FMS-like tyrosine Kinase-3 (FLT3)

Top PDF FMS-like tyrosine Kinase-3 (FLT3):

Virtual Screening Identifies Irreversible FMS-like Tyrosine Kinase 3 Inhibitors with Activity toward Resistance-Conferring Mutations

Virtual Screening Identifies Irreversible FMS-like Tyrosine Kinase 3 Inhibitors with Activity toward Resistance-Conferring Mutations

dependency of kinase inhibition by compounds 4a, b and 3m, n in FLT3(D835Y) representing the most active compounds from both series. As expected, the reversible binding FLT3 inhibitors crenolanib and sunitinib show a strong dependency on ATP concentration, where a 10-fold ATP increase (100 µM to 1 mM) leads to an approximately 10-fold decrease of inhibition whereas crenolanib (4.2 nM to 42 nM) remains more potent than sunitinib (8.2 nM to 105 nM, Figure 3d, e). Since irreversible bond formation leads to non-equilibrium-binding only the initial reversible complex, preceding covalent bond formation, is ATP-competitive. Indeed, inhibition of FLT3(D835Y) by 4a (67 nM at low ATP, 58 nM at high ATP) as well as 4b (11.6 nM at low ATP to 18.6 nM at high ATP) is less vulnerable to increased ATP concentrations. To our surprise, 3m and 3n are highly susceptible to high ATP concentrations suggesting low inactivation kinetics and thus high competitivity to ATP (Figure S35). Additionally, we determined the influence of the pre-incubation time of inhibitor and kinase alone where ATP is not competing with inhibitor binding. As expected, the indolinones 4a and 4b as well as the bisaminopyrimidines 3m and 3n show increased inhibition of FLT3(D835Y) and FLT3(ITD) when pre-incubation time is increased from 5 min to 30 min (Table S8). To determine the K i and
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Der Einfluss des Fms-like tyrosine kinase 3-Liganden (Flt3L) im mütterlichen Blut in der Schwangerschaft und im Nabelschnurblut auf die Reife des kindlichen Immunsystems zum Zeitpunkt der Geburt

Der Einfluss des Fms-like tyrosine kinase 3-Liganden (Flt3L) im mütterlichen Blut in der Schwangerschaft und im Nabelschnurblut auf die Reife des kindlichen Immunsystems zum Zeitpunkt der Geburt

Obgleich die Analysen von Nabelschnurblut und damit die Betrachtung des Flt3L im Hinblick auf Stammzelltransplantationen und hämatoonkologische Fragestellungen einen großen Raum in der Forschung einnehmen, ist wenig bekannt über den Einfluss des Flt3L auf die Reife des kindlichen Immunsystems im Verlauf der Schwangerschaft und zum Zeitpunkt der Geburt. Wenige Studien haben sich mit mütterlichem und kindlichem Flt3L und dessen Rezeptor beschäftigt (Malamitsi- Puchner et al. 2005, Gonzalez et al. 2009). Malamitsi-Puchner et al. beschrieben beispielsweise einen Zusammenhang zwischen mütterlichen Flt3-Level kurz vor der Entbindung, jenen im Nabelschnurblut und den entsprechenden Level des Neurotrophin-3 (NT-3), einem Faktor, dem neben seinem Einfluss auf die
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Functional characterization of non-receptor tyrosine kinase dependent signal transduction in acute lymphoblastic leukemia of childhood

Functional characterization of non-receptor tyrosine kinase dependent signal transduction in acute lymphoblastic leukemia of childhood

