@article{AdakuChilakaMally2020, author = {Adaku Chilaka, Cynthia and Mally, Angela}, title = {Mycotoxin Occurrence, Exposure and Health Implications in Infants and Young Children in Sub-Saharan Africa: A Review}, series = {Foods}, volume = {9}, journal = {Foods}, number = {11}, issn = {2304-8158}, doi = {10.3390/foods9111585}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-219250}, year = {2020}, abstract = {Infants and young children (IYC) remain the most vulnerable population group to environmental hazards worldwide, especially in economically developing regions such as sub-Saharan Africa (SSA). As a result, several governmental and non-governmental institutions including health, environmental and food safety networks and researchers have been proactive toward protecting this group. Mycotoxins, toxic secondary fungal metabolites, contribute largely to the health risks of this young population. In SSA, the scenario is worsened by socioeconomic status, poor agricultural and storage practices, and low level of awareness, as well as the non-establishment and lack of enforcement of regulatory limits in the region. Studies have revealed mycotoxin occurrence in breast milk and other weaning foods. Of concern is the early exposure of infants to mycotoxins through transplacental transfer and breast milk as a consequence of maternal exposure, which may result in adverse health effects. The current paper presents an overview of mycotoxin occurrence in foods intended for IYC in SSA. It discusses the imperative evidence of mycotoxin exposure of this population group in SSA, taking into account consumption data and the occurrence of mycotoxins in food, as well as biomonitoring approaches. Additionally, it discusses the health implications associated with IYC exposure to mycotoxins in SSA.}, language = {en} } @article{AdamAhrweilerSahaMoelleretal.1993, author = {Adam, W. and Ahrweiler, M. and Saha-M{\"o}ller, C. R. and Sauter, M. and Sch{\"o}nberger, A. and Epe, B. and M{\"u}ller, E. and Schiffmann, D. and Stopper, Helga and Wild, D.}, title = {Genotoxicity studies of benzofuran dioxetanes and epoxides with isolated DNA, bacteria and mammalian cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63420}, year = {1993}, abstract = {1.2-Dioxetanes, very reactive and high energy molecules. are involved as labile intermediates in dioxygenase- activated aerobic metabolism and in physiological processes. Various toxico1ogica1 tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonucleasesensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, fonnamidopyrimidines). Pyrimidinedimersand sites ofbase loss (AP sites) which were probed by UV endonuclease and exonuclease 111 are minor lesions in this system. While the alky1-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA I 00. DNA adducts formed with an intermediary alkyJating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter. since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TAIOO and they form DNA adducts. as detected by the 212Ppostlabelling technique. Our results imply that the type of D NA darnage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA darnage by energy transfer. i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA. while radical darnage and alkylation prevail in the cellular system.}, subject = {Toxikologie}, language = {en} } @article{AdamiDragstedEnigetal.1993, author = {Adami, Hans-Olov and Dragsted, Lars and Enig, Bent and Hansen, Jens and Haraldsd{\´o}ttir, J{\´o}hanna and Hill, Michael J. and Holm, Lars Erik and Knudsen, Ib and Larsen, Jens-Jorgen and Lutz, Werner K. and Osler, Merete and Overvad, Kim and Sabroe, Svend and Sanner, Tore and Strube, Michael and Sorensen, Thorkild I. A. and Thorling, Eivind B.}, title = {Report from the working group on diet and cancer.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71601}, year = {1993}, abstract = {No abstract available.}, subject = {Krebs }, language = {en} } @phdthesis{Adelhardt2007, author = {Adelhardt, Melanie}, title = {Einfluß der Dialysetherapie auf den Genomschaden von Nierenpatienten in einer prospektiven Studie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-23123}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Patienten mit terminaler Niereninsuffizienz haben im Vergleich zur Normalbev{\"o}lkerung eine deutlich erh{\"o}hte Inzidenz maligner Erkrankungen. Fr{\"u}here Untersuchungen zeigten, dass periphere Blutlymphozyten dieser Patienten einen h{\"o}heren genetischen Schaden aufweisen, wodurch das Risiko einer malignen Entartung steigt. In dieser Arbeit wurde der genetische Schaden mithilfe zweier Testverfahren, Comet Assay und Mikrokerntest, untersucht. Es handelte sich um eine prospektive Studie mit zwei Patientenkollektiven. Die erste Gruppe bestand aus Patienten, die aufgrund einer terminalen Niereninsuffizienz innerhalb der n{\"a}chsten Monate eine Dialysetherapie mittels konventioneller H{\"a}modialyse beginnen mußten. Die zweite Gruppe bildeten Dialysepatienten, die im Verlauf von konventioneller Dialyse auf H{\"a}modiafiltration umgestellt wurden. Bei allen Patienten wurde der genetische Schaden der peripheren Blutlymphozyten in den Monaten vor und nach Therapiebeginn bzw. Therapieumstellung regelm{\"a}ßig untersucht. Unsere Ergebnisse zeigen, dass 4 der 10 Pr{\"a}dialysepatienten nach Beginn der Dialyse einen niedrigeren genetischen Schaden hatten, 2 Patienten hatten unterschiedliche Werte in Comet Assay und Mikrokerntest und bei 2 Patienten ergab sich im Verlauf eine h{\"o}here DNA-Sch{\"a}digung. Die verbliebenen 2 Patienten mußten aufgrund einer konstant bleibenden Niereninsuffizienz nicht mit der Dialysetherapie beginnen. Bei Zusammenfassung aller Einzelwerte zeigte sich, dass das Kollektiv der Pr{\"a}dialysepatienten insgesamt vom Beginn der Behandlung profitiert hat. In der Gruppe der Dialysepatienten hatte 2 von 7 Patienten nach Umstellung auf H{\"a}modiafiltration eine geringere DNA-Sch{\"a}digung, 2 Patienten zeigten unterschiedliche Ergebnisse im Comet Assay und Mikrokerntest und 2 weitere Patienten wiesen eine h{\"o}heren genetischen Schaden in den Lymphozyten auf. Ein Patient konnte bei fehlenden Vorwerten nicht ber{\"u}cksichtigt werden. Im Gruppenvergleich zeigte sich f{\"u}r alle Dialysepatienten ein gleichbleibender DNA-Schaden, gemessen mithilfe des Comet Assays bei leicht erh{\"o}hten Mikrokernraten. Jedoch hatte sich die Zellproliferation ebenfalls etwas verbessert. Zusammenfassend ergibt sich somit in beiden Gruppen kein eindeutiges Ergebnis, woraus neue Therapieempfehlungen f{\"u}r Patienten mit terminaler Niereninsuffizienz abzuleiten w{\"a}ren. Um weiter Einflußvariablen auf die H{\"o}he des genetischen Schadens festzustellen, sind weiter Untersuchungen mit gr{\"o}ßeren Patientenkollektiven erforderlich.}, language = {de} } @article{AgarwalYangRiceetal.2014, author = {Agarwal, Shailesh R. and Yang, Pei-Chi and Rice, Monica and Singer, Cherie A. and Nikolaev, Viacheslav O. and Lohse, Martin J. and Clancy, Colleen E. and Harvey, Robert D.}, title = {Role of Membrane Microdomains in Compartmentation of cAMP Signaling}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {4}, issn = {1932-6203}, doi = {10.1371/journal.pone.0095835}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116673}, pages = {e95835}, year = {2014}, abstract = {Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.}, language = {en} } @article{AlldrickLutz1989, author = {Alldrick, A. J. and Lutz, Werner K.}, title = {Covalent binding of [2-\(^{14}\)C]2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) to mouse DNA in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60832}, year = {1989}, abstract = {Fernale BALB/c mice were administered intragastrically with equimolar amounts of either [2-\(^{14}\)C]2-amino-3,8-dimethyi[ 4,5-J]qulnoxaline (MeiQx) or 2-acetylamino[9-\(^{14}\)C]fluorene (2AAF). DNA was isolated from tissues of mice killed either 6 or 24 h after administration. Analysis of liver DNA nucleotide digests by HPLC analysis revealed that all of the radioactivity was attributable to adduct formation. Tbe specific activities of DNA samples were converted to covalent bindlog indices (CBI, J.LIDOI adduct per mol DNA nucleotides/mmol chemical app6ed per kg animal body weight). CBI values of 25 and 9 were detennined for 2AAF and MeiQx in tbe llvers of mice killed 6 h after dosing. The values were in general agreement with the moderate carcinogenic potency of these compounds. The specific activities of DNA preparations obtained from the lddneys, spleens, stomachs, small intestines and large intestlnes of mice treated witb MeiQx and killed 6 h after doslng were S- to 35-times less tban those obtained witb the llver. DNA isolated from tbe lungs (a target organ for MeiQx tumorigenicity) of MeiQx-treated mice was not radiolabeUed at tbe limit of detection (CBI <0.3). With tbe exception of tbe gastrolntestinal tract, the specific activities of DNA samples isolated from mice killed 6 h after administration were higher than those from mice killed after 24 h.}, subject = {Toxikologie}, language = {en} } @phdthesis{Anton2021, author = {Anton, Selma}, title = {Characterization of cAMP nanodomains surrounding the human Glucagon-like peptide 1 receptor using FRET-based reporters}, doi = {10.25972/OPUS-19069}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-190695}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Cyclic adenosine monophosphate (cAMP), the ubiquitous second messenger produced upon stimulation of GPCRs which couple to the stimulatory GS protein, orchestrates an array of physiological processes including cardiac function, neuronal plasticity, immune responses, cellular proliferation and apoptosis. By interacting with various effector proteins, among others protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), it triggers signaling cascades for the cellular response. Although the functional outcomes of GSPCR-activation are very diverse depending on the extracellular stimulus, they are all mediated exclusively by this single second messenger. Thus, the question arises how specificity in such responses may be attained. A hypothesis to explain signaling specificity is that cellular signaling architecture, and thus precise operation of cAMP in space and time would appear to be essential to achieve signaling specificity. Compartments with elevated cAMP levels would allow specific signal relay from receptors to effectors within a micro- or nanometer range, setting the molecular basis for signaling specificity. Although the paradigm of signaling compartmentation gains continuous recognition and is thoroughly being investigated, the molecular composition of such compartments and how they are maintained remains to be elucidated. In addition, such compartments would require very restricted diffusion of cAMP, but all direct measurements have indicated that it can diffuse in cells almost freely. In this work, we present the identification and characterize of a cAMP signaling compartment at a GSPCR. We created a F{\"o}rster resonance energy transfer (FRET)-based receptor-sensor conjugate, allowing us to study cAMP dynamics in direct vicinity of the human glucagone-like peptide 1 receptor (hGLP1R). Additional targeting of analogous sensors to the plasma membrane and the cytosol enables assessment of cAMP dynamics in different subcellular regions. We compare both basal and stimulated cAMP levels and study cAMP crosstalk of different receptors. With the design of novel receptor nanorulers up to 60nm in length, which allow mapping cAMP levels in nanometer distance from the hGLP1R, we identify a cAMP nanodomain surrounding it. Further, we show that phosphodiesterases (PDEs), the only enzymes known to degrade cAMP, are decisive in constraining cAMP diffusion into the cytosol thereby maintaining a cAMP gradient. Following the discovery of this nanodomain, we sought to investigate whether downstream effectors such as PKA are present and active within the domain, additionally studying the role of A-kinase anchoring proteins (AKAPs) in targeting PKA to the receptor compartment. We demonstrate that GLP1-produced cAMP signals translate into local nanodomain-restricted PKA phosphorylation and determine that AKAP-tethering is essential for nanodomain PKA. Taken together, our results provide evidence for the existence of a dynamic, receptor associated cAMP nanodomain and give prospect for which key proteins are likely to be involved in its formation. These conditions would allow cAMP to exert its function in a spatially and temporally restricted manner, setting the basis for a cell to achieve signaling specificity. Understanding the molecular mechanism of cAMP signaling would allow modulation and thus regulation of GPCR signaling, taking advantage of it for pharmacological treatment.}, language = {en} } @article{ArimanyNardiMinuesaPastorAngladaetal.2016, author = {Arimany-Nardi, Cristina and Minuesa, Gerard and Pastor-Anglada, Mar{\c{c}}al and Keller, Thorsten and Erkizia, Itziar and Koepsell, Hermann and Martinez-Picado, Javier}, title = {Role of Human Organic Cation Transporter 1 (hOCT1) Polymorphisms in Lamivudine (3TC) Uptake and Drug-Drug Interactions}, series = {Frontiers in Pharmacology}, volume = {7}, journal = {Frontiers in Pharmacology}, number = {175}, doi = {10.3389/fphar.2016.00175}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165236}, year = {2016}, abstract = {Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level.}, language = {en} } @phdthesis{Arnaudov2017, author = {Arnaudov, Theresa Irina}, title = {Anthocyane - Modulation oxidativen Stresses in vivo und in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152593}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die menschliche Nahrung enth{\"a}lt antioxidative Stoffe, die den Menschen m{\"o}glicherweise vor oxidativem Stress und seinen Konsequenzen sch{\"u}tzen k{\"o}nnen. Im Fokus der vorliegenden Arbeit standen Anthocyane, die als vielversprechende antioxidative Pflanzenstoffe in unterschiedlichen Obst- und Gem{\"u}sesorten zu finden sind. Im ersten Teil der Arbeit wurden in einem HT-29-Zellkulturmodell die zwei wichtigsten Vertreter der Anthocyanidine, Delphinidin und Cyanidin, untersucht. Es galt zu pr{\"u}fen, ob beide Pflanzenstoffe in geringen Konzentrationen in humanen Zellen antioxidativ wirken und oxidativen Genomschaden verhindern k{\"o}nnen. Im Comet-Assay reduzierten sowohl Delphinidin (ab 3,2 µM) als auch Cyanidin (ab 1 µM) signifikant die durch 100 µM Wasserstoffperoxid induzierten DNA-Sch{\"a}den in den HT-29-Zellen. Im Comet-Assays mit FPG-Enzym wurde deutlich, dass eine Pr{\"a}inkubation mit Cyanidin wirksam die Oxidation der DNA-Basen verringert. Die Auswirkungen auf den Glutathionspiegel wurden mit Hilfe des Glutathion-Recycling-Assays nach Tietze untersucht. Die Pr{\"a}inkubation mit Cyanidin f{\"u}hrte hierbei zu keinen signifikanten Ver{\"a}nderungen. Um die Auswirkungen der Anthocyanidine auf die intrazellul{\"a}re ROS-Produktion zu beobachten, wurde der fluoreszierenden Farbstoffs DHE verwendet. Sowohl Delphinidin (10 und 15 µM) als auch Cyanidin (10 und 20 µM) senkten signifikant die durch 25 µM Antimycin A angeregte ROS-Produktion. Im zweiten Teil der Arbeit wurde ein anthocyanreicher roter Fruchtsaft in einer 10-w{\"o}chigen Interventionsstudie am Menschen getestet. Hieran nahmen sowohl 19 Fibromyalgiepatienten als auch 10 gesunde Probanden teil. Es sollte die Hypothese gepr{\"u}ft werden, dass die konzentrierte und andauernde Einnahme des Saftes messbar oxidative Stressparameter im Blut ver{\"a}ndert. Außerdem sollten m{\"o}gliche Unterschiede im oxidativen Stresslevel zwischen Patienten und gesunden Probanden aufgedeckt werden. Nach jeder Studienphase erfolgte eine Befragung nach klinischen Symptomen und die Abgabe einer Urin- und Blutprobe in der Schmerzambulanz der Uniklinik W{\"u}rzburg (2 Wochen Einwaschphase, 4 Wochen Fruchtsaftphase mit je 750 ml Saft t{\"a}glich, 4 Wochen Auswaschphase). Das ROS-Level wurde mit 2 Methoden in den mononukle{\"a}ren Blutzellen untersucht: In der photometrischen NBT-Messung konnten keine signifikanten Unterschiede zwischen den Gruppen oder Zeitpunkten beobachtet werden. Bei der durchflusszytometrischen Messung mit Hilfe des fluoreszierenden DCF-Farbstoffes lag das ROS-Level der Patientengruppe vor Fruchtsafteinnahme signifikant h{\"o}her als das der Kontrollgruppe. Zur Messung der antioxidativen Kapazit{\"a}t wurde die Eisen-Reduktionsf{\"a}higkeit (FRAP) im Plasma untersucht. In der Patientengruppe zeigte sich eine Steigerung der antioxidativen Kapazit{\"a}t nach Einnahme des Fruchtsaftes. Die Unterschiede zwischen den beiden Gruppen waren gering. Sowohl das Gesamtglutathion als auch die oxidierte und reduzierte Form wurden in den Erythrozyten der Probanden mit dem Glutathion-Recycling-Assay gemessen. Nach der Fruchtsafteinnahme stieg die Konzentration des Gesamtglutathions in der Patientengruppe an. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass Delphinidin und Cyanidin auch in geringen Konzentrationen (1µM - 20µM) einen antioxidativer Effekt in HT-29-Zellen haben und vor oxidativem DNA-Schaden sch{\"u}tzen k{\"o}nnen. Die Ergebnisse der Interventionsstudie unterschieden sich teilweise in den einzelnen Endpunkten. Es war nicht m{\"o}glich, den Fibromyalgiepatienten ein h{\"o}heres oxidatives Stresslevel nachzuweisen. Ein Grund f{\"u}r die geringeren Effekte des Fruchtsaftes k{\"o}nnte in der eher geringen Bioverf{\"u}gbarkeit der Anthocyane liegen. Außerdem k{\"o}nnte die Heterogenit{\"a}t der Fibromyalgieerkrankung genauso wie andere endogene oder exogene Faktoren wie etwa Alter oder Medikamenteneinnahme die teilweise großen interindividuellen Schwankungen der Messergebnisse hinsichtlich der oxidativen Stressparameter bedingen. Klinisch profitierten einige der Fibromyalgiepatienten von der Fruchtsafteinnahme insbesondere hinsichtlich der Reizdarmsymptomatik. Dieses Volksleiden k{\"o}nnte ein interessanter Ansatzpunkt f{\"u}r Folgeuntersuchungen mit einem anthocyanreichen Produkt sein.}, subject = {Oxidativer Stress}, language = {de} } @phdthesis{Arnold2020, author = {Arnold, Charlotte Antonia}, title = {Reduktion des Genomschadens in peripheren Lymphozyten adip{\"o}ser Patienten nach bariatrischer Operation}, doi = {10.25972/OPUS-21174}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211748}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {{\"U}bergewicht, das als Volkskrankheit ein wachsendes globales Problem darstellt, ist mit mehreren folgenreichen Komorbidit{\"a}ten behaftet und die Assoziation der Erkrankung mit nachweisbarer Sch{\"a}digung des Erbguts durch oxidativen Stress ist mittlerweile unangefochten. In der vorliegenden Studie wurden periphere Lymphozyten stark bis morbid adip{\"o}ser Patienten mit Hilfe des Mikrokern-Assays untersucht und es konnte - begleitend zu der zu erwartenden BMI-Abnahme - eine signifikante Reduktion des Genomschadens durch Magenbypass bzw. Sleeve-Gastrektomie 12 Monate postoperativ detektiert werden. Daneben demonstrierte die Analyse zus{\"a}tzlich erhobener Patientendaten, die u. a. N{\"u}chternglucose, HbA1c, Blutdruck und Herzfrequenz sowie ein Blutbild der Patienten (inklusive CRP als Entz{\"u}ndungsmarker, Transaminasen, gGT sowie Lipidprofil) umfasste, eine deutliche Besserung vieler Parameter bis auf teilweise wieder physiologische Normwerte. All diese Ergebnisse st{\"u}tzen die These der metabolischen Wirksamkeit sowohl des Roux-en-Y-Magenbypass als auch der Sleeve- Gastrektomie. Ihre Bedeutung liegt nicht zuletzt in der angenommenen Reduktion des Krebserkrankungsrisikos, da jeweils ein Zusammenhang mit der Adipositas an sich, dem Diabetes und durch oxidativen Stress verursachter DNA-Sch{\"a}digung besteht. Mit Blick auf eine gegenw{\"a}rtig in der Forschung diskutierte potentielle therapeutische Anwendung von Antioxidantien zur Reduktion von Erbgutsch{\"a}digungen, die durch oxidativen Stress zustande kommen, wurden die Substanzen Tricetinidin, Curcumin und Resveratrol im Rahmen des Mikrokerntests an HL60- und NRK-Zellen untersucht, in denen mit der Mischung aus Insulin und Angiotensin II eine gentoxische Wirkung erzielt worden war.}, subject = {Fettsucht}, language = {de} } @article{AwadOthmanStopper2017, author = {Awad, Eman and Othman, Eman M. and Stopper, Helga}, title = {Effects of resveratrol, lovastatin and the mTOR-inhibitor RAD-001 on insulin-induced genomic damage in vitro}, series = {Molecules}, volume = {22}, journal = {Molecules}, number = {12}, doi = {10.3390/molecules22122207}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159260}, pages = {2207}, year = {2017}, abstract = {Diabetes mellitus (DM) is one of the major current health problems due to lifestyle changes. Before diagnosis and in the early years of disease, insulin blood levels are elevated. However, insulin generates low levels of reactive oxygen species (ROS) which are integral to the regulation of a variety of intracellular signaling pathways, but excess levels of insulin may also lead to DNA oxidation and DNA damage. Three pharmaceutical compounds, resveratrol, lovastatin and the mTOR-inhibitor RAD-001, were investigated due to their known beneficial effects. They showed protective properties against genotoxic damage and significantly reduced ROS after in vitro treatment of cultured cells with insulin. Therefore, the selected pharmaceuticals may be attractive candidates to be considered for support of DM therapy.}, language = {en} } @article{BaertschLutzSchlatter1991, author = {Baertsch, A. and Lutz, Werner K. and Schlatter, C.}, title = {Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60743}, year = {1991}, abstract = {1 ,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-tenn bioassay using gavage in com oil (24 and 48 mg/kg/day), but not by inhalation (up to 150-250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Fernale F-344 rats (183-188 g) were exposed to [1,2-14C]DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l = 80 ppm for 4 h) or to a peak concentration (up to 18 mg/1 = 4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/k:g. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymaticaBy hydrolyzed to the 3' -nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density' indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The Ievel of DNA adducts was expressed in the dose-nonnalized units ofthe Covalent Binding Index, CBI = f.Lmol adduct per mol DNA nucleotide/ mmol DCE per kg body wt. In liver DNA, the different exposure regimens resulted in markedly different CBI values of 1.8 and 69, for "constant-low" and ''peak" DCE exposure Ievels. In the Jung, the respective values were 0.9 and 31. It is concluded that the DNA darnage by DCE depends upon the concentration-time profile and that the carcinogenic potency determined in the gavage study should not be used for low-Ievel inhalation exposure.}, subject = {Toxikologie}, language = {en} } @article{BalasubramanianOthmanKampiketal.2017, author = {Balasubramanian, Srikkanth and Othman, Eman M. and Kampik, Daniel and Stopper, Helga and Hentschel, Ute and Ziebuhr, Wilma and Oelschlaeger, Tobias A. and Abdelmohsen, Usama R.}, title = {Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation}, series = {Frontiers in Microbiology}, volume = {8}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2017.00236}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171844}, year = {2017}, abstract = {Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections.}, language = {en} } @article{BanachBuenemannHueseretal.1993, author = {Banach, Katrin and B{\"u}nemann, Moritz and H{\"u}ser, J{\"o}rg and Pott, Lutz}, title = {Serum contains a potent factor that decreases \(\beta\)-adrenergic receptor-stimulated L-type Ca\(^{2+}\) current in cardiac myocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-32027}, year = {1993}, abstract = {No abstract available}, language = {en} } @article{BankogluArnoldHeringetal.2018, author = {Bankoglu, Ezgi Eyluel and Arnold, Charlotte and Hering, Ilona and Hankir, Mohammed and Seyfried, Florian and Stopper, Helga}, title = {Decreased chromosomal damage in lymphocytes of obese patients after bariatric surgery}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, number = {11195}, doi = {10.1038/s41598-018-29581-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177090}, year = {2018}, abstract = {The number of bariatric surgeries being performed worldwide has markedly risen. While the improvement in obesity-associated comorbidities after bariatric surgery is well-established, very little is known about its impact on cancer risk. The peripheral lymphocyte micronucleus test is a widely used method for the monitoring of chromosomal damage levels in vivo, and micronucleus frequency positively correlates with cancer risk. Therefore, the aim of this study was to compare the micronucleus frequency before and after bariatric surgery in obese subjects. Peripheral blood mononuclear cells were collected from 45 obese subjects before and at two time-points after bariatric surgery (6 and 12 months) to assess spontaneous micronucleus frequency. Consistent with the increased cancer risk previously shown, bariatric surgery-induced weight loss led to a significant reduction in lymphocyte micronucleus frequency after 12 months. Interestingly, comorbidities such as type 2 diabetes mellitus and metabolic syndrome further seemed to have an impact on the lymphocyte micronucleus frequency. Our findings may indicate a successful reduction of cancer risk in patients following weight loss caused by bariatric surgery.}, language = {en} } @article{BankogluSchueleStopper2021, author = {Bankoglu, Ezgi Eyluel and Schuele, Carolin and Stopper, Helga}, title = {Cell survival after DNA damage in the comet assay}, series = {Archives of Toxicology}, volume = {95}, journal = {Archives of Toxicology}, number = {12}, doi = {10.1007/s00204-021-03164-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265339}, pages = {3803-3813}, year = {2021}, abstract = {The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H\(_{2}\)O\(_{2}\)) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30\% DNA in tail caused the death of more than 50\% of the cells, with etoposide causing slightly more cell death than H\(_{2}\)O\(_{2}\) or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20\% DNA in tail, survival data for the cells are provided.}, language = {en} } @article{BankogluStippGerberetal.2021, author = {Bankoglu, Ezgi Eyluel and Stipp, Franzisca and Gerber, Johanna and Seyfried, Florian and Heidland, August and Bahner, Udo and Stopper, Helga}, title = {Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay}, series = {Archives of Toxicology}, volume = {95}, journal = {Archives of Toxicology}, number = {5}, doi = {10.1007/s00204-021-03012-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265326}, pages = {1831-1841}, year = {2021}, abstract = {The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.}, language = {en} } @article{BankogluTschoppSchmittetal.2016, author = {Bankoglu, Ezgi Eyluel and Tschopp, Oliver and Schmitt, Johannes and Burkard, Philipp and Jahn, Daniel and Geier, Andreas and Stopper, Helga}, title = {Role of PTEN in Oxidative Stress and DNA Damage in the Liver of Whole-Body Pten Haplodeficient Mice}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {11}, doi = {10.1371/journal.pone.0166956}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146970}, pages = {e0166956}, year = {2016}, abstract = {Type 2 diabetes (T2DM) and obesity are frequently associated with non-alcoholic fatty liver disease (NAFLD) and with an elevated cancer incidence. The molecular mechanisms of carcinogenesis in this context are only partially understood. High blood insulin levels are typical in early T2DM and excessive insulin can cause elevated reactive oxygen species (ROS) production and genomic instability. ROS are important for various cellular functions in signaling and host defense. However, elevated ROS formation is thought to be involved in cancer induction. In the molecular events from insulin receptor binding to genomic damage, some signaling steps have been identified, pointing at the PI3K/AKT pathway. For further elucidation Phosphatase and Tensin homolog (Pten), a tumour suppressor phosphatase that plays a role in insulin signaling by negative regulation of PI3K/AKT and its downstream targets, was investigated here. Dihydroethidium (DHE) staining was used to detect ROS formation in immortalized human hepatocytes. Comet assay and micronucleus test were performed to investigate genomic damage in vitro. In liver samples, DHE staining and western blot detection of HSP70 and HO-1 were performed to evaluate oxidative stress response. DNA double strand breaks (DSBs) were detected by immunohistostaining. Inhibition of PTEN with the pharmacologic inhibitor VO-OHpic resulted in increased ROS production and genomic damage in a liver cell line. Knockdown of Pten in a mouse model yielded increased oxidative stress levels, detected by ROS levels and expression of the two stress-proteins HSP70 and HO-1 and elevated genomic damage in the liver, which was significant in mice fed with a high fat diet. We conclude that PTEN is involved in oxidative stress and genomic damage induction in vitro and that this may also explain the in vivo observations. This further supports the hypothesis that the PI3K/AKT pathway is responsible for damaging effects of high levels of insulin.}, language = {en} } @article{BarileBerryBlaauboeretal.2021, author = {Barile, Frank A. and Berry, Colin and Blaauboer, Bas and Boobis, Alan and Bolt, Herrmann M. and Borgert, Christopher and Dekant, Wolfgang and Dietrich, Daniel and Domingo, Jose L. and Galli, Corrado L. and Gori, Gio Batta and Greim, Helmut and Hengstler, Jan G. and Heslop-Harrison, Pat and Kacew, Sam and Marquardt, Hans and Mally, Angela and Pelkonen, Olavi and Savolainen, Kai and Testai, Emanuela and Tsatsakis, Aristides and Vermeulen, Nico P.}, title = {The EU chemicals strategy for sustainability: in support of the BfR position}, series = {Archives of Toxicology}, volume = {95}, journal = {Archives of Toxicology}, number = {9}, issn = {0340-5761}, doi = {10.1007/s00204-021-03125-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-307154}, pages = {3133-3136}, year = {2021}, abstract = {The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.}, language = {en} } @phdthesis{Basali2017, author = {Basali, Timo}, title = {Untersuchung der Nierensch{\"a}digung durch Aldosteron am Rattenmodell {\"u}ber die Quantifizierung von Sch{\"a}digungsmarkern mittels Real-Time PCR-Technik}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151311}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die Breite der Wirkungen von Aldosteron auf Nierenzellen wurde lange Zeit untersch{\"a}tzt. Inzwischen zeigte sich ein nicht unerheblicher Anteil des Hyperaldosteronismus an arterieller Hypertonie und ebenso mehren sich die Hinweise auf damit assoziierter erh{\"o}hter Inzidenz f{\"u}r maligne Entartung von Nierengewebe. In dieser Arbeit wurde der Effekt von Hyperaldosteronismus auf Nierenzellen von Ratten in vivo untersucht. Mittels real time quantitative PCR wurden die relative Expressionsver{\"a}nderungen der mRNA von validierten Nierensch{\"a}digungsmarkern im Hyperaldosteronismusmodell kontrolliert beobachtet und statistisch ausgewertet. Anders als im analog durchgef{\"u}hrten Vorversuch mit DOCA an der Stelle von Aldosteron, ließ sich gr{\"o}ßtenteils kein {\"u}ber der nat{\"u}rlichen Streuung der Daten liegender, signifikanter Effekt der Nierensch{\"a}digung durch {\"u}berh{\"o}hte Aldosteronspiegel nachweisen. Hierf{\"u}r kommen vielf{\"a}ltige Gr{\"u}nde in Frage. Neben der technischen Variabilit{\"a}t, der Beschaffenheit der internen Kontrolle, potentiell vorhandenen Inhibitoren und der Qualit{\"a}t der mRNA, konnten eine Reihe von weiteren Gr{\"u}nden als Ursache f{\"u}r die Diskrepanz zu den Ergebnissen der mit DOCA behandelten Tiere ausgeschlossen werden. Neben der theoretischen M{\"o}glichkeit inter-methodischer Differenzen und sich daraus ergebender Variationen, sowie der noch weiter zu untersuchenden Rolle des Glukokortikoidrezeptors durch dessen variable gleichzeitige Aktivierung, ist die Interpretation im Sinne eines zu gering ausgepr{\"a}gten Sch{\"a}digungseffektes durch den Hyperaldosteronismus f{\"u}r den gew{\"a}hlten Stichprobenumfang naheliegend. Hiermit stimmt auch die Tatsache {\"u}berein, dass der Effekt der Behandlung mit Aldosteron im Vergleich zur Behandlung mit DOCA von vorne herein deutlich geringer ausfallend erwartet wurde.}, subject = {Aldosteron}, language = {de} } @phdthesis{BathePeters2022, author = {Bathe-Peters, Marc}, title = {Spectroscopic approaches for the localization and dynamics of β\(_1\)- and β\(_2\)-adrenergic receptors in cardiomyocytes}, doi = {10.25972/OPUS-25812}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-258126}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {In the heart the β\(_1\)-adrenergic receptor (AR) and the β\(_2\)-AR, two prototypical G protein-coupled receptors (GPCRs), are both activated by the same hormones, namely adrenaline and noradrenaline. Both receptors couple to stimulatory G\(_s\) proteins, mediate an increase in cyclic adenosine monophosphate (cAMP) and influence the contractility and frequency of the heart upon stimulation. However, activation of the β\(_1\)-AR, not the β\(_2\)-AR, lead to other additional effects, such as changes in gene transcription resulting in cardiac hypertrophy, leading to speculations on how distinct effects can arise from receptors coupled to the same downstream signaling pathway. In this thesis the question of whether this distinct behavior may originate from a differential localization of these two receptors in adult cardiomyocytes is addressed. Therefore, fluorescence spectroscopy tools are developed and implemented in order to elucidate the presence and dynamics of these endogenous receptors at the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. This allows the visualization of confined localization and diffusion of the β\(_2\)-AR to the T-tubular network at endogenous expression. In contrast, the β\(_1\)-AR is found diffusing at both the outer plasma membrane and the T-tubules. Upon overexpression of the β\(_2\)-AR in adult transgenic cardiomyocytes, the receptors experience a loss of this compartmentalization and are also found at the cell surface. These data suggest that distinct signaling and functional effects can be controlled by specific cell surface targeting of the receptor subtypes. The tools at the basis of this thesis work are a fluorescent adrenergic antagonist in combination of fluorescence fluctuation spectroscopy to monitor the localization and dynamics of the lowly expressed adrenergic receptors. Along the way to optimizing these approaches, I worked on combining widefield and confocal imaging in one setup, as well as implementing a stable autofocus mechanism using electrically tunable lenses.