@article{LempiaeinenCouttetBolognanietal.2012, author = {Lempi{\"a}inen, Harri and Couttet, Philippe and Bolognani, Federico and M{\"u}ller, Arne and Dubost, Val{\´e}rie and Luisier, Rapha{\"e}lle and Rio-Espinola, Alberto del and Vitry, Veronique and Unterberger, Elif B. and Thomson, John P. and Treindl, Fridolin and Metzger, Ute and Wrzodek, Clemens and Hahne, Florian and Zollinger, Tulipan and Brasa, Sarah and Kalteis, Magdalena and Marcellin, Magali and Giudicelli, Fanny and Braeuning, Albert and Morawiec, Laurent and Zamurovic, Natasa and L{\"a}ngle, Ulrich and Scheer, Nico and Sch{\"u}beler, Dirk and Goodman, Jay and Chibout, Salah-Dine and Marlowe, Jennifer and Theil, Dietlinde and Heard, David J. and Grenet, Olivier and Zell, Andreas and Templin, Markus F. and Meehan, Richard R. and Wolf, Roland C. and Elcombe, Clifford R. and Schwarz, Michael and Moulin, Pierre and Terranova, R{\´e}mi and Moggs, Jonathan G.}, title = {Identification of Dlk1-Dio3 imprinted gene cluster non-coding RNAs as novel candidate biomarkers for liver tumor promotion}, series = {Toxicological Sciences}, volume = {131}, journal = {Toxicological Sciences}, number = {2}, publisher = {Oxford University Press}, address = {Oxford}, issn = {1094-2025}, doi = {10.1093/toxsci/kfs303}, pages = {375 -- 386}, year = {2012}, abstract = {The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, sug- gesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.}, language = {en} } @article{ScheerKapelyukhMcEwanetal.2012, author = {Scheer, Nico and Kapelyukh, Yury and McEwan, Jillian and Beuger, Vincent and Stanley, Lesley A. and Rode, Anja and Wolf, C. Roland}, title = {Modeling Human Cytochrome P450 2D6 Metabolism and Drug-drug Interaction by a Novel Panel of Knockout and Humanized Mouse Lines}, series = {Molecular Pharmacology}, volume = {81}, journal = {Molecular Pharmacology}, number = {1}, publisher = {ASPET}, address = {Bethesda, Md.}, issn = {1521-0111}, doi = {10.1124/mol.111.075192}, pages = {63 -- 72}, year = {2012}, abstract = {The highly polymorphic human cytochrome P450 2D6 enzyme is involved in the metabolism of up to 25\% of all marketed drugs and accounts for significant individual differences in response to CYP2D6 substrates. Because of the differences in the multiplicity and substrate specificity of CYP2D family members among species, it is difficult to predict pathways of human CYP2D6-dependent drug metabolism on the basis of animal studies. To create animal models that reflect the human situation more closely and that allow an in vivo assessment of the consequences of differential CYP2D6 drug metabolism, we have developed a novel straightforward approach to delete the entire murine Cyp2d gene cluster and replace it with allelic variants of human CYP2D6. By using this approach, we have generated mouse lines expressing the two frequent human protein isoforms CYP2D6.1 and CYP2D6.2 and an as yet undescribed variant of this enzyme, as well as a Cyp2d cluster knockout mouse. We demonstrate that the various transgenic mouse lines cover a wide spectrum of different human CYP2D6 metabolizer phenotypes. The novel humanization strategy described here provides a robust approach for the expression of different CYP2D6 allelic variants in transgenic mice and thus can help to evaluate potential CYP2D6-dependent interindividual differences in drug response in the context of personalized medicine.}, language = {en} } @article{ScheerKapelyukhRodeetal.2012, author = {Scheer, Nico and Kapelyukh, Yury and Rode, Anja and Buechel, Sandra and Wolf, C. Roland}, title = {Generation and characterization of novel cytochrome P450 Cyp2c gene cluster knockout and CYP2C9 humanized mouse lines}, series = {Molecular Pharmacology}, volume = {82}, journal = {Molecular Pharmacology}, number = {6}, publisher = {ASPET}, address = {Bethesda, Md.