Reactive oxygen species-linked regulation of the multidrug resistance transporter P-glycoprotein in Nox-1 overexpressing prostate tumor spheroids

AbstractExpression of the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) has been demonstrated to be regulated by hypoxia-inducible factor-1α (HIF-1α) and inhibited by intracellular reactive oxygen species (ROS). Herein, P-gp and HIF-1α expression were investigated in multicellular prostate tumor spheroids overexpressing the ROS-generating enzyme Nox-1 in comparison to the mother cell line DU-145. In Nox-1-overexpressing tumor spheroids (DU-145Nox1) generation of ROS as well as expression of Nox-1 was significantly increased as compared to DU-145 tumor spheroids. ROS generation was significantly inhibited in the presence of the NADPH-oxidase antagonists diphenylen-iodonium chloride (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Albeit growth kinetic of DU-145Nox1 tumor spheroids was decreased as compared to DU-145 spheroids, elevated expression of Ki-67 was observed indicating increased cell cycle activity. In DU-145Nox1 tumor spheroids, expression of HIF-1α as well as P-gp was significantly decreased as compared to DU-145 spheroids, which resulted in an increased retention of the anticancer agent doxorubicin. Pretreatment with the free radical scavengers vitamin E and vitamin C increased the expression of P-gp as well as HIF-1α in Nox-1-overexpressing cells, whereas no effect of free radical scavengers was observed on mdr-1 mRNA expression. In summary, the data of the present study demonstrate that the development of P-gp-mediated MDR is abolished under conditions of elevated ROS levels, suggesting that the MDR phenotype can be circumvented by modest increase of intracellular ROS generation

Differential regulation of GPCRs: Are GRK expression levels the key?

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and their signal transduction is tightly regulated by GPCR kinases (GRKs) and β-arrestins. In this review, we discuss novel aspects of the regulatory GRK/β-arrestin system. Therefore, we briefly revise the origin of the “barcode” hypothesis for GPCR/β-arrestin interactions, which states that β-arrestins recognize different receptor phosphorylation states to induce specific functions. We emphasize two important parameters which may influence resulting GPCR phosphorylation patterns: (A) direct GPCR–GRK interactions and (B) tissue-specific expression and availability of GRKs and β-arrestins. In most studies that focus on the molecular mechanisms of GPCR regulation, these expression profiles are underappreciated. Hence we analyzed expression data for GRKs and β-arrestins in 61 tissues annotated in the Human Protein Atlas. We present our analysis in the context of pathophysiological dysregulation of the GPCR/GRK/β-arrestin system. This tissue-specific point of view might be the key to unraveling the individual impact of different GRK isoforms on GPCR regulation

How Carvedilol activates β₂-adrenoceptors

Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β(1)-adrenoceptors, arrestin-biased signalling via β(2)-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol’s cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β(2)ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system

Constraint-Based Modeling and Kinetic Analysis of the Smad Dependent TGF-β Signaling Pathway

Background Investigation of dynamics and regulation of the TGF-β signaling pathway is central to the understanding of complex cellular processes such as growth, apoptosis, and differentiation. In this study, we aim at using systems biology approach to provide dynamic analysis on this pathway. Methodology/Principal Findings We proposed a constraint-based modeling method to build a comprehensive mathematical model for the Smad dependent TGF-β signaling pathway by fitting the experimental data and incorporating the qualitative constraints from the experimental analysis. The performance of the model generated by constraint-based modeling method is significantly improved compared to the model obtained by only fitting the quantitative data. The model agrees well with the experimental analysis of TGF-β pathway, such as the time course of nuclear phosphorylated Smad, the subcellular location of Smad and signal response of Smad phosphorylation to different doses of TGF-β. Conclusions/Significance The simulation results indicate that the signal response to TGF-β is regulated by the balance between clathrin dependent endocytosis and non-clathrin mediated endocytosis. This model is useful to be built upon as new precise experimental data are emerging. The constraint-based modeling method can also be applied to quantitative modeling of other signaling pathways

16p11.2 600 kb Duplications confer risk for typical and atypical Rolandic epilepsy

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65 046 European population controls (5/393 cases versus 32/65 046 controls; Fisher's exact test P = 2.83 × 10−6, odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10−4). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical R

An investigation into advertisements published in the popular medical journals in 1900-1930 based on the example of Homöopathische Monatsblätter