Primary ALL blasts and cell lines either stimulated with the indicated ligands (shown in brackets) or unstimulated were analysed by quantitative WB. Following cell lysis, 20 µg of whole protein lysate were separated by SDS-PAGE and transferred to nitrocellulose membranes. A) The PTK candidate, CSK, was selected as a representative WB for the 7 PTK randomly chosen and two independent experiments are shown as I and II, respectively. The green fluorescents bands represent the target protein. β-actin was used as a loading control (red fluorescents bands). The remaining PTK were blotted accordingly. B) The arbitrary protein target signal of the selected PTK was quantified by using Li-Cor imaging and heat maps were designed with the software Multi Experiment Viewer (MeV 4.8). Heat maps from two independent experiments are shown as PTK expression-I and -II, respectively. C) Ultimately, the Spearman rank was used to cluster the generated heat maps of two independent experiments shown as I and II, respectively. PTK are displayed in columns and primary ALL blasts and cell lines are displayed in rows. Highly expressed TK are shown in shades of yellow, and non expressed TKs are shown in black, according to the color scale. Description of the primary ALL blasts (109, 113, 114, 115, 119, 22089) and ALL cell lines (CEM, Nalm6, SupB15, REH, 697, CALL3, CALL2) are listed in sections 3.2.2 and 3.2.3, respectively. PDGF= platelet-derived growth factor ligand, IGF1= Insulin-like growth factor 1, FLT3= fms-related tyrosine kinase 3 ligand.
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Regulation der Aktivität des fms-like-Thyrosinkinaserezeptor-1-Promotors

Regulation der Aktivität des fms-like-Thyrosinkinaserezeptor-1-Promotors

VEGF bindet und aktiviert v.a. zwei Tyrosinkinaserezeptoren: VEGF-Rezeptor-1 (VEGFR-1, FLT1 = Fms-like tyrosine kinase receptor 1) und VEGF-Rezeptor-2 (VEGFR-2, FLK1/KDR = fetal liver kinase 1 / kinase insert domaine protein receptor). Die meisten Effekte von VEGF werden durch FLK1 vermittelt. FLT1-Aktivierung spielt eine wichtige Rolle bei der Bildung des Gefäßlumens in der Vaskulogenese und ist im Rahmen maligner Wachstumsprozesse mit Progress und Metastasierung assoziiert. Zellen des Monozyten-Makrophagensystems, welche FLT1 exprimieren, steigern ihre chemotaktische Aktivität nach Bindung von VEGF an den FLT1-Rezeptor. Von FLT1 existiert eine lösliche Variante (sFLT1 = soluble FLT1), die im Blut zirkuliert und die Konzentration von ungebundenem VEGF vermindern und somit seine Bindung an die membranständigen Rezeptoren FLT1 und KDR vermindern kann [1,3,4,9].
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myo-Inositolpentakisphosphat-(3/5)-Kinase in Dictyostelium discoideum

myo-Inositolpentakisphosphat-(3/5)-Kinase in Dictyostelium discoideum

phatgruppen) genannt, stellen neben den Phosphoinositiden die biologisch relevanten myo-Inositolderivate dar. In dieser Arbeit stehen die Inositol(poly)phosphate im Mit- telpunkt. Der unterschiedliche Phosphorylierungsgrad und die verschiedenen mögli- chen Stellungen der Phosphatgruppen am myo-Inositolring führen zu 63 verschiede- nen Inositolphosphaten. Um diese eindeutig zu bezeichnen, werden die Isomere nach der IUPAC-Nomenklatur [2] benannt. Zur Vereinfachung der Inositolphosphatbezeich- nung kann bei der Durchnummerierung der Phosphatgruppen die Agranoff Schildkrö- te (Abb.1.1a, [3]), die das myo-Inositol in der Sesselkonformation darstellt, verwen- det werden. Das rechte Vorderbein der Schildkröte markiert die D-1-Position. Von hier aus erfolgt die Nummerierung nach der inzwischen allgemein verwendeten D- Nomenklatur gegen den Uhrzeigersinn, so dass der Kopf der Schildkröte der D-2- Position entspricht. Bei der thermodynamisch stabileren myo-Inositol Sesselkonforma- tion (Abb. 1.1 b) steht die Hydroxylgruppe an Position 2 axial und die verbleibenden fünf Hydroxylgruppen äquatorial. Das Molekül besitzt eine Spiegelebene, die durch die Kohlenstoffatome 2 und 5 verläuft (Abb. 1.1 c).
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Ischemia-associated complications in patients with hepatocellular carcinoma undergoing treatment with the tyrosine-kinase inhibitor Sorafenib

