TY  - JOUR
A1  - Koch, R.
A1  - Deger, A.
A1  - Klotz, Karl-Norbert
A1  - Schenzle, D.
A1  - Krämer, H.
A1  - Kelm, S.
A1  - Müller, G.
A1  - Rapp, R.
A1  - Weber, U.
T1  - Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with  different binding properties
N2  - Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak li) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak Il). Both peaks were glycoproteins. At 4°C peak 1 showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak li bad its binding optimum at pH 7.0 and low ionic strength, where peak I bindingwas minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak 11 an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 oc the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfatejpolyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400000, 365000, 320000, 290000, and 245000 under non-reducing conditions. For peak II two major receptor bands with M\(_r\) 210000 and 115000 were found. The peak II receptor bands were also obtained aftermild reduction of peak I. After complete reduction both peaks showed one major receptor band with M\(_r\) 130000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.
KW  - Toxikologie
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60215
ER  - 
TY  - JOUR
A1  - Heisig, Martin
A1  - Frentzen, Alexa
A1  - Bergmann, Birgit
A1  - Gentschev, Katharina Ivaylo
A1  - Hotz, Christian
A1  - Schoen, Christoph
A1  - Stritzker, Jochen
A1  - Fensterle, Joachim
A1  - Rapp, Ulf R.
A1  - Goebel, Werner
T1  - Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines
N2  - Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.
KW  - Listeria monocytogenes
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68705
ER  - 
TY  - JOUR
A1  - Sanges, C.
A1  - Scheuermann, C.
A1  - Zahedi, R. P.
A1  - Sickmann, A.
A1  - Lamberti, A.
A1  - Migliaccio, N.
A1  - Baljuls, A.
A1  - Marra, M.
A1  - Zappavigna, S.
A1  - Reinders, J.
A1  - Rapp, U.
A1  - Abbruzzese, A.
A1  - Caraglia, M.
A1  - Arcari, P.
T1  - Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells
JF  - Cell Death and Disease
N2  - We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
KW  - EF-1A
KW  - Raf kinases
KW  - signal transduction
KW  - apoptosis
KW  - ubiquitin
KW  - mass spectrometry
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124149
VL  - 3
IS  - e276
ER  - 
TY  - JOUR
A1  - Basset, Yves
A1  - Cizek, Lukas
A1  - Cuénoud, Philippe
A1  - Didham, Raphael K.
A1  - Novotny, Vojtech
A1  - Ødegaard, Frode
A1  - Roslin, Tomas
A1  - Tishechkin, Alexey K.
A1  - Schmidl, Jürgen
A1  - Winchester, Neville N.
A1  - Roubik, David W.
A1  - Aberlenc, Henri-Pierre
A1  - Bail, Johannes
A1  - Barrios, Hector
A1  - Bridle, Jonathan R.
A1  - Castaño-Meneses, Gabriela
A1  - Corbara, Bruno
A1  - Curletti, Gianfranco
A1  - da Rocha, Wesley Duarte
A1  - De Bakker, Domir
A1  - Delabie, Jacques H. C.
A1  - Dejean, Alain
A1  - Fagan, Laura L.
A1  - Floren, Andreas
A1  - Kitching, Roger L.
A1  - Medianero, Enrique
A1  - de Oliveira, Evandro Gama
A1  - Orivel, Jerome
A1  - Pollet, Marc
A1  - Rapp, Mathieu
A1  - Ribeiro, Servio P.
A1  - Roisin, Yves
A1  - Schmidt, Jesper B.
A1  - Sørensen, Line
A1  - Lewinsohn, Thomas M.
