Biomedical subjects

Calin-Aurel Dragan

Publications and source records attributed to Calin-Aurel Dragan.

3 recordsLinked to original sources

PubMed2006

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450-dependent biotransformation in fission yeast.

Cytochrome P450s are haem-containing monooxygenases that catalyse a variety of oxidations utilizing a large substrate spectrum and are therefore of interest for biotechnological applications. We expressed human CYP21 in fission yeast Schizosaccharomyces pombe as a eukaryotic model for P450-dependent whole-cell biotransformation. The resulting strain displayed strong steroid hydroxylase activity that was accompanied by contrary effects on respiration and non-respiratory oxygen consumption, which combined to a significant decline in total oxygen consumption of the cells. While production of ROS (reactive oxygen species) decreased, the TCA cycle activity increased, as was shown by metabolic flux (METAFoR) analysis. Pentose phosphate pathway (PPP) activity was found to be negligible, regardless of growth phase, CYP21 expression or biocatalytic activity, indicating that NADPH levels in Sz. pombe are sufficiently high to support an exogenous P450 without adaptations of central carbon metabolism. We conclude from these data that neither oxygen supply nor NADPH availability are limiting factors in P450-dependent biocatalysis in Sz. pombe.

Biotransformation↗

PubMed2005

A fluorimetric assay for cortisol.

A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 muM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at lambda (ex): 460 nm; lambda (em): 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.

Biotransformation↗

PubMed2004

Development of test systems for the discovery of selective human aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) inhibitors. Discovery of a new lead compound for the therapy of congestive heart failure, myocardial fibrosis and hypertension.

Two key players in adrenal steroid hormone biosynthesis are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2 that catalyze the final steps in the biosynthesis of cortisol and aldosterone, respectively. Overproduction of both hormones contributes to a number of severe diseases, as illustrated by the association of elevated aldosterone levels with hypertension and higher mortality in congestive heart failure, and by Cushing's syndrome as the clinical correlate of chronic hypercortisolism. Thus, CYP11B1 and CYP11B2 comprise new targets for drug treatment and selective inhibitors of both enzymes are of high pharmacological interest. To facilitate the search for such compounds, we have established novel test procedures using recombinant fission yeast strains that stably express these enzymes. The aim of this study was to compare the inhibition profiles displayed by these enzymes in established mammalian cell culture test systems to those obtained with the new fission yeast assays, and to evaluate the usefulness of the Schizosaccharomyces pombe strains as screening systems for the identification of novel lead compounds. Using these test systems, we were able to identify a new and very selective CYP11B2 inhibitor (SIAS-1) that displayed no detectable interference with CYP11B1 activity.

Cushing Syndrome↗