Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules

AbstractNon-hydrolysable analogues of GTP, such as GTPγS and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [AIF4], a compound known to act on trimeric G-proteins. Addition of highly purified G-protein βγ subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein α subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN

Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia.

Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5'-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy

Negative feedback regulation of MAPK signaling is an important driver of chronic lymphocytic leukemia progression

Despite available targeted treatments for the disease, drug-resistant chronic lymphocytic leukemia (CLL) poses a clinical challenge. The objective of this study is to examine whether the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate mitogen-activated protein kinases (MAPKs) and thus counterbalance excessive MAPK activity. We show that high expression of DUSP6 in CLL correlates with poor clinical prognosis. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small-molecule inhibitor reduces CLL cell survival in vitro and in vivo. Using global phospho-proteome approaches, we observe acute activation of MAPK signaling by DUSP1/6 inhibition. This promotes accumulation of mitochondrial reactive oxygen species and, thereby, DNA damage and apoptotic cell death in CLL cells. Finally, we observe that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising treatment concept to eliminate drug-resistant CLL cells

Epithelial IL-23R Signaling Licenses Protective IL-22 Responses in Intestinal Inflammation.

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.This work was supported by DFG Excellence Cluster Inflammation at Interfaces; the SFB877 B9, the SFB 1182 C2 project, and the BMBF IHEC DEEP project TP2.3 and 5.2 (to P.R.); the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007- 2013)/ERC grant agreement 260961 (to A.K.); the National Institute for Health Research Cambridge Biomedical Research Centre, ERC CoG GA 648889, and WTIA 106260-Z-14-Z (to A.K.); NIH DK53056, DK44319, and DK088199 (to R.S.B.); and the Fondation pour la Recherche Medicale (to M.C.).This is the final version of the article. It first appeared from Cell/Elsevier via http://dx.doi.org/10.1016/j.celrep.2016.07.05

Correlation chemical shift imaging with low-power adiabatic pulses and constant-density spiral trajectories

In this work we introduce the concept of correlation chemical shift imaging (CCSI). Novel CCSI pulse sequences are demonstrated on clinical scanners for two-dimensional Correlation Spectroscopy (COSY) and Total Correlation Spectroscopy (TOCSY) imaging experiments. To date there has been limited progress reported towards a feasible and robust multivoxel 2D COSY. Localized 2D TOCSY imaging is shown for the first time in this work. Excitation with adiabatic GOIA-W(16,4) pulses (Gradient Offset Independent Adiabaticity Wurst modulation) provides minimal chemical shift displacement error, reduced lipid contamination from subcutaneous fat, uniform optimal flip angles, and efficient mixing for coupled spins, while enabling short repetition times due to low power requirements. Constant-density spiral readout trajectories are used to acquire simultaneously two spatial dimensions and f2 frequency dimension in (kx,ky,t2) space in order to speed up data collection, while f1 frequency dimension is encoded by consecutive time increments of t1 in (kx,ky,t1,t2) space. The efficient spiral sampling of the k-space enables the acquisition of a single-slice 2D COSY dataset with an 8 × 8 matrix in 8:32 min on 3 T clinical scanners, which makes it feasible for in vivo studies on human subjects. Here we present the first results obtained on phantoms, human volunteers and patients with brain tumors. The patient data obtained by us represent the first clinical demonstration of a feasible and robust multivoxel 2D COSY. Compared to the 2D J-resolved method, 2D COSY and TOCSY provide increased spectral dispersion which scales up with increasing main magnetic field strength and may have improved ability to unambiguously identify overlapping metabolites. It is expected that the new developments presented in this work will facilitate in vivo application of 2D chemical shift correlation MRS in basic science and clinical studies.National Institutes of Health (U.S.) (NIH grant R01 1200-206456)National Institutes of Health (U.S.) (NIH grant R01 EB007942)Siemens Aktiengesellschaft (Siemens-MIT Alliance