Ischemia-associated complications in patients with hepatocellular carcinoma undergoing treatment with the tyrosine-kinase inhibitor Sorafenib

There are several signaling pathways that play an important role in HCC progression. The Ras/Raf/MEK/ERK signaling pathway regulates proliferation, differentiation and apoptosis and is activated by extracellular signals, mostly growth factors, hormones or inducers of differentiation binding to receptor tyrosine kinases (RTK) (118). Mutations can cause the constitutive activation of the signaling cascade and overexpression of c-Raf, MEK and ERK was observed in > 50% of HCC patients (119). RTK-ligand interaction activates another pathway, involving phosphatidylinositide 3-kinases (PI3K) and mTOR (mammalian trarget of rapamycin), which mediates cell growth and metabolism (120). PI3K is usually very low in unstimulated, normal cells (121). The PIP3-AKT-mTOR pathway is frequently deregulated in HCC patients and the overexpression of mTOR seems to correlate with HCC invasion and metastasis (122). Transforming growth factor-β (TGF-β) regulates proliferation, cell death, cytoskeleton, cellular adhesion and would healing. During fibrosis, TGF-β is responsible for the recruitment of immune cells to the inflammation site and matrix deposition (123). In HCC, TGF-β expression was shown to be downregulated and associated with increased tumor size and proliferation (124). The JAK/STAT pathway transfers the signal of growth factors, interleukins and interferons to the nucleus and regulates proliferation, differentiation and apoptosis. In HCC, the JAK/STAT signaling is disturbed (125). In hepatocytes, Wnt/β-Catenin signaling pathway is one of the most important ones for liver development and regeneration and activating mutations in β-Catenin have been frequently observed in HCC patients (126,127). Cellular damages such as hypoxia or viral infection can lead to cell cycle arrest or apoptosis via p53, which is a frequently mutated tumor suppressor in numerous cancer entities. Aflatoxin B1, HBV and HCV can cause direct mutations of p53, especially of codon 249. As a result, G:C is converted to T:A, which is detectable in the serum of HCC patients. A correlation of p53 has been shown with histological grade, survival and response to therapy (128–130). Viral infections such as HBV infection can interrupt genomic stability by integration into the host genome. The viral protein HBx has the ability to inhibit p53 entering the nucleus and leads to accumulations of errors in the DNA. Furthermore, HBx interacts with transcription factors that are involved in cell growth and apoptosis (131,132).
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Zur Bedeutung von Phosphatidylinositol-3-Kinase-Mutationen in der Hepatokarzinogenese – ein Mausmodell

Zur Bedeutung von Phosphatidylinositol-3-Kinase-Mutationen in der Hepatokarzinogenese – ein Mausmodell

Das Exon 1α (E1α) ist nur notwendig für die Expression von RASSF1a, alle anderen bekannten Mitglieder der RASSF1-Familie können bei Entfernung von E1α immer noch exprimiert werden. Darum wurde nur dieses Exon bei den Knockout-Mäusen entfernt. RSF-5 (5'-CTC GCC CCT GTC AGA CCT CAA TTT CCC-3') erzeugt in diesem Modell zusammen mit dem Rückwärts-Primer RSF-3 (5'-CCA GGC TTC CTT CTC ACT CCT CTG CCG C3') bei den RASSF1a-Knockout-Mäusen ein ca. 400 bp langes Produkt. Bei den Wildtypen ist dieser Teil der DNA mit RSF-5 nicht nachweisbar, da das erzeugte Produkt E1α enthält, somit für diese PCR-Bedingungen zu groß ist und instabil wird. RSF-C (5'-AAG GAG AAA GAA AGC TGC TCT GGG GTT CT-3') kann dagegen nur beim Wildtyp ein Produkt zusammen mit RSF-3 erzeugen, da RSF-C in E1 α bindet. Bei den Knockout-Mäusen ist E1α entfernt worden, sodass kein entsprechendes Produkt entstehen kann.
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Die Expression von Doublecortin like kinase 1 (DCLK1) und seine prognostische Relevanz bei sinunasalen Plattenepithelkarzinomen.