}, subject = {G-Protein gekoppelte Rezeptoren}, language = {en} } @article{BauerMallyLiedtke2021, author = {Bauer, Benedikt and Mally, Angela and Liedtke, Daniel}, title = {Zebrafish embryos and larvae as alternative animal models for toxicity testing}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {24}, issn = {1422-0067}, doi = {10.3390/ijms222413417}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284225}, year = {2021}, abstract = {Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.}, language = {en} } @phdthesis{Baumann2009, author = {Baumann, Klaus}, title = {Modulierende Effekte von Kaffee auf die Induktion von Mikrokernen durch mutagene Substanzen in Mauslymphomzellen L5178Y}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-36996}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Kaffee, die in der westlichen Welt am h{\"a}ufigsten verwendete psychoaktive Substanz, erwies sich im Mikrokern-Testes an Maus-Lymphomzellen L 5178Y als modulierend auf bekannte mutagene Agentien. In dieser Arbeit wurde meist Instantkaffee verwendet, der gegen N-Methyl-N-Nitro-N-Nitrosoguanidin (MNNG), Mitomycin C (MMC), Genistein, und Methylmethansulfonat getestet wurde. Bei MMC und Genistein erwies sich Kaffee als antimutagen. Bei MNNG hatte Kaffee keinen klaren Einfluss auf die Gentoxizit{\"a}t, ebenso blieb die Kaffee-Wirkung bez{\"u}glich MMS unklar. Kaffee minderte dosisabh{\"a}ngig das Wachstum und die {\"U}berlebensrate von L 5178Y - Zellen. Es wurde die Frage nach den Ursachen der modulierenden Effekte diskutiert. Insbesondere wurde die Hypothese er{\"o}rtert, dass f{\"u}r die Richtung der Modulation nicht so sehr die Konzentration des Kaffees, sondern die mutagene Potenz der koinkubierten Substanz eine entscheidende Rolle spielen k{\"o}nnte. Ggf. wirkt Kaffee - im Sinne eines "abh{\"a}rtenden" Effektes - bei schwach mutagenen Substanzen anitmutagen, bei stark mutagenen Substanzen hingegen synergistisch mutagen.}, subject = {Kaffee}, language = {de} } @phdthesis{Bayer2005, author = {Bayer, Tanja}, title = {Toxicity and biotransformation of 1,1,1,3,3-Pentafluoropropane, 3,3,3-Trifluoropropionic acid and 1,1,1,3-Tetrachloropropane}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-15731}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {The biotransformation of 1,1,1,3,3-pentafluoropropane was investigated in rats and in in vitro systems. First, the metabolites were identified in vivo using GC/MS and 19F NMR analysis. The main metabolite was identified as trifluoroacetic acid, the minor metabolite as 3,3,3-trifluoropropionic acid and as a cleavage product, inorganic fluoride was found. As the in vitro system, liver microsomes from rat and human samples and rat liver homogenates were used. Trifluoroacetic acid and 3,3,3 trifluoropropionic acid were confirmed in vitro as metabolic intermediates, following biotransformation of 1,1,1,3,3-pentafluoropropane by the cytochrome P-450-system. Studies, designed for clarifying the cardiotoxicity of 1,1,1,3,3-pentafluoropropane were driven by the hypothesis that 3,3,3-trifluoropropionic acid is the toxic agent. This was based on the lethal toxicity, which was observed in previous in vivo experiments. In addition, the point of its structural similarity to toxic agents as for example monofluoroacetic acid or of possible metabolic intermediates like difluoroacrylic acid with known toxicity were considered to support this assumption. However, trifluoroacetic acid was neglected as the sought-after toxic agent because of its different toxic effects, known from literature. Investigations on the biotransformation of 3,3,3-trifluoropropionic acid were performed and resulted in no metabolic activity and in poor elimination of 3,3,3-trifluoropropionic acid in vivo. The histopathological effects on the heart, which were observed in the 90-day oral toxicity study of 1,1,1,3,3-pentafluoropropane in rats, namely mononuclear inflammatory cell infiltrations and degenerated myocardial fibers, were not observed after a 28 day repeated exposure of up to 10 mg/kg b.w. of 3,3,3-trifluoropropionic acid. However, a single high dose of 3,3,3-trifluoropropionic acid lead to severe toxicological effects. The difference in the observed toxic effects after a single and repeated administration may be due to adaptive mechanisms in rats. The toxicological effects included clinical signs like ataxia, coma and cramps. The conditions of the rats suggested possible inhibition of the energy supply to the organism. Furthermore, the interference of 3,3,3-trifluoropropionic acid in the functionality of the organism was investigated. Experiments were performed in vitro in rat liver and heart mitochondria to investigate effects on the mitochondrial ß-oxidation. However, the transformation of the substrate [U14C] palmitic acid in the ß oxidation pathway was not inhibited by 3,3,3-trifluoropropionic acid. In addition, no cytotoxicity of 3,3,3 trifluoropropionic acid was observed in the cell culture systems. The main effect after a single dose of 3,3,3-trifluoropropionic acid was seen in clinical pathology and metabonomic analysis. The decrease in blood glucose is considered to have the most far-reaching consequences for the toxicity of 3,3,3-trifluoropropionic acid. If considering this change as the primary effect after a single dose, secondary effects, for example, the above-mentioned clinical signs could be explained. In addition, the observed high level of ketone bodies might have been responsible for life-threatening possible ketoacidosis. In general, ketoacidosis occurs after an imbalance between glycolysis, lipolysis, TCA cycle activity and respiratory function. Based on the results, ß-oxidation of fatty acids was not affected, and due to the decrease in glucose levels and the high levels of acetyl CoA, glycolysis was considered not to be impaired. Increased amounts of acetyl CoA might be a result of insufficient activity of the TCA cycle. However, the inhibition of the TCA cycle can be based on the impairment of specific enzymes and/or on the involvement of messenger substrates like insulin. Supporting the first mentioned aspect are decreased levels of TCA cycle intermediates, like \&\#945;-ketoglutarate or citrate, as seen in 1H-NMR spectra of urine. However, the second aspect would explain the drop in blood glucose with the impairment of glucose transporters or the impairment of the insulin balance. If a single dose of 3,3,3-trifluoropropionic acid had stimulated the insulin release, glycolysis would be activated, and high amounts of acetyl CoA would be produced. In case of impaired use by the TCA cycle, levels of ketone bodies would be increased. Experiments were designed to characterize the direct effect of 3,3,3-trifluoropropionic acid on rat insulinoma-derived INS-1 cells as possible increase in insulin release. Further investigations are necessary to answer in which step of the metabolic pathway 3,3,3-trifluoropropionic acid interferes or finally which specific enzyme is inhibited or activated by 3,3,3-trifluoropropionic acid, leading to the drop in blood glucose and finally in lethal toxicity.}, subject = {Fluorkohlenwasserstoffe}, language = {en} } @phdthesis{Behr2006, author = {Behr, Bj{\"o}rn}, title = {Neue Mutanten des humanen beta1-adrenergen Rezeptors zeigen f{\"u}r die Rezeptoraktivierung relevante Aminos{\"a}uren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19074}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Eine durch Aktivierung eines G-Protein gekoppelten Rezeptors induzierte Konfirmations{\"a}nderung resultiert in einer Signaltransduktion durch das G-Protein zu einem Effektorenzym wie der Adenylylcyclase. In dieser Arbeit konnten Aminos{\"a}uren in dem zur G-Protein gekoppelten Rezeptorfamilie zugeh{\"o}rigen beta1-adrenergen Rezeptor identifiziert werden, welche f{\"u}r dessen Aktivierung von Bedeutung sind. Der Analyse von beta1-adrenergen Rezeptormutanten lag die Erkenntnis zu Grunde, dass therapeutisch genutzte Liganden wie Terbutalin, oder das experimentell eingesetzte Broxaterol Agonisten am beta2- und beta3-adrenergen Rezeptor, jedoch Antagonisten am beta1-adrenergen Rezeptor sind. Dieses Verhalten wurde zum Anlass genommen spezifische Aminos{\"a}uren zu identifizieren, welche eine bedeutende Funktion in der Aktivierung von beta -Rezeptorsubtypen haben k{\"o}nnten. Nach einem Aminos{\"a}urevergleich innerhalb der Familie der beta-adrenergen Rezeptoren konnten Aminos{\"a}urepositionen identifiziert werden, die identisch im beta2- bzw. beta3-Rezeptor sind und sich von denen des beta1- Rezeptors unterscheiden und damit das Aktivierungsprofil von Broxaterol und Terbutalin widerspiegeln. Mit zielgerichteten Punktmutationen wurden nun insbesondere im Bereich der Transmembranregionen solche Aminos{\"a}uren im beta1-adrenergen Rezeptor durch die entsprechende des beta2- (beta3-) Rezeptors ersetzt. Obwohl keine der getesteten Mutanten Unterschiede im pharmakologischen Bindungsprofil zeigten, konnten vier Mutanten gefunden werden, welche partiell oder vollst{\"a}ndig durch Broxaterol oder Terbutalin aktiviert wurden. Die beiden Mutanten I185L sowie D212N konnten mit Broxaterol und Terbutalin aktiviert werden, zwei Liganden, die Antagonisten am beta1- Wildtyprezeptor sind. Außerdem konnten zwei weitere Mutanten, V120L und K253R, durch Terbutalin aktiviert werden. Betrachtet man die Struktur von Terbutalin, so ist dieser Ligand den endogenen Katecholaminen {\"a}hnlicher als Broxaterol. Ein Rezeptormodell zeigt, dass die vier relevanten Aminos{\"a}uren außerhalb der Ligandenbindungsregion liegen und somit eine direkte Interaktion mit dem Liganden unwahrscheinlich erscheint. Diese These wird durch das im Vergleich zum Wildtyp nicht ver{\"a}nderte Bindungsprofil der beta1-Rezeptormutanten unterst{\"u}tzt. Diese Ergebnisse legen nahe, dass die Aminos{\"a}uren V120, I185, D212 und K253 in der ligandeninduzierten Konfirmations{\"a}nderung des beta1-Rezeptors von Bedeutung sind.}, language = {de} } @phdthesis{Bellwon2015, author = {Bellwon, Patricia}, title = {Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122693}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program "Predict-IV"; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of "omics" technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling. This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day. Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors. Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling. Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions. Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo. Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound's distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.}, subject = {Zellkultur}, language = {en} } @phdthesis{Berisha2019, author = {Berisha, Filip}, title = {Molekulare Wirkmechanismen von Sulfonylharnstoffen: Direkte Epac-Aktivierung oder Hemmung der Phosphodiesterasen}, doi = {10.25972/OPUS-17653}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176535}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Diabetes mellitus ist die h{\"a}ufigste Stoffwechselerkrankung in Deutschland. Sulfonylharnstoffe (SH) stellen die {\"a}lteste und eine sehr prominente Gruppe in der oralen Therapie des Diabetes mellitus Typ II dar, die eine verst{\"a}rkte Insulinfreisetzung vorrangig durch die Hemmung eines ATP-sensitiven Kaliumkanals (K+ATPKanal) erreichen. Daneben konnten weitere Proteine identifiziert werden, die mit SH interagieren und zu deren Effekten beitragen. W{\"a}hrend bereits in fr{\"u}hen Arbeiten gezeigt werden konnte, dass SH Vertreter der Phosphodiesterasen (PDE)Familie in ihrer Funktion behindern k{\"o}nnen, wurde k{\"u}rzlich Epac2 (exchange protein directly activated by cAMP 2) als weiteres Zielprotein f{\"u}r SH angef{\"u}hrt. Insbesondere die F{\"a}higkeit von SH, direkt an Epac2 zu binden, wird in der Literatur kontrovers diskutiert und eine indirekte Aktivierung durch eine PDE-Hemmung und einen erh{\"o}hten cAMP-Spiegel als Mechanismus vermutet. Zur weiteren Untersuchung wurden in dieser Arbeit FRET-basierte Biosensoren verwendet, um die Wirkung von SH auf Epac und PDEs n{\"a}her zu untersuchen. Dabei konnte sowohl in einem photometrischen Ansatz als auch in lebenden Zellen, die einen Epac2-basierten Sensor enthalten, gezeigt werden, dass eine Aktivierung durch SH stattfindet. Da sowohl Epac2-camps, der von allen hier verwendeten Sensoren mit der h{\"o}chsten Sensitivit{\"a}t f{\"u}r cAMP, als auch CFP-Epac1δDEPYFP nicht auf SH reagieren, ist diese Aktivierung selektiv f{\"u}r die Isoform Epac2 und wird vorrangig nicht durch eine PDE-Hemmung verursacht. Die Verwendung weiterer Sensoren mit verschiedenen Varianten von Epac2 (verl{\"a}ngerte Version von Epac2-camps) zeigen mit zunehmender L{\"a}nge {\"u}ber die cAMP-Bindedom{\"a}ne hinaus eine beginnende Reaktion im Sinne einer instabilen FRET-Kurve (Epac2camps long) bzw. eine deutliche Aktivierung durch den SH (Epac2-camps superlong), wodurch eine direkte Aktivierung best{\"a}tigt wird, und suggerieren eine Bindestelle f{\"u}r SH, die sich von denen von cAMP unterschiedet und weiter eingeengt werden konnte (im n{\"a}heren Bereich von Q454 bzw. E460). Obwohl hierdurch eine direkte Aktivierung gezeigt werden konnte, ist die grunds{\"a}tzliche F{\"a}higkeit der SH, PDE zu beeinflussen, keineswegs gekl{\"a}rt. Daher wurden weitere Sensoren konstruiert bzw. verwendet, die basierend auf Epac1-camps und Epac2-camps verschiedene PDEs enthalten. Dabei konnte durch die Zugabe von SH eine deutliche Aktivierung des jeweiligen Sensors und somit eine PDEHemmung nachgewiesen werden. Dies konnte sowohl f{\"u}r PDE4A als auch f{\"u}r die in Inselzellen {\"u}berwiegend vorkommende PDE3B gezeigt werden. Dadurch ergeben sich einige (klinisch relevante) Implikationen. Zum einen stellt neben der direkten Epac-Aktivierung auch die direkte Hemmung der PDE einen wichtigen Mechanismus f{\"u}r die Sekretion von Insulin dar. Außerdem sind bei PDEHemmung und direkter Epac-Aktivierung außerhalb der Inselzellen auch Nebenwirkungen in anderen Organen zu erwarten wie z.B. die Entstehung lebensgef{\"a}hrlicher Rhythmusst{\"o}rungen in Herzmuskelzellen.