Zellteilung und Chromosomensegregation in Corynebacterium glutamicum

Das Gram-positive, stäbchenförmige Bodenbakterium Corynebacterium glutamicum gehört zu den Mykolsäure-haltigen Actinomyceten und durchläuft eine für Coryneforme typische V-förmige Zellteilung. Zellteilungsmechanismen sind im Wesentlichen in den stäbchenförmigen Modellorganismen Escherichia coli und Bacillus subtilis untersucht worden. Im Rahmen dieser Arbeit wurden Mechanismen der Chromosomentrennung und des Chromosomenschutzes gegen eine Teilung durch das Septum sowie Regulatoren der Lokalisation der Zellteilungsebene in C. glutamicum untersucht. Da C. glutamicum keinerlei Gene für bisher beschriebene Proteine des Chromosomenschutzes besitzt, wurde die Frage bearbeitet, inwiefern dieser Organismus die korrekte Aufteilung des Erbguts auf die beiden Tochterzellen und die Lokalisation des Septums zwischen den beiden segregierten Chromsomen gewährleistet. So konnte gezeigt werden, dass das Par System in C. glutamicum, welches aus einem parAB Operon und einer zusätzlichen ATPase ParA2 besteht, als negativer Regulator der FtsZ Polymerisation agiert und somit die Platzierung des Septums negativ reguliert. Eine Überexpression von ParA1 und ParA2 führte zu signifikant verlängerten Zellen. Zudem konnte gereinigtes ParA1 und ParA2 die Polymerisation von FtsZ in vitro inhibieren. So schützen beide ParA ATPasen, die unspezifisch an DNS binden, das Chromosom gegen fatale Septumbildung. Dabei bildet ParA1 innerhalb der Zelle große Strukturen über dem gesamten Chromosom aus, während ParA2 hauptsächlich in kleinen Bereichen am Septum und an den Polen lokalisiert ist. Weiterhin konnte nachgewiesen werden, dass ParB eine hoch-konservierte parS Sequenz spezifisch bindet, welche dreimal auf dem Genom nahe der oriC Region von C. glutamicum identifiziert werden konnte. ParB kolokalisierte mit der oriC Region, was das Modell, nach dem ParB in vivo an die parS Sequenzen bindet und so die Segregation initiiert, unterstützt. Weiterhin steigerte ParB die ATPase Aktivität von ParA2, während es die enzymatische Aktivität von ParA1 fast vollständig inhibierte. Dieses Ergebnis verstärkt die Annahme, dass beide ATPasen während der Zellteilung verschiedene Aufgaben wahrnehmen. So scheint ParA1 für den Schutz und die Segregation des Chromosoms und ParA2 für die Verankerung der Chromosome an den Zellpolen verantwortlich zu sein, was durch Untersuchungen mittels eines bakteriellen Zwei-Hybrid-Systems bestärkt wurde

Exercise as an add-on treatment in individuals with schizophrenia: results from a large multicenter randomized controlled trial

Current treatment methods do not achieve recovery for most individuals with schizophrenia, and symptoms such as negative symptoms and cognitive deficits often persist. Aerobic endurance training has been suggested as a potential add-on treatment targeting both physical and mental health. We performed a large-scale multicenter, rater-blind, parallel-group randomized controlled clinical trial in individuals with stable schizophrenia. Participants underwent a professionally supervised six-month training comprising either aerobic endurance training (AET) or flexibility, strengthening, and balance training (FSBT, control group), follow-up was another six months. The primary endpoint was all-cause discontinuation (ACD); secondary endpoints included effects on psychopathology, cognition, functioning, and cardiovascular risk. In total, 180 participants were randomized. AET was not superior to FSBT in ACD and most secondary outcomes, with dropout rates of 59.55% and 57.14% in the six-month active phase, respectively. However, both groups showed significant improvements in positive, general, and total symptoms, levels of functioning and in cognitive performance. A higher training frequency additionally promoted further memory domains. Participants with higher baseline cognitive abilities were more likely to respond to the interventions. Our results support integrating exercise into schizophrenia treatment, while future studies should aim to develop personalized training recommendations to maximize exercise-induced benefits

Subcellular Localization and Characterization of the ParAB System from Corynebacterium glutamicum▿

