Aufbau eines pharma-/toxikologischen Testsystems für die Säuger-Kaliumkanäle <em>mKir2.1</em> und hERG mit Hilfe des Modellorganismus <i>Saccharomyces cerevisiae</i>

Die humanen Kaliumkanäle hERG und Kir2.1 tragen wesentlich zur Repolarisierung des kardialen Membranpotentials bei (Sanguinetti et al., 1995; Wible et al., 1995). Fehlfunktionen in diesen Kanälen können zu einer Verzögerung der kardialen Repolarisation, einer Verlängerung des QT-Intervalls im Oberflächen-EKG und zu lebensbedrohlichen Herzrhythmusstörungen führen (Tristani-Firouzi et al., 2002; Sanguinetti & Tristani-Firouzi, 2006). Ziel der Arbeit war die Charakterisierung von hERG und dem zum humanen Kir2.1-Protein orthologen Kir2.1-Protein aus der Maus in Saccharomyces cerevisiae K+-Transportmutanten. Die funktionelle Expression von chromosomal als auch episomal exprimierter mKir2.1-cDNA komplementierte den mutanten Wachstumsphänotyp unter nicht permissiven Bedingungen (3-10 mM KCl) in Abhängigkeit vom externen pH. GFP-mKir2.1 Fusionsproteine konnten in der Plasmamembran oder zumindest in unmittelbarer Nähe zu dieser lokalisiert werden. Unter nicht permissiven (20 mM KCl) wie auch permissiven Bedingungen (100 mM KCl) zeigten mKir2.1 exprimierende Zellen, verglichen mit einem Kontrollstamm ohne mKir2.1, eine deutlich verringerte Sensitivität gegenüber dem als Indikator für das Membranpotential beschriebenen Antibiotikum Hygromycin B, was auf eine Membrandepolarisierung mit funktionellem mKir2.1 hindeutet. Die Untersuchung des Wachstums von mKir2.1 exprimierenden Stämmen bei Zugabe der bekannten Blocker von Kir2.1 Ag+, Cs+, Ba2+ und 48F10 zeigte unter K+-limitierenden Bedingungen eine signifikante Inhibierung von mKir2.1 spezifischem Wachstum. Im Zuge der Entwicklung eines standardisierten Testsystems zur Durchmusterung von potentiellen Modulatoren von Kir2.1 wurde in einem Ringtest die Übertragbarkeit dieser Wachstumstests auf andere Labore am Beispiel eines standardisierten CsCl-Test untersucht und bestätigt. Unter permissiven Bedingungen (≥ 50 mM KCl) und pH 7 führte darüber hinaus die Expression von mKir2.1 zu einer signifikanten Wachstumsinhibierung und damit zu einem weiteren mKir2.1 spezifischen Phänotyp. Diese Wachstumsinhibierung konnte durch Zugabe von CsCl und in K+-Effluxmutanten durch Zugabe von BaCl2 aufgehoben werden. Damit konnte für diesen K+-Kanal die funktionelle heterologe Expression gezeigt werden und es wurden zwei neue, bisher nicht beschriebene Phänotypen identifiziert. Mit den konstruierten Hefestämmen steht ein pharma-/toxikologisches Testsystem zur Verfügung, für das die Sensitivität, Spezifität und der Vergleich mit dem ‚Gold Standard’ unter standardisierten Bedingungen gezeigt werden konnte. Die Expression von HERG-cDNA führte in S. cerevisiae K+-Aufnahmemutanten zu keiner Komplementation des mutanten Wachstumsphänotyps. Bei der Expression von Fusionskonstrukten aus GFP und HERG konnte keine Lokalisation an der Zelloberfläche beobachtet werden. Auch die Konstruktion verschiedener HERG-Modifikationen im Sinne eines verbesserten Membran- ‚Targeting’ und/oder verbessertem ER-Exports konnten keine Lokalisierung in oder in der Nähe der Plasmamembran sowie keine erfolgreiche Komplementation bewirken

Synthesis and spectroscopic/DFT structural characterization of coordination compounds of Nb(V) and Ti(IV) with bioactive carboxylic acids

The reactions are reported of NbX5(X = Cl, Br), TiCl4and Ti(OiPr)4with a selection of carboxylic acids exhibiting a known biological role, in a chlorinated solvent. The reactions of NbX5with acetylsalicylic acid (aspirin) proceeded with selective deacetylation of the organic reactant and formation of the salicylate complexes NbX4(C7H5O3) (1a, X = Cl; 1b, X = Br) in 60–65% yields. NbCl5reacted with diclofenac and ethacrynic acid (EA-CO2H) to give NbCl3[κ3O,O,N-O2CCH2(C6H4)NC6H3Cl2], 2 (80% yield), and NbCl4(O2C-EA), 3 (72% yield), respectively. Ti(OiPr)4reacted with ethacrynic acid giving Ti(OiPr)2(O2C-EA)2, 4, in 74% yield, as a mixture of two isomers. All the products were characterized by means of analytical and spectroscopic methods, moreover DFT studies were carried out to give insight into structural features