}, subject = {Sulfonylharnstoffe}, language = {de} } @phdthesis{Berlin2017, author = {Berlin, Christopher}, title = {Die Untersuchung der kardialen Folgen einer ubiquit{\"a}ren Deletion von RKIP in M{\"a}usen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152882}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die Herzinsuffizienz, eine der h{\"a}ufigsten chronischen Krankheiten in der westlichen Welt, ist als Folge einer Myokardsch{\"a}digung durch eine verschlechterte Pumpfunktion des Herzens charakterisiert, die der K{\"o}rper durch verschiedene Kompensationsmechanismen zur Kontraktilit{\"a}tssteigerung auszugleichen versucht. Wichtiger Mechanismus hierf{\"u}r ist die Kontraktilit{\"a}ts- und Frequenzsteigerung {\"u}ber ß-adrenerge Rezeptorsignale, welche bei langfristiger Stimulation allerdings zu einer Abnahme der Funktionalit{\"a}t und Minderexpression eben dieses Rezeptorsystems, sowie der gleichzeitigen Verschlechterung der Herzinsuffizienz f{\"u}hrt. Interessanterweise wird parallel zur verminderten Rezeptorexpression bei Herzinsuffizienzpatienten eine Zunahme der GRK-Aktivit{\"a}t beobachtet. Diese Kinase ist in der Lage, ß-adrenerge GPCR-Signale durch Phosphorylierung des membranst{\"a}ndigen Rezeptors herunterzuregulieren. Durch einen PKC-abh{\"a}ngigen switch von Raf1 zu GRK2 konnte mit RKIP ein kardialer, endogener Inhibitor der GRK2 identifiziert werden. Es wurde in vitro und in vivo in M{\"a}usen mit myokardialer {\"U}berexpression von RKIP gezeigt, dass RKIP f{\"a}hig ist, die kontraktile Funktion von Herzmuskelzellen zu verbessern, negative kardiale Langzeitfolgen wie eine Verschlechterung der Insuffizienz, Remodeling-Prozesse wie Zunahme der Fibrosierung und eine gesteigerte Apoptoserate, sowie kardiale Rhythmusst{\"o}rungen protektiv zu beeinflussen. Um die endogene Rolle von RKIP weiter zu er{\"o}rtern, wurde in dieser Arbeit der Knockout von RKIP unter basalen Bedingungen, als auch nach transverser Aortenkonstriktion (TAC) untersucht. Zur Untersuchung physiologischer Parameter wie der Verk{\"u}rzungsfraktion, oder dem linksventrikul{\"a}rem diastolischen Durchmesser wurden echokardiographische Verfahren herangezogen. In diesen Untersuchungen zeigte sich nach dreiw{\"o}chiger TAC eine Verschlechterung der Pumpfunktion, sowie eine verst{\"a}rkte Dilatation des linken Ventrikels in RKIP-/--M{\"a}usen. Gest{\"u}tzt wurden diese Ergebnisse durch einen erh{\"o}hten pulmonalen Blutr{\"u}ckstau in RKIP-/--M{\"a}usen nach chronischer Druckbelastung. Zudem wurde an isolierten Kardiomyozyten die Kinetik von Kalzium als f{\"u}r die Kontraktion verantwortlichen Botenstoff durch intrazellul{\"a}re Fluoreszenz-Echtzeit-Messungen, sowie die Kontraktion und Relaxation auf Zell- und Sarkomerebene durch ein optisches Kamerasystem untersucht. Hier zeigte sich ohne den Einfluss β-adrenerger Stimulantien {\"a}quivalent zum basalen Ph{\"a}notyp dieser Tiere in RKIP-/--Kardiomyozyten keine Ver{\"a}nderung der Kalzium-Kinetik, sowie der Kontraktion und Relaxation auf Zell- und Sarkomerebene. Des Weiteren wurden mittels realtime PCR die Expressionslevels von Insuffizienzmarkern wie BNP und ANP, sowie von Kollagen 3 bestimmt. Der Grad der Fibrosierung wurde zus{\"a}tzlich durch Quantifizierung der fibrosierten Areale in histologischen Querschnitten untersucht. Apoptotische Ver{\"a}nderungen wurden mittels TUNEL-Assay auf histologischer Ebene bestimmt. In all diesen Untersuchungen zeigte sich ein fortgeschrittenes kardiales Remodeling in RKIP-/--M{\"a}usen nach TAC im Vergleich zu Wildtyptieren. Hand in Hand mit dem Bild einer fortgeschrittenen Herzinsuffizienz in RKIP-/--M{\"a}usen nach TAC konnte zudem in diesen Tieren eine gesteigerte Mortalit{\"a}t nach chronischer Hochdruckbelastung festgestellt werden. In Kombination mit den protektiven Eigenschaften einer kardialen RKIP-{\"U}berexpression, sowie dem positiven Effekt einer retroviralen RKIP-Transfektion sprechen diese Ergebnisse f{\"u}r RKIP als einen interessanten k{\"o}rpereigenen Angriffspunkt f{\"u}r die kontraktilit{\"a}tssteigernde Therapie der Herzinsuffizienz, den es in weiteren klinischen Studien zu untersuchen gilt.�}, subject = {RKIP}, language = {de} } @phdthesis{Bermel2006, author = {Bermel, Christina Maria}, title = {Regulation von G-beta-gamma-Untereinheiten durch Phosducin-{\"a}hnliche Proteine (PhLP)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19981}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Phosducin-like protein (PhLP) geh{\"o}rt zur Phosducinfamilie der G-Protein-betagamma-Regulatoren und kommt in zwei Spleißvarianten vor. Die lange Isoform PhLPlong und die kurze Isoform PhLPshort unterscheiden sich allein durch das Vorhandensein des 81 Aminos{\"a}uren langen N-Terminus von PhLPlong. In Versuchen mit gereinigten Proteinen erwies sich PhLPlong als der st{\"a}rkere Gbetagamma-Inhibitor, w{\"a}hrend die Bedeutung von PhLPshort nicht bekannt war. In dieser Arbeit wird gezeigt, daß in transfizierten HEK 293-Zellen PhLPshort die Gbetagamma-vermittelte Signalbildung 20mal st{\"a}rker hemmt als PhLPlong. Da der zus{\"a}tzliche N-Terminus von PhLPlong mehrere potentielle Phosphorylierungsstellen f{\"u}r die konstitutiv aktive Caseinkinase 2 besitzt, wurde angenommen, daß die lange Spleißvariante in Zellen einer solchen Regulation unterliegt, was sich auf seine Funktion als Gbetagamma-Inhibitor auswirkt. Durch schrittweise Trunkierungen bzw. Serin-/Threonin-Alaninmutationen wurden die potentiellen Phosphorylierungsstellen entfernt, was zur Verbesserung der Gbetagamma-Hemmf{\"a}higkeit f{\"u}hrte. Der N-terminale Aminos{\"a}urenabschnitt Ser-18/Thr-19/Ser-20 wurde dabei als die verantwortliche Stelle identifiziert. In unserer Arbeitsgruppe wurde gezeigt, daß PhLPlong in HEK 293-Zellen im Gegensatz zu PhLPshort einer konstitutiven Phosphorylierung unterliegt und durch die Caseinkinase2 katalysiert wird. In dieser Arbeit werden die Phosphorylierung von PhLPlong durch die Caseinkinase2 anhand von rekombinanten Proteinen sowie ihre Kinetik dargestellt. Das Vorhandensein von mRNA der Caseinkinase2 in HEK 293-Zellen zeigt, daß sie in der Zelle exprimiert und somit aktiv ist, was ihre physiologische Bedeutung herausstellt. Eine direkte Gbetagamma-Bindung konnte durch Immunpr{\"a}zipitation nur f{\"u}r PhLPlong nachgewiesen werden, weswegen f{\"u}r PhLPshort ein alternativer Mechanismus der Gbetagamma-Hemmung angenommen wurde, was in folgenden Arbeiten n{\"a}her untersucht wurde.}, language = {de} } @phdthesis{Bertelsmann2022, author = {Bertelsmann, Dietmar}, title = {Analysis of the Frequency of Kidney Toxicity in Preclinical Safety Studies using the eTOX Database}, doi = {10.25972/OPUS-25710}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257104}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {This research aimed to obtain reliable data on the frequency of different types of renal toxicity findings in 28-day oral gavage studies in Wistar rats, their consistency across species and study duration, as well as the correlation between histopathological endpoints and routinely used clinical chemistry parameters indicative of kidney injury. Analysis of renal histopathological findings was carried out through extraction of information from the IMI eTOX database. Spontaneous renal histopathological findings in 28-day oral gavage studies in control Wistar rats and beagle dogs confirmed tubular basophilia and renal dilation as the most frequent incidental findings in controls, whereas necrosis and glomerulosclerosis were not identified at all or only rarely as a background lesion. Histopathological evidence of necrosis and glomerulosclerosis was associated with changes in clinical chemistry parameters in 28-day oral gavage Wistar rat studies. Necrosis was frequently accompanied by a statistically significant rise in serum creatinine and serum urea, whereas serum albumin was frequently found to decrease statistically significantly in treatment groups in which necrosis was recorded. In contrast to necrosis, glomerulosclerosis was not associated with statistically significant changes in serum creatinine and urea in any of the 28-day oral gavage Wistar rat treatment groups, but appears to be best reflected by a pattern of statistically significantly lowered serum albumin and serum protein together with a statistically significant increase in serum cholesterol. As might have been expected based on the high background incidences of tubular basophilia and dilation, no consistent changes in any of the clinical chemistry parameters were evident in animals in which renal lesions were con� fined to renal tubular basophilia or dilation. In summary, the routinely provided clinical chemistry parameters are rather insensitive - novel kidney biomarkers such as Cystatin C, β-trace protein and Kidney injury molecule 1 should further be evaluated and integrated into routine preclinical and clinical practice. However, evaluation of clinical chemistry data was limited by the lack of individual animal data. Even though an extensive amount of preclinical studies is accessible through the eTOX database, comparison of consistency across time was limited by the limited number of shorter- and longer term studies conducted with the compounds identified as causing renal histopathological changes within a 28- day study in rats. A high consistency across time for both treatment-related tubular basophilia and treatment-related dilation cannot be confirmed for either of the two effects as these two findings were both induced only rarely in studies over a different treatment-duration other than 28 days after administration of the compounds which provoked the respective effect in a 28-day study. For the finding of necrosis consistency across time was low with the exception of "AZ_GGA_200002321", in which renal papillary necrosis was identified consist� ently throughout different treatment durations (2, 4, 26, 104 weeks). No shorter and longer-term studies were available for the compounds identified as causing glomerulosclerosis within a 28-day study in rats. No consistent findings of the selected histopathological endpoints were identified in any of the corresponding 28-day oral gavage beagle dog studies after treatment with the identical compounds, which caused the respective ef� fect after 28-day treatment in rats. However, in the overwhelming majority of cases, beagle dogs were administered lower doses in these studies in compar� ison to the corresponding 28-day Wistar rat studies. Searching the eTOX database yielded no 28-day oral gavage studies in Wistar and Wistar Han rats in which accumulation of hyaline droplets, tubular atrophy or hyperplasia was recorded. Only one 28-day oral gavage Wistar rat study was identified with the histopathological result of neutrophilic inflammation. Consequently, evaluation of these four renal findings in relation to clinical chemistry parameters and consistency across time and species cannot be made. In summary, this work contributes knowledge through mining and evaluating the eTOX database on a variety of specific renal endpoints that frequently occur after administration of trial substances in 28-day oral gavage studies in Wistar rats in the field of preclinical toxicity with specific focus on their frequency relation to background findings, as well as consistency across time and species. Targeted statistical evaluation of in vivo data within joint research ventures such as the eTOX project, presents an enormous opportunity for an innovative future way of aiding preclinical research towards a more efficient research in the preclinical stage of drug development. This could be achieved through the aug� mentation of methodological strategies and possibly novel software tools in order to predict in vivo toxicology of new molecular entities by means of information that is already available before early stages of the drug development pipeline begin.}, language = {en} } @phdthesis{Bieber2010, author = {Bieber, Daniela}, title = {Der A2B-Adenosinrezeptor und MAP-Kinase Aktivit{\"a}t in MDA-MB-231 Brustkrebszellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65707}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Sowohl MAPK als auch Adenosin werden mit Tumorproliferation und Angiogenese in Verbindung gebracht. MDA-MB-231 {\"O}strogenrezeptor-negative Brustkrebszellen zeigen eine sehr starke Expression des A2BAR, der außerdem der einzige von dieser Zelllinie exprimierte Adenosinrezeptor ist. Es konnte gezeigt werden, dass MDA-MB-231-Brustkrebszellen eine hohe basale MAPK-Aktivit{\"a}t aufweisen, welche durch Stimulation mit FCS nicht weiter gesteigert werden kann. Diese hohe basale MAPK-Aktivit{\"a}t wird durch die src-Kinase und Her2 verursacht, da eine Inhibition dieser beiden Tyrosinkinasen eine Hemmung der basalen ERK-Phosphorylierung induziert. Interessanterweise f{\"u}hrt die Stimulation des A2BAR der MDA-MB-231-Brustkrebszellen mit dem unselektiven Agonisten NECA zu einer zeitanh{\"a}ngigen Inhibition der ERK-1/2-Phosphorylierung. Eine Behandlung der Brustkrebszelllinie mit 10 µM CGS 21680 zeigten keinen Einfluss auf die ERK-Aktivit{\"a}t, weshalb davon ausgegangen werden kann, dass die zeitabh{\"a}ngige Inhibition der ERK-1/2-Phosphorylierung durch den A2BAR vermittelt wird. Eine Beteiligung von cAMP an der MAPK-Signaltransduktion des A2BAR scheint insofern wahrscheinlich, als sowohl eine Behandlung der Zellen mit Forskolin als auch der Kombination aus cAMP-AM und dem PDE4-Inhibitor Rolipram eine zeitabh{\"a}ngige Hemmung der ERK-1/2-Phosphorylierung induzieren. Jedoch scheint weder die PKA noch die PI3K an dieser Signaltransduktion des A2BAR beteiligt zu sein, da die A2BAR-vermittelte Inhibition der MAPK auch in Anwesenheit von PKA- und PI3K-Inhibitoren bestehen bleibt. Auch scheinen cAMP-GEFs wie beispielsweise Epac in diesem Zusammenhang keine Rolle zu spielen. In Gegenwart des PLC-Inhibitors U-73122 und des Ca2+-Chelators BAPTA verschwand die NECA-induzierte Hemmung der ERK-1/2-Phosphorylierung, was f{\"u}r eine Beteiligung der PLC und des Ca2+ an der A2BAR-vermittelten Hemmung der MAPK-Aktivit{\"a}t spricht. Letzten Endes konnte jedoch kein Mechanismus eruiert werden, welcher diese A2BAR-vermittelte, Ca2+-abh{\"a}ngige MAPK-Hemmung mediiert, da weder eine Inhibition der PKC, der CamKII oder des Calcineurins Einfluss auf die NECA-induzierte MAPK-Hemmung hatten. Was Wachstum und Proliferation der {\"O}strogenrezeptor-negativen Brustkrebszelllinie MDA-MB-231 anbelangt, so konnte gezeigt werden, dass der unselektive Agonist NECA zu einer signifikanten Wachstumshemmung dieser Brustkrebszelllinie f{\"u}hrt. Allerdings kommt es aufgrund einer Desensitisierung der A2BAR in MDA-MB-231-Brustkrebszellen lediglich zu einem transienten proliferationshemmenden Effekt nach Stimulation mit NECA.}, subject = {Adenosinrezeptor}, language = {de} } @article{BittnerBoonDelbancoetal.2022, author = {Bittner, Nataly and Boon, Andy and Delbanco, Evert H. and Walter, Christof and Mally, Angela}, title = {Assessment of aromatic amides in printed food contact materials: analysis of potential cleavage to primary aromatic amines during simulated passage through the gastrointestinal tract}, series = {Archives of Toxicology}, volume = {96}, journal = {Archives of Toxicology}, number = {5}, doi = {10.1007/s00204-022-03254-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-324697}, pages = {1423-1435}, year = {2022}, abstract = {Recent analyses conducted by German official food control reported detection of the aromatic amides N-(2,4-dimethylphenyl)acetamide (NDPA), N-acetoacetyl-m-xylidine (NAAX) and 3-hydroxy-2-naphthanilide (Naphthol AS) in cold water extracts from certain food contact materials made from paper or cardboard, including paper straws, paper napkins, and cupcake liners. Because aromatic amides may be cleaved to potentially genotoxic primary amines upon oral intake, these findings raise concern that transfer of NDPA, NAAX and Naphthol AS from food contact materials into food may present a risk to human health. The aim of the present work was to assess the stability of NDPA, NAAX and Naphthol AS and potential cleavage to 2,4-dimethylaniline (2,4-DMA) and aniline during simulated passage through the gastrointestinal tract using static in vitro digestion models. Using the digestion model established by the National Institute for Public Health and the Environment (RIVM, Bilthoven, NL) and a protocol recommended by the European Food Safety Authority, potential hydrolysis of the aromatic amides to the respective aromatic amines was assessed by LC-MS/MS following incubation of the aromatic amides with digestive fluid simulants. Time-dependent hydrolysis of NDPA and NAAX resulting in formation of the primary aromatic amine 2,4-DMA was consistently observed in both models. The highest rate of cleavage of NDPA and NAAX was recorded following 4 h incubation with 0.07 M HCl as gastric-juice simulant, and amounted to 0.21\% and 0.053\%, respectively. Incubation of Naphthol AS with digestive fluid simulants did not give rise to an increase in the concentration of aniline above the background that resulted from the presence of aniline as an impurity of the test compound. Considering the lack of evidence for aniline formation from Naphthol AS and the extremely low rate of hydrolysis of the amide bonds of NDPA and NAAX during simulated passage through the gastrointestinal tract that gives rise to only very minor amounts of the potentially mutagenic and/or carcinogenic aromatic amine 2,4-DMA, risk assessment based on assumption of 100\% cleavage to the primary aromatic amines would appear to overestimate health risks related to the presence of aromatic amides in food contact materials.}, language = {en} } @phdthesis{Blankenburg2010, author = {Blankenburg, Robert}, title = {Longitudinale Untersuchungen der kardialen Morphologie von knockin-M{\"a}usen mit humanen Myosinmutationen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71417}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Longitudinale Untersuchungen der kardialen Morphologie von knockin-M{\"a}usen mit humanen Myosinmutationen}, subject = {Kardiomyopathie}, language = {de} } @article{BoivinBeyersdorfPalmetal.2015, author = {Boivin, Val{\´e}rie and Beyersdorf, Niklas and Palm, Dieter and Nikolaev, Viacheslav O. and Schlipp, Angela and M{\"u}ller, Justus and Schmidt, Doris and Kocoski, Vladimir and Kerkau, Thomas and H{\"u}nig, Thomas and Ertl, Georg and Lohse, Martin J. and Jahns, Roland}, title = {Novel Receptor-Derived Cyclopeptides to Treat Heart Failure Caused by \(Anti-β_1-Adrenoceptor\) Antibodies in a Human-Analogous Rat Model}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {2}, doi = {10.1371/journal.pone.0117589}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126028}, pages = {e0117589}, year = {2015}, abstract = {Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the \(β_1\) adrenergic receptor \((β_1EC2)\). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195-225) every 4 weeks; n = 38/114 rats were control-injected with 0.9\% NaCl. Intravenous application of a novel cyclic peptide mimicking \(β_1EC2\) (\(β_1EC2-CP\), 1.0 mg/kg every 4 weeks) or administration of the \(β_1-blocker\) bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received \(β_1EC2-CP/bisoprolol\) co-treatment. We found that \(β_1EC2-CP\) prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, \(β_1EC2-CP\) mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free \(anti-β_1EC2-antibodies\) and by targeting \(β_1EC2\)-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful \(anti-β_1EC2-antibodies\) and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to \(β_1\)-blockade represents a promising new therapeutic option in immune-mediated heart failure.}, language = {en} } @article{BommakantiBokochTolleyetal.1992, author = {Bommakanti, R. and Bokoch, G. M. and Tolley, J. O. and Schreiber, R. E. and Siemsen, D. W. and Klotz, Karl-Norbert and Jesaitis, A. J.}, title = {Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein: Inhibition by pertussis toxin-catalyzed ADP ribosylation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60406}, year = {1992}, abstract = {Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent Sedimentation coefficients of approximately 4 and 7 S. Tbe 7 S form can be converted to the 4 S form by guanosine 5' -0- (3-thiotriphosphate) (GTP-yS) with an EC\&o of -20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. 0., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP-yS-treated neutrophil plasma membranes, was incubated with purified (>95\%) G. protein from bovine brain (containing both G\(_{ia1}\) and G\(_{ia2}\)) or with neutrophil G protein (G\(_a\)), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC\(_{50}\) of 7 S complex formation induced by the two G proteins was 70 \(\pm\) 25 and 170 \(\pm\) 40 DM for G\(_a\) and G\(_1\), respectively. No complexation was measurable when bovine transducin (G\(_t\)) was used up to 30 times the EC\(_{50\) for G\(_a\). The EC\(_{50}\) for G\(_t\) was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 \(\mu\)M GTP-yS to the reconstituted 7 S complex caused a complete reversion of the receptor to the 4 S form, and anti-G\(_1\) peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gt prevented formation of the 7 S form even at 20 times the concentration of unribosylated G. normally used to attain 50\% conversion to the 7 S form. These observations suggest that the 7 S species is a pbysical complex containing N-formyl chemotactic peptide receptor and G protein.}, subject = {Toxikologie}, language = {en} } @article{BommakantiKlotzDratzetal.1993, author = {Bommakanti, R. K. and Klotz, Karl-Norbert and Dratz, E. A. and Jesaitis, A. J.}, title = {A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60456}, year = {1993}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @phdthesis{Bossle2002, author = {Bossle, Franz}, title = {Zur Pharmakologie von meta-Iodbenzylguanidin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-3717}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2002}, abstract = {Obwohl radioaktiv markiertes meta-Iodbenzylguanidin (MIBG) h{\"a}ufig in der Diagnose und bei der Behandlung von Neuroblastomen in der Klink Verwendung findet, ist bis heute sehr wenig {\"u}ber seine Pharmakologie bekannt. In der Literatur finden sich {\"o}fters Andeutungen, die aber nicht belegt wurden. So fehlten Untersuchungen {\"u}ber indirekt-sympathomimetische Wirkungen von MIBG. Vor diesem Hintergrund untersuchten wir am isoliert perfundierten Kaninchenherzen die Wirkung von MIBG im Vergleich zum Prototyp eines indirekten Sympathomimetikums (Tyramin). Dabei zeigte sich, daß MIBG zwar etwas potenter aber nicht so effektiv wie Tyramin war. Dies zeigte sich sowohl beim Paramter Herzfrequenz als auch beim Parameter Noradrenalin-Freisetzung. Im Gegensatz dazu zeigte sich im Zeitverlauf, daß die Wirkung von MIBG wesentlich l{\"a}nger anhielt als die von Tyramin. Der Unterschied zwischen MIBG und Tyramin bez{\"u}glich der Effektivit{\"a}t als indirekte Sympathomimetika konnte mit unterschiedlichen Wirkst{\"a}rken beider Substanzen als Hemmstoff des vesikul{\"a}ren Monoamin-Transporters erkl{\"a}rt werden. Tyramin und MIBG wurden in Versuchen mit Neuroblastomzellen mit gleicher Geschwindigkeit durch Uptake1 aufgenommen, Tyramin war aber ein wesentlich potenterer Hemmstoff des vesikul{\"a}ren Monoamin-Transporters als MIBG. Da aber MIBG im Gegensatz zu Tyramin kein Substrat der neuronalen Monoaminoxidase ist, hielt seine Wirkung auch deutlich l{\"a}nger an als die von Tyramin. Die indirekt sympathomimetische Wirkung von MIBG wurde anschließend auch in-vivo untersucht. Dort zeigte sich auch, daß MIBG trotz im Vergleich zu klinischen Anwendungen hoher Dosen wesentlich schw{\"a}cher indirekt-sympathomimetisch wirkt als Tyramin. In diesen Versuchen wurde auch beobachtet, daß die indirekt-sympathomimetische Wirkung auf die Herzfrequenz durch eine Gegenregulation des Nervensystems (n{\"a}mlich den Barorezeptor-Reflex) maskiert wurde. Obwohl MIBG in der Literatur von Anfang an als adrenerger Neuronenblocker bezeichnet wurde, fand sich in der Literatur kein direkter Beweis f{\"u}r diese Behauptung. Mit Hilfe eines in-vitro Modells konnte in der vorliegenden Arbeit der Beweis erbracht werden, daß MIBG ein adrenerger Neuronenblocker ist. Dazu benutzten wir als Parameter die durch elektrische Stimulation induzierte Freisetzung von Noradrenalin im spontan schlagenden, perfundierten Kaninchenherzen. Die stimulationsbedingte Abgabe von Noradrenalin ins Perfusat wurde durch MIBG zeit- und konzentrationsabh{\"a}ngig blockiert. Da viele adrenerge Neuronenblocker das Enzym Monoaminoxidase (MAO) hemmen, wurde in-vitro untersucht, ob MIBG die beiden Iso-Enzyme MAO-A und MAO-B hemmt. Es konnte gezeigt werden, daß MIBG die MAO kompetitiv hemmt und zwar bevorzugt die Isoform MAO-A. Diese MAO-Hemmung wurde auch in-vivo in den Versuchen mit narkotisierten Kaninchen beobachtet. MIBG verminderte n{\"a}mlich dosisabh{\"a}ngig die Konzentration des desaminierten Noradrenalin-Metaboliten DOPEG im Blutplasma der Tiere. Die Beobachtung, daß f{\"u}r die Hemmung der MAO-A im perfundierten Herzen eine IC50 von 17 nM, im Gewebehomogenat von Herzen dagegen eine IC50 von 18 µM gefunden wurde, spricht daf{\"u}r, daß MIBG als Substrat von Uptake1 im Axoplasma der sympathischen Neurone des Herzens um den Faktor 1000 angereichert wird. Somit konnten in der vorliegenden Arbeit einige offene Fragen zur Pharmakologie von MIBG im Bereich des sympathomimetischen Nervensystems beantwortet werden, die auch f{\"u}r den klinischen Einsatz von MIBG wichtig sein k{\"o}nnten.}, subject = {MIBG}, language = {de} } @phdthesis{Boullay2002, author = {Boullay, Felix}, title = {Quantifizierung von DNA-Sch{\"a}den peripherer Lymphozyten bei Patienten mit chronischer Niereninsuffizienz}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6722}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2002}, abstract = {Schon vor mehr als zwei Jahrzehnten wurde eine erh{\"o}hte Tumorentstehungsrate bei Patienten mit chronischer Nierenerkrankung unter Dialysebehandlung festgestellt. Eine der wahrscheinlichsten Erkl{\"a}rungen f{\"u}r dieses Ph{\"a}nomen ist die klinische Manifestation eines Immundefektes innerhalb dieses Patientenkollektives. Lymphozyten von Patienten mit chronischen Nierenerkrankungen ohne Dialyse und Dialysepatienten mit einer Behandlungsdauer von mehr als 120 Monaten verf{\"u}gen nachweislich {\"u}ber eine reduzierte DNA-Reparaturf{\"a}higkeit. Zus{\"a}tzlich weisen sie eine erh{\"o}hte Rate von Mikrokernen auf, was f{\"u}r verst{\"a}rkte gentoxische Einfl{\"u}sse im Patientenblut spricht. In dieser Arbeit wurde mittels Comet Assay, einem sensiblen Testverfahren zur Quantifizierung von DNA-Sch{\"a}den auf Einzellzellniveau, aus verschiedenen Gruppen von chronisch Nierenkranken die Zellkern-DNA von peripheren Lymphozyten auf Sch{\"a}den untersucht. Neben Patienten mit leicht bis stark erh{\"o}hten Kreatininspiegeln wurden auch Kollektive mit H{\"a}modialyse und H{\"a}modiafiltrationsbehandlung auf DNA-Sch{\"a}den untersucht und miteinander verglichen. In den Untersuchungen konnte gezeigt werden, dass in der Gruppe der chronisch Nierenkranken ohne Dialysebehandlung offensichtlich ein Zusammenhang zwischen H{\"o}he des Kreatininspiegels und einer durch den Comet Assay feststellbaren DNA-Sch{\"a}digung besteht: im Kollektiv der H{\"a}modialysepatienten ist mit der Dauer der Behandlung ein Anstieg des Schadens zu verzeichnen. Bei Patienten mit H{\"a}modiafiltrationsbehandlung hingegen war kein Anstieg der DNA-Sch{\"a}den mit der L{\"a}nge der Behandlung feststellbar. Bei gleicher Behandlungsdauer bestehen zwischen H{\"a}modialyse- und H{\"a}modiafiltrationsgruppe nur unwesentliche Schadensdifferenzen. Dies war nicht vorhersehbar, da besonders Patienten mit st{\"a}rkeren gesundheitlichen Einschr{\"a}nkungen in den Vorzug der H{\"a}modiafiltration gelangen. Insgesamt zeigten jedoch alle untersuchten Gruppen einen signifikanten Anstieg der DNA-Sch{\"a}digung gegen{\"u}ber den Kontrollen. Da der Comet Assay derzeit noch mit methodischen und patientenbedingten Ergebniss-Schwankungen behaftet ist, muss jede Interpretation mit Zur{\"u}ckhaltung erfolgen. Insbesondere muss anhand eines Zusammenhanges hinsichtlich Gentoxizit{\"a}t und vorliegender Erkrankung untersucht und kritisch hinterfragt werden, ob ein fr{\"u}herer Beginn der Dialyse-Behandlung f{\"u}r den Patienten von Vorteil sein k{\"o}nnte. Inwieweit eine Umstellung von H{\"a}modialyse auf H{\"a}modiafiltration die Sch{\"a}den der lymphozyt{\"a}ren Zellkern DNA und somit eventuell auch die Tumorentstehungsraten beeinflusst, ist durch weitere Forschungen auf diesem Gebiet zu kl{\"a}ren.}, language = {de} } @phdthesis{Brand2012, author = {Brand, Susanne}, title = {Oxidativer Stress und DNA-Sch{\"a}den induziert durch das Peptidhormon Angiotensin II in vivo : Identifizierung des AT1-Rezeptors und reaktiver Sauerstoffspezies als urs{\"a}chliche Faktoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77573}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Das Renin-Angiotensin-Aldosteron-System (RAAS) reguliert den Blutdruck und den Wasser- und Elektrolythaushalt des K{\"o}rpers. Angiotensin II (Ang II), das aktive Peptid des RAAS, bewirkt eine Vasokonstriktion und in h{\"o}heren Konzentrationen Bluthochdruck. Epidemiologische Studien haben gezeigt, dass eine Verbindung zwischen Hypertonie und dem geh{\"a}uften Auftreten von Krebs besteht. Eine Metaanalyse von 13 Fall-Kontroll-Studien konnte einen Zusammenhang zwischen Hypertonie und einem erh{\"o}hten Risiko, an einem Nierenzellkarzinom zu erkranken nachweisen. In vitro-Studien und Studien an der isolierten Niere konnten bereits genotoxische Effekte des blutdruckregulierenden Hormons Ang II zeigen. Zielsetzung dieser Arbeit war es, zun{\"a}chst in vivo zu pr{\"u}fen, ob steigende Ang II-Konzentrationen einen Einfluss auf die genomische Stabilit{\"a}t von Nieren- und Herzzellen besitzen. Hierzu wurden im Dosisversuch m{\"a}nnliche C57BL/6-M{\"a}use mit osmotischen Minipumpen ausgestattet, die Ang II in vier verschiedenen Konzentrationen zwischen 60 ng/kg min und 1 µg/kg min {\"u}ber einen Zeitraum von 28 Tagen abgeben sollten. W{\"a}hrend des Versuchszeitraums fanden regelm{\"a}ßige, nicht-invasive Blutdruckmessungen an der Maus statt. Die Behandlung mit Ang II f{\"u}hrte zu einem signifikanten Anstieg des Blutdrucks und zu histopathologischen Ver{\"a}nderungen der Glomeruli und des Tubulussystems, was sich in einer verschlechterten Albumin-Ausscheidung wiederspiegelte. Außerdem induzierte die Behandlung mit Ang II die dosisabh{\"a}ngige Bildung von reaktiven Sauerstoffspezies, DNA-Doppelstrangbr{\"u}chen und oxidativer DNA-Sch{\"a}den. Diese Parameter waren bereits in Tieren erh{\"o}ht, die keinen Bluthochdruck entwickelten und stiegen mit der h{\"o}chsten Ang II-Konzentration noch an, obwohl hier im Vergleich zur Vorg{\"a}ngergruppe, die eine geringere Ang II-Konzentration erhielt, kein h{\"o}herer Blutdruck vorlag. Diese Beobachtung deutet auf eine m{\"o}gliche Unabh{\"a}ngigkeit des entstandenen Schadens vom Bluthochdruck hin und lenkt die Aufmerksamkeit auf Ang II als genomsch{\"a}digenden Faktor. Der folgende Interventionsversuch sollte Aufschluss {\"u}ber die m{\"o}gliche blutdruckunabh{\"a}ngige genomsch{\"a}digende Wirkung von Ang II geben. Dazu wurden C57BL/6-M{\"a}use neben der Ang II-Behandlung in einer Konzentration von 600 ng/kg min zus{\"a}tzlich {\"u}ber einen Zeitraum von 28 Tagen mit 5 verschiedenen Substanzen behandelt: Candesartan, Ramipril, Hydralazin, Eplerenon und Tempol. Candesartan ist ein Ang II-Rezeptor-Antagonist, der selektiv den AT1-Rezeptor blockiert. Ramipril wirkt als Hemmer des Angiotensin-Konversions-Enzyms und verhindert die Bildung von endogenem Ang II aus Ang I. Hydralazin, als Vasodilatator, greift nicht in das Renin-Angiotensin-Aldosteron-System ein. Eplerenon blockiert als selektiver Aldosteronantagonist den Mineralkortikoidrezeptor. Tempol wirkt als Antioxidans. Die Behandlung mit Ang II in einer Konzentration von 600 ng/kg min im Interventionsversuch f{\"u}hrte zur Hochregulierung der NADPH-Oxidase 4 und zur Produktion reaktiver Sauerstoffspezies in der Niere und im kardiovaskul{\"a}ren Gewebe. Der entstandene oxidative Stress f{\"u}hrte wiederum zu DNA-Sch{\"a}den und einer Aktivierung der Transkriptionsfaktoren Nrf2 und NF-B. Nrf2-vermittelt wurde die Induktion antioxidativer Gene ausgel{\"o}st, was allerdings nicht ausreichend war, um vor Ang II-induzierten ROS und DNA-Sch{\"a}den zu sch{\"u}tzen. Eine l{\"a}ngerfristige NF-B-Aktivierung durch hohe Ang II-Spiegel kann das {\"U}berleben und die Proliferation von Zellen, die DNA-Sch{\"a}den in Form von Doppelstrangbr{\"u}chen tragen, f{\"o}rdern, was eine Tumor-initiierende Wirkung haben k{\"o}nnte. Die beschriebenen Effekte erh{\"o}hter Ang II-Spiegel konnten durch die Intervention mit dem AT1-Rezeptorblocker Candesartan verhindert werden, was die Beteiligung des Rezeptors nachweist. Eine blutdruckunabh{\"a}ngige, genomsch{\"a}digende Wirkung von Ang II konnte leider durch die Intervention mit Hydralazin nicht verdeutlicht werden, da die erw{\"u}nschte langfristige Blutdrucksenkung ausblieb. Allerdings zeigte die Intervention mit Tempol eine Abnahme an oxidativem Stress und DNA-Sch{\"a}den trotz ausbleibender Blutdrucksenkung. Die Bedeutung von ROS in der Bildung von DNA-Sch{\"a}den und die Unabh{\"a}ngigkeit dieser Sch{\"a}den vom Blutdruck konnten somit hervorgehoben werden. Die Tatsache, dass die Intervention mit Ramipril den Blutdruck nicht senken konnte, der oxidative Stress und die DNA-Sch{\"a}den durch m{\"o}gliche antioxidative Eigenschaften aber vermindert wurden, unterst{\"u}tzt diese Beobachtung. Die Intervention mit Eplerenon f{\"u}hrte zum Teil zu einer Verminderung an ROS und DNA-Sch{\"a}den, brachte diese Parameter aber nicht auf Kontrollniveau zur{\"u}ck. Somit ist eine Beteiligung von Aldosteron nicht auszuschließen.}, subject = {Oxidativer Stress}, language = {de} } @article{BrandAmannMandeletal.2014, author = {Brand, Susanne and Amann, Kerstin and Mandel, Philipp and Zimnol, Anna and Schupp, Nicole}, title = {Oxidative DNA Damage in Kidneys and Heart of Hypertensive Mice Is Prevented by Blocking Angiotensin II and Aldosterone Receptors}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {12}, issn = {1932-6203}, doi = {10.1371/journal.pone.0115715}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118011}, pages = {e115715}, year = {2014}, abstract = {INTRODUCTION: Recently, we could show that angiotensin II, the reactive peptide of the blood pressure-regulating renin-angiotensin-aldosterone-system, causes the formation of reactive oxygen species and DNA damage in kidneys and hearts of hypertensive mice. To further investigate on the one hand the mechanism of DNA damage caused by angiotensin II, and on the other hand possible intervention strategies against end-organ damage, the effects of substances interfering with the renin-angiotensin-aldosterone-system on angiotensin II-induced genomic damage were studied. METHODS: In C57BL/6-mice, hypertension was induced by infusion of 600 ng/kg • min angiotensin II. The animals were additionally treated with the angiotensin II type 1 receptor blocker candesartan, the mineralocorticoid receptor blocker eplerenone and the antioxidant tempol. DNA damage and the activation of transcription factors were studied by immunohistochemistry and protein expression analysis. RESULTS: Administration of angiotensin II led to a significant increase of blood pressure, decreased only by candesartan. In kidneys and hearts of angiotensin II-treated animals, significant oxidative stress could be detected (1.5-fold over control). The redox-sensitive transcription factors Nrf2 and NF-κB were activated in the kidney by angiotensin II-treatment (4- and 3-fold over control, respectively) and reduced by all interventions. In kidneys and hearts an increase of DNA damage (3- and 2-fold over control, respectively) and of DNA repair (3-fold over control) was found. These effects were ameliorated by all interventions in both organs. Consistently, candesartan and tempol were more effective than eplerenone. CONCLUSION: Angiotensin II-induced DNA damage is caused by angiotensin II type 1 receptor-mediated formation of oxidative stress in vivo. The angiotensin II-mediated physiological increase of aldosterone adds to the DNA-damaging effects. Blocking angiotensin II and mineralocorticoid receptors therefore has beneficial effects on end-organ damage independent of blood pressure normalization.