TY - JOUR A1 - Donat, Ulrike A1 - Rother, Juliane A1 - Schäfer, Simon A1 - Hess, Michael A1 - Härtl, Barbara A1 - Kober, Christina A1 - Langbein-Laugwitz, Johanna A1 - Stritzker, Jochen A1 - Chen, Nanhai G. A1 - Aguilar, Richard J. A1 - Weibel, Stephanie A1 - Szalay, Alandar A. T1 - Characterization of Metastasis Formation and Virotherapy in the Human C33A Cervical Cancer Model JF - PLoS ONE N2 - More than 90% of cancer mortalities are due to cancer that has metastasized. Therefore, it is crucial to intensify research on metastasis formation and therapy. Here, we describe for the first time the metastasizing ability of the human cervical cancer cell line C33A in athymic nude mice after subcutaneous implantation of tumor cells. In this model, we demonstrated a steady progression of lumbar and renal lymph node metastases during tumor development. Besides predominantly occurring lymphatic metastases, we visualized the formation of hematogenous metastases utilizing red fluorescent protein (RFP) expressing C33A-RFP cells. RFP positive cancer cells were found migrating in blood vessels and forming micrometastases in lungs of tumor-bearing mice. Next, we set out to analyze the influence of oncolytic virotherapy in the C33A-RFP model and demonstrated an efficient virus-mediated reduction of tumor size and metastatic burden. These results suggest the C33A-RFP cervical cancer model as a new platform to analyze cancer metastases as well as to test novel treatment options to combat metastases. KW - metastasis KW - renal cancer KW - oncolytic viruses KW - lymph nodes KW - kidneys KW - lung and intrathoracic tumors KW - secondary lung tumors KW - cancer treatment Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119674 SN - 1932-6203 VL - 9 IS - 6 ER - TY - JOUR A1 - Koelmel, Wolfgang A1 - Kuper, Jochen A1 - Kisker, Caroline T1 - Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem JF - Scientific Reports N2 - Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a "quick-soak"-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy. KW - structural biology KW - X-ray crystallography Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261644 VL - 11 IS - 1 ER - TY - THES A1 - Lorenz, Viola T1 - Cellular regulation of the hemITAM-coupled platelet receptor C-type lectin-like receptor 2 (CLEC-2): In vitro and in vivo studies in mice T1 - Zelluläre Regulation des hemITAM-gekoppelten Thrombozytenrezeptors C-type lectin-like receptor 2 (CLEC-2): In vitro und in vivo Studien in Mäusen N2 - Platelet aggregation at sites of vascular injury is essential to limit posttraumatic blood loss, but may also cause acute ischemic disease states such as myocardial infarction or stroke. Stable thrombus formation requires a series of molecular events involving platelet receptors and intracellular signal transduction, which contribute to adhesion, activation and aggregation of platelets. In this thesis, the cellular regulation of platelet surface receptors and their involvement in thrombus formation was investigated using genetically modified mice. In the first part of the study, the functional relevance of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and of the recently identified hemITAM-bearing C-type lectin-like receptor 2 (CLEC-2) for in vivo thrombus formation was analyzed. Megakaryocyte/ platelet-specific CLEC-2 knock out mice displayed a defective lymphatic development and were protected from occlusive arterial thrombus formation. These phenotypes were more pronounced in mice with a GPVI/CLEC-2 double deficiency. Hemostasis was not compromised in CLEC-2 or GPVI single-deficient animals, as they showed only mildly prolonged tail bleeding times. Combined depletion of both receptors resulted in markedly prolonged bleeding times revealing an unexpected redundant function of the two receptors in hemostasis as well as thrombosis. These findings might have important implications for the development of anti-CLEC-2/ anti-GPVI agents as therapeutics. In the second part, mechanisms underlying the cellular regulation of CLEC-2 were studied. Previous studies have shown that injection of the anti-CLEC-2 antibody INU1 results in complete immunodepletion of platelet CLEC-2 in mice, which is preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. It is demonstrated that INU1-induced CLEC-2 immunodepletion occurs through Src family kinase (SFK)-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with spleen tyrosine kinase (Syk) deficiency, INU1-induced CLEC-2 internalization/ degradation was fully preserved, whereas the associated thrombocytopenia was largely prevented. These results show that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia. Since INU1 IgG induced a pronounced thrombocytopenia, the in vivo effects of monovalent INU1 F(ab) fragments were analyzed. Very unexpectedly, injection of the F(ab) fragments resulted in widespread thrombus formation leading to persistent neurological deficits of the animals. This intravascular thrombus formation is the result of CLEC-2-dependent platelet activation and aggregation. The mechanism underlying the thrombus formation is still unknown and depends potentially on binding of a yet unidentified ligand to F(ab)-opsonized CLEC-2 on platelets. N2 - Die Aggregation von Thrombozyten ist ein essentielle Prozess, um Blutungen nach einer Gefäßverletzung zu stoppen. Sie kann aber auch zu akuten thrombotischen Erkrankungen, wie Herzinfarkt und Schlafanfall, führen. Die Bildung eines stabilen Thrombus ist ein dynamischer Prozess, der ein definiertes Zusammenspiel von thrombozytären Rezeptoren und intrazellulären Signalen benötigt, die zur Adhäsion, Aktivierung und Aggregation der Thrombozyten beitragen. In der hier vorliegenden Dissertation wurde zellulär Regulation von thrombozytären Oberflächenrezeptoren und ihre Beteiligung an der Thromben-Bildung mittels genetisch veränderter Mäuse untersucht. Im ersten Teil der Arbeit wurde die funktionelle Relevanz des immunoreceptor tyrosine-based activation motif (ITAM)-gekoppelten Kollagenrezeptors GPVI und des vor kurzem entdeckten hemITAM-gekoppelten C-type lectin-like receptor 2 (CLEC-2) für die in vivo Thromben-Bildung charakterisiert. Mäuse mit einer Megakaryozyten/ Thrombozyten-spezifischen CLEC-2-Defizienz weisen Defekte in der Entwicklung ihrer lymphatischen Gefäße auf und sind vor arteriellen Gefäßverschlüssen geschützt. Dieser Phänotyp war in Mäusen mit einer Defizient GPVI und CLEC-2 verstärkt. GPVI oder CLEC-2-defiziente Mäuse zeigen eine normale Hämostase, da ihre Schwanzblutungszeiten nur minimal verlängert waren. Die gleichzeitige Defizienz beider Rezeptoren verlängerte die Blutungszeiten der Tiere allerdings erheblich. Das spricht dafür, dass beiden Rezeptoren eine unerwartete redundante Funktion sowohl in der Hämostase als auch während der pathologischen Thromben-Bildung haben. Diese Ergebnisse könnten für die Entwicklung neuer therapeutischen Wirkstoffen, die gegen GPVI und/ oder CLEC-2 gerichtet sind, bedeutsam sein. Im zweiten Teil der Arbeit wurde die zelluläre Regulation von CLEC-2 untersucht. Frühere Studien haben gezeigt, dass die Behandlung von Mäusen mit dem gegen CLEC-2 gerichteten Antikörper INU1 zu einem spezifischen Verlust des Rezeptors in zirkulierenden Thrombozyten führt. Dieser Prozess, der „Immunodepletion“ genannt wird, ist von einer Thrombozytopenie begleitet, die das therapeutische Potential eines solchen Ansatzes reduziert. Im Verlauf der Arbeit konnte gezeigt werden, dass die INU1-abhängige Immunodepletion in vitro und in vivo mittels src family kinase (SFK)-abängiger Internalisierung geschieht. Der Internalisierung ist vermutlich ein intrazellulärer Abbau nachgeschaltet. In Mäusen mit einer spleen tyrosine kinase (Syk) Defizienz war die INU1-abhängige Thrombozytopenie zum größten Teil verhindert, aber die Herabregulation durch Internalisierung von CLEC-2 blieb erhalten. Diese Ergebnisse zeigen, dass CLEC-2 in vitro und in vivo durch Internalisierung von der Thrombozyten-Oberfläche herabreguliert werden kann. Darüber hinaus kann dies von der Antikörper-vermittelten Thrombozytopenie mechanistisch entkoppelt werden. Da die Injektion von INU1 IgG eine starke Thrombozytopenie in der Tieren verursacht, sollen die in vivo Effekte von monovalenten INU1 F(ab) Fragmenten getestet werden. Unerwarteter weise führte die Injektion dieser F(ab) Fragmente zu einer systemischen Thromben-Bildung, die unter anderem neurologische Defizite in den Tieren auslösten. Diese intravaskuläre Thromben-Bildung ist ein Ergebnis von CLEC-2-abhängier Thrombozyten Aktivierung und Aggregation. Der Mechanismus, der dieser Thromben-Bildung zugrunde liegt, ist bisher noch nicht aufgeklärt. Möglicherweise wird er durch die Bindung eines unbekannten Liganden an das F(ab)-gebunde CLEC-2 auf Thrombozyten ausgelöst. KW - Thrombozytenaggregation KW - CLEC-2 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116724 ER - TY - THES A1 - Rabie, Tamer T1 - Cellular regulation of platelet glycoprotein VI : in vivo and in vitro studies in mice T1 - Zelluläre Regulation von Plättchen Glykoprotein VI : in vivo und in vitro Studien in der Maus N2 - Platelet interaction with the subendothelium is essential to limit blood loss after tissue injury. However, upon rupture of atherosclerotic plaques, this interaction may result in blood vessel occlusion leading to life threatening diseases such as myocardial infarction or stroke. Among the subendothelial matrix proteins, collagen is considered to be the most thrombogenic component as it directly activates platelets. Platelets interact with collagen, either indirectly through glycoprotein (GP) Ib-V-IX receptor complex, or directly through the major collagen receptor on the platelet surface, GPVI. The work presented here focused on studying the cellular regulation of GPVI. In addition, a possible role for GPVI in thrombus formation induced by atherosclerotic plaque material was investigated and it was found that GPVI plays an important role in this process. Using a recently published mitochondrial injury model, it was found that GPVI contains a cleavage site for a platelet-expressed metalloproteinase. Further studies showed that platelet activation by CRP, or thrombin induced down-regulation of GPIb, but not GPVI. In parallel, cellular regulation of GPV was studied and it was found that GPV is cleaved in vitro by the metalloproteinase ADAM17. In previous studies it was shown that injection of mice with the anti-GPVI mAb, JAQ1, induces GPVI down-regulation, which is associated with a strong, but transient, thrombocytopenia. Using new anti-GPVI mAbs, which bind different epitopes on the receptor, it is shown in this study that GPVI down-regulation occurs in an epitope-independent manner. Further experiments showed that antibody treatment induces a transient, but significant increase in bleeding time. Using different genetically modified mice, it is shown that, upon antibody injection, GPVI is both, shed from the platelet surface and internalized into the platelet. Signaling through the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR chain is essential for both processes, while LAT and PLC2 are essential for the shedding process only. Antibody-induced increase in bleeding time and thrombocytopenia were absent in LAT deficient mice, showing that it is possible to uncouple the associated side effects from the down-regulation process. As antibody-induced GPVI internalization still occurs in LAT and PLC2 deficient mice, this suggests a novel signaling pathway downstream of GPVI that has not been described so far. N2 - Plättchen Interaktion mit dem Subendothel ist für die Blutstillung essentiell. Dies kann jedoch nach dem Aufbrechen atherosklerotischer Plaques zu lebensbedrohlicher Erkrankungen wie Infarkt oder Schlaganfall führen. Kollagen, welches die Plättchen dirket aktiviert, ist der thrombogenste Bestandteil der Extrazellularmatrix (EZM). Die Bindung zwischen Plättchen und Kollagen wird sowohl indirekt durch den Glykoprotein (GP) Ib-V-IX Rezeptorkomplex, als auch direkt durch den Kollagenrezeptor GPVI, auf der Plättchenoberfläche vermittelt. In der vorliegenden Arbeit wurde die zelluläre Regulation von GPVI untersucht. Des Weiteren wurde die Rolle von GPVI in durch atheroklerotisches Plaquematerial induzierter Thrombusbildung studiert. Hierbei wurde festgestellt, dass GPVI eine wichtige Funktion in diesem Prozess spielt. Mittels eines jüngst publizierten mitochondrialen Verletzungsmodels, konnte gezeigt werden, dass GPVI eine Erkennungsstelle für eine in den Plättchen exprimierte Metalloproteinase besitzt. Mehrere Versuche haben gezeigt, dass Plättchenaktivierung durch CRP, und Thrombin zur Runterregulierung von GPIb aber nicht von GPVI führt. Parallellaufende Untersuchungen zeigten, dass GPV durch die Metalloproteinase ADAM17 in vitro abgespalten wird. Vorherige Studien ergaben, dass die in vivo Behandlung von Mäusen mit dem anti-GPVI Antikörper, JAQ1, zur Runterregulierung des Rezeptors führt. Dieses ist mit einer starken, transienten Thrombozytopenie assoziiert. Mittels neu generierte anti-GPVI Antikörper (JAQ2, 3), die unterschiedliche Bindungsstellen auf GPVI erkennen, konnte demonstriert werden, dass die Antikörper vermittele GPVI Runterregulierung Epitop unabhängig ist. Weitere Untersuchungen ergaben, dass Anitkörperinjektion eine transiente Erhöhung der Blutungszeit verursacht. Mittels genetisch modifizierter Mäuse konnte dargestellt werden, dass die Antikörpergabe GPVI sowohl von der Plättchenoberfläche abgespalten, als auch internalisiert wird. Während die Signaltransduktion durch das ITAM Motif der FcR Kette essentiell für beide Prozesse ist, sind LAT und PLC2 nur für das Abspalten wichtig. Antikörper induzierte Erhöhung der Blutungszeit und Thrombozytopenie sind abwesend in LAT-defizienten Mäuse, was zeigt, dass möglicherweise die GPVI Runterregulierung von den assoziierten Nebenwirkungen zu trennen ist. Da die GPVI Runterregulierung in LAT und –PLC2 defizienten Mäusen weiterhin stattfindet, zeigt dies einen neuen GPVI Signalweg, der bisher noch nicht beschrieben wurde. KW - Maus KW - Thrombozyt KW - Glykoproteine KW - Regulation KW - Biologie KW - Plättchen KW - Maus KW - Thrombose KW - Kardiovaskulär KW - maus KW - platelets KW - thrombosis KW - cardiovascular Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-14267 ER - TY - THES A1 - Gotru, Sanjeev Kiran T1 - Cation Homeostasis in Platelets T1 - Kationen-Homöostase in Thrombozyten N2 - Divalent cations are important second messengers triggering various signal transduction events in platelets. Whereas calcium channel blockers have an established antithrombotic effect and the regulation of Ca2+ homeostasis has been elucidated in platelets, the molecular regulation of Mg2+ and Zn2+ homeostasis has not been investigated so far. In the first part of the thesis, the role of -type serine-threonine kinase linked to transient receptor potential cation channel, subfamily M, member 7 (TRPM7) in platelets was investigated. Using Trpm7R/R mice with a point mutation deleting the kinase activity, we showed that the TRPM7 kinase regulates platelet activation via immunoreceptor tyrosine-based activation motif (ITAM), hem(ITAM) and protease-activated receptor (PAR) signaling routes. Furthermore, Trpm7R/R mice were protected from in vivo thrombosis and stroke, thus establishing TRPM7 kinase as a promising anti-thrombotic target. In the second part of the thesis, the role of TRPM7 channel in a megakaryocyte (MK) and platelet-specific knockout mouse, Trpm7fl/fl-Pf4Cre, was investigated. Here, we observed that depending on the type of stimulation, Trpm7fl/fl-Pf4Cre platelets showed either enhanced or inhibited responses. Although Trpm7fl/fl-Pf4Cre mice were thrombocytopenic, no differences to wildtype mice were observed in models of in vivo thrombosis and stroke. The above two studies highlight that inhibition of TRPM7 kinase but not the channel itself (in MKs and platelets) may be a promising anti-thrombotic strategy. Besides TRPM7, we investigated the role of magnesium transporter 1 (MAGT1) in platelet Mg2+ homeostasis and found that MAGT1 primarily regulates receptor-operated calcium entry (ROCE) in platelets specifically upon GPVI activation. This physiological crosstalk is triggered by protein kinase C (PKC) isoforms. Platelets from Magt1-/y mice hyper-reacted to GPVI and thromboxane A2 (TXA2) receptor stimulation in vitro. Consequently, Magt1-/y platelets were found to be pro-thrombotic in disease models of thrombosis and stroke. To compare platelet ITAM-signaling to the immune system, we further investigated the role of MAGT1 in T and B cells. We described the primary role of MAGT1 in mice under pathogen-free conditions. Magt1-/y B cells showed dysregulated Mg2+ and Ca2+ homeostasis upon B-cell receptor activation, thereby altering Syk, LAT, phospholipase C (PLC)2 and PKC phosphorylation. In contrast to human MAGT1-deficient T cells, development and effector functions of mouse Magt1-/y T cells showed no alterations. Finally, in the last part of the thesis, we described methods to measure intracellular free zinc [Zn2+]i in human and mouse platelets with storage pool disease (SPD). We propose to measure the [Zn2+]i status in SPD platelets as a relatively easy diagnostic to screen platelet granule abnormalities. N2 - Zweiwertige Kationen sind wichtige sekundäre Botenstoffe, welche verschiedene Signaltransduktionsereignisse in Thrombozyten initiieren. Zwar wurde die Regulation der Ca2+Homöostase in Blutplättchen bereits aufgeklärt und der Einsatz von Calciumkanalblockern zur antithrombotischen Therapie ausführlich diskutiert, die molekulareRegulation der Mg2+und Zn2+Homöostase in Thrombozyten und Megakaryozyten (MK) wurdebisher jedoch nicht untersucht.Im ersten Teil dieser Thesis wurde die Rolle der -Typ Serin-Threonin Kinase des transienten Rezeptortyp Kation Kanals, Unterfamilie M, 7 (TRPM7) in Thrombozyten untersucht. Unter Verwendung von Trpm7R/RMäusen mit einer Punktmutation in der Kinasedomäne, welche die Aktivität der Kinase blockiert, konnten wir zeigen, dass die TRPM7-Kinase die Thrombozytenaktivierung über Immunorezeptor-Tyrosin-basierte Aktivierungsmotive (ITAM), Hem(ITAM) und Protease-aktivierte Rezeptoren (PAR) reguliert. Trpm7R/RMäuse waren vor in vivoThrombose und Schlaganfall geschützt, was die TRPM7 Kinase als vielversprechendes antithrombotisches Zielprotein etabliert.Im zweiten Teil wurde die Rolle des TRPM7 Kanals in einer Megakaryozyten (MK)-und Plättchen-spezifischen Knockout Maus(Trpm7fl/fl-Pf4Cre) untersucht. Wir konnten zeigen, dass Trpm7fl/fl-Pf4CrePlättchen je nach Art der Stimulation entweder erhöhte oder verminderte Reaktionen zeigten. Obwohl Trpm7fl/fl-Pf4Cre-Mäuse thrombozytopen waren, wurden keine Unterschiede in in vivoThrombosemodellen und Schlaganfall beobachtet. Diese Studien heben hervor, dass die Hemmung der TRPM7 Kinase, aber nicht die des Kanal selbst (inMKs und Plättchen), eine vielversprechende anti-thrombotische Therapie sein könnte. Neben TRPM7 untersuchten wir die Rolle von Magnesium Transporter 1 (MAGT1)in der Mg2+-Homöostase in Thrombozyten und konnten zeigen, dass MAGT1 primär den Rezeptor-gesteuerten Calciuminflux (ROCE) spezifisch nach GPVI Aktivierung reguliert. Dieser physiologische Crosstalk wird durch Proteinkinase C (PKC) Isoformen vermittelt. Thrombozyten von Magt1-/yMäusen reagierten in vitrohyperreaktiv auf GPVI und ThromboxanA2(TXA2) Rezeptor Stimulation. Dementsprechend konnte auch gezeigt werden, dass Magt1-/yPlättchen in Modellen von Thrombose und Schlaganfall pro-thrombotisch wirkten.Um die ITAM-Signalübertragung in Thrombozyten mit der in T und B Zellen zu vergleichen, untersuchten wir die Rolle von MAGT1 in Immunzellen. Wir überprüften die Rolle von MAGT1 in Mäusen unter pathogen-freien Bedingungen. Magt1-/yB Zellen zeigten eine dysregulierte Mg2+und Ca2+Homöostase nach Aktivierung des B Zell Rezeptors, wodurch Syk, LAT, PLCγ2 und PKC Phosphorylierung beeinflusst wurde. Im Gegensatz zu menschlichen MAGT1-defizienten T Zellen, zeigten Magt1-/yT Zellen keine Veränderungen in Entwicklung und Effektorfunktion. Schließlich beschrieben wir im letzten Teil der Arbeit Methoden zur Messung des intrazellulären freien Zinks [Zn2+]iin humanen und murinen Thrombozyten mit Storage-Pool-Defekt (SPD). Wir unterbreiten in dieser Thesis, den [Zn2+]iStatus in SPD Thrombozyten zu messenum nachAnomalien in den Thrombozyten-Granula zu suchen. KW - Thrombozyt KW - Kationen-Homöostase KW - Homöostase KW - Cation Homeostasis KW - Platelets Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176616 ER - TY - JOUR A1 - Maiellaro, Isabella A1 - Lohse, Martin J. A1 - Kitte, Robert J. A1 - Calebiro, Davide T1 - cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons JF - Cell Reports N2 - The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons. KW - cAMP KW - synaptic plasticity KW - PDE KW - octopamine KW - FRET KW - active zone KW - dunce KW - GPCR Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162324 VL - 17 IS - 5 ER - TY - JOUR A1 - Calebiro, Davide A1 - Maiellaro, Isabella T1 - cAMP signaling microdomains and their observation by optical methods JF - Frontiers in Cellular Neuroscience N2 - The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains. KW - G protein-coupled receptor KW - cyclic AMP KW - signaling microdomain KW - fluorescence resonance energy transfer KW - neurons Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118252 SN - 1662-5102 VL - 8 ER - TY - JOUR A1 - Jeanclos, Elisabeth A1 - Knobloch, Gunnar A1 - Hoffmann, Axel A1 - Fedorchenko, Oleg A1 - Odersky, Andrea A1 - Lamprecht, Anna‐Karina A1 - Schindelin, Hermann A1 - Gohla, Antje T1 - Ca\(^{2+}\) functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin JF - FEBS Letters N2 - Pyridoxal 5′‐phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ‐aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)‐ and integrin‐binding protein 1 (CIB1) as a PDXP interactor by yeast two‐hybrid screening and find a calmodulin (CaM)‐binding motif that overlaps with the PDXP‐CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP‐CIB1 complex may functionally differ from the PDXP‐Ca\(^{2+}\)‐CaM complex. KW - calmodulin KW - chronophin KW - CIB1 KW - haloacid dehalogenase KW - pyridoxal phosphatase KW - vitamin B6 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217963 VL - 594 IS - 13 SP - 2099 EP - 2115 ER - TY - JOUR A1 - Tilstam, Pathricia V. A1 - Gijbels, Marion J. A1 - Habbeddine, Mohamed A1 - Cudejko, Celine A1 - Asare, Yaw A1 - Theelen, Wendy A1 - Zhou, Baixue A1 - Döring, Yvonne A1 - Drechsler, Maik A1 - Pawig, Lukas A1 - Simsekyilmaz, Sakine A1 - Koenen, Rory R. A1 - de Winther, Menno P. J. A1 - Lawrence, Toby A1 - Bernhagen, Jürgen A1 - Zernecke, Alma A1 - Weber, Christian A1 - Noels, Heidi T1 - Bone Marrow-Specific Knock-In of a Non-Activatable Ikkα Kinase Mutant Influences Haematopoiesis but Not Atherosclerosis in Apoe-Deficient Mice JF - PLOS ONE N2 - Background: The Ikkα kinase, a subunit of the NF-kappa B-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. However, the role of Ikkα-mediated phosphorylation in haematopoiesis and atherogenesis remains unexplored. In this study, we investigated the effect of a bone marrow (BM)-specific activation-resistant Ikk alpha mutant knock-in on haematopoiesis and atherosclerosis in mice. Methods and Results: Apolipoprotein E (Apoe)-deficient mice were transplanted with BM carrying an activation-resistant Ikkα gene (Ikkα(AA/AA) Apoe(-/-)) or with Ikkα(+/+) Apoe(-/-) BM as control and were fed a high-cholesterol diet for 8 or 13 weeks. Interestingly, haematopoietic profiling by flow cytometry revealed a significant decrease in B-cells, regulatory T-cells and effector memory T-cells in