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Kaasik K.,University of California at San Francisco | Kivimae S.,University of California at San Francisco | Allen J.J.,University of California at San Francisco | Chalkley R.J.,University of California at San Francisco | And 9 more authors.
Cell Metabolism | Year: 2013

Posttranslational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β; hence, OGT and GSK3β exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock. © 2013 Elsevier Inc.


Bouwman P.,Netherlands Cancer Institute | van der Gulden H.,Netherlands Cancer Institute | van der Heijden I.,Netherlands Cancer Institute | Drost R.,Netherlands Cancer Institute | And 8 more authors.
Cancer Discovery | Year: 2013

Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely conducted in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUSs have been described, thus far it has not been possible to conduct a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUSs based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay, we have analyzed 74 unclassified BRCA1 missense mutants for which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains. SIGNIFICANCE: BRCA1 VUSs are frequently found in patients with hereditary breast or ovarian cancer and present a serious problem for clinical geneticists. This article describes the generation, validation, and application of a reliable high-throughput assay for the functional classification of BRCA1 sequence variants of uncertain significance. © 2013 American Association for Cancer Research.


Gebeshuber C.A.,Medical University of Vienna | Gebeshuber C.A.,IMBA Institute of Molecular Biotechnology | Kornauth C.,Medical University of Vienna | Dong L.,Fritz Lipmann Institute e.V. | And 13 more authors.
Nature Medicine | Year: 2013

Focal segmental glomerulosclerosis (FSGS) is a frequent and severe glomerular disease characterized by destabilization of podocyte foot processes. We report that transgenic expression of the microRNA miR-193a in mice rapidly induces FSGS with extensive podocyte foot process effacement. Mechanistically, miR-193a inhibits the expression of the Wilms' tumor protein (WT1), a transcription factor and master regulator of podocyte differentiation and homeostasis. Decreased expression levels of WT1 lead to downregulation of its target genes PODXL (podocalyxin) and NPHS1 (nephrin), as well as several other genes crucial for the architecture of podocytes, initiating a catastrophic collapse of the entire podocyte-stabilizing system. We found upregulation of miR-193a in isolated glomeruli from individuals with FSGS compared to normal kidneys or individuals with other glomerular diseases. Thus, upregulation of miR-193a provides a new pathogenic mechanism for FSGS and is a potential therapeutic target. © 2013 Nature America, Inc. All rights reserved.


Van Riggelen J.,Stanford University | Muller J.,University of Würzburg | Otto T.,Dana-Farber Cancer Institute | Beuger V.,University of Würzburg | And 7 more authors.
Genes and Development | Year: 2010

The Myc protein suppresses the transcription of several cyclin-dependent kinase inhibitors (CKIs) via binding to Miz1; whether this interaction is important for Myc's ability to induce or maintain tumorigenesis is not known. Here we show that the oncogenic potential of a point mutant of Myc (MycV394D) that is selectively deficient in binding to Miz1 is greatly attenuated. Binding of Myc to Miz1 is continuously required to repress CKI expression and inhibit accumulation of trimethylated histone H3 at Lys 9 (H3K9triMe), a hallmark of cellular senescence, in T-cell lymphomas. Lymphomas that arise express high amounts of transforming growth factor β-2 (TGFβ-2) and TGFβ-3. Upon Myc suppression, TGFβ signaling is required to induce CKI expression and cellular senescence and suppress tumor recurrence. Binding of Myc to Miz1 is required to antagonize growth suppression and induction of senescence by TGFβ. We demonstrate that, since lymphomas express high levels of TGFβ, they are poised to elicit an autocrine program of senescence upon Myc inactivation, demonstrating that TGFβ is a key factor that establishes oncogene addiction of T-cell lymphomas. © 2010 by Cold Spring Harbor Laboratory Press.


Ramchandani V.A.,U.S. National Institutes of Health | Umhau J.,U.S. National Institutes of Health | Pavon F.J.,Scripps Research Institute | Ruiz-Velasco V.,Pennsylvania State University | And 14 more authors.
Molecular Psychiatry | Year: 2011

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and 11 C-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward. © 2011 Macmillan Publishers Limited.