}, language = {en} } @phdthesis{Brede2002, author = {Brede, Anja}, title = {{\"U}ber die Beteiligung EGF-Rezeptor-vermittelter Signaltransduktionswege an den tumorpromovierenden Effekten von 2-Acetylaminofluoren, 2-Nitrosofluoren und Phenobarbital in HepG2-Zellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-4024}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2002}, abstract = {In dieser Arbeit wurden die tumorpromovierenden Effekte von 2-Acetylaminofluoren (2-AAF), 2-Nitrosofluoren (2-NOF) und Phenobarbital (PB) auf die Expression des EGF-Rezeptors (EGF-R), der Proteinkinase C (PKC), der Protoonkogene c-FOS und c-JUN in HepG2 Zellen bestimmt. Nur PB hemmte die Expression des EGF-R, wohingegen die PKC, c-FOS und c-JUN nicht beeinflusst wurden. 2-AAF, 2-NOF und PB wirkten konzentrationsabh{\"a}ngig zytotoxisch und antiproliferativ auf HepG2-Zellen. Die PKC scheint an diesem Effekt beteiligt zu sein. Die Bindungsaktivit{\"a}t der Transkriptionsfaktoren AP1 und NFkappa B wurde durch 2-NOF und Phenobarbital erh{\"o}ht, wohingegen der Effekt von 2-AAF nicht endeutig zu kl{\"a}ren war.}, language = {de} } @phdthesis{Brede2001, author = {Brede, Marc}, title = {Kardiovaskul{\"a}re Ph{\"a}notypisierung von Angiotensin II AT2-Rezeptor- und adrenergen Rezeptor-"Knockout"-M{\"a}usen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-1179726}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2001}, abstract = {Die Deletion des AT2-Rezeptors (AT2-KO) f{\"u}hrt zu erh{\"o}hter Blutdruckempfindlichkeit und vaskul{\"a}rer Hypertrophie durch Aktivit{\"a}tszunahhme der P70S6-Kinase. Die Vasodilatation von Blutgef{\"a}ßen wird maßgeblich durch beta1-adrenerge Rezeptoren vermittelt. Die Deletion von alpha2-adrenergen Rezeptoren (alpha2-KO) f{\"u}hrt zur Entwicklung einer Herzinsuffizienz nach Aortenstenose. Der Mortalit{\"a}tanstieg ist mit erh{\"o}hten Plasmanoradrenalin-Spiegeln (a2A-KO), bzw. Plasmaadrenalin-Spiegeln (a2C-KO) assoziiert.}, language = {de} } @article{BreitenbachLorenzDandekar2019, author = {Breitenbach, Tim and Lorenz, Kristina and Dandekar, Thomas}, title = {How to steer and control ERK and the ERK signaling cascade exemplified by looking at cardiac insufficiency}, series = {International Journal of Molecular Sciences}, volume = {20}, journal = {International Journal of Molecular Sciences}, number = {9}, issn = {1422-0067}, doi = {10.3390/ijms20092179}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285164}, year = {2019}, abstract = {Mathematical optimization framework allows the identification of certain nodes within a signaling network. In this work, we analyzed the complex extracellular-signal-regulated kinase 1 and 2 (ERK1/2) cascade in cardiomyocytes using the framework to find efficient adjustment screws for this cascade that is important for cardiomyocyte survival and maladaptive heart muscle growth. We modeled optimal pharmacological intervention points that are beneficial for the heart, but avoid the occurrence of a maladaptive ERK1/2 modification, the autophosphorylation of ERK at threonine 188 (ERK\(^{Thr188}\) phosphorylation), which causes cardiac hypertrophy. For this purpose, a network of a cardiomyocyte that was fitted to experimental data was equipped with external stimuli that model the pharmacological intervention points. Specifically, two situations were considered. In the first one, the cardiomyocyte was driven to a desired expression level with different treatment strategies. These strategies were quantified with respect to beneficial effects and maleficent side effects and then which one is the best treatment strategy was evaluated. In the second situation, it was shown how to model constitutively activated pathways and how to identify drug targets to obtain a desired activity level that is associated with a healthy state and in contrast to the maleficent expression pattern caused by the constitutively activated pathway. An implementation of the algorithms used for the calculations is also presented in this paper, which simplifies the application of the presented framework for drug targeting, optimal drug combinations and the systematic and automatic search for pharmacological intervention points. The codes were designed such that they can be combined with any mathematical model given by ordinary differential equations.}, language = {en} } @unpublished{BrennerZinkWitzingeretal.2024, author = {Brenner, Marian and Zink, Christoph and Witzinger, Linda and Keller, Angelika and Hadamek, Kerstin and Bothe, Sebastian and Neuenschwander, Martin and Villmann, Carmen and von Kries, Jens Peter and Schindelin, Hermann and Jeanclos, Elisabeth and Gohla, Antje}, title = {7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase}, series = {eLife}, journal = {eLife}, doi = {10.7554/eLife.93094.2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350446}, year = {2024}, abstract = {Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.}, language = {en} } @phdthesis{Brink2007, author = {Brink, Andreas}, title = {The biological significance of chemically-induced DNA adducts in relation to background DNA damage}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-23850}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {No abstract available}, subject = {DNS-Sch{\"a}digung}, language = {en} } @article{BussCaviezelLutz1990, author = {Buss, P. and Caviezel, M. and Lutz, Werner K.}, title = {Linear dose-response relationship for DNA adducts in rat liver from chronic exposure to aflatoxin B1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60779}, year = {1990}, abstract = {Male F-344 rats were given eH]aßatoxin B1 (AFB1) in the drinking water at three exposure Ievels (0.02, 0.6, 20 J,Lgll, resulting in average dose Ievels of 2.2, 73, 2110 nglkg per day). After 4, 6 and 8 weeks, DNA was ~ted frorn the livers and analyzed for aßatoxin-DNA adducts. Tbe Ievel of DNA adducts did not increase significantly after 4 weeks, indicating that a steady-state for adduct formation and removal had nearly been reached. At 8 weeks, the adduct Ievels were 0.91, 32 and 850 nucleotide-aßatoxin adducts per to' nucleotides, i.e. clearly proportional to the dose. At the high dose Ievel, a near SO\% tumor incidence would be expected in a 2-year bioassay with F -344 rats while the low dose used is within the range of estlmated human dietary exposures to aßatoxin in W estem countries. The proportionality seen between exposure and steady-state DNA adduct Ievel is discussed with respect to a linear extrapolation of the tumor risk to low dose.}, subject = {Toxikologie}, language = {en} } @phdthesis{Baetz2012, author = {B{\"a}tz, Julia}, title = {FRET-basierte Untersuchungen zur ligandenselektiven Beeinflussung der Rezeptorkonformation durch orthosterische und allosterische Liganden am Beispiel des muskarinischen M2 Acetylcholinrezeptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72836}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Zahlreiche experimentelle Befunde lassen vermuten, dass G-Protein gekoppelte Rezeptoren (GPCR) nach ihrer Aktivierung einer ligandenselektiven {\"A}nderung der Rezeptorkonformation unterliegen. Ziel der vorliegenden Arbeit war es dieses Ph{\"a}nomen am Subtyp 2 der muskarinischen Acetylcholinrezeptoren (M2 AChR) zu untersuchen. Muskarinische Acetylcholinrezeptoren (mAChR) k{\"o}nnen in f{\"u}nf Subtypen (M1-M5) unterschieden werden. Durch die Beteiligung der mAChR an zahlreichen physiologischen Prozessen stellen sie wichtige Zielstrukturen pharmakologischer Therapien dar. Da die orthosterische Ligandenbindestelle (= Bindestelle des endogenen Liganden) in allen f{\"u}nf Subtypen hoch konserviert ist, wird ihr pharmakologischer Nutzen derzeit allerdings durch die unselektive Rezeptormodulation und dem damit verbundenen Auftreten unerw{\"u}nschter Arzneimittelwirkungen stark limitiert. Ein Ansatz zur Erzielung subtypselektiver Effekte besteht in der Verwendung allosterischer Modulatoren. Da die allosterische Bindestelle der mAChR eine geringere Sequenzhomologie aufweist, k{\"o}nnen so gezielt einzelne Subtypen der mAChR reguliert werden. Der M2 AChR stellt hinsichtlich allosterischer Modulation ein gut charakterisiertes Modellsystem dar. F{\"u}r ihn wurde bereits eine Vielzahl allosterischer Liganden entwickelt. Auch bitopische Liganden, die sowohl einen allosterischen als auch einen orthosterischen Anteil enthalten, wurden f{\"u}r den M2 AChR bereits beschrieben. Im ersten Teil der vorliegenden Arbeit wurden verschiedene FRET-Sensoren des M2 AChR generiert und charakterisiert. Als FRET-Paar wurden das cyan fluoreszierende Protein (CFP) und der niedermolekulare fluorescein-basierte Fluorophor FlAsH (fluorescein arsenical hairpin binder) gew{\"a}hlt. CFP wurde in den Sensoren am Ende des C-Terminus angef{\"u}gt. Die zur Markierung mit FlAsH n{\"o}tige Tetracysteinsequenz wurde in verschiedenen Bereichen der dritten intrazellul{\"a}ren Rezeptorschleife (IL) eingebracht. Die auf diese Weise erstellten Re-zeptorsensoren trugen das Tetracysteinmotiv in der N terminalen (M2i3-N) bzw. in der C terminalen Region (M2i3-C) von IL 3. Die Charakterisierung der Rezeptorsensoren bez{\"u}glich Ligandenbindung, Gi-Protein Aktivierung und β-Arrestin2 Translokation ergab keine signifikanten Unterschiede zwischen M2i3-N, M2i3 C und M2CFP oder Wildtyp M2 AChR. Zun{\"a}chst wurden sowohl unterschiedliche orthosterische, als auch allosterische Liganden hinsichtlich ihrer mittleren effektiven Konzentration und ihrer maximalen Wirkst{\"a}rke an den Rezeptorsensoren untersucht. Mit Hilfe von FRET-Messungen konnte ein superago-nistisches Verhalten des orthosterischen Testliganden Iperoxo gezeigt werden. Die Eigenschaften der allosterischen Substanzen wurden durch Messung der Rezeptordeakti-vierungskinetik und des maximalen Hemmeffekts auf einen vorstimulierten Rezeptor charakterisiert. Alle allosterischen Liganden deaktivierten den vorstimulierten M2 AChR mit einer schnelleren Kinetik als Atropin. Die EC50-Werte der unterschiedlichen Substanzen waren unabh{\"a}ngig von der Markierungsposition im verwendeten Rezeptorsensor vergleich-bar. Ausnahmen bildeten die allosterischen Liganden JK 289, JK 338, ½ W84 und EHW 477, die liganden- und sensorabh{\"a}ngig unterschiedliche mittlere effektive Konzentrationen aufwie-sen. Bei der Untersuchung der Konformations{\"a}nderung des M2 AChR konnte kein liganden-selektiver Unterschied zwischen den FRET-Signalen f{\"u}r 19 getestete orthosterische Liganden beobachtet werden. Dies deutet darauf hin, dass alle orthosterischen Testliganden eine dem Acetylcholin (ACh) vergleichbare {\"A}nderung der M2 AChR Konformation induzier-ten. Um zu untersuchen, ob f{\"u}r die orthosterischen Testliganden eine Korrelation zwischen ihrer maximalen Wirkst{\"a}rke hinsichtlich Rezeptoraktivierung in FRET-Experimenten und der Aktivierung nachgeschalteter Signalwege besteht, wurde die orthosterisch-vermittelte Translokation von β-Arrestin2 mit Hilfe der Konfokalmikroskopie bestimmt. Bis auf 5-Methyl-furmethiodid translozierten alle orthosterischen Liganden β-Arrestin2 in einem Ausmaß, das mit der maximalen Rezeptoraktivierung vergleichbar war. Dagegen rief 5 Methylfurmethiodid verglichen mit dem endogenen Liganden ACh zwar eine ca. halbmaximale Rezeptorakti-vierung, aber nur eine {\"a}ußerst geringe β-Arrestin2 Translokation hervor. Im zweiten Teil der Arbeit wurde der Einfluss verschiedener Allostere auf eine ligandenselektive Konformations{\"a}nderung des M2 AChR untersucht. Die allosterischen Liganden JK 337 und Seminaph beeinflussten den M2i3-C Sensor signifikant st{\"a}rker, als das M2i3-N Konstrukt. Dagegen zeigte EHW 477 eine st{\"a}rkere Beeinflussung der Rezeptorkon-formation des M2i3-N-, als des M2i3-C Sensors. Dies erlaubt die Vermutung, dass JK 337 und Seminaph eine st{\"a}rkere Bewegung unterhalb von Transmembrandom{\"a}ne (TM) 6, als unterhalb von TM 5 hervorriefen. Die Ergebnisse f{\"u}r EHW 477 legen nahe, dass TM 5 eine gr{\"o}ßere Bewegung eingeht, als TM 6. FRET-basierte Untersuchungen der Einfl{\"u}sse der allosterischen Testliganden auf nachgeschaltete Signalwege ergaben, dass sowohl die Akti-vierung des Gi Proteins, als auch die β-Arrestin2 Translokation selektiv durch einzelne allosterische Liganden beeinflusst werden. Auch ein Zusammenhang zwischen Rezeptor-aktivierung und der Regulation nachgeschalteter Signalwege war erkennbar. Allerdings waren auf Grund der Versuchsbedingungen keine quantitativen Aussagen m{\"o}glich. Im Folgenden wurden die bitopischen Liganden Hybrid 1 und 2 (H 1, H 2) hinsichtlich ihres Effekts auf die Konformations{\"a}nderung des M2 AChR untersucht. W{\"a}hrend eine Stimulation mit H 1 vergleichbare FRET-Signale an beiden Sensoren ergab, konnte mit H 2 weder am M2i3-N-, noch an M2i3-C Sensor eine FRET-{\"A}nderung detektiert werden. Um den mangeln-den Effekt der Hybridsubstanzen in FRET-mikroskopischen Untersuchungen aufzukl{\"a}ren, wurden verschiedene Ans{\"a}tze gew{\"a}hlt: Mit kettenverl{\"a}ngerten Derivaten der Hybridsubstanzen konnte in FRET-Messungen eine {\"A}nderung des FRET-Signals detektiert werden. Die Entfernung des allosterischen Bausteins f{\"u}hrte in FRET-Experimenten zu einer verglichen mit dem endogenen Liganden ACh etwa halbmaximalen Aktivierung beider Sensoren. Dagegen resultierte die Mutation der alloste-rischen Bindestelle in nachfolgenden FRET-Untersuchungen mit H 1 und H 2 in keiner Signal{\"a}nderung des FRET-Ratio. Diese Beobachtungen f{\"u}hrten zu der Annahme, dass die Linkerkette, die orthosterischen und allosterischen Baustein der Hybride miteinander verbindet, zu kurz war um eine gleichzeitige Bindung an die allosterische und orthosterische Bindestelle zu erm{\"o}glichen. Ein anderer Erkl{\"a}rungsansatz besteht darin, dass nach Bindung des Orthosters der Kanal zwischen orthosterischer und allosterischer Bindestelle durch die Konformations{\"a}nderung des Rezeptors verschlossen wird, weshalb keine dauerhafte, dualsterische Bindung der Hybridsubstanzen an den M2 AChR m{\"o}glich ist. Im Rahmen der vorliegenden Arbeit ist es gelungen mittels FRET-Experimenten die Existenz einer ligandenselektiven Rezeptorkonformation des M2 AChR mit allosterischen Liganden nachzuweisen. Dar{\"u}ber hinaus konnte auch ein Bezug zum Auftreten einer funktionellen Selektivit{\"a}t mit allosterischen Liganden hergestellt werden. Die Untersuchung von 19 orthosterischen Liganden hinsichtlich ihres Einflusses auf die Rezeptorkonformation des M2 AChR ergab keinen Hinweis auf eine ligandenselektive Konformations{\"a}nderung. Die Betrachtung der orthosterisch-vermittelten Translokation von β-Arrestin2 zeigte, dass zwischen der Effizienz der orthosterischen Testliganden, den M2 AChR zu aktivieren und dem Ausmaß, in dem sie eine β Arrestin2 Translokation induzierten eine direkte Korrelation besteht. Lediglich 5-Methylfurmethiodid rief eine ungleich geringere β-Arrestin2 Translokation hervor, verglichen mit dem Ausmaß an Rezeptoraktivierung. Diese Beobachtung deutet auf die Existenz eines signaling-bias f{\"u}r diesen Liganden hin. Die Untersuchung der dualsterischen Liganden H 1 und 2 bez{\"u}glich ihrer F{\"a}higkeit zur Rezeptoraktivierung ergab, dass erst durch eine Verl{\"a}ngerung der Linkerkette, durch die orthosterischer und alloste-rischer Baustein miteinander verbunden sind eine Konformations{\"a}nderung des M2 AChR hin zu einer aktiven Konformation erreicht werden kann. Es kann somit angenommen werden, dass in den urspr{\"u}nglichen Hybridsubstanzen H 1 und H 2 eine zu kurze Linkerkette, durch die keine dualsterische Bindung der Hybride an die allosterische und orthosterische Bindestelle m{\"o}glich ist, urs{\"a}chlich f{\"u}r die mangelnde Rezeptoraktivierung des M2 AChR war.