Ikkα(AA/AA) Apoe(-/-) BM-chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences were observed in the size, stage or cellular composition of atherosclerotic lesions in the aorta and aortic root of Ikkα(AA/AA) Apoe(-/-) vs Ikkα(+/+) Apoe(-/-) BM-transplanted mice, as shown by histological and immunofluorescent stainings. Necrotic core sizes, apoptosis, and intracellular lipid deposits in aortic root lesions were unaltered. In vitro, BM-derived macrophages from Ikkα(AA/AA) Apoe(-/-) vs Ikkα(+/+) Apoe(-/-) mice did not show significant differences in the uptake of oxidized low-density lipoproteins (oxLDL), and, with the exception of Il-12, the secretion of inflammatory proteins in conditions of Tnf-α or oxLDL stimulation was not significantly altered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array were comparable. Conclusion: Our data reveal an important and previously unrecognized role of haematopoietic Ikkα kinase activation in the homeostasis of B-cells and regulatory T-cells. However, transplantation of Ikkα AA mutant BM did not affect atherosclerosis in Apoe(-/-) mice. This suggests that the diverse functions of Ikkα in haematopoietic cells may counterbalance each other or may not be strong enough to influence atherogenesis, and reveals that targeting haematopoietic Ikkα kinase activity alone does not represent a therapeutic approach. KW - NF-KAPPA-B KW - regulatory T cells KW - indoleamine 2,3-dioxygenase KW - dendritic cells KW - gene expression KW - increases atherosclersosis KW - receptor KW - inhibition KW - pathway KW - beta Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117450 VL - 9 IS - 2 ER - TY - JOUR A1 - Göbel, Kerstin A1 - Pankratz, Susann A1 - Asaridou, Chloi-Magdalini A1 - Herrmann, Alexander M. A1 - Bittner, Stefan A1 - Merker, Monika A1 - Ruck, Tobias A1 - Glumm, Sarah A1 - Langhauser, Friederike A1 - Kraft, Peter A1 - Krug, Thorsten F. A1 - Breuer, Johanna A1 - Herold, Martin A1 - Gross, Catharina C. A1 - Beckmann, Denise A1 - Korb-Pap, Adelheid A1 - Schuhmann, Michael K. A1 - Kuerten, Stefanie A1 - Mitroulis, Ioannis A1 - Ruppert, Clemens A1 - Nolte, Marc W. A1 - Panousis, Con A1 - Klotz, Luisa A1 - Kehrel, Beate A1 - Korn, Thomas A1 - Langer, Harald F. A1 - Pap, Thomas A1 - Nieswandt, Bernhard A1 - Wiendl, Heinz A1 - Chavakis, Triantafyllos A1 - Kleinschnitz, Christoph A1 - Meuth, Sven G. T1 - Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells JF - Nature Communications N2 - Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein–kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders. KW - blood coagulation KW - factor XII KW - neuroinflammation KW - dendric cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165503 VL - 7 IS - 11626 ER - TY - JOUR A1 - Bieber, Michael A1 - Schuhmann, Michael K. A1 - Bellut, Maximilian A1 - Stegner, David A1 - Heinze, Katrin G. A1 - Pham, Mirko A1 - Nieswandt, Bernhard A1 - Stoll, Guido T1 - Blockade of platelet glycoprotein Ibα augments neuroprotection in Orai2-deficient mice during middle cerebral artery occlusion JF - International Journal of Molecular Sciences N2 - During ischemic stroke, infarct growth before recanalization diminishes functional outcome. Hence, adjunct treatment options to protect the ischemic penumbra before recanalization are eagerly awaited. In experimental stroke targeting two different pathways conferred protection from penumbral tissue loss: (1) enhancement of hypoxic tolerance of neurons by deletion of the calcium channel subunit Orai2 and (2) blocking of detrimental lymphocyte–platelet responses. However, until now, no preclinical stroke study has assessed the potential of combining neuroprotective with anti-thrombo-inflammatory interventions to augment therapeutic effects. We induced focal cerebral ischemia in Orai2-deficient (Orai2\(^{-/-}\)) mice by middle cerebral artery occlusion (MCAO). Animals were treated with anti-glycoprotein Ib alpha (GPIbα) Fab fragments (p0p/B Fab) blocking GPIbα–von Willebrand factor (vWF) interactions. Rat immunoglobulin G (IgG) Fab was used as the control treatment. The extent of infarct growth before recanalization was assessed at 4 h after MCAO. Moreover, infarct volumes were determined 6 h after recanalization (occlusion time: 4 h). Orai2 deficiency significantly halted cerebral infarct progression under occlusion. Inhibition of platelet GPIbα further reduced primary infarct growth in Orai2\(^{-/-}\) mice. During ischemia–reperfusion, upon recanalization, mice were likewise protected. All in all, we show that neuroprotection in Orai2\(^{-/-}\) mice can be augmented by targeting thrombo-inflammation. This supports the clinical development of combined neuroprotective/anti-platelet strategies in hyper-acute stroke. KW - ischemic penumbra KW - Orai2 KW - glycoprotein receptor Ibα KW - ischemic stroke KW - thrombo-inflammation KW - middle cerebral artery occlusion Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286038 SN - 1422-0067 VL - 23 IS - 16 ER - TY - THES A1 - Beitzinger, Christoph T1 - Binding-, Blocking- and Translocation-Processes Concerning Anthrax-Toxin and Related Bacterial Protein-Toxins of the AB7-Family T1 - Charakterisierung von Bindungs-, Blockierungs- und Translokationsprozessen am Anthrax-Toxin und verwanten Toxinen der AB7-Gruppe N2 - Bacterial protein toxins belong to the most potent toxins which are known. They exist in many different forms and are part of our every day live. Some of them are spread by the bacteria during infections and therefore play a crucial role in pathogenicity of these strains. Others are secreted as a defense mechanism and could be uptaken with spoiled food. Concerning toxicity, some of the binary toxins of the AB7-type belong to the most potent and dangerous toxins in the world. Even very small amounts of these proteins are able to cause severe symptoms during an infection with pathogen species of the genus Clostridium or Bacillus. Apart from the thread the toxins constitute, they exhibit a unique way of intoxication. Members of the AB7-toxin family consist of a pore-forming subunit B, that acts as a molecular syringe to translocate the enzymatic moieties A into the cytosol of target cells. This complex mechanism does not only kill cells with high efficiency and therefore should be studied for treatment, but also displays a possibility to address certain cells with a specific protein cargo if used as a molecular delivery tool. Concerning both issues, binding and translocation of the channel are the crucial steps to either block or modify the system in the desired way. To gain deeper insight into the transport of binary toxins the structure of the B subunit is of great importance, but being a membrane protein, no crystal could be obtained up to now for either protective antigen (PA) of Anthrax toxin or any other AB7-type binding domain. Therefore, the method of choice in this work is an electro-physical approach using the so-called black-lipid-bilayer system for determination of biophysical constants. Additionally, diverse cell based assays serve as a proving method for the data gained during in vitro measurements. Further information was gathered with specially designed mutants of the protein channel. The first part of this thesis focuses on the translocation process and its possible use as a molecular tool to deliver protein cargo into special cell types. The task was addressed by measuring the binding of different effector proteins related and unrelated to the AB7 toxin family. These proteins were tested in titration experiments for the blockage of the ion current through a membrane saturated with toxin channels. Especially the influence of positively charged His-tags has been determined in detail for PA and C2II. As described in chapter 2, a His-tag transferred the ability of being transported by PA, but not by C2II, to different proteins like EDIN (from S. aureus) in vitro and in cell-based experiments. This process was found to change the well-known voltage-dependency of PA to a huge extend and therefore is related to membrane potentials which play a crucial role in many processes in living cells. Chapter 3 sums up findings, which depict that binding partners of PA share certain common motives. These could be detected in a broad range of substrates, ranging from simple ions in an electrolyte over small molecules to complex protein effectors. The gathered information could be further used to design blocker-substrates for treatment of Anthrax infections or tags, which render PA possible as a molecular syringe for cargo proteins. The deeper insight to homologies and differences of binary toxin components is the core of chapter 4, in which the cross-reactivity of Anthrax and C2-toxin was analyzed. The presented results lead to a better understanding of different motives involved in binding and translocation to and via the B components PA and C2II, as well as the enzymatically active A moieties edema factor (EF), lethal factor (LF) and C2I. In the second part of the thesis, the blockage of intoxication is the center of interest. Therefore, chapter 5 focuses on the analysis of specially designed blocker-substrate molecules for PA. These molecules form a plug in the pore, abolishing translocation of the enzymatic units. Especially, if multi-resistant strains of Anthrax (said to be already produced in Russia as a biological weapon) are taken into consideration, these substrates could stop intoxication and buy time, to deal with the infection. Chapter 6 describes the blockage of PA-channels by anti-His antibody from the trans-side of the porin, an effect which was not described for any other antibody before. Interestingly, even mutation of the estimated target amino acid Histidine 310 to Glycine could not interfere with this ionic strength dependent binding. N2 - Bakterielle Protein-Toxine gehören zu den wirksamsten bekannten Toxinen. In vielfältigen Variationen findet man sie in allen Bereichen des Lebens. Einige werden von den Bakterien während einer Infektion freigesetzt und übernehmen einen wichtigen Part in der Pathogenität. Andere werden zu Verteidigungszwecken sekretiert und können in verdorbenen Lebensmitteln gefunden werden. Was die Wirkung binärer Toxine der AB7-Gruppe angeht, so gehören diese zu den potentesten und gefährlichsten Giften weltweit. Selbst kleine Mengen dieser Proteine können schwerste Symptome während einer Infektion mit Bakterien der Gattung Clostridium oder Bacillus verursachen. Abgesehen von der Bedrohung die durch die Toxine ausgeht, zeichnen sie sich durch einen einzigartigen Intoxikationsmechanismus aus. AB7-Toxine sind aus einer porenformenden Domäne B, die als eine Art molekulare Injektionskanüle fungiert, und enzymatisch aktiven Proteinen A zusammengesetzt. Der komplexe Wirkmechanismus ermöglicht es nicht nur Zellen in höchst effektiver Weise abzutöten und sollte deswegen zu Behandlungszwecken untersucht werden, sondern könnte auch als molekulares Werkzeug umfunktioniert werden, um spezielle Zellen mit gewünschten Proteinen zu beladen. Für beide Zwecke (Blockierung und gezielter Transport) ist die Bindung an, und der Transport durch die porenformende Domäne von größter Bedeutung. Die Struktur der B-Domäne ist wichtig um tiefere Einsicht in den Transportprozess der binären Toxine zu ermöglichen. Leider ist es bisher nicht gelungen die Kristallstruktur des Membranproteins protective antigen (PA) von Anthrax oder irgendeiner anderen Bindedomäne eines AB7-Toxins zu lösen. Deshalb wurde in dieser Arbeit ein elektrophysiologischer Ansatz zur Bestimmung der biophysikalischen Konstanten des Prozesses gewählt, die Black-lipid-Bilayer Methode. Zusätzliche Versuche an Zellen und mit Mutanten der Proteine dienen zur Absicherung der in vitro Ergebnisse. Im ersten Teil der Arbeit wird der Translokationsmechanismus, und die mögliche Nutzung dessen als molekulares Werkzeug näher behandelt. Dies erfolgte durch Bindungsstudien an PA und