}, issn = {1521-0111}, doi = {10.1124/mol.112.080036}, pages = {1022 -- 1029}, year = {2012}, abstract = {Compared with rodents and many other animal species, the human cytochrome P450 (P450) Cyp2c gene cluster varies significantly in the multiplicity of functional genes and in the substrate specificity of its enzymes. As a consequence, the use of wild-type animal models to predict the role of human CYP2C enzymes in drug metabolism and drug-drug interactions is limited. Within the human CYP2C cluster CYP2C9 is of particular importance, because it is one of the most abundant P450 enzymes in human liver, and it is involved in the metabolism of a wide variety of important drugs and environmental chemicals. To investigate the in vivo functions of cytochrome P450 Cyp2c genes and to establish a model for studying the functions of CYP2C9 in vivo, we have generated a mouse model with a deletion of the murine Cyp2c gene cluster and a corresponding humanized model expressing CYP2C9 specifically in the liver. Despite the high number of functional genes in the mouse Cyp2c cluster and the reported roles of some of these proteins in different biological processes, mice deleted for Cyp2c genes were viable and fertile but showed certain phenotypic alterations in the liver. The expression of CYP2C9 in the liver also resulted in viable animals active in the metabolism and disposition of a number of CYP2C9 substrates. These mouse lines provide a powerful tool for studying the role of Cyp2c genes and of CYP2C9 in particular in drug disposition and as a factor in drug-drug interaction.}, language = {en} } @article{ScheerBalimaneHaywardetal.2012, author = {Scheer, Nico and Balimane, Praveen and Hayward, Michael D. and Buechel, Sandra and Kauselmann, Gunther and Wolf, C. Roland}, title = {Generation and Characterization of a Novel Multidrug Resistance Protein 2 Humanized Mouse Line}, series = {Drug Metabolism and Disposition}, volume = {40}, journal = {Drug Metabolism and Disposition}, number = {11}, publisher = {ASPET}, address = {Bethesda, Md.}, issn = {1521-0111}, doi = {10.1124/dmd.112.047605}, pages = {2212 -- 2218}, year = {2012}, abstract = {The multidrug resistance protein (MRP) 2 is predominantly expressed in liver, intestine, and kidney, where it plays an important role in the excretion of a range of drugs and their metabolites or endogenous compounds into bile, feces, and urine. Mrp knockout [Mrp2(-/-)] mice have been used recently to study the role of MRP2 in drug disposition. Here, we describe the first generation and initial characterization of a mouse line humanized for MRP2 (huMRP2), which is nulled for the mouse Mrp2 gene and expresses the human transporter in the organs and cell types where MRP2 is normally expressed. Analysis of the mRNA expression for selected cytochrome P450 and transporter genes revealed no major changes in huMRP2 mice compared with wild-type controls. We show that human MRP2 is able to compensate functionally for the loss of the mouse transporter as demonstrated by comparable bilirubin levels in the humanized mice and wild-type controls, in contrast to the hyperbilirubinemia phenotype that is observed in MRP2(-/-) mice. The huMRP2 mouse provides a model to study the role of the human transporter in drug disposition and in assessing the in vivo consequences of inhibiting this transporter by compounds interacting with human MRP2.}, language = {en} } @article{PaulssenNgyugenKahlckeetal.2012, author = {Paulßen, Elisabeth and Ngyugen, Hung Huy and Kahlcke, Nils and Deflon, Victor M. and Abram, Ulrich}, title = {Tricarbonyltechnetium(I) and -rhenium(I) complexes with N′-thiocarbamoylpicolylbenzamidines}, series = {Polyhedron}, volume = {40}, journal = {Polyhedron}, number = {1}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0277-5387}, doi = {10.1016/j.poly.2012.04.008}, pages = {153 -- 158}, year = {2012}, abstract = {N,N-Dialkylamino(thiocarbonyl)-N′-picolylbenzamidines react with (NEt4)2[M(CO)3X3] (M = Re, X = Br; M = Tc, X = Cl) under formation of neutral [M(CO)3L] complexes in high yields. The monoanionic NNS ligands bind in a facial coordination mode and can readily be modified at the (CS)NR1R2 moiety. The complexes [99Tc(CO)3(LPyMor)] and [Re(CO)3(L)] (L = LPyMor, LPyEt) were characterized by X-ray diffraction. Reactions of [99mTc(CO)3(H2O)3]+ with the N′-thiocarbamoylpicolylbenzamidines give the corresponding 99mTc complexes. The ester group in HLPyCOOEt allows linkage between biomolecules and the metal core.}, language = {en} } @article{PaulssenKongArciszewskietal.2012, author = {Paulßen, Elisabeth and Kong, Shushu and Arciszewski, Pawel and Wielbalck, Swantje and Abram, Ulrich}, title = {Aryl and NHC Compounds of Technetium and Rhenium}, series = {Journal of the American Chemical Society}, volume = {134}, journal = {Journal of the American Chemical Society}, number = {22}, publisher = {ACS Publications}, address = {Washington, DC}, issn = {1520-5126}, doi = {10.1021/ja3033718}, pages = {9118 -- 9121}, year = {2012}, abstract = {Air- and water-stable phenyl complexes with nitridotechnetium(V) cores can be prepared by straightforward procedures. [TcNPh2(PPh3)2] is formed by the reaction of [TcNCl2(PPh3)2] with PhLi. The analogous N-heterocyclic carbene (NHC) compound [TcNPh2(HLPh)2], where HLPh is 1,3,4-triphenyl-1,2,4-triazol-5-ylidene, is available from (NBu4)[TcNCl4] and HLPh or its methoxo-protected form. The latter compound allows the comparison of different Tc-C bonds within one compound. Surprisingly, the Tc chemistry with such NHCs does not resemble that of corresponding Re complexes, where CH activation and orthometalation dominate.