Die Expression von Doublecortin like kinase 1 (DCLK1) und seine prognostische Relevanz bei sinunasalen Plattenepithelkarzinomen.

Die entparaffinisierten und rehydrierten Gewebeproben wurden in einer PBS (Phosphate Buffered Saline)-Lösung gewaschen. Um die endogene Peroxidaseaktivität zu hemmen wurden die Proben 10 Minuten lang in 3%igem Wasserstoffperoxid blockiert. Danach wurden den Proben 4 Mal je 3 Minuten eine Pufferlösung (0.1% Tween 20-Lösung) zugefügt und diese anschließend mittels Ultra V Block (Thermo Scientific, Fremont, CA) für 5 Minuten bei Raumtemperatur inkubiert. Nach Inkubation wurden die Präparate erneut in einer PBS-Lösung gewaschen. Nach Aufbringen des Primärantikörpers gegen DCLK1 (1: 400, Abcam, Cambridge, UK) erfolgte, gemäß Protokoll, eine einstündige Inkubation bei Raumtemperatur. Weiters wurden die Gewebeproben erneut in einer PBS-Lösung gewaschen. Unter Vermeidung unnötiger Lichteinwirkung erfolgte das Auftragen der lichtempfindlichen HPR-Polymere für 15 Minuten. Nach Auftragen der HPR-Polymere wurden die Gewebeproben in deionisiertem Wasser für insgesamt 30 Sekunden gewaschen. Für die Sichtbarmachung der DCLK1-Färbung wurde ein Tropfen (40 yL) DAB Plus Chromogen und 2 ml DAB Plus-Substrat vermengt, auf die Proben aufgetragen und dann für 5 Minuten inkubiert. Die Präparate wurden im weiteren Schritt für insgesamt 30 Sekunden je 4 Mal in deionisiertem Wasser gewaschen. Die Gegenfärbung erfolgte mittels Hematoxylin Gill III (Merck, Darmstadt, GER). Die fertig gefärbten Gewebeproben wurden mit einem Deckglas versehen auf ein dauerhaftes Montagemedium (Entellan, Merck, Darmstadt, GER) fixiert. Anschließend wurden die Proben mit dem Olympus BH-2-Mikroskop (Olympus, Tokio, JPN) analysiert und fotografiert.
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Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia

Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia

32Dcl3 (here after named as 32D) and BA/F3 cells (ACC-411, ACC-300, DSMZ, 2018–01) were cultured as described previously [ 25 , 26 ]. TF-1 cells (ACC-334, DSMZ, 2018–01) were cultured using RPMI 1640/ 10%FCS/GM-CSF (5 ng/ml). All cell lines were routinely tested for mycoplasma using PCR. Authentication of cell lines was performed using qRT-PCR for murine or human housekeeping gene as well as cell surface expres- sion of characteristic receptor expression pattern (CD34, CD11b, Gr-1) using FACS analysis. Primary murine cells were cultured in serum-free BIT9500 cell culture medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with mIL-3 (10 ng/ml), mIL-6 (5 ng/ml) and mSCF (50 ng/ml). All cytokines were purchased form ImmunoTools, (Friesoythe, Germany). Further, lineage negative transgenic SCLtTA/Bcr-Abl BM cells were retrovirally infected using MSCV-ER- Hoxb8-Neo plasmid as described previously [ 27 ]. ER- HoxB8 derived immortalized progenitor cells were cultured in IMDM containing 10% FBS, 5% SCF- supernatant and 1% Pen-Strep and selected with G418 (1 mg/ml) for 1 week. FACS analysis for Gr-1, CD11b, B220, CD3 and Ter119 (BioLegend, Fell, Germany) were performed demonstrated the absence of mature cell surface markers.
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Analysis of the impact of Bruton tyrosine kinase on myeloid cell development and function

Analysis of the impact of Bruton tyrosine kinase on myeloid cell development and function