K(2P)18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K(2P)18.1 is a relevant regulator. Here, we identify K(2P)18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-kappa B-mediated K(2P)18.1 upregulation in tTreg progenitors. K(2P)18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-kappa B- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K(2P)18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K(2P)18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K(2P)18.1 variant that is associated with poor clinical outcomes indicate that K(2P)18.1 also plays a role in human Treg development. Pharmacological modulation of K(2P)18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K(2P)18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K(2P)18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.Peer reviewe

European Recovery System (ERS)

Up to the TEXUS-44 microgravity research mission, successfully launched in February 2008, the payloads of the TEXUS vehicles were exclusively equipped with the Magellan (former Bristol Aerospace Ltd.) ORSA recovery system, integrated into the ogive nose cone. With the intention to gain more independency from the North American market and the inherent procurement and ITAR regulations problems, the European Space Agency (ESA) has taken initiative to contract industry for the development and built-up of a new European Recovery System (ERS) in 2006. For the design, manufacturing and qualification task sharing, a cooperation of DLR Moraba and the Kayser-Threde GmbH has been initialized. The ERS is designed to recover payloads of up to 450 kg mass and 17 inch (438 mm) diameter by a two-stage subsonic parachute system. It features a separating ogive nose tip with a forward deploying recovery parachute. The assembly incorporates a 3:1 fineness ratio ogive which forward portion is ejected exo-atmospherically to permit subsequent parachute recovery system operation. The aft ogive houses the parachute system, autonomous redundant pyrotechnic ignition system, housekeeping electronics, TM interface, beacon system, camera system and pyrotechnic and electronic batteries. The parachute system activation is controlled by barometric switches on the descent trajectory at a nominal altitude of 15 kft (4.6 km) in the combination of an electronic timing activation unit (ignition unit). Together with the heat shield ejection the drogue parachute is deployed and the payload is mainly stabilized from flat spin and decelerated. After complete stabilization the drogue parachute is separated and extracts the main parachute out of the deployment bag. At fully opened main parachute the final sink rate is around 8 m/sec

The Demand for Regulation for the German Legislator to Adjust the Stock Companies Law to the SE-Regulation Regarding the Board of Management

Die Dissertation behandelt den Regelungsbedarf des deutschen Gesetzgebers zur Anpassung des deutschen Aktienrechts im Hinblick auf den Vorstand vor dem Hintergrund der am 08. Oktober 2001 verabschiedeten und am 10. November desselben Jahres im Amtsblatt der EG veröffentlichten Verordnung (EG) Nr. 2157/2001 über das Statut der Europäischen Gesellschaft (SE). Die Arbeit betrachtet zunächst die genannte Verordnung im System des Europäischen sowie des deutschen Rechts und stellt anschließend die allgemeinen Grundlagen des Regelungsbedürfnisses des deutschen Gesetzgebers dar, so insbesondere Grundsätzliches zur Verweisungstechnik und zu den einzelnen Verweisungsarten. Nach Darlegung der Beschränkungen und der zu beachtenden Grundsätze im Rahmen der Anpassungen des nationalen Rechts werden die zu regelnden Bereiche und der konkrete Regelungsbedarf des deutschen Gesetzgebers in einem SE-Ausführungsgesetz dargestellt. Dabei werden sowohl das dualistische wie auch das im deutschen Aktienrecht bisher unbekannte monistische Verwaltungsmodell untersucht und verschiedene Änderungen und Ergänzungen des bestehenden deutschen Rechts erörtert.The thesis looks at the demand for regulation for the German legislator to adjust the German stock companies law regarding the board of management to the EC-Regulation No. 2157/2001 on the Societas Europaea. The thesis first deals with the mentioned EC Regulation and, then, describes the basic demands for adjustment, fundamental techniques and ways of reference. After explaining the limits and principles which have to be considered adjusting German national law, the scope and demands for regulation in a German SE-Execution Act is described, in particular the so far in German stock companies law unknown monistic model of administration as well as other necessary alterations and additions to German law