}, language = {en} } @article{ImmelGruetzkeSpaeteetal.2012, author = {Immel, Timo and Gr{\"u}tzke, Martin and Sp{\"a}te, Anne-Katrin and Groth, Ulrich and {\"O}hlschl{\"a}ger, Peter and Huhn, Thomas}, title = {Synthesis and X-ray structure analysis of a heptacoordinate titanium(IV)-bis-chelate with enhanced in vivo antitumor efficacy}, series = {Chemical Communications}, volume = {48}, journal = {Chemical Communications}, number = {46}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1364-548X}, doi = {10.1039/C2CC31624B}, pages = {5790 -- 5792}, year = {2012}, abstract = {Chelate stabilization of a titanium(IV)-salan alkoxide by ligand exchange with 2,6-pyridinedicarboxylic acid (dipic) resulted in heptacoordinate complex 3 which is not redox-active, stable on silica gel and has increased aqueous stability. 3 is highly toxic in HeLa S3 and Hep G2 and has enhanced antitumor efficacy in a mouse cervical-cancer model.}, language = {en} } @article{HenkenOosterhuisOehlschlaegeretal.2012, author = {Henken, F. E. and Oosterhuis, K. and {\"O}hlschl{\"a}ger, Peter and Bosch, L. and Hooijberg, E. and Haanen, J. B. A. G. and Steenbergen, R. D. M.}, title = {Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7}, series = {Vaccine}, volume = {30}, journal = {Vaccine}, number = {28}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0264-410X}, doi = {10.1016/j.vaccine.2012.04.013}, pages = {4259 -- 4266}, year = {2012}, abstract = {Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of 'gene-shuffled' (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.}, language = {en} } @article{RibitschHeumannKarletal.2012, author = {Ribitsch, D. and Heumann, S. and Karl, W. and Gerlach, J. and Leber, R. and Birner-Gruenberger, R. and Gruber, K. and Eiteljoerg, I. and Remler, P. and Siegert, Petra and Lange, J. and Maurer, Karl-Heinz and Berg, G. and Guebitz, G. M. and Schwab, H.}, title = {Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli}, series = {Journal of biotechnology}, volume = {157}, journal = {Journal of biotechnology}, number = {1}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1873-4863 (E-Journal); 0168-1656 (Print)}, doi = {10.1016/j.jbiotec.2011.09.025}, pages = {140 -- 147}, year = {2012}, abstract = {A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.}, language = {en} } @article{BorgmeierBongaertsMeinhardt2012, author = {Borgmeier, Claudia and Bongaerts, Johannes and Meinhardt, Friedhelm}, title = {Genetic analysis of the Bacillus licheniformis degSU operon and the impact of regulatory mutations on protease production}, series = {Journal of biotechnology}, volume = {159}, journal = {Journal of biotechnology}, number = {1-2}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1873-4863 (E-Journal); 0168-1656 (Print)}, doi = {10.1016/j.jbiotec.2012.02.011}, pages = {12 -- 20}, year = {2012}, abstract = {Disruption experiments targeted at the Bacillus licheniformis degSU operon and GFP-reporter analysis provided evidence for promoter activity immediately upstream of degU. pMutin mediated concomitant introduction of the degU32 allele - known to cause hypersecretion in Bacillus subtilis - resulted in a marked increase in protease activity. Application of 5-fluorouracil based counterselection through establishment of a phosphoribosyltransferase deficient Δupp strain eventually facilitated the marker-free introduction of degU32 leading to further protease enhancement achieving levels as for hypersecreting wild strains in which degU was overexpressed. Surprisingly, deletion of rapG - known to interfere with DegU DNA-binding in B. subtilis - did not enhance protease production neither in the wild type nor in the degU32 strain. The combination of degU32 and Δupp counterselection in the type strain is not only equally effective as in hypersecreting wild strains with respect to protease production but furthermore facilitates genetic strain improvement aiming at biological containment and effectiveness of biotechnological processes.}, language = {en} }