Furthermore, the need for C/EBPα during the transition from CMP to GMP is possibly due to the transcriptional upregulation of PU.1, since forced C/EBPα expression in hematopoietic progenitors favors monopoiesis and not granulopoiesis, whereas exogenous C/EBPα in myeloid cell lines directs granulopoiesis 78 . Nevertheless, C/EBPα is probably indispensable for granulocyte development due to the transcriptional upregulation of several granulocyte-specific factors. One of these factors is the transcription factor growth factor independent-1 (Gfi-1), which is necessary for the repression of proliferation as well as monocyte lineage-promoting factors such as M-CSF, miR21 or miR196b 79 . Another important target of C/EBPα is the transcription factor C/EBPε that is important for terminal granulocyte differentiation, because of the transcriptional control of granule-specific genes (lactoferrin and gelatinase) as well as cell cycle regulation 79 . Besides the upregulation of other transcription factors, C/EBPα force granulocyte development additionally by transactivation of various genes, such as G-CSF receptor 80,81 or myeloperoxidase (MPO) 82 as well as miR223 83 , and downregulation of proliferation by direct interaction with the cell cycle regulator E2F 84,85 . In line with these experimental results is the association of inactivating C/EBPα mutations with hematopoietic malignancies like acute myeloid leukemia and high-risk myelodysplastic syndrome proposing that C/EBPα possesses the ability to arrest cell proliferation and to drive terminal differentiation 86 .
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Preclinical positron emission tomography imaging with the carbon-11-labelled tyrosine kinase inhibitor erlotinib

Preclinical positron emission tomography imaging with the carbon-11-labelled tyrosine kinase inhibitor erlotinib

Positron emission tomography (PET) is a non-invasive imaging method which is used as a diagnostic tool in nuclear medicine. For this purpose, radioactively labeled compounds (so- called radiotracers), which are able to visualize disease-related molecular targets with high affinity and selectivity, are required. A relatively new and not so widely used approach is to perform PET measurements with radiolabeled drugs. This very powerful method has the potential to assess, whether and to which extent a drug of interest reaches its target tissue (e.g. brain, lung, primary tumors and metastases). Other than assessing drug distribution to tissues targeted for treatment, PET with radiolabeled drugs potentially also offers the possibility to determine, if a certain therapeutic molecular target is present or not. In this doctoral thesis small-animal PET experiments were performed with radiolabeled erlotinib, a first-generation tyrosine-kinase inhibitor (TKI) targeting the epidermal growth factor receptor (EGFR) which is used in the clinic for the treatment of non-small cell lung cancer (NSCLC) and pancreatic cancer patients. Factors were investigated which may cause variability in the tissue distribution and excretion of erlotinib. Furthermore, it was investigated, whether PET with radiolabeled erlotinib is able to predict response to erlotinib therapy.
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Macrophage migration inhibitory factor führt in humanen Endothelzellen zur Aktivierung der SRC-Kinase, der PI-3-Kinase und des MAP-Kinase Signalwegs

Macrophage migration inhibitory factor führt in humanen Endothelzellen zur Aktivierung der SRC-Kinase, der PI-3-Kinase und des MAP-Kinase Signalwegs