Carnosol controls the human glioblastoma stemness features through the epithelial-mesenchymal transition modulation and the induction of cancer stem cell apoptosis

A high cell proliferation rate, invasiveness and resistance to chemotherapy are the main features of glioblastoma (GBM). GBM aggressiveness has been widely associated both with a minor population of cells presenting stem-like properties (cancer stem-like cells, CSCs) and with the ability of tumor cells to acquire a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Carnosol (CAR), a natural inhibitor of MDM2/p53 complex, has been attracted attention for its anti-cancer effects on several tumor types, including GBM. Herein, the effects of CAR on U87MG-derived CSC viability and stemness features were evaluated. CAR decreased the rate of CSC formation and promoted the CSC apoptotic cell death through p53 functional reactivation. Moreover, CAR was able to control the TNF-α/TGF-β-induced EMT, counteracting the effects of the cytokine on EMT master regulator genes (Slug, Snail, Twist and ZEB1) and modulating the activation of miR-200c, a key player in the EMT process. Finally, CAR was able to increase the temozolomide (TMZ) anti-proliferative effects. These findings demonstrate that CAR affected the different intracellular mechanism of the complex machinery that regulates GBM stemness. For the first time, the diterpene was highlighted as a promising lead for the development of agents able to decrease the stemness features, thus controlling GBM aggressiveness

New insights into the anticancer activity of carnosol: P53 reactivation in the U87MG human glioblastoma cell line

Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- A nd chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes' transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells' proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule

Gene-expression analysis of adult-onset Still’s disease and systemic juvenile idiopathic arthritis is consistent with a continuum of a single disease entity

Background: Adult-onset Still’s disease (AOSD), a rare autoinflammatory disorder, resembles systemic juvenile idiopathic arthritis (SJIA). The superimposable systemic clinical features of AOSD and SJIA suggest both clinical phenotypes represent the same disease continuum with different ages of onset. To further characterize the similarity between AOSD and SJIA at the molecular level, 2 previously identified response gene sets in SJIA were used to investigate how genes that respond to interleukin (IL)-1β inhibition with canakinumab in SJIA patients behave in AOSD patients with active disease prior to IL-1β targeting therapy, relative to healthy subjects. Findings: All genes downregulated in SJIA patients following canakinumab treatment were upregulated in most patients with active AOSD prior to canakinumab treatment, relative to healthy subjects. A few patients with milder AOSD had expectedly gene-expression patterns that resembled those in healthy subjects. Comparison of the gene-expression patterns with neutrophil counts showed a correlation between elevated neutrophil numbers and upregulation of canakinumab-responsive genes. Correspondingly, most genes upregulated following canakinumab treatment in patients with SJIA patients were downregulated in the majority of AOSD patients. Conclusions: These results further support the concept of a Still’s disease continuum that includes both a pediatric/juvenile onset (SJIA) and adult onset (AOSD) form

Kdm7a expression is spatiotemporally regulated in developing Xenopus laevis embryos, and its overexpression influences late retinal development

Background: Post-translational histone modifications are among the most common epigenetic modifications that orchestrate gene expression, playing a pivotal role during embryonic development and in various pathological conditions. Among histone lysine demethylases, KDM7A, also known as KIAA1718 or JHDM1D, catalyzes the demethylation of H3K9me1/2 and H3K27me1/2, leading to transcriptional regulation. Previous data suggest that KDM7A plays a central role in several biological processes, including cell proliferation, commitment, differentiation, apoptosis, and maintenance. However, information on the expression pattern of KDM7A in whole organisms is limited, and its functional role is still unclear. Results: In Xenopus development, kdm7a is expressed early, undergoing spatiotemporal regulation in various organs and tissues, including the central nervous system and the eye. Focusing on retinal development, we found that kdm7a overexpression does not affect the expression of genes critically involved in early neural development and eye-field specification, whereas unbalances the distribution of neural cell subtypes in the mature retina by disfavoring the development of ganglion cells while promoting that of horizontal cells. Conclusions: Kdm7a is dynamically expressed during embryonic development, and its overexpression influences late retinal development, suggesting a potential involvement in the molecular machinery regulating the spatiotemporally ordered generation of retinal neuronal subtypes

Lung MRI as a Possible Alternative to CT Scan for Patients With Primary Immune Deficiencies and Increased Radiosensitivity

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