A1  - Leponce, Maurice
T1  - Arthropod Distribution in a Tropical Rainforest: Tackling a Four Dimensional Puzzle
JF  - PLoS ONE
N2  - Quantifying the spatio-temporal distribution of arthropods in tropical rainforests represents a first step towards scrutinizing the global distribution of biodiversity on Earth. To date most studies have focused on narrow taxonomic groups or lack a design that allows partitioning of the components of diversity. Here, we consider an exceptionally large dataset (113,952 individuals representing 5,858 species), obtained from the San Lorenzo forest in Panama, where the phylogenetic breadth of arthropod taxa was surveyed using 14 protocols targeting the soil, litter, understory, lower and upper canopy habitats, replicated across seasons in 2003 and 2004. This dataset is used to explore the relative influence of horizontal, vertical and seasonal drivers of arthropod distribution in this forest. We considered arthropod abundance, observed and estimated species richness, additive decomposition of species richness, multiplicative partitioning of species diversity, variation in species composition, species turnover and guild structure as components of diversity. At the scale of our study (2km of distance, 40m in height and 400 days), the effects related to the vertical and seasonal dimensions were most important. Most adult arthropods were collected from the soil/litter or the upper canopy and species richness was highest in the canopy. We compared the distribution of arthropods and trees within our study system. Effects related to the seasonal dimension were stronger for arthropods than for trees. We conclude that: (1) models of beta diversity developed for tropical trees are unlikely to be applicable to tropical arthropods; (2) it is imperative that estimates of global biodiversity derived from mass collecting of arthropods in tropical rainforests embrace the strong vertical and seasonal partitioning observed here; and (3) given the high species turnover observed between seasons, global climate change may have severe consequences for rainforest arthropods.
KW  - trees
KW  - species richness
KW  - beta-diveristy
KW  - strategy
KW  - turnover
KW  - similarity
KW  - biodiversity
KW  - specialization
KW  - herbivorous insects
KW  - assemblages
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136393
VL  - 10
IS  - 12
ER  - 
TY  - JOUR
A1  - Sanges, C.
A1  - Scheuermann, C.
A1  - Zahedi, R. P.
A1  - Sickmann, A.
A1  - Lamberti, A.
A1  - Migliaccio, N.
A1  - Baljuls, A.
A1  - Marra, M.
A1  - Zappavigna, S.
A1  - Rapp, U.
A1  - Abbruzzese, A.
A1  - Caraglia, M.
A1  - Arcari, P.
T1  - Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells
JF  - Cell Death & Disease
N2  - We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B-and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
KW  - signal transduction
KW  - mass spectrometry
KW  - elongation
KW  - protein docking
KW  - factor EEF1A2
KW  - cancer-cells
KW  - lung cancer
KW  - EF-1A
KW  - Raf kinases
KW  - aminoacyl-transfer-RNA
KW  - tyrosine phosphorylation
KW  - factor 1-alpha
KW  - nucleotide exchange
KW  - polyarcylamide gels
KW  - chain
KW  - apoptosis
KW  - ubiquitin
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134673
VL  - 3
IS  - e276
ER  - 
TY  - JOUR
A1  - Ceteci, Fatih
A1  - Ceteci, Semra
A1  - Zanucco, Emanuele
A1  - Thakur, Chitra
A1  - Becker, Matthias
A1  - El-Nikhely, Nefertiti
A1  - Fink, Ludger
A1  - Seeger, Werner
A1  - Savai, Rajkumar
A1  - Rapp, Ulf R.
T1  - E-Cadherin Controls Bronchiolar Progenitor Cells and Onset of Preneoplastic Lesions in Mice
JF  - Neoplasia
N2  - Although progenitor cells of the conducting airway have been spatially localized and some insights have been gained regarding their molecular phenotype, relatively little is known about the mechanisms regulating their maintenance, activation, and differentiation. This study investigates the potential roles of E-cadherin in mouse Clara cells, as these cells were shown to represent the progenitor/stem cells of the conducting airways and have been implicated as the cell of origin of human non-small cell lung cancer. Postnatal inactivation of E-cadherin affected Clara cell differentiation and compromised airway regeneration under injury conditions. In steady-state adult lung, overexpression of the dominant negative E-cadherin led to an expansion of the bronchiolar stem cells and decreased differentiation concomitant with canonical Wnt signaling activation. Expansion of the bronchiolar stem cell pool was associated with an incessant proliferation of neuroepithelial body-associated Clara cells that ultimately gave rise to bronchiolar hyperplasia. Despite progressive hyperplasia, only a minority of the mice developed pulmonary solid tumors, suggesting that the loss of E-cadherin function leads to tumor formation when additional mutations are sustained. The present study reveals that E-cadherin plays a critical role in the regulation of proliferation and homeostasis of the epithelial cells lining the conducting airways.