Seibler J.,TaconicArtemis GmbH | Schwenk F.,TaconicArtemis GmbH | Schwenk F.,Gelsenkirchen University of Applied Sciences
Methods in Enzymology | Year: 2010

Within the past 10 years, RNA interference has emerged as a powerful experimental tool as it allows rapid gene function analysis. Unique features such as reversibility of gene silencing and simultaneous targeting of several genes characterize the approach. In this chapter, transgenic RNAi techniques in reverse mouse genetics are discussed and protocols are provided. © 2010 Elsevier Inc.


Kauselmann G.,FARO | Dopazo A.,Genomics Unit | Link W.,TaconicArtemis GmbH
Current Cancer Drug Targets | Year: 2012

The current paradigm for cancer therapy is undergoing a change from non-specific cytotoxic agents to more specific approaches based on unique molecular features of cancer cells. The identification and validation of disease relevant targets are crucial for the development of molecularly targeted anticancer therapies. Advances in our understanding of the molecular basis of cancer together with novel approaches to interfere with signal transduction pathways have opened new horizons for anticancer target discovery. In particular, the image-based large scale analysis of cellular phenotypes that arise from genetic or chemical perturbations paved the way for the identification and validation of disease relevant molecular targets independent of preconceived notions of mechanistic relationships. In addition, novel and sophisticated techniques of genome manipulation allow for the use of mouse models that faithfully recapitulate critical elements of human cancer for target validation in vivo. We believe that these advances will translate into more and better validated drug targets. © 2012 Bentham Science Publishers.


Scheer N.,TaconicArtemis GmbH | Roland Wolf C.,University of Dundee
Drug Metabolism Reviews | Year: 2013

The nuclear receptors pregnane X receptor, constitutive androstane receptor, and peroxisome proliferator-activated receptor alpha have important endogenous functions and are also involved in the induction of drug-metabolizing enzymes and transporters in response to exogenous xenobiotics. Though not belonging to the same protein family, the Per-Sim-ARNT domain receptor aryl hydrocarbon receptor functionally overlaps with the three nuclear receptors in many aspects and is therefore included in this review. Significant species differences in ligand affinity and biological responses as a result of activation of these receptors have been described. Several xenobiotic receptor humanized mice have been created to overcome these species differences and to provide in vivo models that are more predictive for human responses. This review provides an overview of the different xenobiotic receptor humanized mouse models described to date and will summarize how these models can be applied in basic research and improve drug discovery and development. Some of the key applications in the evaluation of drug induction, drug-drug interactions, nongenotoxic carcinogenicity, other toxicity, or efficacy studies are described. We also discuss relevant considerations in the interpretation of such data and potential future directions for the use of xenobiotic receptor humanized mice. © 2013 Informa Healthcare USA, Inc.


Walkley C.R.,University of Melbourne | Liddicoat B.,University of Melbourne | Hartner J.C.,TaconicArtemis GmbH
Current Topics in Microbiology and Immunology | Year: 2012

RNA editing by deamination of adenosine to inosine (A-to-I editing) is a physiologically important posttranscriptional mechanism that can regulate expression of genes by modifying their transcripts. A-to-I editing is mediated by adenosine deaminases acting on RNA (ADAR) that can catalytically exchange adenosines to inosines, with varying efficiency, depending on the structure of the RNA substrates. Significant progress in understanding the biological function of mammalian ADARs has been made in the past decade by the creation and analysis of gene-targeted mice with disrupted or modified ADAR alleles. These studies have revealed important roles of ADARs in neuronal and hematopoietic tissue during embryonic and postnatal stages of mouse development. © 2011 Springer-Verlag Berlin Heidelberg.


Patent
TaconicArtemis GmbH | Date: 2010-03-24

The invention provides particular lentiviral vectors that are useful for the transfection of cells with shRNAs. Moreover, cells transfected with such vectors and non-human vertebrates having such cells are provided.

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