C2II mit Effektoren (sowohl aus der AB7-Familie, als auch nicht näher verwandt). In Kapitel 2 wird beschrieben, dass ein His-Tag es EDIN (von S. aureus) und anderen Effektoren ermöglicht, dass ein Transport durch PA-Poren in vitro und in vivo stattfindet (nicht aber durch C2II). Ebenfalls konnte eine starke Abweichung in der bekannten Spannungsabhängigkeit von PA festgestellt werden, die den Prozess eindeutig mit den Membranpotentialen in Verbindung bringt, die häufig eine wichtige Rolle im Metabolismus spielen. Kapitel 3 fasst zusammen, dass Bindungspartner von PA bestimmte Motive beinhalten, die von Substraten wie Ionen in Elektrolyten, über kleine Moleküle, bis hin zu komplexen Proteinen reichen. Diese Erkenntnisse könnten genutzt werden um Blockersubstanzen zur Behandlung von Anthrax, oder Tags zur Aufnahme durch Anthrax zu designen. Neueste Befunde zu Homologien und Unterschieden zwischen den Komponenten der binären Toxine sind der Kern von Kapitel 4, in dem die Kreuzreaktivität von Anthrax und C2-Toxin analysiert wurde. Die enthaltenen Daten ermöglichen einen tieferen Einblick in die verschiedenen Stufen der Bindung und Translokation des edema factor (EF), des lethal factor (LF) und von C2I an und durch PA und C2II. Im zweiten Teil rückt die Blockierung der Intoxikation in den Fokus. Die Analyse speziell designter Blockersubstanzen für PA wird in Kapitel 5 behandelt. Diese formen einen Porenverschluss, der weiteren Transport von Toxinkomponenten verhindert. Eine besondere Bedeutung könnten diese Substanzen im Zusammenhang mit Multiresitenz bei Anthrax Stämmen (vermutlich in Russland als biologische Waffe hergestellt) zur Verhinderung von Symptomen und der Verlängerung der Zeit spielen, die man hat um neue Antibiotika zu erzeugen. Kapitel 6 beschreibt zum ersten Mal die Blockierung von PA-Poren mittels eines Anti-His Antikörpers von der trans-Seite aus. Interessanterweise trat diese Ionenstärke abhängige Blockierung, auch bei einer Histidin zu Glycin Mutation an der Stelle 310 (vermutete Bindeposition) auf. KW - Bacillus anthracis KW - Translokation KW - Toxin KW - Protective antigen KW - Bindungsprozess KW - Black-lipid-bilayer KW - Bacillus anthracis KW - protective antigen KW - Anthrax Toxin KW - translocation Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70052 ER - TY - JOUR A1 - Makbul, Cihan A1 - Kraft, Christian A1 - Grießmann, Matthias A1 - Rasmussen, Tim A1 - Katzenberger, Kilian A1 - Lappe, Melina A1 - Pfarr, Paul A1 - Stoffer, Cato A1 - Stöhr, Mara A1 - Wandinger, Anna-Maria A1 - Böttcher, Bettina T1 - Binding of a pocket factor to Hepatitis B virus capsids changes the rotamer conformation of Phenylalanine 97 JF - Viruses N2 - (1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses. KW - Hepatitis B Virus KW - pocket factor KW - Triton X 100 KW - envelopment KW - maturation signal KW - single strand blocking KW - electron cryo-microscopy KW - isothermal titration calorimetry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248565 SN - 1999-4915 VL - 13 IS - 11 ER - TY - THES A1 - Batsching, Sophie Johanna T1 - Behavior under uncontrollable stress in \(Drosophila\) \(melanogaster\) - Learned Helplessness revisited T1 - Verhalten unter nicht kontrollierbarem Stress - Neubetrachtung der Erlernten Hilflosigkeit bei \(Drosophila\) \(melanogaster\) N2 - In order to select the appropriate behavior, it is important to choose the right behavior at the right time out of many options. It still remains unclear nowadays how exactly this is managed. To address this question, I expose flies (Drosophila melanogaster) to uncontrollable stress to study their behavior under restrictive circumstances by using the so-called shock box. Exposing animals to uncontrollable stress may have an impact on subsequent behavior and can last for some time. The animal learns that whatever it does, it cannot change the situation and therefore can develop something called learned helplessness. The term was first conceptualized by two American psychologists Maier and Seligman (1967), who discovered this phenomenon while doing experiments with dogs. They found out that dogs which are exposed to inescapable stress, later fail in a learning task (‘shuttle box’). In this work the walking patterns of three different types of experimental flies, walking in a small dark chamber, were evaluated. Using the triadic design (Seligman and Maier, 1967), flies were either exposed to electric shock randomly (yoked), could turn it off by being active (master) or did not receive punishment at all (control). Master flies were shocked whenever they sat for more than 0.9 seconds. At the same time yoked flies received a shock as well independent of what they were doing, to ensure the same amount of shocks received and to create random punishment pattern for the yoked group. With this so-called no-idleness paradigm flies were conditioned either 10 minutes, which resulted in a short (3 minutes) after-effect, or 20 minutes that turned out to be more stable (10 minutes). In a second part, the behavior during the 20 minute conditioning and a 10 minutes post-test was described in detail. Female flies of the yoked group developed lower activity levels, longer pauses and walked more slowly than master and control flies during conditioning. In the time after the shocks while still in the box, the yoked flies also reduced the frequency and duration of walking bouts as well as their walking speed. Additionally, they took more time to resume walking after the onset of an electric shock than master flies (escape latency) and turned out to make less pauses lasting between 1-1.5 seconds which supports the finding concerning the escape latency. Male flies, tested under the same conditions, showed a slightly weaker after-effect regarding the difference between master and yoked during conditioning and post-test when compared to female flies. When comparing the 20 minutes conditioning with subsequent 10 minutes test in the heat and the shock box in parallel, one finds the same effect: Flies which do not have control over the shocks, lower their activity, make less but longer pauses and walk more slowly than their respective master flies. Despite the similar effect of heat and shock on the flies, some differences between the devices occurred, which can partly be explained by different humidity conditions as well as by different surfaces within the chambers. When the control over the shocks is given back to the yoked flies, it takes them about seven minutes to realize it. One could also show that dopamine levels in the brain were reduced in comparison to flies which did not receive shocks. Yoked flies also were impaired in a place learning task (place learning) and their reaction to light (exit from the box towards the light) directly after conditioning. After characterizing the walking behavior in the chambers, the study deals with the question whether the effects observed in the chambers transfer to different environments. In free walk they only differed from flies which did not receive electric shocks and no effect of uncontrollability was transferred to courtship behavior. Handling as the cause could be excluded. Since handling could be exclude to be the cause of losing the effect, I assumed that the behavior shown in the boxes are context depend. Not only were the after-effects of inescapable shock subject of the current research also the impact of the rearing situation on the response to electric shock was investigated in the present study. Flies which grew up in a single-reared situation turned out to be less affected by inescapable stress in both sexes. In the next part, the first steps to unravel the neuronal underpinning were taken. A mutant – fumin – which is defective in the dopamine re-uptake transporter showed less reaction to inescapable foot shocks, while a mutant for the gene which encodes an adenylate cyclase (rutabaga2080) resulted in a good score during conditioning, but showed no stable after-effect. Downregulating the expression of the adenylate cyclase gene (rutabaga) in different parts of the mushroom bodies showed, that rutabaga is necessary in the α’β’-lobes for expressing the differences between master and yoked flies in the no-idleness paradigm. The study further confirmed previous findings, that rutabaga is needed in operant but not in classical conditioning. As a result, the study could show that not the stimulus itself causes the state of uncontrollability but the fact that the fly learned that it was not in control of the stimulus. This state turned out to be context and time dependent. N2 - Eine wichtige Aufgabe für ein Tier ist es, das passende Verhalten zur richtigen Zeit zu wählen. Heutzutage ist immer noch unklar, wie dieser Prozess exakt abläuft. Zur Untersuchung dieser Frage werden Fliegen (Drosophila melanogaster) in der so genannten Schockbox unkontrollierbarem Stress ausgesetzt um auf diesem Weg Verhaltenskontrolle unter stressigen und stark restriktiven Umständen untersuchen zu können. Wenn Tiere unkontrollierbarem Stress ausgesetzt sind, kann dieser Zustand sowohl langanhaltend sein als auch Einfluss auf das Folgeverhalten haben. Das Tier lernt, dass alle Aktivitäten, die es in dieser Situation unternimmt keinen Einfluss auf die Situation haben. Dadurch kann das Tier einen Zustand der sogenannten Erlernten Hilflosigkeit entwickeln. Dieser Begriff wurde von zwei amerikanischen Psychologen, Maier und Seligman (1976), geprägt, die dieses Phänomen während Experimenten mit Hunden entdeckten und konzipierten. Sie fanden heraus, dass Hunde, die unkontrollierbarem Stress ausgesetzt waren, an einer anschließend gestellten Lernaufgabe scheiterten (‚shuttle-box‘). Gegenstand der vorliegenden Arbeit ist es, das Laufmuster in einer schmalen und kleinen Kammer an drei verschiedenen Versuchsgruppen von Fliegen zu analysieren. Unter Verwendung des sogenannten triadischen Konzepts (Seligman and Maier, 1967) wurden die Fliegen drei unterschiedlichen Situationen ausgesetzt: Zufällige Elektroschocks (Yoked-Gruppe), durch Laufen abschaltbare Elektroschocks (Master-Gruppe) oder keine Bestrafung (Kontroll-Gruppe). Master-Fliegen wurden immer dann geschockt, wenn sie für länger als 0,9 Sekunden saßen. Unabhängig ihres Verhaltens erhielten die Yoked-Fliegen zeitgleich einen Schock um einen zufälligen Bestrafungsreiz zu generieren. Mit diesem so genannten ‚no-idleness‘ (nicht ruhen dürfen) Paradigma wurden die Fliegen entweder zehn Minuten oder 20 Minuten konditioniert. Während eine zehnminütige Konditionierung zu einem kurzen Nacheffekt führte (Nacheffekt von drei Minuten), stellte sich die zwanzigminütige Konditionierung als nachhaltiger heraus (Nacheffekt von zehn Minuten). In einem zweiten Teil der Arbeit wurde das Verhalten der Fliegen sowohl während der zwanzig Minuten andauernden Konditionierung also auch im nachfolgenden zehnminütigen Test im Detail beschrieben. Während der zwanzigminütigen Konditionierung zeigten weibliche Yoked-Fliegen eine geringere Aktivität, saßen länger und liefen langsamer als Master- oder Kontroll-Fliegen. In der Zeit nach den Schocks, zeigten sie immer noch eine verminderte Lauffrequenz sowie kürzere und langsamere Laufphasen. Zusätzlich benötigten sie länger um nach dem Einsetzten eines Elektroschocks loszulaufen (Flucht-Latenzzeit) und machten weniger Kurzpausen die zwischen 1 bis 1,5 Sekunden lang waren. Dies unterstützt das Ergebnis der verlängerten Flucht-Latenzzeit. Männchen, die unter gleichen Bedingungen getestet wurden, wiesen im Vergleich zu weiblichen Fliegen eine leicht abgeschwächte Reaktion bezüglich des Master-Yoked-Unterschieds auf. Wenn die Konditionierung mit dem anschließenden Test in der Schock- und der Hitzekammer gleichzeitig durchgeführt wurde, resultierte dies in vergleichbaren Ergebnissen: Fliegen, die keine Kontrolle über den Reiz haben, vermindern ihr Aktivitätslevel, sitzen seltener aber