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Hornig J.,Friedrich - Alexander - University, Erlangen - Nuremberg | Frob F.,Friedrich - Alexander - University, Erlangen - Nuremberg | Vogl M.R.,Friedrich - Alexander - University, Erlangen - Nuremberg | Hermans-Borgmeyer I.,Universitatsklinikum Eppendorf | And 2 more authors.
PLoS Genetics | Year: 2013

Myelin is essential for rapid saltatory conduction and is produced by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system. In both cell types the transcription factor Sox10 is an essential component of the myelin-specific regulatory network. Here we identify Myrf as an oligodendrocyte-specific target of Sox10 and map a Sox10 responsive enhancer to an evolutionarily conserved element in intron 1 of the Myrf gene. Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes. This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination. Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented. © 2013 Hornig et al.


Bhuwania R.,Universitatsklinikum Eppendorf | Cornfine S.,Universitatsklinikum Eppendorf | Fang Z.,University of Massachusetts Medical School | Kruger M.,Max Planck Institute for Heart and Lung Research | And 3 more authors.
Journal of Cell Science | Year: 2012

Podosomes are actin-rich adhesion and invasion structures. Especially in macrophages, podosomes exist in two subpopulations, large precursors at the cell periphery and smaller podosomes (successors) in the cell interior. To date, the mechanisms that differentially regulate these subpopulations are largely unknown. Here, we show that the membrane-associated protein supervillin localizes preferentially to successor podosomes and becomes enriched at precursors immediately before their dissolution. Consistently, podosome numbers are inversely correlated with supervillin protein levels. Using deletion constructs, we find that the myosin II regulatory Nterminus of supervillin [SV(1-174)] is crucial for these effects. Phosphorylated myosin light chain (pMLC) localizes at supervillinpositive podosomes, and time-lapse analyses show that enrichment of GFP-supervillin at podosomes coincides with their coupling to contractile myosin-IIA-positive cables. We also show that supervillin binds only to activated myosin IIA, and a dysregulated N-terminal construct [SV(1-830)] enhances pMLC levels at podosomes. Thus, preferential recruitment of supervillin to podosome subpopulations might both require and induce actomyosin contractility. Using siRNA and pharmacological inhibition, we demonstrate that supervillin and myosin IIA cooperate to regulate podosome lifetime, podosomal matrix degradation and cell polarization. In sum, we show here that podosome subpopulations differ in their molecular composition and identify supervillin, in cooperation with myosin IIA, as a crucial factor in the regulation of podosome turnover and function. © 2012.


In order to identify requirements of quality assurance in this field, a general description of prevention, health promotion and education is outlined, based upon healthcare supply analyses and 158 interviews with experts. Prominent features are, among others, a distinctive heterogeneity and complexity of settings, suppliers, and interventions; supply of coverage is below public health criteria (needs); weak outcome evaluation; competition among suppliers, providers, and political agencies and decision makers; need for intra-and intersectoral coordination; the lack of evidence-based healthcare planning; and hurdles for quality assurance. A structural taxonomy of quality assurance systems is then developed, consisting of three dimensions: validity (quality of information), regularity, and degree of commitment and obligation. In terms of these dimensions, important systems of quality assurance in prevention, health promotion and education in Germany are analyzed. A number of different systems and approaches can be found. However, most of them share questionable validity, regularity, and degree of obligation. Increased commitment on behalf of providers, suppliers, and political institutions and decision makers for the quality of preventive interventions is inevitable in order to raise the performance of prevention and health promotion in Germany to their potential effectiveness. © Springer-Verlag 2011.


Cervero P.,Universitatsklinikum Eppendorf | Himmel M.,Universitatsklinikum Eppendorf | Kruger M.,Max Planck Institute for Heart and Lung Research | Linder S.,Universitatsklinikum Eppendorf
European Journal of Cell Biology | Year: 2012

Podosomes are multifunctional organelles of invasive cells that combine several key abilities, including adhesion, matrix degradation and mechanosensing. The necessary spatiotemporal fine-tuning of podosome structure, turnover and function implies the existence of an intricate network of proteins, comparable to other integrin-based adhesions. However, no systematic effort has yet been made to map the podosome proteome. Here, we describe the purification of podosome-enriched fractions from primary human macrophages, labelled with isotopically stable amino acids, and the subsequent mass spectrometric analysis of these fractions. We present a consensus list of 203 proteins, comprising 33 known podosome proteins and 170 potential novel components. We also present second-level analyses of the podosome proteome, as well as proof-of-principle experiments by showing that the newly identified components WDR1/AIP-1 and hnRNP-K localise to the core structure of macrophage podosomes. Comparisons with other adhesion structure proteomes confirm that the podosome proteome shares components with focal adhesions and invadopodia, but also reveal an extensive overlap with spreading initiation centres (SICs). We suggest that the consensus list comprises a significant part of the podosome proteome and will be helpful for future studies on podosome structure, composition and function, and also for detailed classification of adhesion structure subtypes. © 2012 Elsevier GmbH.


Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders.


Cervero P.,Universitatsklinikum Eppendorf | Panzer L.,Universitatsklinikum Eppendorf | Linder S.,Universitatsklinikum Eppendorf
Methods in Molecular Biology | Year: 2013

Podosomes are multifunctional organelles of invasive cells that combine several key abilities including cell-matrix adhesion, extracellular matrix degradation, and mechanosensing. In combination with their high turnover rates that allow quick adaptation to the pericellular environment, podosomes are likely to play important roles during invasive migration of cells. Primary human macrophages constitutively form numerous podosomes and are thus an ideal system for the quantitative study of podosome dynamics. This protocol describes assays for the study of podosome dynamics, namely, reformation of podosomes, in fixed and living cells, with subsequent software-based analyses allowing the extraction of quantitative parameters such as the number of podosomes per cell, podosome density, and half times for podosome disruption and reformation. Moreover, we describe the preparation of podosome-enriched cell fractions and their analysis by immunoblotting. © Springer Science+Business Media, LLC 2013.


Stummer W.,University of Munster | Van Den Bent M.J.,Erasmus University Rotterdam | Westphal M.,Universitatsklinikum Eppendorf
Acta Neurochirurgica | Year: 2011

Background: This article discusses data from 3 randomized phase 3 trials, supporting a role for surgery in glioblastoma. Methods: Data were reviewed by extent of resection during primary surgery from the ALA-Glioma Study (fluorescence-guided versus conventional resection), the BCNU wafer study (BCNU wafer versus placebo), and the EORTC Study 26981-22981 (radiotherapy versus chemoradiotherapy with temozolomide). Results: For glioblastoma patients in the ALA study, median survival was 16.7 and 11.8 months for complete versus partial resection, respectively (P<0.0001). Survival effects were maintained after correction for differences in age and tumor location. For glioblastoma patients who received ≥90% resection in the BCNU wafer study, median survival increased for BCNU wafer versus placebo (14.5 versus 12.4 months, respectively; P=0.02), but no survival increase was found for <90% resection (11.7 versus 10.6 months, respectively; P=0.98). In the EORTC study, absolute median gain in survival with chemoradiotherapy versus radiotherapy was greatest for complete resections (+4.1 months; P=0.0001), compared with partial resections (+1.8 months; P=0.0001), or biopsies (+1.5 months; P=0.088), suggesting surgery enhanced adjuvant treatment. Conclusion: Complete resection appears to improve survival and may increase the efficacy of adjunct/adjuvant therapies. If safely achievable, complete resection should be the surgical goal for glioblastoma. © 2011 Springer-Verlag.