}, subject = {Muscarinrezeptor}, language = {de} } @article{BoeschFriederichLutzetal.1987, author = {B{\"o}sch, R. and Friederich, U. and Lutz, Werner K. and Brocker, E. and Bachmann, M. and Schlatter, C.}, title = {Investigations on DNA binding in rat liver and in Salmonella and on mutagenicity in the Ames test by emodin, a natural anthraquinone}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60913}, year = {1987}, abstract = {Emodin (1,6,8-trihydroxy-3-methylanthraquinone), an important aglycone found in natural anthraquinone glycosides frequently used in Iaxative drugs, was mutagenic in the Salmonellajmammalian microsome assay (Ames test) with a specificity for strain TA1537. The mutagenic activity was activationdependent with an optimal amount of S9 from Aroclor 1254-treated male Sprague-Dawley rats of 20\% in the S9 mix (v jv) for 10 p.g emodin per plate. Heat inactivation of the S9 for 30 min at 60 ° C prevented mutagenicity. The addition of the cytochrome P-448 inhibitor 7,8-benzoflavone (18.5 nmoles per plate) reduced the mutagenic activity of 5.0 p.g emodin per plate to about one third, whereas the P-450 inhibitor metyrapone (up to 1850 nmoles per plate) was without effect. To test whether a metabolite" binds covalently to Salmonella DNA, [10-\(^{14}\)C]emodin was radiosynthesized, large batches of bacteria were incubated with [10-\(^{14}\)C]emodin and DNA was isolated. [G- \(^{3}\)H]Aflatoxin B1 (AFB1) was used as a positive control mutagen known to act via DNA binding. DNA obtained after aflatoxin treatment could be purified to constant specific activity. With emodin, the specific activity of DNA did not remain constant after repeated precipitations so that it is unlikely that the mutagenicity of emodin is due to covalent interaction of a metabolite with DNA. The antioxidants vitamin C and E or glutathione did not reduce the mutagenicity. Emodin was also negative with strain TA102. Thus, oxygen radicals are probably not involved. When emodin was incubated with S9 alone for up to 50 h before heat-inactivation of the enzymes and addition of bacteria, the mutagenic activity did not decrease. It is concluded that the mutagenicity of emodin is due to a chemically stable, oxidized metabolite forming physico-chemical associations with DNA, possibly of the intercalative type. In order to check whether an intact mammalian organism might be able to activate emodin to a DNA-binding metabolite, radiolabelled emodin was administered by oral gavage to male SD rats and liver DNA was isolated after 72 h. Very little radioactivity was associated with the DNA. Considering that DNA radioactivity could also be due to sources other than covalent interactions, an upper limit for the · covalent binding index, CBI = (p.moles chemical bound per moles DNA nucleotides)/(mmoles chemical administered per kg body weight) of 0.5 is deduced. This is 104 times below the CBI of AFB1. The demonstration of a lack of covalent interaction with DNA bothin Salmonellaandin rat liver is discussed in terms of a reduced hazard posed by emodin as a mutagenic drug in use in humans.}, subject = {Toxikologie}, language = {en} } @phdthesis{Buecheler2003, author = {B{\"u}cheler, Markus}, title = {Regulation der Neurotransmission im zentralen Nervensystem durch alpha2-adrenerge Rezeptoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-7803}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2003}, abstract = {Die Gruppe der adrenergen Rezeptoren (AR) umfasst neun Rezeptoren (3 alpha1-, 3 alpha2-, 3 beta-AR), die alle durch die physiologischen Liganden Adrenalin und Noradrenalin aktiviert werden k{\"o}nnen. Eine Subgruppe der AR bilden die drei alpha2-AR alpha2A, alpha2B und alpha2C. Sie k{\"o}nnen pr{\"a}- oder postsynaptisch lokalisiert sein. Pr{\"a}synaptisch lokalisierte alpha2-AR hemmen die Transmitterfreisetzung im Sinne einer negativen R{\"u}ckkopplung. Die Hauptrolle bei der pr{\"a}synaptischen Hemmung der Transmitterfreisetzung spielen alpha2-AR vom Subtyp alpha2A. Es lagen zu Beginn dieser Arbeit auch Hinweise vor, daß noch weitere alpha2-Rezeptorsubtypen an dieser Funktion beteiligt sind. Eine eindeutige Zuordnung dieser alpha2-AR zu den Subtypen alpha2B oder alpha2C gelang aber bisher nicht. In dieser Arbeit sollte deshalb die Frage beantwortet werden, welche alpha2-AR neben dem alpha2A-AR an der pr{\"a}synaptischen Hemmung der Transmitterfreisetzung im zentralen Nervensystem beteiligt sind. Zur Subtypunterscheidung wurden "knockout"-M{\"a}use verwendet, die nur einen oder zwei alpha2-Rezeptorsubtypen exprimierten. Gehirnschnitte aus dem Neokortex und den Basalganglien dieser Mauslinien wurden mit radoaktiv markiertem Noradrenalin bzw. Dopamin inkubiert. Anschließend wurde in Transmitterfreisetzungsexperimenten mit den so behandelten Gehirnschnitten Konzentrations-Wirkungskurven mit verschiedenen Liganden erstellt. Auf diese Weise konnte gezeigt werden, daß neben den alpha2A-AR auch alpha2C-AR pr{\"a}synaptisch die Transmitterfreisetzung von Noradrenalin und Dopamin hemmen. In einem weiteren Schritt wurde die Aktivierungs- und Deaktivierungskinetik der alpha2A- und alpha2C-AR im heterologen Expressionssystem untersucht. Hierzu wurden stabile HEK293-Zellinien generiert, die entweder alpha2A- oder alpha2C-AR unterschiedlich stark exprimierten. Diese Zellinien wurden transient mit GIRK-Kan{\"a}len transfiziert, um die durch Stimulation mit Noradrenalin resultierenden Kaliumstr{\"o}me mit der "patch-clamp"-Technik zu messen. Dabei ergab sich kein signifikanter Unterschied zwischen alpha2A- und alpha2C-AR bez{\"u}glich der Aktivierungskinetik. Alpha2C-AR deaktivierten jedoch deutlich langsamer als alpha2A-AR. Diese Befunde belegen, daß zwei der drei alpha2-AR-Subtypen, alpha2A und alpha2C, als pr{\"a}synaptische Autorezeptoren (Noradrenalin) bzw. Heterorezeptoren (Dopamin) die Neurotransmission modulieren. Dies k{\"o}nnte in der Zukunft f{\"u}r die Entwicklung neuer, subtypspezifischer Pharmaka von großer Bedeutung sein.}, language = {de} } @article{BuenemannPott1993, author = {B{\"u}nemann, Moritz and Pott, Lutz}, title = {Membrane-delimited activation of muscarinic K current by an albumin-associated factor in guinea-pig atrial myocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-31300}, year = {1993}, abstract = {No abstract available}, language = {en} } @article{BuesserLutz1987, author = {B{\"u}sser, M. T. and Lutz, Werner K.}, title = {Stimulation of DNA synthesis in rat and mouse liver by various tumor promoters}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60908}, year = {1987}, abstract = {In order to investigate whether the Stimulation of liver DNA synthesis might be used to detect one class of hepatic tumor promoters, the incorporation of orally administered radiolabelled thymidine into liver DNA was detennined in rats and mice 24 h after a single oral gavage of test compounds at various dose Ievels. Three DNA-binding hepatocarcinogens, aflatoxin B1; benzidine and carbon tetrachloride, did not stimulate but rather inhibited DNA synthesis (not for CCla). Four hepatic tumor promoters, clofibrate, DDT, phenobarbital and thioacetamide, gave rise to a Stimulation in a dosedependent manner. Single oral doses between 0.02 and 0.3 mmol/kg were required to double the level of thymidine incorporation into liver DNA (= doubling dose, DD). Differentes between species or sex as obsprved in long-term carcinogenicity studies were reflected by a different stimulation of liver DNA synthesis. In agreement with the bioassay data, aldrin was positive only in male mice (DD = 0.007 mmol/kg) but not in male rats or female mice. 2,3, 7,8-TCDD was positive in male mice (DD = 10\(^{-6}\) mmol/kg) andin female rats (DD = 2 x 10\(^{-6}\) mmol/kg) but not in male rats. The assay was also able to distinguish between structural isomers with different carcinogenicities. [alpha]Hexachlorocyclohexane stimulated Iiver DNA synthesis with a doubling dose of about 0.2 mmol/kg in male rats whereas the [gamma]isomer was ineffective even at l mmol/kg. So far, only one result was inconsistent with carcinogenicity bioassay data. The different carcinogenicity of di(2-ethylhexyl)adipate (negative in rats) and di(2-ethylhe.xyl)phthalate (positive) was not detectable. 8oth plasticizers were positive in.this short-term system with DD's of 0. 7 mmol/kg for DEHA and 0.5 mmol/kg for DEHP. The proposed assay is discussed as an attempt to devise short-term assays for carcinogens not detected by the routine genotoxicity test systems.}, subject = {Toxikologie}, language = {en} } @article{CalebiroMaiellaro2014, author = {Calebiro, Davide and Maiellaro, Isabella}, title = {cAMP signaling microdomains and their observation by optical methods}, series = {Frontiers in Cellular Neuroscience}, volume = {8}, journal = {Frontiers in Cellular Neuroscience}, issn = {1662-5102}, doi = {10.3389/fncel.2014.00350}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118252}, pages = {350}, year = {2014}, abstract = {The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains.}, language = {en} } @article{CantoreggiDietrichLutz1993, author = {Cantoreggi, S. and Dietrich, D. R. and Lutz, Werner K.}, title = {Induction of cell proliferation in the forestomach of F344 rats following subchronic administration of styrene 7,8-oxide and butylated hydroxyanisole}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60669}, year = {1993}, abstract = {The question addressed was whether Stimulation of cell proliferation could be responsible for tumor induction in the torestornach by styrene 7,8-oxide (SO). Male F344 rats were treated for 4 weeks with 0, 137,275, and 550 mglkg SO by p.o. gavage 3 times/week. Positive controls received 0, 0.5, I, and 2\% butylated hydroxyanisole (BHA) in the diet for 4 weeks. Twenty-four h before termination of the experlment, the rats were implanted s.c. with an osmotic minipump deliverlog S-bromo-2'-deoxyuri· dine (BrdU). Cell proliferation in the forestomach was assessed by immunohistochemistry for BrdU incorporated into DNA. Cell number/mm section length and fraction of replicating cells (labeling Index) were determined in 3 domains of the forestomach, the saccus caecus, the midregion, and the prefundic region. With the exception of the prefundic reglon of the low-dose SO group, a significant increase of the labeling index was found in all regions both with SO and BHA. Rats treated with BHA showed, in addition, a dose-dependent increase in number and size of hyperplastic lesions. This was most pronounced in the prefundic region where carcinomas were reported to be localized. In this region, the number of dividing cells/mm section length was increased up to 17-fold. With SO, only marginal morphological changes were occasionally observed, despite the fact that the respective long-term treatment bad been reported to result in a higher carcinoma incidence than treatment with BHA. It ls concluded that the rate of replicating cells alone, numerically expressed by the labeling Index, is an lnsufficient tool for interpretlog the role of cell division in carcinogenesis. It is postulated that SO and BHA induce forestomach tumors via different mechanisms. While hyperplasia in the prefundic region most likely dominates the carcinogenicity of BHA, a mechanism combining marginal genotoxicity with strong promotion by increased cell proliferation appears to be involved in the tumorigenic action of SO.}, subject = {Toxikologie}, language = {en} } @incollection{CantoreggiGuptaLutz1993, author = {Cantoreggi, S. and Gupta, R. C. and Lutz, Werner K.}, title = {An improved 32P-postlabelling assay for detection and quantitation of styrene 7,8-oxide-DNA adducts}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86305}, publisher = {Universit{\"a}t W{\"u}rzburg}, year = {1993}, abstract = {Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P 1-enriched adducts were 32P-labelled and purified by high-salt ( 4.0 M ammonium formate, pH 6.1} C1s reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H20 (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected. The efficiency of the kinase reaction depended on the ATP concentration. Use of standard labelling conditions (['Y· 32P]ATP, <3000 Ci/mmol; <2 Mikromol) resulted in poor ( 4-7\%) adduct recovery. An ATP concentration of 40 Mikromol, however, increased the labeJling efficiency by a factor of 5-8 (35-55\% based on 3H-SO labelied DNA). The results indicate that the new separation technique is suitable for the relatively polar SO-DNA adducts and that high labelling efficiency can be achieved.}, subject = {Medizin}, language = {en} } @article{CantoreggiLutz1993, author = {Cantoreggi, S. and Lutz, Werner K.}, title = {Covalent binding of styrene to DNA in rat and mouse}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60693}, year = {1993}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{CantoreggiLutz1992, author = {Cantoreggi, S. and Lutz, Werner K.}, title = {Investigation of the covalent binding of styrene-7,8-oxide to DNA in rat and mouse}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60721}, year = {1992}, abstract = {Styrene-7,8-oxide (SO), the main intennediate metabolite of styrene, induces hyperkeratosis and tumors in the forestomach of rats and mice upon chronic administration by gavage. The aim of this study was to investigate wbether DNA binding could be responsible for the carcinogenic effect observed. [7-\(^3\)H]SO was administered by oral gavage in com oll to male CD rats at two dose levels (1.65 or 240 mg/kg). After 4 or 24 h, forestomach, glandular stomach and Uver were exclsed, DNA was isolated and its radioactivity detennined. At the 4 h time polnt, the DNA radioactivity was below the Iimit of detection in the torestornach and the liver. Expressed in the units of the covalent bindlng Index, CBI = (pmol adduct/mol DNA nucleotide)/(mmol cbemical administeredlkg body wt), the DNA-binding potency was below 2.6 and 2.0 respectively. In the glandular stomach at 4 b, and in most 24 b samples, DNA was slightly radiolabeled. Enzymatic degradation of the DNA and separation by HPLC ofthe normal nucleotides sbowed that the DNA rad.ioactivity represented biosynthetic incorporation of radlolabel into newly synthesized DNA. The Iimit of detection of DNA adducts in the glandular stomach was 1.0. In a second experlment, [7-\(^3\)H]SO was administered by i.p. injection to male 86C3Fl rnice. Liver DNA was analyzed after 2 h. No radloactivity was detectable at a Iimit of detection of CBI < 0.6. In agreement with the relatively long half-life of SO in animals, the cbemical reactivity of SO appears to be too low to result in a detectable production of DNA adducts in an in vivo situation. Upon comparison with the DNA-binding of other carcinogens, a purely genotoxic mechanism of tumorigenJc action of SO is unlikely. The observed tumorigenic potency in the forestomach could be the result of strong tumor promotion by high-dose cytotoxicity foUowed by regenerative hyperplasia.}, subject = {Toxikologie}, language = {en} } @article{CapraBusnelliPerennaetal.2013, author = {Capra, Val{\´e}rie and Busnelli, Marta and Perenna, Alessandro and Ambrosio, Manuela and Accomazzo, Maria Rosa and Gal{\´e}s, Celine and Chini, Bice and Rovati, G. Enrico}, title = {Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0060475}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131013}, pages = {e60475}, year = {2013}, abstract = {The intrahelical salt bridge between \(E/D^{3.49}\) and \(R^{3.50}\) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of \(E^{3.49/6.30}\) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.}, language = {en} } @article{CaviezelAeschbachLutzetal.1984, author = {Caviezel, M. and Aeschbach, A. P. and Lutz, Werner K. and Schlatter, C.}, title = {Reduction of covalent binding of aflatoxin B1 to rabbit liver DNA after immunization against this carcinogen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-80116}, year = {1984}, abstract = {The covalent binding of [3H]aflatoxin B1 (AF) to liver DNA was determined, 6 h after oral administration to male rabbits. A Covalent Binding Index, CBI (flmol AF/mol DNA-P)/(mmol AF/kg b. w.) = 8,500 was found. Pretreatment of rabbits with AF coupled to bovine serum albumin in Freund's adjuvant led to the production of AF-directed antibodies. Administration of [3H]AF to such immunized rabbits resulted in a CJH of only 2,500, i.e., the iiDJ{.lUnization provided a protection by a factor of more than 3. Although this is encouraging evidence for the potential of active immunization against genotoxic carcinogens, a nurober of pointswill have to be clarified, such as the time course for the DNA binding and the question of a possible shift to other target cells.}, subject = {Krebs}, language = {en} } @article{CaviezelLutzMininietal.1984, author = {Caviezel, M. and Lutz, Werner K. and Minini, U. and Schlatter, C.