Faithful segregation of chromosomes and plasmids is a vital prerequisite to produce viable and genetically identical progeny. Bacteria use a specialized segregation system composed of the partitioning proteins ParA and ParB to segregate certain plasmids. Strikingly, homologues of ParA and ParB are found to be encoded in many chromosomes. Although mutations in the chromosomal Par system have effects on segregation efficiency, the exact mechanism by which the chromosomes are segregated into the daughter cells is not fully understood. We describe the polar localization of the ParB origin nucleoprotein complex in the actinomycete Corynebacterium glutamicum. ParB and the origin of replication were found to be stably localized to the cell poles. After replication, the origins move toward the opposite pole. Purified ParB was able to bind to the parS consensus sequence in vitro. C. glutamicum possesses two ParA-like partitioning ATPase proteins. Both proteins interact with ParB but show a slightly different subcellular localization and phenotype. While ParA might be part of a conventional partitioning system, PldP seems to play a role in division site selection

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+.

The possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.Thus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice.Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-.Our results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling

Identification of the NADPH Oxidase 4 Inhibiting Principle of Lycopus europaeus

NADPH oxidase 4 (Nox4) has recently been implicated as driving force in cellular senescence. Thus, there is growing interest to develop Nox4 inhibitors, which might be valuable agents for cosmeceutical applications. Alpine plants represent a valuable source for the identification of novel bioactive natural products with anti-ageing effects, especially substances that protect plants against UV radiation, which is also known to contribute to the ageing of human skin. Therefore, the aim of this study was to identify novel Nox4 inhibitors from alpine plants. Within an initial screening of extracts of alpine plants on their ability to inhibit Nox4 activity in HEK cells, the methanolic extract of the subaerial parts of Lycopus europaeus showed a strong inhibition of Nox4 (81% chemiluminescence quenching) and a simultaneously high cell viability (91% vitality). Rosmarinic acid was isolated and identified as the major compound in this bioactive extract. It showed a dose dependent inhibitory activity on Nox4 with an IC50 of 1 µM. Moreover, it also showed a significant inhibitory activity on Nox2 in the low micromolar range, whereas no inhibition of Nox5 was detected. Further investigations confirmed that the observed effects of rosmarinic acid on Nox2 and Nox4 are real inhibitory activities, and not due to ROS scavenging effects. Therefore, L. europaeus, which we demonstrated to be a good source of rosmarinic acid, has great potential for usage in cosmeceutical products with anti-ageing activity

Eligibility of Qualification Obtained in another Member State

My thesis summarizes the legal documents relating to eligibility and possibility of legal practice in another state than the home member state. European Union Law in this regard is binding for the Czech Republic since its accession to the EU in 2004. The work is divided into chapters giving comprehensive and coherent information. The introduction justifies my choice of the topic and outlines particular chapters. The first chapter is devoted to cross-border advocacy and the related terms of a European lawyer or Union element. The second chapter begins with the interpretation of the content of primary law in relation to advocates and their activity. The third chapter presents relevant legal documents of secondary union law - directives. The fourth chapter describes fundamental Czech legislation, namely the Law on Advocacy. The fifth chapter discusses the legislation in France. The sixth chapter contains two examples of Czech and EU case law. In the chapter of annexe there is a list of the professional designation of the home state set by Ministry of Justice, so it is a list of titles of persons authorized to provide legal services, and the second annexe is a bar chart showing the number of European lawyers established in the Czech Republic. The thesis provides a comprehensive set of information for..

Connexin 43 acts as a cytoprotective mediator of signal transduction by stimulating mitochondrial KATP channels in mouse cardiomyocytes

Potassium (K+) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive K+ (mitoKATP) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of mitoKATP channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, mitoKATP channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide 43GAP27 each substantially reduced diazoxide-mediated stimulation of mitoKATP channels. Suppression of mitochondrial Cx43 inhibited mitoKATP channel activation by PKC. MitoKATP channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in mitoKATP channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and mitoKATP channel opening, making Cx43 an attractive therapeutic target for protection against cell injury

Gating parameters of mCa1 in UCP2^-/-.

<p>Gating parameters of mCa1 in UCP2^-/-.</p