länger und laufen langsamer als die dazugehörigen Master-Fliegen. Neben der Tatsache, dass ein ähnlicher Effekt auftritt, weisen die Apparaturen dennoch kleine Unterschiede auf. Diese können zu Teilen mit den unterschiedlichen Luftfeuchtigkeitsniveaus als auch durch die Verschiedenheit der Laufoberfläche der jeweiligen Kammern erklärt werden. Wird den Fliegen die Kontrolle über die Schocks zurückgegeben, benötigen sie etwa sieben Minuten um dies zu erkennen. Zudem konnte gezeigt werden, dass die Dopaminkonzentration in den Köpfen, im Vergleich zu Tieren die keine Schocks erhalten haben, vermindert war. Yoked-Fliegen wiesen außerdem unmittelbar nach der Konditionierung Defekte im Ortslernen und in ihrer positiven Reaktion auf Licht auf. Nachdem das Laufverhalten innerhalb der Kammern ausführlich charakterisiert wurde, geht diese Studie darauf ein, ob die Effekte, die in den Kammern gemessen wurden, auch in anderen Umgebungen zu beobachten sind. Im freien Lauf unterschieden sie sich lediglich von Fliegen, die keine Schocks erhalten hatten und es sind keine Auswirkungen durch Kontrollverlust im Paarungsverhalten festzustellen. Da die Handhabung der Tiere als Grund für den Verlust des Nacheffektes ausgeschlossen werden konnte, lässt sich schlussfolgern, dass das Verhalten das in den Kammern gemessen wurde, kontextabhängig ist. Zusätzlich zur Untersuchung der Auswirkungen unausweichlichen Stresses, wurde der Einfluss, der Aufzuchtbedingungen auf die Stress-Antwort in der vorliegenden Studie untersucht. Fliegen, die einzeln aufgezogen wurden, weisen bei beiden Geschlechtern eine verminderte Antwort auf Stress auf. Im darauffolgenden Abschnitt wurden erste Schritte unternommen, um die neuronalen Grundlagen der Erlernten Hilflosigkeit zu untersuchen. Eine Mutante – fumin – die ein defektes Wiederaufnahmetransporter-Gen für Dopamin besitzt, wies eine verminderte Stressantwort auf. Während eine Mutante des Adenylatzyklasegens (rutabaga2080) normale Ergebnisse während der Konditionierung aufzeigten, war im Post-test kein signifikanter Nacheffekt messbar. Das Herunterregulieren des Adenylatcyclasengens (rutabaga), in verschiedenen Teilen der Pilzkörper, zeigte dass die Expression von rutabaga in den α’β’-Loben für die Entwicklung der Erlernten Hilflosigkeit im no-idleness Paradigma benötigt wird. Zudem konnten vorangegangene Studien bestätigt werden, die rutabaga eine Rolle im operanten Lernen jedoch nicht im klassischen Lernen zuordnen. Als Fazit zeigt die Studie, dass nicht der Stressor selbst, sondern die Unkontrollierbarkeit des Stressors der Grund für die Entwicklung der Erlernten Hilflosigkeit darstellt und das Phänomen, innerhalb der hier gewählten Zeitspanne (20 Minuten Stress), kontextabhängig zu sein scheint. KW - Taufliege KW - Stress KW - Verhalten KW - Gelernte Hilflosigkeit KW - Erlernte Hilflosigkeit KW - Learned Helplessness KW - Behavior KW - Drosophila melanogaster Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145416 ER - TY - JOUR A1 - Wippel, Carolin A1 - Maurer, Jana A1 - Fortsch, Christina A1 - Hupp, Sabrina A1 - Bohl, Alexandra A1 - Ma, Jiangtao A1 - Mitchell, Timothy J. A1 - Bunkowski, Stephanie A1 - Brück, Wolfgang A1 - Nau, Roland A1 - Iliev, Asparouh I. T1 - Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage JF - PLoS Pathogens N2 - Abstract Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage. Author Summary Bacterial meningitis is one of the most devastating brain diseases. Among the bacteria that cause meningitis, Streptococcus pneumoniae is the most common. Meningitis predominantly affects children, especially in the Third World, and most of them do not survive. Those that do survive often suffer permanent brain damage and hearing problems. The exact morphological substrates of brain damage in Streptococcus pneumoniae meningitis remain largely unknown. In our experiments, we found that the brain cortex of patients with meningitis demonstrated a loss of synapses (the contact points among neurons, responsible for the processes of learning and memory), and we identified the major pneumococcal neurotoxin pneumolysin as a sufficient cause of this loss. The effect was not direct but was mediated by the brain neurotransmitter glutamate, which was released upon toxin binding by one of the non-neuronal cell types of the brain – the astrocytes. Pneumolysin initiated calcium influx in astrocytes and subsequent glutamate release. Glutamate damaged the synapses via NMDA-receptors – a mechanism similar to the damage occurring in brain ischemia. Thus, we show that synaptic loss is present in pneumococcal meningitis, and we identify the toxic bacterial protein pneumolysin as the major factor in this process. These findings alter our understanding of bacterial meningitis and establish new therapeutic strategies for this fatal disease. KW - synapses KW - brain damage KW - astrocytes KW - neuronal dendrites KW - meningitis KW - glutamate KW - bacterial meningitis KW - neocortex Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130462 VL - 9 IS - 6 ER - TY - JOUR A1 - Bangalore, Disha M. A1 - Heil, Hannah S. A1 - Mehringer, Christian F. A1 - Hirsch, Lisa A1 - Hemmen, Katharina A1 - Heinze, Katrin G. A1 - Tessmer, Ingrid T1 - Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases JF - Scientific Reports N2 - Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions. KW - atomic-force microscopy KW - base pairs KW - molecular structure KW - crystal structure KW - structural basis KW - repair KW - recognition KW - 8-oxoguanine KW - thymine KW - mismatches Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231338 VL - 10 ER - TY - THES A1 - Busch, Martin T1 - Aortic Dendritic Cell Subsets in Healthy and Atherosclerotic Mice and The Role of the miR-17~92 Cluster in Dendritic Cells T1 - Subsets dendritischer Zellen in der Aorta gesunder und atherosklerotischerMäuse und die Rolle des miR-17~92 Clusters in dendritischen Zellen N2 - Atherosclerosis is accepted to be a chronic inflammatory disease of the arterial vessel wall. Several cellular subsets of the immune system are involved in its initiation and progression, such as monocytes, macrophages, T and B cells. Recent research has demonstrated that dendritic cells (DCs) contribute to atherosclerosis, too. DCs are defined by their ability to sense and phagocyte antigens, to migrate and to prime other immune cells, such as T cells. Although all DCs share these functional characteristics, they are heterogeneous with respect to phenotype and origin. Several markers have been used to describe DCs in different lymphoid and non-lymphoid organs; however, none of them has proven to be unambiguous. The expression of surface molecules is highly variable depending on the state of activation and the surrounding tissue. Furthermore, DCs in the aorta or the atherosclerotic plaque can be derived from designated precursor cells or from monocytes. In addition, DCs share both their marker expression and their functional characteristics with other myeloid cells like monocytes and macrophages. The repertoire of aortic DCs in healthy and atherosclerotic mice has just recently started to be explored, but yet there is no systemic study available, which describes the aortic DC compartment. Because it is conceivable that distinct aortic DC subsets exert dedicated functions, a detailed description of vascular DCs is required. The first part of this thesis characterizes DC subsets in healthy and atherosclerotic mice. It describes a previously unrecognized DC subset and also sheds light on the origin of vascular DCs. In recent years, microRNAs (miRNAs) have been demonstrated to regulate several cellular functions, such as apoptosis, differentiation, development or proliferation. Although several cell types have been characterized extensively with regard to the miRNAs involved in their regulation, only few studies are available that focus on the role of miRNAs in DCs. Because an improved understanding of the regulation of DC functions would allow for new therapeutic options, research on miRNAs in DCs is required. The second part of this thesis focuses on the role of the miRNA cluster miR- 17~92 in DCs by exploring its functions in healthy and atherosclerotic mice. This thesis clearly demonstrates for the first time an anti-inflammatory and atheroprotective role for the miR17-92 cluster. A model for its mechanism is suggested. N2 - Atherosklerose ist eine chronisch-entzündliche Erkrankung der arteriellen Gefäßwand und zahlreiche Zellen des Immunsystems, wie zum Beispiel Monozyten, Makrophagen, T und B Zellen sind an der Entstehung und Entwicklung beteiligt. Aktuelle Forschungsergebnisse haben gezeigt, dass auch dendritische Zellen (DCs) zur Atherosklerose beitragen. DCs sind durch ihre Fähigkeit gekennzeichnet, Antigene zu erkennen, aufzunehmen, zu migrieren und andere Immunzellen, wie zum Beispiel T Zellen, zu aktivieren. Auch wenn alle DCs diese funktionellen Merkmale teilen, so sind sie in Bezug auf ihren Phänotyp oder Ursprung eine eher heterogene Gruppe. Zahlreiche Oberflächenmoleküle wurden in der Vergangenheit genutzt, um DCs in lymphatischen und nicht-lymphatischen Geweben zu beschreiben. Allerdings hat sich keines dieser Moleküle als spezifisch und unverwechselbar erwiesen. Die Expression von Oberflächenmolekülen ist sehr variabel und hängt nicht nur vom Aktivierungszustand der DCs, sondern auch vom umliegenden Gewebe ab. Dazu kommt, dass DCs in der Aorta, beziehungsweise im atherosklerotischen Plaque, von designierten Vorläuferzellen, aber auch von Monozyten abstammen können und DCs das Profil ihrer Oberflächenmoleküle, sowie ihre funktionellen Eigenschaften, mit anderen myeloiden Zellen wie Monozyten und Makrophagen teilen. Neuere Arbeiten haben damit begonnen das Repertoire an DCs in der Aorta von gesunden und atherosklerotischen Mäusen zu untersuchen. Da es naheliegt, dass verschiedene DC Untergruppen ganz bestimmte Funktionen ausüben, wird eine detaillierte Beschreibung vaskulärer DCs in der Forschung benötigt. Weil es hierzu allerdings bislang kaum Studien gibt, untersucht der erste Teil dieser Arbeit zum ersten Mal systematisch die in gesunden und atherosklerotischen Mäusen vorkommenden Gruppen an DCs. Sie beschreibt außerdem eine zuvor nicht beachtete DC-Untergruppe und gibt Aufschluss über den Ursprung vaskulärer DCs. In den letzten Jahren wurde gezeigt, dass microRNAs (mirRNAs) zahlreiche zelluläre Vorgänge wie Apoptose, Differenzierung, Entwicklung und Proliferation regulieren. Obwohl viele Zelltypen in Bezug auf die in ihrer Regulation eingebundenen mirRNAs charakterisiert wurden, gibt es nur wenige Studien, die sich mit der Rolle von mirRNAs in DCs beschäftigen. Der zweite Teil dieser Arbeit konzentriert sich auf die Rolle der miRNA Gruppe miR-17~92 in DCs und untersucht deren Rolle in gesunden und atherosklerotischen Mäusen. Diese Arbeit zeigt erstmals eine deutliche anti-inflammatorische und protektive Rolle dieser miRNA und schlägt ein Modell für die entdeckten Mechanismen vor. KW - Aorta KW - Maus KW - Zelle KW - Cluster KW - miRNS KW - Dendritische Zelle KW - Arteriosklerose KW - miR-17~92 KW - dendritic cells KW - atherosclerosis KW - mice KW - murine Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71683 ER - TY - THES A1 - Gschmack, Eva Maria T1 - Anti-Gehirn-Autoantikörper und deren Bedeutung bei Morbus Parkinson T1 - Anti-brain-Autoantibodies and their role in Parkinson's Disease N2 - Hintergrund: Die der Pathogenese von Morbus Parkinson (PD, Parkinson’s disease) zugrunde liegenden Mechanismen sind bis heute nur unvollständig verstanden. Insbesondere ist unklar, durch welche ursächlichen Faktoren Parkinson ausgelöst wird. Bei der HIV-Infektion treten bei vielen Patienten neurologische Störungen auf (HIV-Associated Neurological Disorders, HAND), die in der klinischen Symptomatik und der Lokalisation der betroffenen Gehirnareale dem Morbus Parkinson