Biomedical subjects

Simon S Jones

Publications and source records attributed to Simon S Jones.

3 recordsLinked to original sources

PubMed2003

A high-throughput method for enzyme kinetic studies.

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.

Aryl Hydrocarbon Hydroxylases↗

PubMed2002

BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells.

BACKGROUND: Proximal tubule epithelial cells (PTEC) play a central role in the response of the kidney to insult by virtue of their production of chemokines and cytokines that signal an inflammatory response. Bone morphogenic protein-7 (BMP-7/OP-1), a member of the transforming growth factor-beta (TGF-beta) superfamily, has previously been demonstrated to reduce macrophage infiltration and tissue damage in animal models of acute and chronic renal failure. The present study was designed to define the molecular mechanism of BMP-7 action in human PTEC. METHODS: Expression of BMP-7 in the adult mouse kidney was determined indirectly through X-gal staining of heterozygous BMP-7/lacZ mice in combination with cell-type specific markers. Primary human PTEC were cultured in the presence of the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), with and without BMP-7. RNA isolated from these two populations was then used to identify differentially regulated genes via gene-array analysis. Modulation of potential target genes was subsequently confirmed through ELISA and/or quantitative PCR. RESULTS: Expression from the BMP-7/lacZ transgene was detected in the collecting duct, thick ascending limb, distal convoluted tubule, and podocytes within glomeruli. No expression was detected within PTEC; however, these cells were found to express mRNA for BMP receptors including, ActR-I, BMPR-IA, ActR-II, ActR-IIB, and BMPR-II. BMP-7 significantly reduced TNF-alpha stimulated increases in mRNA for the pro-inflammatory genes, interleukin-6 (IL-6) and interleukin-1beta (IL-1beta), and the chemoattractants monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in primary human PTEC. In addition, BMP-7 also reduced the expression of mRNA for endothelin-2 (ET-2), a vasoconstrictor, and increased the expression of mRNA for heme oxygenase-1 (HO-1), a vasodilator, although the latter was not statistically significant. In experiments designed to examine MCP-1 and IL-6 protein levels in response to additional TGF-beta superfamily members, TGF-beta1 was unable to mimic the effects of BMP-7 in reducing IL-6 production. However, the closely related BMP-6 exhibited similar properties to those of BMP-7. Each of the factors reduced MCP-1 expression. CONCLUSIONS: BMP-7 represses the basal and TNF-alpha-stimulated expression of the pro-inflammatory cytokines IL-6 and IL-1beta, the chemokines MCP-1 and IL-8, and the vasoconstrictor ET-2 in PTEC. This data are consistent with the in vivo observations that BMP-7 administration in a model of chronic and acute renal failure results in a reduction in the infiltration of macrophages in the renal interstitium. Taken together, these observations suggest that BMP-7 may be a novel therapeutic agent for kidney disorders involving inflammation and ischemic damage of PTEC.

Animals↗

PubMedDate not supplied

Affinity ligand selection from a library of small molecules: assay development, screening, and application.

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.

Chromatography, Affinity↗