal responses of equimolar amounts of QA-GSH standards were compared to assess the feasibility of using a QA-GSH IS approach to semiquantify reactive metabolite levels in vitro. The full scan Q1 MS response for each standard was within 3.3-fold of one another even though the "parent" moiety structure of each QA-GSH conjugate standard differed significantly. Standard curve analysis using selected reaction monitoring for each QA-GSH standard gave slope values that differed by only 1.5-fold. The QA-GSH IS semiquantitation method was tested by determining the level of QA-GS-acetaminophen conjugate formation at three different concentrations of acetaminophen and comparing the results to those from linear regression of authentic standards. The calculated levels of conjugate formed compared closely with those calculated from linear regression data of authentic standard curves. These results show that the QA-GSH semiquantitation assay described herein is a viable method for semiquantitatively assessing the bioactivation potential in vitro and is well-suited for use in early drug discovery high throughput screening paradigms.

Acetaminophen↗

PubMed2004

Selection of active ScFv to G-protein-coupled receptor CCR5 using surface antigen-mimicking peptides.

This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library. Antibodies reactive with CCR5 on cells were detected using a homogeneous high throughput assay. Of 19 isolated ScFv antibodies that bound to CCR5+ cells, three inhibited CCR5-mediated but not CXCR4-mediated HIV infection. Only ScFvs selected by binding to cyclic constrained peptides exhibited inhibitory activity. Our results demonstrate that surface-antigen mimetics of a GPCR are effective tools for selecting active, site-specific ScFv antibodies that hold promise as immunological reagents and therapeutics.

Amino Acid Sequence↗

PubMed2004

The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent.

An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry (microLC-microESI-MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH+ to [MH - 129]+ transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH+ to [MH - 129]+ transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen-GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).

Biological Assay↗

PubMedDate not supplied

Cyclization of N-terminal S-carbamoylmethylcysteine causing loss of 17 Da from peptides and extra peaks in peptide maps.

Enzymatic digests of proteins S-alkylated with iodoacetamide may contain peptides with N-terminal S-carbamoylmethylcysteine. These can be partly converted to a form with 17 Da lower mass and increased HPLC retention. Proof by synthesis supported by MS/MS and NMR spectroscopy was used to show that N-terminal S-carbamoylmethyl-L-cysteine can cyclize, losing NH3 to form an N-terminal residue of (R)-5-oxoperhydro-1,4-thiazine-3-carboxylic acid. The abbreviation Otc is proposed for the (R)-5-oxoperhydro-1,4-thiazine-3-carbonyl residue. The rate of cyclization is significant in 0.1 M NH4HCO3 at 37 degrees C, with the half-life of the acyclic form being 10-12 h for several peptides tested. This is similar to the rate at which N-terminal pyroglutamate forms from N-terminal glutamine.

Chromatography, High Pressure Liquid↗