156 results on '"SEIFERT, Oliver"'
Search Results
152. Identification and characterization of RNA guanine-quadruplex binding proteins.
- Author
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von Hacht A, Seifert O, Menger M, Schütze T, Arora A, Konthur Z, Neubauer P, Wagner A, Weise C, and Kurreck J
- Subjects
- Actin-Related Protein 2 metabolism, HEK293 Cells, HeLa Cells, Humans, Matrix Metalloproteinase 16 metabolism, Protein Biosynthesis, RNA-Binding Proteins analysis, 5' Untranslated Regions, Actin-Related Protein 2 genetics, G-Quadruplexes, Matrix Metalloproteinase 16 genetics, RNA-Binding Proteins metabolism
- Abstract
Guanine quadruplex (G-quadruplex) motifs in the 5' untranslated region (5'-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2014
- Full Text
- View/download PDF
153. Expression and purification of recombinant antibody formats and antibody fusion proteins.
- Author
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Siegemund M, Richter F, Seifert O, Unverdorben F, and Kontermann RE
- Subjects
- Antibodies genetics, Cell Line, Escherichia coli genetics, Escherichia coli metabolism, Humans, Recombinant Fusion Proteins genetics, Recombinant Proteins genetics, Antibodies metabolism, Recombinant Fusion Proteins metabolism, Recombinant Proteins metabolism
- Abstract
In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains.
- Published
- 2014
- Full Text
- View/download PDF
154. [Psoriasis is more than just a skin disease. Patients should be monitored for cardiovascular risk factor].
- Author
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Duvetorp A, Ljungberg A, and Seifert O
- Subjects
- Comorbidity, Diabetes Mellitus epidemiology, Early Diagnosis, Humans, Hyperlipidemias epidemiology, Hypertension epidemiology, Metabolic Syndrome epidemiology, Patient Education as Topic, Psoriasis classification, Psoriasis diagnosis, Risk Factors, Cardiovascular Diseases epidemiology, Psoriasis epidemiology
- Published
- 2013
155. Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion.
- Author
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Wäster P, Rosdahl I, Gilmore BF, and Seifert O
- Subjects
- Becaplermin, Cell Line, Cell Movement radiation effects, Endopeptidases, Humans, Keratinocytes metabolism, Keratinocytes radiation effects, Melanocytes metabolism, Melanocytes radiation effects, Platelet-Derived Growth Factor metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-sis, Transforming Growth Factor beta1 metabolism, Wnt Proteins metabolism, Wnt-5a Protein, Fibroblasts metabolism, Fibroblasts radiation effects, Gelatinases metabolism, Gene Expression Regulation, Neoplastic radiation effects, Melanoma pathology, Membrane Proteins metabolism, Neoplasm Invasiveness pathology, Serine Endopeptidases metabolism, Ultraviolet Rays adverse effects
- Abstract
Fibroblast activation protein-α (FAP-α) promotes tumor growth and cell invasiveness through extracellular matrix degradation. How ultraviolet radiation (UVR), the major risk factor for malignant melanoma, influences the expression of FAP-α is unknown. We examined the effect of UVR on FAP-α expression in melanocytes, keratinocytes and fibroblasts from the skin and in melanoma cells. UVR induces upregulation of FAP-α in fibroblasts, melanocytes and primary melanoma cells (PM) whereas keratinocytes and metastatic melanoma cells remained FAP-α negative. UVA and UVB stimulated FAP-α-driven migration and invasion in fibroblasts, melanocytes and PM. In co-culture systems UVR of melanocytes, PM and cells from regional metastases upregulated FAP-α in fibroblasts but only supernatants from non-irradiated PM were able to induce FAP-α in fibroblasts. Further, UV-radiated melanocytes and PM significantly increased FAP-α expression in fibroblasts through secretory crosstalk via Wnt5a, PDGF-BB and TGF-β1. Moreover, UV radiated melanocytes and PM increased collagen I invasion and migration of fibroblasts. The FAP-α/DPPIV inhibitor Gly-ProP(OPh)2 significantly decreased this response implicating FAP-α/DPPIV as an important protein complex in cell migration and invasion. These experiments suggest a functional association between UVR and FAP-α expression in fibroblasts, melanocytes and melanoma cells implicating that UVR of malignant melanoma converts fibroblasts into FAP-α expressing and ECM degrading fibroblasts thus facilitating invasion and migration. The secretory crosstalk between melanoma and tumor surrounding fibroblasts is mediated via PDGF-BB, TGF-β1 and Wnt5a and these factors should be evaluated as targets to reduce FAP-α activity and prevent early melanoma dissemination.
- Published
- 2011
- Full Text
- View/download PDF
156. Downregulation of SMAD2, 4 and 6 mRNA and TGFbeta receptor I mRNA in lesional and non-lesional psoriatic skin.
- Author
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Yu H, Mrowietz U, and Seifert O
- Subjects
- Adult, Female, Humans, Male, Protein Serine-Threonine Kinases genetics, RNA, Messenger genetics, Receptor, Transforming Growth Factor-beta Type I, Receptors, Transforming Growth Factor beta genetics, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction genetics, Smad7 Protein genetics, Smad7 Protein metabolism, Transforming Growth Factor beta genetics, Down-Regulation physiology, Protein Serine-Threonine Kinases metabolism, Psoriasis genetics, Psoriasis metabolism, RNA, Messenger metabolism, Receptors, Transforming Growth Factor beta metabolism, Smad2 Protein metabolism, Smad4 Protein metabolism, Smad6 Protein genetics, Transforming Growth Factor beta metabolism
- Abstract
Transforming growth factor beta (TGFbeta) has been suggested to be an effective inhibitor of the increased keratinocyte proliferation in psoriasis. Three TGFbeta isoforms are described (TGFbeta1, 2 and 3), signalling via a heteromeric receptor complex of TGFbetaRI and TGFbetaRII. Receptor binding activates SMAD2, 3 and 4, which translocate into the nucleus and regulate TGFbeta-responsive genes. SMAD6 and 7 proteins represent a negative feedback loop inhibiting the TGFbeta-SMAD signalling path-way. As TGFbeta1 overexpression inhibits keratinocyte proliferation, the aim of this study was to investigate with real-time RT-PCR the expression of TGFbeta1, 2 and 3, TGFbetaRI and TGFbetaRII and SMAD2, 3, 4, 6 and 7 in lesional and non-lesional psoriatic skin from 13 patients with chronic plaque-type psoriasis as compared to skin from 10 healthy subjects . The study data demonstrate significantly downregulated TGFbetaRI and SMAD2, 4 and 6 mRNA expression in lesional and non-lesional psoriatic skin. SMAD7 mRNA expression was significantly decreased in lesional psoriatic skin compared with both non-lesional psoriatic skin and healthy skin. A significant TGFbeta3 and TGFbetaRII mRNA upregulation exclusively in non-lesional psoriatic skin but no significant difference in the expression of TGFbeta1 and 2 was found. The results of this study suggest that the expression of TGFbeta isoforms, receptors and SMADs may be involved in the increased proliferation of keratinocytes in psoriatic skin.
- Published
- 2009
- Full Text
- View/download PDF
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