ähneln. Möglicherweise könnte eine Fehlregulation der Immunantwort eine Rolle als Auslöser beider Erkrankungen spielen. In dieser Arbeit wurde die Autoimmunantwort von PD- und HAND-Patienten und gesunden Kontrollen gegen verschiedene Gehirnhomogenate untersucht, die während der Parkinsonerkrankung in unterschiedlichem Ausmaß geschädigt werden. Das Autoimmun-Signal wurde quantifiziert und prominente Autoantigene wurden identifiziert. Methoden: In dieser Arbeit wurde ein Western-Blot-basiertes Verfahren zum Nachweis von Autoantikörpern gegen Gehirngewebe entwickelt. Dieses Verfahren wurde nach Optimierung mit Plasmaproben von gesunden Kontrollen, PD-Patienten und Patienten mit HIV-Infektion insbesondere an einer Gruppe von 40 Parkinson-Patienten (Durchschnittsalter 65 Jahre, 45 % weiblich) und 40 alters- und geschlechtsgemachten Kontrollen (Durchschnittsalter 62 Jahre, 50 % weiblich) angewendet und die humorale Autoimmunität gegen verschiedene Gehirnareale untersucht. Dazu wurden die verschiedenen Areale (dorsaler Motornucleus des Glossopharynx- und Vagusnervs (dm), Substantia nigra (SN), anteromedialer temporaler Mesocortex (MC), high order sensorische Assoziations- und präfrontale Felder (HC), first oder sensorische Assoziations- und prämotorische Felder, primäre sensorische und motorische Felder (FC)) von post-mortem Gehirnen homogenisiert, auf SDS-Gradienten-Gelen elektrophoretisch aufgetrennt und auf Nitrocellulose geblottet. Die Membranen wurden mit den Plasmen inkubiert und gebundene Autoantikörper immunologisch detektiert. Die Signale wurden qualitativ und quantitativ ausgewertet. Mit Hilfe einer zweidimensionalen Elektrophorese und anschließender Immunfärbung wurden prominente Autoantigene durch Massenspektroskopie identifiziert. Ergebnisse: Mit dem in dieser Arbeit entwickelten Assay lässt sich die humorale Autoimmunantwort gegen Gehirngewebe semiquantitativ bestimmen. In allen untersuchten Proben konnten verschiedene Autoantikörper gegen unterschiedliche Antigene nachgewiesen werden. Der Gesamt-IgG-Gehalt der Plasmen unterscheidet sich weder zwischen PD-Patienten und gesunden Kontrollen, noch zwischen Männern und Frauen signifikant. Weibliche PD-Patienten zeigen signifikant stärkere Signale gegen dm als männliche (p = 0.02, Mann-Whitney-U-Test), der wiederum in jedem Patienten - unabhängig vom Geschlecht - von den untersuchten Hirnarealen signifikant stärker autoimmunologisch erkannt wird, als die übrigen Hirnareale (p < 0.0001, Friedman-ANOVA). In jedem Hirnareal wurden drei Banden besonders häufig erkannt (45, 40 und 37 kDa), jede davon am stärksten im dm (p < 0.0001, Friedman-ANOVA). Die Einzelanalysen der Signalintensitäten zeigt, dass PD-Patienten signifikant weniger Autoreaktivität gegen die 45 kDa-Bande in der SN (p = 0.056), im MC (p = 0.0277) und im FC (p = 0.0188) zeigen, als Kontrollen. Weitere Analysen zeigen, dass männliche PD-Patienten hochsignifikant weniger das 45 kDa-Protein im SN (p < 0.0001), MC (p = 0.0042) und FC (p = 0.0088) erkennen als Kontrollen, wohingegen bei den weiblichen Kontroll- und PD-Plasmen kein Unterschied festzustellen war. Ein weiteres Protein bei 160 kDa wird signifikant unterschiedlich stark in allen Gehirnarealen erkannt (p < 0.0001, Friedman-ANOVA), wobei die stärkste Immunreaktivität gegen FC besteht. Basierend auf dem Nachweis der 45 kDa-Bande aus der SN ergibt sich eine Odds Ratio für das Merkmal Parkinson von 3.38 (CI 1.11 – 10.30). Bei Männern ist diese Odds Ratio sogar 53.12 (CI 2.79 - 1012), bei Frauen 0.44 (CI 0.09 – 2.09). Die Sensitivität dieses Tests liegt bei Männern bei 1 (CI 0.84 – 1), die Spezifität bei 4.41 (0.31 – 0.78). Die negativ prädiktiven Werte liegen in allen Gruppen über 99.15 %. Die Identifizierung der Proteine mittels Massenspektroskopie ergab, dass es sich bei den 37 – 45 kDa Banden um Isoformen oder posttranslational modifizierte Formen des GFAP (glial fibrillary acidic protein), einem Bestandteil von Neurofilamenten v.a. in Astrozyten handelt. Außerdem wurde Fructose-Bisphosphate Aldolase A und Aspartat-Aminotransferase (mitochondriale Isoform 1 Vorläufer), beides Proteine des Kohlenhydrat-Stoffwechsels und der Glykolyse, als weitere Proteine mit ebenfalls 45 kDa identifiziert. Bei dem identifizierten Protein mit dem Molekulargewicht von 160 kDa handelt es sich wahrscheinlich um Dihydropyrimidinase-related protein 2, wie GFAP ebenfalls bei der Bildung des Zytoskeletts beteiligt. Diskussion: Autoantikörper gegen Gehirnantigene sind ein physiologisches Phänomen, das unabhängig von dem Vorliegen einer neurologischen Erkrankung besteht. Gehirnareale, die bei Parkinson besonders stark geschädigt werden, werden von dieser humoralen Autoimmunantwort besonders stark erkannt. Eine vorübergehende Permeabilisierung der Blut-Hirn-Schranke durch Infektion oder Trauma könnte den Zutritt der Autoantikörper zum Gehirn erlauben und so autoreaktive Prozesse in Gang setzen und zum Untergang dopaminerger Neuronen führen. Bei den identifizierten Proteinen handelt es sich um grundlegende Bestandteile eukaryotischer Zellen, was die Hypothese eines Art Beseitigungsmechanismus der Autoantikörper und damit die Aufgabe der Aufrechterhaltung der Homöostase darstellen könnte. Bei männlichen PD Patienten wird die 45 kDa Bande signifikant weniger stark von Auto-IgGs erkannt; dieser Mechanismus könnte somit in den männlichen PD-Patienten vermindert sein. Als Folge wäre die Ablagerung von Zelltrümmern im Gehirn vorstellbar, die dann auch langfristig eine Angriffsfläche für Autoimmunprozesse mit dem Verlust dopaminerger Neuronen bieten könnte. N2 - Background: The pathogenic mechanisms of Parkinson’s disease (PD) are not yet fully understood. Particularly the basic cause of the disease remains unclear. In HIV-Infection many patients show neurologic impairments (HIV-associated neurological disorders, HAND), which are similar in clinic symptoms and localization of the affected brain areas to these observed in Parkinson’s disease. Possibly a dysregulation of immunologic responses might play a role as a cause for PD. In this work autoimmune processes in PD- and HAND-patients as well as in healthy controls against different brain homogenates were measured, which are damaged in variable extent during pathogenesis of PD. The autoimmune-signal was quantified and prominent autoantigens were identified. Methods: In this work we established a Western-Blot-based method to detect autoantibodies against brain homogenates. After the optimization with plasma of healthy controls, PD-patients and HIV-infected patients, the technique was used to measure the humoral autoimmunity against different brain areas in a group of 40 PD-patients (mean age 65, 45 % females), and 40 age- and sex-matched controls (mean age 62 years, 50 % female). Different brain areas (of the dorsal motor nucleus of the glossopharyngeal and vagal nerves (dm), substantia nigra (SN), anteromedial temporal mesocortex (MC), high order sensory association areas and prefrontal fields (HC), first order sensory association areas, premotor areas, as well as primary sensory and motor fields (FC)) of post-mortem brains were homogenized, separated by gel electrophoresis and blotted on a nitrocellulose membrane. The membranes were incubated with plasma and bound antibodies were detected immunologically. The signals were analyzed by quality and quantity. With a two-dimensional electrophoresis and following immune staining prominent autoantigens were identified by mass spectroscopy. Results: With the established assay, the humoral autoimmunity against brain tissue can be analyzed semi-quantitatively. In all investigated samples several autoantibodies against different antigens could be proven. The overall IgG-content did not differ significantly between PD-patients and healthy controls, nor between men and women. Female PD-patients show significant stronger signals against dm than male (p = 0.02, Mann-Whitney-U-test), which was recognized significantly stronger than the other brain areas in each patient, independently from sex (p < 0.0001, Friedman-ANOVA). In each brain area 3 bands were recognized stronger than others (45, 40 and 37 kDa), each of them the strongest in dm (p < 0.0001, Friedman-ANOVA). The single analysis of the signal intensities show that PD-patients display significantly less autoreactivity against the 45 kDa-band in SN (p = 0.056), MC (p = 0.0277) and FC (p = 0.0188) than controls. Further analyses show that plasma of male PD-patients detect highly significantly less the 45 kDa-protein in SN (p < 0.0001), MC (p = 0.0042) and FC (p = 0.0088) than controls, whereas there was no difference measurable in female control- and PD-plasma. Another protein at 160 kDa is recognized at significantly different intensities in all brain areas (p < 0.0001, Friedman-ANOVA), whereas the strongest immunoreactivity is shown against FC. Based on the detection of the 45 kDa-band from the SN the odds ratio for the feature PD is 3.38 (CI 1.11 – 10.30). In men the odds ratio is even 53.12 (CI 2.79 - 1012), in female 0.44 (CI 0.09 – 2.09). The sensitivity for the test in the group of men displays 1 (CI 0.84 – 1), the specificity is 4.41 (0.31 – 0.78). The negative predictive values exceed 99.15 % in all investigated groups. The identification of the proteins by mass spectroscopy showed that the 37 – 45 kDa-bands might be isoforms of posttranslational modified forms of GFAP (glial fibrillary acidic protein), a part of neurofilaments in astrocytes. Furthermore, fructose-bisphosphate aldolase A and aspartate aminotransferase (mitochondrial isoform 1 precursor), both in the metabolism of carbon hydrates and glycolysis, have been identified for further 45 kDa-bands. The identified protein with a molecular weight of 160 kDa is probably dihydropyrimidinase-related protein 2, like GFAP involved in the formation of the cytoskeleton. Conclusion: The presence of anti-brain-autoantibodies are a physiological occurrence in human plasma, independent of the presence of neurological disease. Brain areas that are damaged prominently during the pathogenesis of PD are recognized stronger by humoral autoimmunity. A temporal permeabilization of the blood brain barrier through infection or trauma might allow autoantibodies access to the brain and initiate autoreactive processes that lead to the demise of dopaminergic neurons. The identified proteins are basic members of eukaryotic cells. This could suggest the hypothesis of sort of a cleaning mechanism of the antibodies, which might have the function of sustaining homeostasis. Male PD patients showed significantly decreased levels of autoreactive antibodies against the 45 kDa band in SN, MC and FC, which could enhance the accumulation of protein debris due to a missing removal mechanism and in long-distance might be responsible for neurodegenerative processes. KW - Parkinson-Krankheit KW - Autoantikörper KW - Parkinson Disease KW - anti-brain autoantibodies KW - GFAP KW - Gliafaserprotein KW - Anti-Gehirn Autoantikörper Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149348 ER - TY - JOUR A1 - Busch, Albert A1 - Busch, Martin A1 - Scholz, Claus-Jürgen A1 - Kellersmann, Richard A1 - Otto, Christoph A1 - Chernogubova, Ekaterina A1 - Maegdefessel, Lars A1 - Zernecke, Alma A1 - Lorenz, Udo T1 - Aneurysm miRNA Signature Differs, Depending on Disease Localization and Morphology JF - International Journal of Molecular Science N2 - Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways. KW - AAA KW - miRNA expression KW - pathway analysis KW - histologic diversity KW - popliteal aneurysm Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146422 SN - International Journal of Molecular Science VL - 17 IS - 1 ER - TY - JOUR A1 - Brünnert, Daniela A1 - Seupel, Raina A1 - Goyal, Pankaj A1 - Bach, Matthias A1 - Schraud, Heike A1 - Kirner, Stefanie A1 - Köster, Eva A1 - Feineis, Doris A1 - Bargou, Ralf C. A1 - Schlosser, Andreas A1 - Bringmann, Gerhard A1 - Chatterjee, Manik T1 - Ancistrocladinium A induces apoptosis in proteasome inhibitor-resistant multiple myeloma cells: a promising therapeutic agent candidate JF - Pharmaceuticals N2 - The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent. KW - multiple myeloma KW - ancistrocladinium A KW - naphthylisoquinoline alkaloids KW - proteasome inhibitor resistance KW - RNA splicing KW - cellular stress response KW - proteasome subunit beta type-5 (PSMB5) KW - activating transcription factor 4 (ATF4) KW - ataxia teleagiectasia mutated (ATM) KW - H2A histone family member X (H2AX) Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-362887 SN - 1424-8247 VL - 16 IS - 8 ER - TY - JOUR A1 - Ries, Lena K. A1 - Sander, Bodo A1 - Deol, Kirandeep K. A1 - Letzelter, Marie-Annick A1 - Strieter, Eric Robert A1 - Lorenz, Sonja T1 - Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity JF - Journal of Biological Chemistry N2 - Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin-conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases. KW - ubiquitin KW - ubiquitin ligase KW - ubiquitylation (ubiquitination) KW - post-translational modification KW - enzyme mechanism Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226207 VL - 294 IS - 15 ER - TY - JOUR A1 - Mietrach, Nicole A1 - Schlosser, Andreas A1 - Geibel, Sebastian T1 - An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization JF - Acta Crystallographica Section F N2 - The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α‐helices with a melting point of 34.5°C. Size‐exclusion chromatography combined with multi‐angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging‐drop vapor‐diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit‐cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°. KW - ESAT‐6‐like secretion system KW - ESS KW - type VII secretion system KW - EsaA KW - extracellular domain KW - Staphylococcus aureus USA300 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213681 VL - 75 IS - 12 ER - TY - JOUR A1 - Sirén, Anna-Leena A1 - Stetter, Christian A1 - Hirschberg, Markus A1 - Nieswandt, Bernhard A1 - Ernestus, Ralf-Ingo A1 - Heckmann, Manfred T1 - An experimental protocol for in vivo imaging of neuronal structural plasticity with 2-photon microscopy in mice JF - Experimental & Translational Stroke Medicine N2 - Introduction Structural plasticity with synapse formation and elimination is a key component of memory capacity and may be critical for functional recovery after brain injury. Here we describe in detail two surgical techniques to create a cranial window in mice and show crucial points in the procedure for long-term repeated in vivo imaging of synaptic structural plasticity in the mouse neocortex. Methods Transgenic Thy1-YFP(H) mice expressing yellow-fluorescent protein (YFP) in layer-5 pyramidal neurons were prepared under anesthesia for in vivo imaging of dendritic spines in the parietal cortex either with an open-skull glass or thinned skull window. After a recovery period of 14 days, imaging sessions of 45–60 min in duration were started under fluothane anesthesia. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate fixed to metal base. The animals were set under a two-photon microscope with multifocal scanhead splitter (TriMScope, LaVision BioTec) and the Ti-sapphire laser was tuned to the optimal excitation wavelength for YFP (890 nm). Images were acquired by using a 20×, 0.95 NA, water-immersion objective (Olympus) in imaging depth of 100–200 μm from the pial surface. Two-dimensional projections of three-dimensional image stacks containing dendritic segments of interest were saved for further analysis. At the end of the last imaging session, the mice were decapitated and the brains removed for histological analysis. Results Repeated in vivo imaging of dendritic spines of the layer-5 pyramidal neurons was successful using both open-skull glass and thinned skull windows. Both window techniques were associated with low phototoxicity after repeated sessions of imaging. Conclusions Repeated imaging of dendritic spines in vivo allows monitoring of long-term structural dynamics of synapses. When carefully controlled for influence of repeated anesthesia and phototoxicity, the method will be suitable to study changes in synaptic structural plasticity after brain injury. KW - 2-photon microscopy KW - Fluorescence KW - In vivo imaging KW - Neurons KW - Cranial window KW - Mouse model Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96908 UR - http://www.etsmjournal.com/content/5/1/9 ER - TY - JOUR A1 - Segerer, Gabriela A1 - Hadamek, Kerstin A1 - Zundler, Matthias A1 - Fekete, Agnes A1 - Seifried, Annegrit A1 - Mueller, Martin J. A1 - Koentgen, Frank A1 - Gessler, Manfred A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation JF - Scientific Reports N2 - Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive Pgp\(^{D34N}\) mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation. KW - cell proliferation KW - DNA metabolism KW - lipidomics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181094 VL - 6 ER - TY - THES A1 - El Merahbi, Rabih T1 - Adrenergic-induced ERK3 pathway drives lipolysis and suppresses energy dissipation T1 - Der adrenerge induzierte ERK3-Signalweg verstärkt Lipolyse und unterdrückt Energiedissipation N2 - Obesity-induced diabetes affects over 400 million people worldwide. Obesity is a complex metabolic disease and is associated with several co-morbidities, all of which negatively affect the individual’s quality of life. It is commonly considered that obesity is a result of a positive energy misbalance, as increased food intake and lower expenditure eventually lead to the development of this disease. Moreover, the pathology of obesity is attributed to several genetic and epigenetic factors that put an individual at high risk compared to another. Adipose tissue is the main site of the organism’s energy storage. During the time when the nutrients are available in excess, adipocytes acquire triglycerides, which are released during the time of food deprivation in the process of lipolysis (free fatty acids and glycerol released from adipocytes). Uncontrolled lipolysis is the consequent event that contributes to the development of diabetes and paradoxically obesity. To identify the genetic factors aiming for future therapeutic avenues targeting this pathway, we performed a high-throughput screen and identified the Extracellular-regulated kinase 3 (ERK3) as a hit. We demonstrate that β-adrenergic stimulation stabilizes ERK3 leading to the formation of a complex with the co-factor MAP kinase-activated protein kinase 5 (MK5) thereby driving lipolysis. Mechanistically, we identify a downstream target of the ERK3/MK5 pathway, the transcription factor FOXO1, which promotes the expression of the major lipolytic enzyme ATGL. Finally, we provide evidence that targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, but elevates energy dissipation, promoting lean phenotype and ameliorating diabetes. Moreover, we shed the light on our pharmacological approach in targeting ERK3/MK5 pathways using MK5 specific inhibitor. Already after 1 week of administering the inhibitor, mice showed signs of improvement of their metabolic fitness as showed here by a reduction in induced lipolysis and the elevation in the expression of thermogenic genes. Taken together, our data suggest that targeting the ERK3/MK5 pathway, a previously unrecognized signaling axis in adipose tissue, could be an attractive target for future therapies aiming to combat obesity-induced diabetes. N2 - Adipositas-induzierter Diabetes betrifft weltweit über 400 Millionen Menschen. Adipositas ist eine komplexe Stoffwechselerkrankung und geht mit mehreren Komorbiditäten einher, die sich alle negativ auf die Lebensqualität der Betroffenen auswirken. Es wird generell angenommen, dass Adipositas aus einem positiven Energieungleichgewicht resultiert, da eine erhöhte Nahrungsaufnahme und ein geringerer Verbrauch zu der Ausbildung dieser Krankheit führen. Darüber hinaus ist die Pathologie von Adipositas auf mehrere genetische und epigenetische Faktoren zurückzuführen, wodurch Individuen einem erhöhtem Risiko ausgesetzt sein können. Das Fettgewebe ist der vorwiegende Energiespeicher des Organismus. In Zeiten eines Nährstoffüberschusses speichern Adipozyten Triglyceride, die im Falle eines Nahrungsmangels durch den Prozess der Lipolyse in Form von freien Fettsäuren und Glycerin freigesetzt werden. Unkontrollierte Lipolyse ist ein Folgeereignis, welches zur Entwicklung von Diabetes und paradoxerweise zu Adipositas beiträgt. Um die genetischen Faktoren zu identifizieren, die in Zukunft therapeutische Angriffspunkte darstellen könnten, haben wir ein Hochdurchsatz-Screening durchgeführt und die extrazellulär regulierte Kinase 3 (ERK3) als Treffer identifiziert. Wir zeigen, dass β-adrenerge Stimulation ERK3 stabilisiert, was zur Bildung eines Komplexes mit dem Cofactor MAP-Kinase-aktivierte Proteinkinase 5 (MK5) führt und dadurch die Lipolyse vorantreibt. Mechanistisch identifizieren wir den Transkriptionsfaktor FOXO1, der dem ERK3/MK5-Signalweg nachgeschaltet ist und die Expression des wichtigsten lipolytischen Enzyms ATGL fördert. Darüber hinaus belegen wir, dass die gezielte Deletion von ERK3 in Maus-Adipozyten die Lipolyse hemmt, aber die Energiedissipation erhöht, den mageren Phänotyp fördert und Diabetes lindert. Außerdem nutzen wir einen pharmakologischen Ansatz durch Verwendung eines MK5 spezifischen Inhibitors, um auf den ERK3/MK5-Signalweg abzuzielen. Bereits eine Woche nach Verabreichung des Inhibitors zeigen Mäuse Anzeichen einer verbesserten metabolischen Fitness, die sich durch einer Verringerung der induzierten Lipolyse und eine verstärkte Expression von thermogenen Genen auszeichnet. Zusammenfassend legen unsere Daten nahe, dass der ERK3/MK5-Signalweg, eine zuvor nicht erkannte Signalachse im Fettgewebe, ein attraktiver Ansatzpunkt für zukünftige Therapien zur Bekämpfung von Adipositas-induziertem Diabetes sein könnte. KW - Metabolism KW - Lipolysis KW - Obesity KW - Adrenalin KW - ATGL KW - Foxo1 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217510 ER - TY - JOUR A1 - Busse, Kathy A1 - Strotmann, Rainer A1 - Strecker, Karl A1 - Wegner, Florian A1 - Devanathan, Vasudharani A1 - Gohla, Antje A1 - Schöneberg, Torsten A1 - Schwarz, Johannes T1 - Adaptive Gene Regulation in the Striatum of RGS9-Deficient Mice JF - PLOS ONE N2 - Background: RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions. Results: Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient mice. Conclusion: Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchallenged conditions. Significance: Identified signaling components may represent novel targets in antidyskinetic therapy. The long splice variant of the regulator of G-protein signaling 9 (RGS9-2) is enriched in striatal medium spiny neurons and dampens dopamine D2 receptor signaling. Lack of RGS9-2 can promote while its overexpression prevents drug-induced dyskinesia. Other animal models of drug-induced dyskinesia rather pointed towards overactivity of dopamine receptor-mediated signaling. To evaluate changes in signaling pathways mRNA expression levels were determined and compared in wild-type and RGS9-deficient mice. Unexpectedly, expression levels of dopamine receptors were unchanged in RGS9-deficient mice, while several genes related to Ca2+ signaling and long-term depression were differentially expressed when compared to wild type animals. Detailed investigations at the protein level revealed hyperphosphorylation of DARPP32 at Thr34 and of ERK1/2 in striata of RGS9-deficient mice. Whole cell patch clamp recordings showed that spontaneous synaptic events are increased (frequency and size) in RGS9-deficient mice while long-term depression is reduced in acute brain slices. These changes are compatible with a Ca2+-induced potentiation of dopamine receptor signaling which may contribute to the drug-induced dyskinesia in RGS9-deficient mice. KW - medium spiny neurons KW - long-term depression KW - dopa-induced dyskinesia KW - adenylyl cyclase KW - Parkinsons disease KW - synaptic plasticity KW - L-3,4-Dihydroxyphenylalanine-induced dyskinesia KW - ampa receptors KW - cholinergic interneurons KW - endocannabinoid release Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117048 VL - 9 IS - 3 ER - TY - JOUR A1 - Li, Cong A1 - Deng, Xiaobing A1 - Xie, Xiaowen A1 - Liu, Ying A1 - Friedmann Angeli, José Pedro A1 - Lai, Luhua T1 - Activation of Glutathione Peroxidase 4 as a Novel Anti-inflammatory Strategy JF - Frontiers in Pharmacology N2 - The anti-oxidative enzyme, glutathione peroxidase 4 (GPX4), helps to promote inflammation resolution by eliminating oxidative species produced by the arachidonic acid (AA) metabolic network. Up-regulating its activity has been proposed as a promising strategy for inflammation intervention. In the present study, we aimed to study the effect of GPX4 activator on the AA metabolic network and inflammation related pathways. Using combined computational and experimental screen, we identified a novel compound that can activate the enzyme activity of GPX4 by more than two folds. We further assessed its potential in a series of cellular assays where GPX4 was demonstrated to play a regulatory role. We are able to show that GPX4 activation suppressed inflammatory conditions such as oxidation of AA and NF-κB pathway activation. We further demonstrated that this GPX4 activator can decrease the intracellular ROS level and suppress ferroptosis. Our study suggests that GPX4 activators can be developed as anti-inflammatory or cyto-protective agent in lipid-peroxidation-mediated diseases. KW - arachidonic acid metabolic network KW - GPX4 KW - enzyme activator KW - allosterism KW - drug discovery KW - anti-inflammatory KW - ferroptosis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195985 SN - 1663-9812 VL - 9 IS - 1120 ER - TY - JOUR A1 - Pfeiffer, Verena A1 - Götz, Rudolf A1 - Xiang, Chaomei A1 - Camarero, Guadelupe A1 - Braun, Attila A1 - Zhang, Yina A1 - Blum, Robert A1 - Heinsen, Helmut A1 - Nieswandt, Bernhard A1 - Rapp, Ulf R. T1 - Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum JF - PLoS ONE N2 - This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures. KW - granule cells KW - hippocampus KW - neurons KW - neuronal dendrites KW - embryos KW - dentate gyrus KW - neuronal differentiation KW - cerebellum Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130304 VL - 8 IS - 3 ER - TY - JOUR A1 - Schihada, Hannes A1 - Vandenabeele, Sylvie A1 - Zabel, Ulrike A1 - Frank, Monika A1 - Lohse, Martin J. A1 - Maiellaro, Isabella T1 - A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics JF - Communications Biology N2 - G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the alpha(2 Lambda)-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning beta(2)-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands. KW - Fluorescence resonance energy transfer KW - G protein-coupled receptors KW - High-throughput screening Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228592 VL - 1 IS - 105 ER - TY - JOUR A1 - Jun, Kyong-Hwa A1 - Gholami, Spedideh A1 - Song, Tae-Jin A1 - Au, Joyce A1 - Haddad, Dana A1 - Carson, Joshua A1 - Chen, Chun-Hao A1 - Mojica, Kelly A1 - Zanzonico, Pat A1 - Chen, Nanhai G. A1 - Zhang, Qian A1 - Szalay, Aladar A1 - Fong, Yuman T1 - A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter JF - Journal of Experimental & Clinical Cancer Research N2 - Background: Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with Tc-99m pertechnetate scintigraphy and I-124 positron emission tomography (PET). Methods: GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. Tc-99m pertechnetate scintigraphy and I-124 microPET imaging were performed. Results: GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK-45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by Tc-99m pertechnetate scintigraphy and I-124 microPET imaging 2 days after treatment. Conclusions: GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings. KW - oncolytic viral therapy KW - GLV-1 h153 KW - gastric cancer KW - human sodium iodide symporter (hNIS) KW - radioiodine therapy KW - gene therapy KW - expression KW - replication KW - stomach KW - tumors KW - surgery Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117716 SN - 1756-9966 VL - 33 IS - 2 ER - TY - JOUR A1 - van Unen, Jakobus A1 - Stumpf, Anette D. A1 - Schmid, Benedikt A1 - Reinhard, Nathalie R. A1 - Hordijk, Peter L. A1 - Hoffmann, Carsten A1 - Gadella, Theodorus W. J. A1 - Goedhart, Joachim T1 - A New Generation of FRET Sensors for Robust Measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) Activation Kinetics in Single Cells JF - PLoS ONE N2 - G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα\(_{i}\) subunit, and cp173Venus fused to the Gγ\(_{2}\) subunit as acceptor. The Gα\(_{i}\) FRET biosensors constructs are expressed together with Gβ\(_{1}\) from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα\(_{i}\) FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα\(_{i}\) FRET sensor in single cells upon stimulation of several GPCRs, including the LPA\(_{2}\), M\(_{3}\) and BK\(_{2}\) receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation will be valuable for live-cell measurements that probe Gα\(_{i}\) activation. KW - FRET sensors KW - G-protein coupled receptors KW - Förster resonance energy transfer KW - Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation KW - biosensors Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167387 VL - 11 IS - 1 ER - TY - JOUR A1 - Zaho, Huaying A1 - Ghirlando, Rodolfo A1 - Alfonso, Carlos A1 - Arisaka, Fumio A1 - Attali, Ilan A1 - Bain, David L. A1 - Bakhtina, Marina M. A1 - Becker, Donald F. A1 - Bedwell, Gregory J. A1 - Bekdemir, Ahmet A1 - Besong, Tabot M. D. A1 - Birck, Catherine A1 - Brautigam, Chad A. A1 - Brennerman, William A1 - Byron, Olwyn A1 - Bzowska, Agnieszka A1 - Chaires, Jonathan B. A1 - Chaton, Catherine T. A1 - Coelfen, Helmbut A1 - Connaghan, Keith D. A1 - Crowley, Kimberly A. A1 - Curth, Ute A1 - Daviter, Tina A1 - Dean, William L. A1 - Diez, Ana I. A1 - Ebel, Christine A1 - Eckert, Debra M. A1 - Eisele, Leslie E. A1 - Eisenstein, Edward A1 - England, Patrick A1 - Escalante, Carlos A1 - Fagan, Jeffrey A. A1 - Fairman, Robert A1 - Finn, Ron M. A1 - Fischle, Wolfgang A1 - Garcia de la Torre, Jose A1 - Gor, Jayesh A1 - Gustafsson, Henning A1 - Hall, Damien A1 - Harding, Stephen E. A1 - Hernandez Cifre, Jose G. A1 - Herr, Andrew B. A1 - Howell, Elizabeth E. A1 - Isaac, Richard S. A1 - Jao, Shu-Chuan A1 - Jose, Davis A1 - Kim, Soon-Jong A1 - Kokona, Bashkim A1 - Kornblatt, Jack A. A1 - Kosek, Dalibor A1 - Krayukhina, Elena A1 - Krzizike, Daniel A1 - Kusznir, Eric A. A1 - Kwon, Hyewon A1 - Larson, Adam A1 - Laue, Thomas M. A1 - Le Roy, Aline A1 - Leech, Andrew P. A1 - Lilie, Hauke A1 - Luger, Karolin A1 - Luque-Ortega, Juan R. A1 - Ma, Jia A1 - May, Carrie A. A1 - Maynard, Ernest L. A1 - Modrak-Wojcik, Anna A1 - Mok, Yee-Foong A1 - Mücke, Norbert A1 - Nagel-Steger, Luitgard A1 - Narlikar, Geeta J. A1 - Noda, Masanori A1 - Nourse, Amanda A1 - Obsil, Thomas A1 - Park, Chad K A1 - Park, Jin-Ku A1 - Pawelek, Peter D. A1 - Perdue, Erby E. A1 - Perkins, Stephen J. A1 - Perugini, Matthew A. A1 - Peterson, Craig L. A1 - Peverelli, Martin G. A1 - Piszczek, Grzegorz A1 - Prag, Gali A1 - Prevelige, Peter E. A1 - Raynal, Bertrand D. E. A1 - Rezabkova, Lenka A1 - Richter, Klaus A1 - Ringel, Alison E. A1 - Rosenberg, Rose A1 - Rowe, Arthur J. A1 - Rufer, Arne C. A1 - Scott, David J. A1 - Seravalli, Javier G. A1 - Solovyova, Alexandra S. A1 - Song, Renjie A1 - Staunton, David A1 - Stoddard, Caitlin A1 - Stott, Katherine A1 - Strauss, Holder M. A1 - Streicher, Werner W. A1 - Sumida, John P. A1 - Swygert, Sarah G. A1 - Szczepanowski, Roman H. A1 - Tessmer, Ingrid A1 - Toth, Ronald T. A1 - Tripathy, Ashutosh A1 - Uchiyama, Susumu A1 - Uebel, Stephan F. W. A1 - Unzai, Satoru A1 - Gruber, Anna Vitlin A1 - von Hippel, Peter H. A1 - Wandrey, Christine A1 - Wang, Szu-Huan A1 - Weitzel, Steven E A1 - Wielgus-Kutrowska, Beata A1 - Wolberger, Cynthia A1 - Wolff, Martin A1 - Wright, Edward A1 - Wu, Yu-Sung A1 - Wubben, Jacinta M. A1 - Schuck, Peter T1 - A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation JF - PLoS ONE N2 - Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304\(\pm\)0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of \(\pm\)0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies. KW - fluorescence-detected sedimentation KW - size exclusion chromatography KW - field flow fractionation KW - spinco ultracentrifuge KW - aggregation KW - bead models KW - velocity KW - hydrodynamics KW - biopharmaceuticals KW - proteins Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151903 VL - 10 IS - 5 ER - TY - JOUR A1 - Sander, Bodo A1 - Xu, Wenshan A1 - Eilers, Martin A1 - Popov, Nikita A1 - Lorenz, Sonja T1 - A conformational switch regulates the ubiquitin ligase HUWE1 JF - eLife N2 - The human ubiquitin ligase HUWE1 has key roles in tumorigenesis, yet it is unkown how its activity is regulated. We present the crystal structure of a C-terminal part of HUWE1, including the catalytic domain, and reveal an asymmetric auto-inhibited dimer. We show that HUWE1 dimerizes in solution and self-associates in cells, and that both occurs through the crystallographic dimer interface. We demonstrate that HUWE1 is inhibited in cells and that it can be activated by disruption of the dimer interface. We identify a conserved segment in HUWE1 that counteracts dimer formation by associating with the dimerization region intramolecularly. Our studies reveal, intriguingly, that the tumor suppressor p14ARF binds to this segment and may thus shift the conformational equilibrium of HUWE1 toward the inactive state. We propose a model, in which the activity of HUWE1 underlies conformational control in response to physiological cues—a mechanism that may be exploited for cancer therapy. KW - Medicine KW - Structural Biology KW - Molecular Biophysics KW - HUWE1 KW - HECT Ligase KW - Ubiquitin KW - P14ARF KW - X-Ray Chrystallography KW - Enzyme Regulation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171862 VL - 6 ER - TY - JOUR A1 - Dütting, Sebastian A1 - Gaits-Iacovoni, Frederique A1 - Stegner, David A1 - Popp, Michael A1 - Antkowiak, Adrien A1 - van Eeuwijk, Judith M.M. A1 - Nurden, Paquita A1 - Stritt, Simon A1 - Heib, Tobias A1 - Aurbach, Katja A1 - Angay, Oguzhan A1 - Cherpokova, Deya A1 - Heinz, Niels A1 - Baig, Ayesha A. A1 - Gorelashvili, Maximilian G. A1 - Gerner, Frank A1 - Heinze, Katrin G. A1 - Ware, Jerry A1 - Krohne, Georg A1 - Ruggeri, Zaverio M. A1 - Nurden, Alan T. A1 - Schulze, Harald A1 - Modlich, Ute A1 - Pleines, Irina A1 - Brakebusch, Cord A1 - Nieswandt, Bernhard T1 - A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis JF - Nature Communications N2 - Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V. KW - megakaryocytes KW - blood platelets KW - regulatory circuit downstream KW - glycoprotein Ib Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170797 VL - 8 IS - 15838 ER - TY - INPR A1 - Brenner, Marian A1 - Zink, Christoph A1 - Witzinger, Linda A1 - Keller, Angelika A1 - Hadamek, Kerstin A1 - Bothe, Sebastian A1 - Neuenschwander, Martin A1 - Villmann, Carmen A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - 7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase T2 - eLife N2 - Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain. KW - 7,8-dihydroxyflavone (7,8-DHF) KW - pyridoxal phosphatase (PDXP) KW - vitamin B6 KW - PDXP inhibitors Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350446 ER -