MIF liegt vorgeformt in intrazellulären Vesikeln vor und wird auf einen Stimulus hin ausgeschüttet. Außerdem zirkuliert MIF im Blutstrom mit einer Konzentration von 3-5 ng/ml [89]. Diese Merkmale unterscheiden MIF von anderen proinflammatorischen Zytokinen, welche auf einen Reiz hin erst synthetisiert werden müssen [8]. Die zellulären Quellen von MIF sind im gesamten Körper verbreitet und reichen von Zellen des Immunsystems über Hypophysenzellen bis hin zu Endothelzellen [28]. Die Makrophagen sind eine der wichtigsten Quellen für MIF und wird aus ihnen nach Stimulierung durch bakterielle Endotoxine und Exotoxine, wie LPS, TSST-1 und SPEA, aber auch durch proinflammatorische Effektormoleküle, wie TNF-α und INF-γ ausgeschüttet [24, 26]. Nach der Freisetzung wirkt MIF wie ein klassisches proinflammatorisches Zytokin, indem es Zellen des Immunsystems aktiviert [86]. Dies ist anhand der steigenden Proliferationsrate von Lymphozyten und einer erhöhten Phagozytoserate der Makrophagen zu sehen [4, 98, 135]. Zudem hemmt MIF die Apoptose von Makrophagen und Neutrophilen [9, 92]. Diese Eigenschaften tragen gemeinsam zu einer verbesserten Immunantwort und wiederum zur Destabilisierung der atherosklrotschen Plaques bei [32]. Zu Beginn einer akuten Entzündung sind erhöhte Gewebe- und Serumspiegel von MIF schädlich [27]. Die proinflammatorische Wirkung von MIF während der akuten Entzündung wird durch die Stimulierung und Ausschüttung vieler entzündungsfördernder Faktoren, wie TNF-α, IL-6, Stickoxid und Produkten der Arachidonsäuremetabolismus deutlich [15, 25, 91]. Des Weiteren ist MIF ein natürlicher Gegenspieler der antiinflammatorischen Glucocorticoide, indem MIF die durch Glucocorticoide induzierte Inhibition der proinflammatorischen Stoffe TNF-α, IL-1, IL-6, IL-8 rückgängig macht [25].
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RIG-I-like-Helikasen als Zielstrukturen für die Immuntherapie des 
hepatozellulären Karzinoms mittels bifunktioneller siRNA gegen Polo-
like-Kinase 1

RIG-I-like-Helikasen als Zielstrukturen für die Immuntherapie des hepatozellulären Karzinoms mittels bifunktioneller siRNA gegen Polo- like-Kinase 1

Die Versuche dieser Arbeit legen eine wichtige Grundlage dafür, dass die RIG-I-basierte Immuntherapie in Kombination mit PLK-1- Hemmung beim HCC als Therapieoption in Zu[r]

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Bruton's Tyrosine Kinase Inhibition Attenuates the Cardiac Dysfunction Caused by Cecal Ligation and Puncture in Mice

Bruton's Tyrosine Kinase Inhibition Attenuates the Cardiac Dysfunction Caused by Cecal Ligation and Puncture in Mice

Keywords: Bruton’s tyrosine kinase (BTK), sepsis, cardiac dysfunction, ibrutinib, acalabrutinib, NLRP3, NF-κB, mice INTRODUCTION Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection ( 1 ), which affects approximately 30 million people worldwide ( 2 ). In the UK, sepsis is the second leading cause of death with 36,000– 64,000 patients dying each year ( 3 ) costing the NHS £2.5 billion annually ( 4 ). The development of cardiac dysfunction affects 40% of septic patients ( 5 ) and is associated with an increased mortality rate of 70–90% in comparison to 20% mortality in patients who do not present with cardiac dysfunction ( 6 ). However, the mechanisms that underlie this cardiac dysfunction are not well-known. Evidence suggests that multiple factors contribute to the pathophysiology of the cardiac dysfunction associated with sepsis. These include the activation of NF-κB and NLRP3 leading to excessive formation of e.g., IL-1 and TNF-α ( 7 , 8 ). There are currently no drugs for the specific treatment of the cardiac dysfunction (or indeed the multiple organ dysfunction) associated with sepsis that specifically target NF-κB and the NLRP3 inflammasome.
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NDEL1-PDGFRB fusion gene in a myeloid malignancy with eosinophilia associated with resistance to tyrosine kinase inhibitors

NDEL1-PDGFRB fusion gene in a myeloid malignancy with eosinophilia associated with resistance to tyrosine kinase inhibitors

