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Berlin, Germany

Wolff M.,Justus Liebig University | Noreikat K.,Justus Liebig University | Noreikat K.,University of Leipzig | Ibanez-Tallon I.,Max Delbrueck Center | And 4 more authors.
Life Sciences | Year: 2012

Aims: In the oviduct, muscarinic acetylcholine receptors (MR) are linked with motility regulation and nicotinic receptors (nAChR) with ectopic pregnancy. We here aimed to determine the repertoire of cholinergic receptor expression in the murine oviduct and their functional coupling to regulation of intracellular calcium concentration ([Ca2+]i). Main methods: Cholinergic receptor transcripts were assessed by RT-PCR in oviductal segments (ampulla, isthmus, uterotubar junction) in all cyclic stages and pregnancy, and in laser-microdissected samples of epithelium and smooth muscle, nAChR subunit α3 distribution in tissue sections using an appropriate genetic reporter mouse strain. [Ca2+]i responses were monitored in ciliated and non-ciliated oviductal cells isolated from wild-type and MR subtypes 1 and 3 gene deficient mice. Key findings: Transcripts for all MR subtypes (M1-M5) are constantly expressed whereas there is some variability in nAChR expression from individual to individual. The qualitative expression pattern is independent from the hormonal status of the animal, except for nAChR α7, which is less present during pregnancy. The epithelium expresses M1, M3, nAChR α7 (data from laser-assisted microdissection) and nAChR α3 (ultrastructural investigation of reporter mice). MR dominate over nAChR in increasing [Ca 2+]i with being M3 the major, but not sole subtype driving this effect. The general nAChR inhibitor mecamylamine enhances muscarinic and purinergic responses. Significance: In conclusion, the murine oviduct is endowed with a multiplicity of muscarinic and nicotinic receptors subtypes that, with respect to regulation of [Ca2+]i, are inversely linked to each other. The major, but not sole, cholinergic receptor driving increase in [Ca2+]i is M3. © 2012 Elsevier Inc. Source

Porras P.,Max Delbrueck Center | McDonagh B.,University of Cordoba, Spain | McDonagh B.,Instituto Maimonides Of Investigacion Biomedica Of Cordoba Imibic | Pedrajas J.R.,University of Jaen | And 4 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2010

We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H2O2 as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide. © 2009 Elsevier B.V. All rights reserved. Source

Klinghammer K.,Charite - Medical University of Berlin | Klinghammer K.,Max Delbrueck Center | Raguse J.-D.,Charite - Medical University of Berlin | Plath T.,Charite - Medical University of Berlin | And 8 more authors.
International Journal of Cancer | Year: 2014

Patient-derived xenograft (PDX) models have shown to reflect original patient tumors better than any other preclinical model. We embarked in a study establishing a large panel of head and neck squamous cell carcinomas PDX for biomarker analysis and evaluation of established and novel compounds. Out of 115 transplanted specimens 52 models were established of which 29 were characterized for response to docetaxel, cetuximab, methotrexate, carboplatin, 5-fluorouracil and everolimus. Further, tumors were subjected to sequencing analysis and gene expression profiling of selected mTOR pathway members. Most frequent response was observed for docetaxel and cetuximab. Responses to carboplatin, 5-fluorouracil and methotrexate were moderate. Everolimus revealed activity in the majority of PDX. Mutational profiling and gene expression analysis did not reveal a predictive biomarker for everolimus even though by trend RPS6KB1 mRNA expression was associated with response. In conclusion we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers. What's new? Preclinical drug evaluation in head and neck squamous cell carcinoma (HNSCC) is challenged by the inability of established cell lines to predict clinical impact. It may be possible to overcome that problem with patient-derived xenografts (PDX), which more closely reflect tumor characteristics. Here, a large collection of PDXs were established for HNSCC and tested for therapeutic response. The mTOR inhibitor everolimus was found to be active in a majority of the models. Biomarkers capable of predicting tumor response to everolimus were not identified, though increased expression of RPS6KB1, a member of the mTOR pathway, was common among responders. © 2014 UICC. Source

Hillmeister P.,Center for Cardiovascular Research | Hillmeister P.,Center for Stroke Research Berlin | Gatzke N.,Center for Cardiovascular Research | Dulsner A.,Center for Cardiovascular Research | And 20 more authors.
Circulation Research | Year: 2011

Rationale: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. Objective: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. Methods and results: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. Conclusions: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways. © 2011 American Heart Association, Inc. Source

LaMarca B.,University of Mississippi Medical Center | Speed J.,University of Mississippi Medical Center | Ray L.F.,University of Mississippi Medical Center | Cockrell K.,University of Mississippi Medical Center | And 3 more authors.
International Journal of Interferon, Cytokine and Mediator Research | Year: 2011

Background: Increases in interleukin 6 (IL-6) and agonistic autoantibodies to the angiotensin IItype 1 receptor (AT1-AA) are proposed to be important links between placental ischemia andhypertension in preeclampsia.Methods: The purpose of this study was to determine whether IL-6 (5 ng/day), infused intonormal pregnant (NP) rats, increased mean arterial pressure (MAP) and AT1-AA. MAP wasanalyzed in the presence and absence of an angiotensin type 1 receptor (AT1R) antagonist,losartan, L.Results: MAP and AT1-AA increased from102 ± 2 to 118 ± 4 mmHg and 0.7 ± 0.3 NP to14.1 ± 1.4 chronotropic units with chronic IL-6 infusion. MAP responses to IL-6 were abolishedin losartan pretreated rats (85 ± 4 in NP + L vs 85 ± 3 mmHg in IL-6 + L).Conclusion: These data indicate that IL-6 stimulates AT1-AA and that activation of the AT1Rmediates IL-6 induced hypertension during pregnancy. © 2011 LaMarca et al, publisher and licensee Dove Medical Press Ltd. Source

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