Thema der Arbeit ist die Betrachtung der Anzeigenwerbung der Laienpresse von 1900-1930. Die Anzeigenwerbung für Arzneimittel veränderte sich im ersten Drittel des 20. Jahrhunderts im Zuge der Industrialisierung umfassend. Die pharmazeutische Industrie produzierte Arzneimittel auf Vorrat, für deren Absatz die Anzeige als Mittler zwischen Produzent und Konsument immer größere Bedeutung erlangte. In der Laienpresse war noch eine Bewerbung aller Arzneimittel möglich. Es konnten darin Werbung für die traditionellen Geheimmittel sowie für die, in rascher Folge entwickelten, synthetisch hergestellten Arzneispezialitäten erscheinen. Die vorliegende Arbeit untersucht die Anzeigen der populärmedizinischen Zeitschrift „Homöopathische Monatsblätter“ von 1900-1930 quantitativ und qualitativ. Ein Schwerpunkt wird auf die Arznei-, Heil- und Nahrungsergänzungsmittel gelegt, die zusammen 21,3% des gesamten Anzeigenvolumens ausmachen. Mit dem Analyseschlüssel der Emnid-Faktoriellen-Anzeigenanalyse aus dem Jahr 1961 werden die formalen und inhaltlichen Veränderungen der Anzeigen im Verlauf des Beobachtungszeitraums aufgezeigt. Die kontinuierliche Zunahme der Bilder und Abbildung von Konsumenten verdeutlicht diese Entwicklung anschaulich. An der Anzeigenserie des Nervenmittels „Biocitin“ wird zudem die suggestive Beeinflussung des Lesers bis zum Kaufwunsch dargestellt. Weiterhin werden die Arzneimittel selbst betrachtet. Nach ihrer Wirkung erfolgt eine Einteilung in Husten-, Magen-Darm-, Nerven-, Herzkreislauf-, Stärkungs-, Schwächemittel und andere mehr. Auf der Grundlage der deklarierten Inhaltsstoffe und der angegebenen Wirkungsbreite wird eine Unterscheidung in Universalheilmittel und Spezifika vorgenommen. Darüber hinaus wird die Frage untersucht, ob Universalheilmittel identisch mit Geheimmitteln und Spezifika mit Spezialitäten sind und welche Anhaltspunkte die Anzeigen liefern. Die Ergebnisse zeigen eine Zuordnung von 57,5% der Arzneimittel zu den spezifisch wirkenden Mitteln und damit zu den Spezialitäten. Die Geheimmittel sind aufgrund der Merkmale in den Anzeigen hingegen retrospektiv nicht eindeutig identifizierbar.The topic of this paper is the analysis of advertising in the lay press in 1900-1930. The print media advertising has fundamentally changed in the course of industrialisation in the first third of the 20th century. The pharmaceutical industry produced pharmaceuticals for stock, and advertising as a link between the manufacturer and the consumer became more an more important for their sale. Moreover, the lay press offered an opportunity to advertise all pharmaceuticals. It was a place for advertising traditional nostrums as well as synthetically manufactured proprietary medicinal products undergoing rapid development. This paper presents a quantitative and qualitative investigation into advertisements published in "Homöopathische Monatsblätter", the popular medical journal, in 1900-1930. The focus is on the pharmaceuticals, remedies and food supplements which together present 21.3% of the total number of advertisements. Analysis keys from the factor analysis of advertising provided by Emnid (market and opinion research institute) in 1961 were used to show changes in form and content during the observation period. The continuous growth in the number of pictures and consumer photos provides a clear illustration of this development. Moreover, the series of advertisements for Biocitin nerve medicine shows suggestive influence on the reader leading to the desire to buy. Furthermore, attention is paid to pharmaceuticals themselves. They are divided into cough suppressants, gastro-intestinal, nerve, cardiovascular drugs, tonics, etc. Depending on the declared ingredients and the spectrum of effect, they are divided into universal remedies and special drugs. Another issue under investigation is whether universal remedies are identical to nostrums and special drugs with proprietary medicinal products and which criteria are shown in the advertisements. The results show that 57.5% of pharmaceuticals are drugs with special effects, and consequently proprietary medicinal products. At the same time, the features described in the advertisements provide no basis for clear retrospective identification of the nostrums

Development and evaluation of the LittlEARS® Early Speech Production Questionnaire – LEESPQ

PeerReviewe

Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity