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Search Results: 1 - 10 of 1504 matches for " Marius Ueffing "
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Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila
Ana Griciuc,Liviu Aron ,Michel J. Roux,Rüdiger Klein,Angela Giangrande,Marius Ueffing
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1001075
Abstract: The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (RhP23H). Unlike the wild-type Rh, mutant RhP23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded RhP23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that RhP23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1P37H (the equivalent of mammalian RhP23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1P37H retina. Despite this, Rh1P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1P37H retina. Pharmacological treatment of Rh1P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration.
Bovine neonatal pancytopenia - Comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK)
Kerstin N Euler, Stefanie M Hauck, Marius Ueffing, Cornelia A Deeg
BMC Veterinary Research , 2013, DOI: 10.1186/1746-6148-9-18
Abstract: By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production.The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.Bovine neonatal pancytopenia (BNP) is a disease transferred by colostral alloantibodies binding to peripheral blood-derived leukocytes and platelet antigens of calves [1]. Remarkably, calves develop a severe thrombocytopenia and leukocytopenia within few hours after passive transfer of colostral antibodies to blood and die within several days from bleeding disorder and bone marrow depletion [1,2]. Respective alloantibodies responsible for BNP can develop in cows previously vaccinated with a specific Bovine Viral Diarrhoea (BVD) vaccine (PregSure BVD; Pfizer, Berlin, Germany; vaccine A) [1]. Colostra of these cows transfer BNP to healthy calves, indicating a commonly expressed target antigen in responding calves [2]. Alloantibodies are also detectable in blood of respective BNP dams [1], suggesting their development to be systemically and not directly in udder. Further immunological characterization of these antibodies revealed that they were of IgG1 subclass [3]. IgG1 antibodies reflect a Th2-response in cows. So far, Major histocompatibility comple
Unraveling the Equine Lymphocyte Proteome: Differential Septin 7 Expression Associates with Immune Cells in Equine Recurrent Uveitis
Roxane L. Degroote, Stefanie M. Hauck, Barbara Amann, Sieglinde Hirmer, Marius Ueffing, Cornelia A. Deeg
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091684
Abstract: Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease.
ARHGEF7 (BETA-PIX) Acts as Guanine Nucleotide Exchange Factor for Leucine-Rich Repeat Kinase 2
Karina Haebig,Christian Johannes Gloeckner,Marta Garcia Miralles,Frank Gillardon,Claudia Schulte,Olaf Riess,Marius Ueffing,Saskia Biskup,Michael Bonin
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0013762
Abstract: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro.
The Enhancer of split transcription factor Her8a is a novel dimerisation partner for Her3 that controls anterior hindbrain neurogenesis in zebrafish
Katharine J Webb, Marion Coolen, Christian J Gloeckner, Christian Stigloher, Brigitte Bahn, Stefanie Topp, Marius Ueffing, Laure Bally-Cuif
BMC Developmental Biology , 2011, DOI: 10.1186/1471-213x-11-27
Abstract: We used a yeast two-hybrid screen with Her5 as bait and recovered a novel zebrafish Hairy/E(spl) factor - Her8a. Using phylogenetic and synteny analyses, we demonstrate that her8a evolved from an ancient duplicate of Hes6 that was recently lost in the mammalian lineage. We show that her8a is expressed across the mid- and anterior hindbrain from the start of segmentation. Through knockdown and misexpression experiments, we demonstrate that Her8a is a negative regulator of neurogenesis and plays an essential role in generating progenitor pools within rhombomeres 2 and 4 - a role resembling that of Her3. Her8a co-purifies with Her3, suggesting that Her8a-Her3 heterodimers may be relevant in this domain of the neural plate, where both proteins are co-expressed. Finally, we demonstrate that her8a expression is independent of Notch signaling at the early neural plate stage but that SoxB factors play a role in its expression, linking patterning information to neurogenesis control. Overall, the regulation and function of Her8a differ strikingly from those of its closest relative in other vertebrates - the Hes6-like proteins.Our results characterize the phylogeny, expression and functional interactions involving a new Her factor, Her8a, and highlight the complex interplay of E(spl) proteins that generates the neurogenesis pattern of the zebrafish early neural plate.Neurogenesis in the early vertebrate neural plate begins at stereotyped loci - termed proneural clusters -, which prefigure the localization of the earliest neuronal groups and the architecture of the primary embryonic neuronal scaffold. These proneural clusters consist of spatially defined progenitor groups engaged in active neurogenesis, within which committed precursors expressing higher levels of proneural genes (such as neurogenin or achaete-scute-like genes, respectively neurog1 and ascl1 in zebrafish) are singled out to differentiate first. An identical scaffold is found in all vertebrate embryos, highlight
The Equine CD4+ Lymphocyte Proteome
Roxane L. Degroote,Sandra Helm,Ute Klein,Ramona Schmitt,Marius Ueffing,Stefanie M. Hauck,Cornelia A. Deeg
Dataset Papers in Science , 2014, DOI: 10.1155/2014/105312
Abstract: CD4+ T cells are key players in immunology and disease pathology, including relapsing autoimmune uveitis. Equine recurrent uveitis is the only spontaneous animal model for this disease in man. Knowledge about the CD4+ cell proteome is crucial for studies on possible changes in proteome expression of CD4+ effector cells in disease. For this purpose, we generated a reference dataset of the equine CD4+ cell proteome by sorting equine CD4+ lymphocytes followed by analysis of whole cell lysate as well as membrane protein fraction using mass spectrometry. 1. Introduction CD4+ lymphocytes play a major role in several immunological processes and diseases, including autoimmune uveitis [1]. Several experimental animal models exist for this disease; however, due to striking immunopathological and clinical similarities, equine recurrent uveitis is the only spontaneous model for relapsing autoimmune uveitis in man [2]. Equine recurrent uveitis is an autoimmune mediated disease affecting horses worldwide [3]. It presents with painful, remitting-relapsing inflammatory attacks of inner eye structures alternating with stages of quiescence [4]. Directly prior to a uveitic attack, immune cells are activated in periphery, migrate into the eye, and attack the retina [5–7]. These cells infiltrating the eye are mainly CD4+ T cells with a Th1 phenotype [7, 8]. Knowledge on the protein repertoire of CD4+ cells is crucial for the investigation of potential changes in protein expression occurring in these cells in course of immune reactions. To create a solid fundament for further studies, we generated a table of all proteins expressed in CD4+ cells (Dataset Item 1 (Table)) as well as a separate table comprising only membrane associated proteins (Dataset Item 2 (Table)). For this purpose, CD4+ lymphocytes were isolated from total equine lymphocytes by fluorescence activated cell sorting. Subsequently, we extracted membrane proteins from these cells and analyzed this protein fraction using mass spectrometry. In parallel, we performed mass spectrometry analysis on whole CD4+ cell lysates (Figure 1). Figure 1: Complete workflow for our study. Left panel shows isolation and staining of equine lymphocytes, middle panel shows cell sorting, and right panel shows further processing of sorted cells for mass spectrometry analysis. The two dataset items presented in this study give a detailed description of the physiological CD4+ immune cell proteome repertoire and set a reference for further comparative proteomic studies on activated cells or those altered in course of disease. 2.
CRALBP is a Highly Prevalent Autoantigen for Human Autoimmune Uveitis
Cornelia A. Deeg,Albert J. Raith,Barbara Amann,John W. Crabb,Stephan R. Thurau,Stefanie M. Hauck,Marius Ueffing,Gerhild Wildner,Manfred Stangassinger
Clinical and Developmental Immunology , 2007, DOI: 10.1155/2007/39245
Abstract: Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P<.01) in human uveitis patients than in normal controls. Thirty out of 56 tested uveitis patient's sera contained autoantibodies reactive against CRALBP, compared to only four out of 23 normal control subjects. The presence of CRALBP autoantibodies in 54% of tested uveitis patients supports CRALBP as a possible autoantigen in human autoimmune uveitis.
Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Christoph M. Szober,Stefanie M. Hauck,Kerstin N. Euler,Kristina J. H. Fr?hlich,Claudia Alge-Priglinger,Marius Ueffing,Cornelia A. Deeg
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms131114053
Abstract: The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.
Successful Subretinal Delivery and Monitoring of MicroBeads in Mice
M. Dominik Fischer, Tobias Goldmann, Christine Wallrapp, Regine Mühlfriedel, Susanne C. Beck, Gabi Stern-Schneider, Marius Ueffing, Uwe Wolfrum, Mathias W. Seeliger
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055173
Abstract: Background To monitor viability of implanted genetically engineered and microencapsulated human stem cells (MicroBeads) in the mouse eye, and to study the impact of the beads and/or xenogenic cells on retinal integrity. Methodology/Principal Findings MicroBeads were implanted into the subretinal space of SV126 wild type mice using an ab externo approach. Viability of microencapsulated cells was monitored by noninvasive retinal imaging (Spectralis? HRA+OCT). Retinal integrity was also assessed with retinal imaging and upon the end of the study by light and electron microscopy. The implanted GFP-marked cells encapsulated in subretinal MicroBeads remained viable over a period of up to 4 months. Retinal integrity and viability appeared unaltered apart from the focal damage due to the surgical implantation, GFAP upregulation, and opsin mistargeting in the immediate surrounding tissue. Conclusions/Significance The accessibility for routine surgery and its immune privileged state make the eye an ideal target for release system implants for therapeutic substances, including neurotrophic and anti-angiogenic compounds or protein based biosimilars. Microencapsulated human stem cells (MicroBeads) promise to overcome limitations inherent with single factor release systems, as they are able to produce physiologic combinations of bioactive compounds.
Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface
Claudia S. Priglinger, Christoph M. Szober, Siegfried G. Priglinger, Juliane Merl, Kerstin N. Euler, Marcus Kernt, Gabor Gondi, Jennifer Behler, Arie Geerlof, Anselm Kampik, Marius Ueffing, Stefanie M. Hauck
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0070011
Abstract: Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.
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