the positively charged side chain of R853. By contrast, the DFG-in model suggested the occurrence of two intriguing amino acid interactions upon transition of the A-loop from inactive to the active state (Figure 1a; green). One interaction implicated the negatively charged D850 and the positively charged, conserved H657 in the αC-helix (Figure 1c), which is expected to stabilize the A-loop in the active conformation. 9 This interaction can be further enhanced by the D850E mutation, because the longer side chain of glutamate in comparison with aspartate brings the negatively charged carboxylic group 1.1 Å closer to the positively charged histidine. This increases the stability of the A-loop in the active conformation (Figure 1d), because the forces of electrostatic interaction between opposite charges increase with the second power of decreasing distance, and become largely ineffective at distances exceeding 4.5 Å. 10 The structural model also suggested that the mutation H657K would have an effect similar to the mutation D850E in terms of stabilizing the active conformation (Figure 1e). The other interaction involved R853 and E946 in the C-lobe of the TKD (Figure 1f). The +3 position to D850 is one of the least conserved positions in the A-loop of RTKs from the PDGFR family (Figure 1h), and the arginine at this position in PDGFR β (R853) has the longest side chain among all members. The DFG-in model suggested that the positively charged side chain of R853 can reach a distance of ~ 2.7 Å to the negatively charged carboxyl group of E946, which may facilitate electrostatic bonds and provide additional stabilization of the DFG-in conformation of the PDGFRβ TKD. The structural model
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Phosphatidylinositol 3-kinase p110δ expression in Merkel cell carcinoma

Phosphatidylinositol 3-kinase p110δ expression in Merkel cell carcinoma

Treatment with PI3K inhibitors The cell lines were tested for their sensitivity towards the PI3K p110δ specific inhibitor idelalisib. A decrease of the cell viability of almost all treated cell lines was observed after 120 h incubation. The best response was observed in the B-ALL cell line REH with an IC 50 of 3.1 µM. The cell lines MKL-2, WaGa, PeTa and MCC13 showed for the idelalisib treatment almost a comparable range of IC 50 50 µM to 63 µM. The MKL-1 cells were 2-fold more sensitive compared to all other MCC cell lines. MCC26 cells showed a 1.4- to 1.6-fold weaker response compared to all other MCC cell lines. The multiple myeloma cell line U266 showed a decrease of the cell viability at a concentration of 12.5 µM, an IC 50 value could not be determined for this PI3K p110δ-negative cell line. In general, all MCC cell lines showed with an overall p-value < 0.001 a significant dose-dependent sensitivity to idelalisib after 120 h incubation (Figure 3 and Table 2). The effect of idelalisib on the PI3K pathway of the treated cell lines was tested by Western blotting analyzing the threonine 308 phosphorylation of protein kinase B (AKT) which is located downstream of PI3K. A significant decrease of AKT phosphorylation was restricted to REH and MKL-1 with increasing concentrations of idelalisib (Figure 4).
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Evolutionary divergent ligand activation of PKA-like kinase from Trypanosoma brucei

Evolutionary divergent ligand activation of PKA-like kinase from Trypanosoma brucei

The mutations discussed in section 3.3.2.1 were introduced into the PBC domains of TbPKAR by PCR site directed mutagenesis of overlap extensions as described by (Ho et al., 1989). Briefly, complementary primer pairs containing the mutations of both PBC domains (see section 2.1.1.1) and two other primers (5'PKARHispETDuet and 3'PKARHispETDuet, section 2.1.1.4) annealing at both the 5' and 3' ends for integration into the pETDuet E. coli expression vector, were used to generate the PKAR PBCmut ORF. The first PCRs were with primer pairs 5'PKARHispETDuet/LowerPBC1mut and 3'PKARHispETDuet / UpperPBC1mut, generating two PCR products with overlapping ends. In the second PCR, only the 5'PKARHispETDuet and 3'PKARHispETDuet primers were used with equimolar concentrations of the PCR products from the first PCR reaction. In the first cycle of the reaction, the overlapping ends of the PCR products annealed and acted as primers for the extension to obtain the entire ORF which could then be amplified with the aforementioned primer pair. The process was repeated for mutation of the second PBC domain, using the second complimentary primer pairs (LowerPBC2mut and UpperPBC2mut) and the previous PCR product (containing the first set of mutations) as template. The final PCR product, containing mutations at both PBCs, was ligated into the MCS1 of pETDuet vector to generate pETDuet.PKAR-6xHis PBCmut as described in section 2.1.1.4. The ligation was transformed into E. coli SURE chemical competent cells. The plasmid DNA extraction and purification was performed as described 2.2.4.7. The pETUpstream and DuetDOWN1 Primers were used to verify the integrity of the PKAR PBCmut ORF by sequencing. This vector was then used as template for swapping mutations into the pBSK.PKAR and pBSK.PKAR-Ty1 vectors to generate the corresponding mutant versions as described in section 2.1.1.2.
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Regulation of the interaction between protein kinase C-related protein kinase 2(PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

