Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

Impact of differential P450c17 phosphorylation by cAMP stimulation and by starvation conditions on enzyme activities and androgen production in NCI-H295R cells

CYP17A1 plays a pivotal role in the biosynthesis of androgens in the adrenals and the gonads. Although this enzyme catalyzes two different reactions on one single active site, its specific activities are regulated independently. Although the 17alpha-hydroxylase activity is rather constant and regulated by gene expression, the 17,20-lyase activity varies significantly with the amount of cofactors or by protein phosphorylation. cAMP increases CYP17A1 expression, P450c17 phosphorylation, and androgen production. However, the exact mechanism(s) and the specific regulators of CYP17A1 remain unknown. Therefore, we studied the regulation of adrenal androgen biosynthesis in human adrenal H295R cells focusing on CYP17A1. We analyzed androgen production and P450c17 activities in H295R cells grown under normal and serum-free conditions and/or after stimulation with 8-bromoadenosine-cAMP. H295R cells grown in starvation medium produced more androgens and had decreased HSD3B2 expression and activity but increased P450c17-17,20-lyase activity and serine phosphorylation. Although starvation increased serine phosphorylation of P450c17 specifically, cAMP stimulation enhanced threonine phosphorylation exclusively. Time-course experiments revealed that a short cAMP stimulation augmented threonine phosphorylation of P450c17 but did not increase 17,20-lyase activity. By contrast, long cAMP stimulation increased androgen production through increased P450c17 activities by enhancing CYP17A1 gene expression. We conclude that serum withdrawal shifts steroidogenesis of H295R cells towards androgen production, providing a suitable model for detailed studies of androgen regulation. In addition, our study shows that starvation and cAMP stimulation regulate P450c17 phosphorylation differentially and that an increase in P450c17 phosphorylation does not necessarily lead to enhanced enzyme activity and androgen production.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.

Retinoic acid reduces glucocorticoid sensitivity in C2C12 myotubes by decreasing 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor activities

Vitamin A is a nutrient with remarkable effects on adipose tissue and skeletal muscles, and plays a role in controlling energy balance. Retinoic acid (RA), the carboxylic form of vitamin A, has been associated with improved glucose tolerance and insulin sensitivity. In contrast, elevated glucocorticoids have been implicated in the development of insulin resistance and impaired glucose tolerance. Here, we investigated whether RA might counteract glucocorticoid effects in skeletal muscle cells by lowering 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local glucocorticoid activation and/or activation of glucocorticoid receptor (GR). We found a dose-dependent down-regulation of 11beta-HSD1 mRNA expression and activity upon incubation of fully differentiated mouse C2C12 myotubes with RA. In addition, RA inhibited GR transactivation by an 11beta-HSD1-independent mechanism. The presence of RA during myogenesis did not prevent myotube formation but resulted in relatively glucocorticoid-resistant myotubes, exhibiting very low 11beta-HSD1 expression and GR activity. The use of selective retinoic acid receptor (RAR) and retinoid X receptor ligands provided evidence that these effects were mediated through RARgamma. Importantly, short hairpin RNA against RARgamma abolished the effect of RA on 11beta-HSD1 and GR. In conclusion, we provide evidence for an important role of RA in the control of glucocorticoid activity during myogenesis and in myotubes. Disturbances of the nutrient and hormonal regulation of glucocorticoid action in skeletal muscles might be relevant for metabolic diseases.

The Swiss Schools have been Governed by Local Lay Authorities

After the introduction of the liberal-democratic constitutions in the Swiss cantons in the first half of the 1830ies the grid of existing schools has been systemized and broadly expanded. The school systems have ever since been characterized by one key element: a special local authority type called „Schulkommission“ or „Schulpflege“. They take the form of committees consisting of laymen that are appointed by democratic elections like all the other executive bodies on the different federal levels in Switzerland. When it comes to their obligations and activities these community level school committees conform very much to the school boards in the American and Canadian school systems. They are accountable for the selection and supervision of the teachers. They approve decisions about the school careers of pupils and about curricular matters like the choice of school books. Normally their members are elected by the local voters for four year terms of office (reelection remains possible) and with regard to pedagogics they normally are non-professionals. The board members are responsible for classes and teachers assigned to them and they have to go to see them periodically. These visitations and the board meetings each month together with the teachers enable the board members to attain a deep insight into what happens in their schools over the course of their term of office. But they are confronted as laymen with a professional teaching staff and with educational experts in the public administration. Nevertheless this form of executive power by non-professionals is constitutive for the state governance in the Swiss as well as in other national political environments. It corresponds to the principles of subsidiarity and militia and therefore allows for a strong accentuation of liberty and the right of self-determination, two axioms at the very base of democratic federalist ideology. This governance architecture with this strong accent on local anchorage features substantial advantages for the legitimacy and acceptability of political and administrative decisions. And this is relevant especially in the educational area because the rearing of the offspring is a project of hope and, besides, quite costly. In the public opinion such supervision bodies staffed by laymen seem to have certain credibility advances in comparison with the professional administration. They are given credit to be capable of impeding the waste of common financial resources and of warranting the protection and the fostering of the community’s children at once. Especially because of their non-professional character they are trusted to be reliably immune against organizational blindness and they seem to be able to defend the interests of the local community against the standardization and centralization aspirations originating from the administrational expertocracy. In the paper these common rationales will be underpinned by results of a comprehensive historical analysis of the Session protocols of three Bernese school commissions from 1835 to 2005.

Sustained impact of a short small group course with systematic feedback in addition to regular clinical clerkship activities on musculoskeletal examination skills-a controlled study.

BACKGROUND The discrepancy between the extensive impact of musculoskeletal complaints and the common deficiencies in musculoskeletal examination skills lead to increased emphasis on structured teaching and assessment. However, studies of single interventions are scarce and little is known about the time-dependent effect of assisted learning in addition to a standard curriculum. We therefore evaluated the immediate and long-term impact of a small group course on musculoskeletal examination skills. METHODS All 48 Year 4 medical students of a 6 year curriculum, attending their 8 week clerkship of internal medicine at one University department in Berne, participated in this controlled study. Twenty-seven students were assigned to the intervention of a 6×1 h practical course (4-7 students, interactive hands-on examination of real patients; systematic, detailed feedback to each student by teacher, peers and patients). Twenty-one students took part in the regular clerkship activities only and served as controls. In all students clinical skills (CS, 9 items) were assessed in an Objective Structured Clinical Examination (OSCE) station, including specific musculoskeletal examination skills (MSES, 7 items) and interpersonal skills (IPS, 2 items). Two raters assessed the skills on a 4-point Likert scale at the beginning (T0), the end (T1) and 4-12 months after (T2) the clerkship. Statistical analyses included Friedman test, Wilcoxon rank sum test and Mann-Whitney U test. RESULTS At T0 there were no significant differences between the intervention and control group. At T1 and T2 the control group showed no significant changes of CS, MSES and IPS compared to T0. In contrast, the intervention group significantly improved CS, MSES and IPS at T1 (p < 0.001). This enhancement was sustained for CS and MSES (p < 0.05), but not for IPS at T2. CONCLUSIONS Year 4 medical students were incapable of improving their musculoskeletal examination skills during regular clinical clerkship activities. However, an additional small group, interactive clinical skills course with feedback from various sources, improved these essential examination skills immediately after the teaching and several months later. We conclude that supplementary specific teaching activities are needed. Even a single, short-lasting targeted module can have a long lasting effect and is worth the additional effort.