Langer F.,Universitatsklinikum Eppendorf | Ruf W.,Scripps Research Institute | Ruf W.,Universitatsmedizin Mainz
Thrombosis and Haemostasis | Year: 2014

Tissue factor (TF), the cellular receptor and cofactor for factor VII/VIIa, initiates haemostasis and thrombosis. Initial tissue distribution studies suggested that TF was sequestered from the circulation and only present at perivascular sites. However, there is now clear evidence that TF also exists as a blood-borne form with critical contributions not only to arterial thrombosis following plaque rupture and to venous thrombosis following endothelial perturbation, but also to various other clotting abnormalities associated with trauma, infection, or cancer. Because thrombin generation, fibrin deposition, and platelet aggregation in the contexts of haemostasis, thrombosis, and pathogen defence frequently occur without TF de novo synthesis, considerable efforts are still directed to understanding the molecular events underlying the conversion of predominantly non-coagulant or cryptic TF on the surface of haematopoietic cells to a highly procoagulant molecule following cellular injury or stimulation. This article will review some of the still controversial mechanisms implicated in cellular TF activation or decryption with particular focus on the coordinated effects of outer leaflet phosphatidylserine exposure and thiol-disulfide exchange pathways involving protein disulfide isomerase (PDI). In this regard, our recent findings of ATP-triggered stimulation of the purinergic P2X7 receptor on myeloid and smooth muscle cells resulting in potent TF activation and shedding of procoagulant microparticles as well as of rapid monocyte TF decryption following antithymocyte globulin-dependent membrane complement fixation have delineated specific PDIdependent pathways of cellular TF activation and thus illustrated additional and novel links in the coupling of inflammation and coagulation. © Schattauer 2014.


Wiesner. C.,Universitatsklinikum Eppendorf | El Azzouzi K.,Universitatsklinikum Eppendorf | Linder S.,Universitatsklinikum Eppendorf
Journal of Cell Science | Year: 2013

The matrix metalloproteinase MT1-MMP has a major impact on invasive cell migration in both physiological and pathological settings such as immune cell extravasation or metastasis of cancer cells. Surface-associated MT1-MMP is able to cleave components of the extracellular matrix, which is a prerequisite for proteolytic invasive migration. However, current knowledge on the molecular mechanisms that regulate MT1-MMP trafficking to and from the cell surface is limited. We have identified three members of the RabGTPase family, Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in primary human macrophages. Both overexpressed and endogenous forms show prominent colocalisation with MT1-MMP-positive vesicles, whereas expression of mutant constructs, as well as siRNA-induced knockdown, reveal that these RabGTPases are crucial in the regulation of MT1-MMP surface exposure, contact of MT1-MMP-positive vesicles with podosomes, extracellular matrix degradation in two and three dimensions, as well as three-dimensional proteolytic invasion of macrophages. Collectively, our results identify Rab5a, Rab8a and Rab14 as major regulators of MT1-MMP trafficking and invasive migration of primary human macrophages, which could be promising potential targets for manipulation of immune cell invasion. © 2013 Published by The Company of Biologists Ltd.


Hummel F.C.,Universitatsklinikum Eppendorf
Nervenarzt | Year: 2014

Modern neuroimaging techniques, such as structural and functional magnetic resonance imaging (MRI) and non-invasive brain stimulation techniques, such as transcranial magnetic stimulation (TMS) are increasingly used in the neuroscientific research of neurological disorders, such as stroke, tinnitus and movement disorders. These methods offer a non-invasive approach and especially in combination, not only the opportunity to add to the pathophysiological understanding of these disorders but also to provide information about the functional recovery and the natural course of the disease in a predictive way. Based on such knowledge therapeutic approaches can be adapted in a patient-tailored fashion to achieve the best therapeutic effects. Furthermore, these methods might provide additional non-invasive information for neurosurgical interventions reducing perioperative interventional risks.In the present article these aspects will be discussed with a focus on the combination of MRI and TMS especially addressed for the topic of recovery from stroke. © Springer-Verlag Berlin Heidelberg 2014.

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