Regulation of the interaction between protein kinase C-related protein kinase 2(PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

SDS-PAGE (“Sodium DodecylSulfate PolyAcrylamide Gel Electrophoresis”) is a common technique used to separate proteins according to their size. For this purpose, we first prepared SDS-PAGE gels, each consisting of a notched alumina plate, a rectangular glass plate and two side spacers. To prepare a higher amount of gels at the same time, we used a Hoefer multiple gel caster and separated the individual plate assemblies from each other with a thin weighing paper. Then, we mixed the ingredients of the resolving gel solution (Table 3) and poured the mix in the plate assembly. To guarantee a plane surface of the gel and avoid an inhibition of the polymerization reaction of acrylamide / bisacrylamide through the influence of O 2 , we covered the gel surface with isobutanol. After a waiting period of about 20 minutes to allow the gel solution to polymerize, we discharged the isobutanol and prepared the stacking solution according to Table 4. Afterwards, we poured the stacking solution onto the polymerized resolving solution in the plate assembly and in addition, we placed appropriate combs on top of the stacking solution to provide bags for sample loading. The total percent of acrylamide in the resolving gel determines the pore size, which is responsible for the separation of the proteins. Actually, smaller proteins migrate more easily through the pores further down the gel, while larger proteins come across more resistance and consequently remain closer to the starting point.
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Die Rolle der Proteine Piwi-like 1 und Piwi-like 3 für die Spermatogenese des Menschen und die männliche Fertilität

Die Rolle der Proteine Piwi-like 1 und Piwi-like 3 für die Spermatogenese des Menschen und die männliche Fertilität

In Studien konnte gezeigt werden, dass sowohl Mili-null als auch Miwi-like2 - null Mäuse steril sind aufgrund der beeinträchtigten Spermatogenese in der pachytänen Phase. Miwi-like 2 defiziente Mäuse zeigen einen Defekt in der Meiose in der frühen Prophase von Meiose I und einen Verlust der Stammzellen. Mili und Miwi sind zytoplasmatische Proteine, wohingegen Miwi-like2 im Kern gefunden wird und nur für eine kurze Zeit der Meiose exprimiert wird. Interessanterweise wird Miwi-like2 bei Mili-Knockout Mäusen im Zytoplasma gefunden. Bei den Miwi-like2 Knockout Mäusen findet sich Mili weiterhin im Zytoplasma, was daraufhin weißt, dass Mili für die Lokalisation von Miwi-like2 im Kern verantwortlich ist (Bak et al. 2011). Es ist bekannt, dass nicht nur die Piwi-Proteine eine essentielle Rolle für die Fertilität spielen, sondern auch der Verlust von Komponenten des piRNA-Silencing Systems führt für gewöhnlich zur Sterilität (Yakushev et al. 2013).
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Functional characterization of a novel Xenopus polo-like kinase interacting protein

Functional characterization of a novel Xenopus polo-like kinase interacting protein

When wildtype XErp1 was incubated in anaphase-arrested egg extract that lacks CaMKII activity it experienced the M-phase specific upshift like in CSF extract, but remained stable over the course of the experiment (see Figure 2.14a, middle). In contrast, wildtype XErp1 was very rapidly degraded in extract in which CaMKII activity was retriggered by the addition of calcium (see Figure 2.14a, right). This degradation could be prevented with a peptide corresponding to the calmodulin-binding region in CaMKII that prevents efficient activation of the kinase (see Figure 2.14a, left). It was shown that CaMKII can phosphorylate XErp1 directly and that phosphorylation on threonine 195 leads to enhanced Plx1 recruitment which triggers XErp1 degradation (Rauh et al., 2005). Consequently, a mutant in this critical residue was resistant to calcium-induced degradation. Interestingly, the previously proposed CSF component Emi1 was unstable in anaphase extract in the presence or absence of CaMKII activity suggesting that its stability might not be regulated by fertilization and the subsequent calcium-signal.
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