KW  - injury
KW  - lung cancer
KW  - stem cells
KW  - clara cell
KW  - gene expression
KW  - basal cell
KW  - in vivo
KW  - epithelium
KW  - airway
KW  - renewal
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135407
VL  - 14
IS  - 12
ER  - 
TY  - JOUR
A1  - Zanucco, Emanuele
A1  - Götz, Rudolf
A1  - Potapenko, Tamara
A1  - Carraretto, Irene
A1  - Ceteci, Semra
A1  - Ceteci, Fatih
A1  - Seeger, Werner
A1  - Savai, Rajkumar
A1  - Rapp, Ulf R.
T1  - Expression of B-RAF V600E in Type II Pneumocytes Causes Abnormalities in Alveolar Formation, Airspace Enlargement and Tumor Formation in Mice
JF  - PLOS ONE
N2  - Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Perturbations of these cascades have severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of human tumorigenesis, where B-RAF V600E has been identified as the prevalent mutant. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. The inflammatory cell infiltration did not precede the formation of the lung lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation.
KW  - obstructive pulmonary-disease
KW  - lung-cancer
KW  - somatic mutations
KW  - epithelial-cells
KW  - mouse models
KW  - protein
KW  - kinase
KW  - inflammation
KW  - activation
KW  - pathway
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137061
VL  - 6
IS  - 12
ER  - 
TY  - JOUR
A1  - Pfeiffer, Verena
A1  - Götz, Rudolf
A1  - Xiang, Chaomei
A1  - Camarero, Guadelupe
A1  - Braun, Attila
A1  - Zhang, Yina
A1  - Blum, Robert
A1  - Heinsen, Helmut
A1  - Nieswandt, Bernhard
A1  - Rapp, Ulf R.
T1  - Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum
JF  - PLoS ONE
N2  - This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.
KW  - granule cells
KW  - hippocampus
KW  - neurons
KW  - neuronal dendrites
KW  - embryos
KW  - dentate gyrus
KW  - neuronal differentiation
KW  - cerebellum
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130304
VL  - 8
IS  - 3
ER  - 
TY  - JOUR
A1  - Ceteci, Fatih
A1  - Xu, Jiajia
A1  - Ceteci, Semra
A1  - Zanucco, Emanuele
A1  - Thakur, Chitra
A1  - Rapp, Ulf R.
T1  - Conditional Expression of Oncogenic C-RAF in Mouse Pulmonary Epithelial Cells Reveals Differential Tumorigenesis and Induction of Autophagy Leading to Tumor Regression
JF  - Neoplasia
N2  - Here we describe a novel conditional mouse lung tumor model for investigation of the pathogenesis of human lung cancer. On the basis of the frequent involvement of the Ras-RAF-MEK-ERK signaling pathway in human non-small cell lung carcinoma (NSCLC), we have explored the target cell availability, reversibility, and cell type specificity of transformation by oncogenic C-RAF. Targeting expression to alveolar type II cells or to Clara cells, the two likely precursors of human NSCLC, revealed differential tumorigenicity between these cells. Whereas expression of oncogenic C-RAF in alveolar type II cells readily induced multifocal macroscopic lung tumors independent of the developmental state, few tumors with type II pneumocytes features and incomplete penetrance were found when targeted to Clara cells. Induced tumors did not progress and were strictly dependent on the initiating oncogene. Deinduction of mice resulted in tumor regression due to autophagy rather than apoptosis. Induction of autophagic cell death in regressing lung tumors suggests the use of autophagy enhancers as a treatment choice for patients with NSCLC.
KW  - Human lung-cancer
KW  - K-RAS
KW  - Induced senescence
KW  - Gene-expression
KW  - In-vivo
KW  - Kinase pathway
KW  - P53
KW  - Activation
KW  - Model
KW  - Adenocarcinomas
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134347
VL  - 13
IS  - 11
ER  -