}, title = {Interaction of estrone and estradiol with DNA and protein of liver and kidney in rat and hamster in vivo and in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60995}, year = {1984}, abstract = {(6,7-\(^3\)H] Estrone (E) and [6,7-\(^3\)H]estradiol-17ß (E\(_2\)) have been synthesized by reduction of 6-dehydroestrone and 6-dehydroestradiol with tritium gas. Tritiated E and E\(_2\) were administered by oral gavage to female rats and to male and female hamsters on a dose level of about 300 \(\mu\)g/kg (54 mCi/kg). After 8 h, the liver was excised from the rats; liver and kidneys were taken from the hamsters. DNA was purified either directly from an organ homogenate or via chromatin. The radioactivity in the DNA was expressed in the units of the Covalent Binding Index, CBI = (\(\mu\)mol chemical bound per mol Similar considerations can be made for the liver where any true covalent DNA binding must be below a Ievel of 0.01. It is concluded that an observable tumor induction by estrone or estradiol is unlikely to be due to DNA binding. DNA-P)/(mmol chemical administered per kg b.w.). Rat liver DNA isolated via chromatin exhibited the very low values of 0.08 and 0.09 for E and E\(_2\) respectively. The respective figures in hamster liver were 0.08 and 0.11 in females and 0.21 and 0.18 in the males. DNA isolated from the kidney revealed a detectable radioactivity only in the female, with values of 0.03 and 0.05 for E and E\(_2\) respectively. The values for male hamster kidney were < 0.01 for both hormones. The minute radioactivity detectable in the DNA samples does not represent covalent binding to DNA, however, as indicated by' two sets of control experiments. (A) Analysis by HPLC of the nucleosides prepared by enzyme digest of liver DNA isolated directly or via chromatin did not reveal any consistent peak which could have been attributed to a nucleoside-steroid adduct. (B) All DNA radioactivity could be due to protein contaminations, because the specific activity of chromatin protein was determined to be more than 3 ,000 tim es high er than of DNA. The high affinity of the hormone to protein was also demonstrated by in vitro incubations, where it could be shown that the specific activity of DNA and protein was essentially proportional to the concentration of radiolabelled hormone in the organ homogenate, regardless of whether the animal was treated or whether the hormone was added in vitro to the homogenate. Carcinogens acting by covalent DNA binding can be classified according to potency on the basis of the Covalent Binding Index. Values of 10\(^3\)-10\(^4\) have been found for potent, 10\(^2\) for moderate, and 1-10 for weak carcinogens. Since estrone is moderately carcinogenic for the kidney of the male hamster, a CBI of about 100 would be expected. The actually measured Iimit of detection of 0.01 places covalent DNA binding among the highly unlikely mechanisms of action.}, subject = {Toxikologie}, language = {en} } @article{ChenGassnerBoerneretal.2012, author = {Chen, Wen and Gaßner, Birgit and B{\"o}rner, Sebastian and Nikolaev, Viacheslav O. and Schlegel, Nicolas and Waschke, Jens and Steinbronn, Nadine and Strasser, Ruth and Kuhn, Michaela}, title = {Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway}, series = {Cardiovascular Research}, volume = {93}, journal = {Cardiovascular Research}, number = {1}, doi = {10.1093/cvr/cvr279}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126562}, pages = {141-151}, year = {2012}, abstract = {Aims Cardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP. Methods and results Intravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1-/- mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains. Conclusion ANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.}, language = {en} } @article{ChengOthmanStopperetal.2017, author = {Cheng, Cheng and Othman, Eman M. and Stopper, Helga and Edrada-Ebel, RuAngelie and Hentschel, Ute and Abdelmohsen, Usama Ramadan}, title = {Isolation of petrocidin A, a new cytotoxic cyclic dipeptide from the marine sponge-derived bacterium \(Streptomyces\) sp. SBT348}, series = {Marine Drugs}, volume = {15}, journal = {Marine Drugs}, number = {12}, doi = {10.3390/md15120383}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172644}, year = {2017}, abstract = {A new cyclic dipeptide, petrocidin A (\(\textbf{1}\)), along with three known compounds—2,3-dihydroxybenzoic acid (\(\textbf{2}\)), 2,3-dihydroxybenzamide (\(\textbf{3}\)), and maltol (\(\textbf{4}\))—were isolated from the solid culture of \(Streptomyces\) sp. SBT348. The strain \(Streptomyces\) sp. SBT348 had been prioritized in a strain collection of 64 sponge-associated actinomycetes based on its distinct metabolomic profile using liquid chromatography/high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR). The absolute configuration of all α-amino acids was determined by HPLC analysis after derivatization with Marfey's reagent and comparison with commercially available reference amino acids. Structure elucidation was pursued in the presented study by mass spectrometry and NMR spectral data. Petrocidin A (\(\textbf{1}\)) and 2,3-dihydroxybenzamide (\(\textbf{3}\)) exhibited significant cytotoxicity towards the human promyelocytic HL-60 and the human colon adenocarcinoma HT-29 cell lines. These results demonstrated the potential of sponge-associated actinomycetes for the discovery of novel and pharmacologically active natural products.}, language = {en} } @article{ChilakaObidiegwuChilakaetal.2022, author = {Chilaka, Cynthia Adaku and Obidiegwu, Jude Ejikeme and Chilaka, Augusta Chinenye and Atanda, Olusegun Oladimeji and Mally, Angela}, title = {Mycotoxin regulatory status in Africa: a decade of weak institutional efforts}, series = {Toxins}, volume = {14}, journal = {Toxins}, number = {7}, issn = {2072-6651}, doi = {10.3390/toxins14070442}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-278941}, year = {2022}, abstract = {Food safety problems are a major hindrance to achieving food security, trade, and healthy living in Africa. Fungi and their secondary metabolites, known as mycotoxins, represent an important concern in this regard. Attempts such as agricultural, storage, and processing practices, and creation of awareness to tackle the menace of fungi and mycotoxins have yielded measurable outcomes especially in developed countries, where there are comprehensive mycotoxin legislations and enforcement schemes. Conversely, most African countries do not have mycotoxin regulatory limits and even when available, are only applied for international trade. Factors such as food insecurity, public ignorance, climate change, poor infrastructure, poor research funding, incorrect prioritization of resources, and nonchalant attitudes that exist among governmental organisations and other stakeholders further complicate the situation. In the present review, we discuss the status of mycotoxin regulation in Africa, with emphasis on the impact of weak mycotoxin legislations and enforcement on African trade, agriculture, and health. Furthermore, we discuss the factors limiting the establishment and control of mycotoxins in the region.}, language = {en} } @article{ChristianSeierDrakopoulosetal.2020, author = {Christian, Gentzsch and Seier, Kerstin and Drakopoulos, Antonios and Jobin, Marie-Lise and Lanoisel{\´e}e, Yann and Koszegi, Zsombor and Maurel, Damien and Sounier, R{\´e}my and H{\"u}bner, Harald and Gmeiner, Peter and Granier, S{\´e}bastien and Calebiro, Davide and Decker, Michael}, title = {Selective and Wash-Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single-Molecule Imaging of μ-Opioid Receptor Dimerization}, series = {Angewandte Chemie International Edition}, volume = {59}, journal = {Angewandte Chemie International Edition}, number = {15}, doi = {10.1002/anie.201912683}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212398}, pages = {5958-5964}, year = {2020}, abstract = {μ-Opioid receptors (μ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of μ-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ-OR pharmacology at single-molecule level.}, language = {en} } @phdthesis{Cirkel2018, author = {Cirkel, Nanett Christin}, title = {Beeinflussung des oxidativen Stress-Status in einem Rattenmodell: Effekt von Selenmangel auf Niere und Leber}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163631}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Das Spurenelement Selen und Vitamin E reduzieren reaktive Sauerstoff Spezies (ROS). Bei Mangel dieser wichtigen Stoffe erh{\"o}ht sich die Konzentration an ROS und der oxidative Stress steigt. Unter erh{\"o}hten ROS entstehen vermehrt DNA-Sch{\"a}den und Lipidperoxidationen. Das ROS Wasserstoffperoxid wird zu Wasser {\"u}ber das Enzym Gluthationperxoidase reduziert. Dessen Aktivit{\"a}t steigert Selen um den Faktor 100-1.000. Das Aktivit{\"a}tsmaximum des Enzyms liegt bei einer t{\"a}glichen Selenaufnahme von 60-80 Mikrogramm/Tag. Dadurch wird die Menge an ROS reduziert und der oxidative Stress in der Zelle nimmt ab. Vitamin E fungiert als Radikalf{\"a}nger. Sein Derivat alpha- Tocopherol besitzt die h{\"o}chste antioxidative Wirkung und kann Lipidperoxidationen unterbrechen. Die vorliegende Arbeit untersucht Auswirkungen von oxidativem Stress, den ein Mangel von Selen und Vitamin E in der Nahrung bei 6 Monate und 12 Monate alten Tieren auf Leber und Niere verursacht. Der Nachweis von oxidativem Stress erfolgte {\"u}ber sogenannte Hitzeschockproteine HSP70 und H{\"a}moxygenase 1. HSP 70 wird auch unter physiologischen Bedingungen exprimiert. Es wirkt als Chaperon und ist u.a. f{\"u}r die korrekte Faltung und Stabilisierung von Proteinen zust{\"a}ndig. Die Versuche zeigten, dass im Alter in der Niere die HSP70 Konzentration ansteigt und die Zelle unter vermehrtem oxidativen Stress leidet. Entsprechende Literaturergebnisse wurden best{\"a}tigt. Die H{\"a}moxygenase 1 (HO-1) ist ein Schl{\"u}sselenzym, das vermehrt bei oxidativem Stress gebildet wird. Hoch reaktionsfreudige und freie Blutbestandteile katalysiert die H{\"a}moxygenase. Einen Abfall der HO- 1 Konzentration zeigten Untersuchungen von Leber und Niere bei Selen, - Vitamin E Mangel und h{\"o}herem Lebensalter. Gr{\"u}nde f{\"u}r die verminderte Expression sind noch wenig erforscht. Die vermehrte Anreicherung von Superoxidanionradikalen wurde in den Geweben von Leber und Niere {\"u}ber Dihydroethidium (DHE) F{\"a}rbung nachgewiesen. Die Hypothese wurde best{\"a}tigt, dass bei Selen, -Vitamin E Mangelnahrung und h{\"o}herem Alter vermehrter oxidativer Stress entsteht. Selenmangel beg{\"u}nstigt die Entstehung verschiedener Krankheiten, z.B. Krebs, koronale Herzerkrankung und vor allem die Keshan-Krankheit, die den Herzmuskel bef{\"a}llt. Selen nimmt positiven Einfluss auf K{\"o}rperfunktionen: Fertilit{\"a}t, embryonalen Entwicklung und Entwicklung eines Neugeborenen. Einige Fragen bleiben ungekl{\"a}rt: Welche physiologischen Entwicklungsprozesse f{\"o}rdert Selen? Nimmt Selen eine wichtige Funktion bei der Befruchtung der Eizelle ein? Wie beeinflusst Selen die Entwicklung des Gehirns? Dem Spurenelement Selen kommen offensichtlich neben seiner Bedeutung zur Minderung des oxidativen Stresses noch weitere wichtige Funktionen zu, die bisher wenig untersucht wurden.}, subject = {Oxidativer Stress}, language = {de} } @phdthesis{Classen2021, author = {Claßen, Alexandra}, title = {The ERK-cascade in the pathophysiology of cardiac hypertrophy}, doi = {10.25972/OPUS-22966}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229664}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {ERK1/2 are known key players in the pathophysiology of heart failure, but the members of the ERK cascade, in particular Raf1, can also protect the heart from cell death and ischemic injury. An additional autophosphorylation (ERK1 at Thr208, ERK2 at Thr188) empowers ERK1/2 translocation to the nucleus and phosphorylation of nuclear targets which take part in the development of cardiac hypertrophy. Thereby, targeting this additional phosphorylation is a promising pharmacological approach. In this thesis, an in silico model of ERK cascade in the cardiomyocyte is introduced. The model is a semi-quantitive model and its behavior was tested with different softwares (SQUAD and CellNetAnalyzer). Different phosphorylation states of ERK1/2 as well as different stimuli can be reproduced. The different types of stimuli include hypertrophic as well as non-hypertrophic stimuli. With the introduced in-silico model time courses and synergistic as well as antagonistic receptor stimuli combinations can be predicted. The simulated time courses were experimentally validated. SQUAD was mainly used to make predictions about time courses and thresholds, whereas CNA was used to analyze steady states and feedback loops. Furthermore, new targets of ERK1/2 which partially contribute, also in the formation of cardiac hypertrophy, were identified and the most promising of them were illuminated. Important further targets are Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 and SPIN90. Cardiomyocyte gene expression data sets were analyzed to verify involved components and to find further significantly altered genes after induced hypertrophy with TAC (transverse aortic constriction). Changes in the ultrastructure of the cardiomyocyte are the final result of induced hypertrophy.}, subject = {Herzhypertrophie}, language = {en} } @article{CristalliEleuteriVittorietal.1992, author = {Cristalli, G. and Eleuteri, A. and Vittori, S. and Volpini, R. and Lohse, M. J. and Klotz, Karl-Norbert}, title = {2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamides as selective agonists at A\(_2\) adenosine receptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60412}, year = {1992}, abstract = {In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A\(_2\) receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cycla.se, and platelet aggregation studies. Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited by compounds 2a-d with K\(_i\) values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affmity binding at solubilized A\(_2\) receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [\(^3\)H]DPCPX and for the agonist [\(^3\)H]CCPA gave K\(_i\) values in the nanomolar range, and the compounds showed moderate A\(_2\) selectivity. In order to improve this selectivity, the correaponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. A\(_1\) expected, the 5'-N-ethyluronamide derivatives retained the A\(_2\) affinity whereas the A\(_1\) affinity was attenuated, resulting in an up to 10-fold increase in A\(_2\) selectivity. A similar patternwas observed in adenylate cyclase assays andin platelet aggregation studies. A 30- to 45-fold selectivity for platelet A\(_2\) receptors compared to A\(_1\) receptors was found for compounds 8a-c in adenylate cyclase studies.}, subject = {Toxikologie}, language = {en} } @article{CristalliFranchettiGrifantinietal.1988, author = {Cristalli, G. and Franchetti, P. and Grifantini, M. and Vittori, S. and Klotz, Karl-Norbert and Lohse, M. J.}, title = {Adenosine receptor agonists: Synthesis and biological evaluation of 1-deaza analogues of adenosine}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60262}, year = {1988}, abstract = {In a search for more selective A\(_1\) adenosine receptor agonists, N\(^6\)-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N\(^6\)-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N\(^6\)-cyclohexyl-l-deazaadenosine (1-deazaCHA, Sc), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-ß-D-ribofuranosyl-3Himidazo[ 4,5-b]pyridine (1). On the other band, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-ß-D-ribofuranuronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-ß-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine (4), in an attempt to find a more selective A\(_2\) agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase andin radioligand binding studies. 1-DeazaNECA (10) proved tobe a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N\(^6\)-substituted 1-deazaadenosines largely retain the A\(_1\) agonist activity of their parent compounds, but lose some of their A\(_2\) agonist activity. This results in A\(_1\)-selective compounds, of which N\(^6\)cyclopentyl- 2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A\(_1\) adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A\(_1\) receptor mediated adenosine actions.}, subject = {Toxikologie}, language = {en} } @phdthesis{Curtaz2019, author = {Curtaz, Carolin Julia}, title = {\(In\) \(vitro\) Analysen der Wechselwirkung erh{\"o}hter Temperatur mit Zytostatika am Beispiel von Cisplatin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174543}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Neben der Chemotherapie ist heutzutage auch die Hyperthermie-Behandlung eine wichtige S{\"a}ule der antitumor{\"o}sen Therapie. W{\"a}hrend der sogenannten HIPEC Therapie (Hypertherme intraperitoneale Chemoperfusion) werden die beiden Arten der Therapieformen kombiniert und in der klinischen Praxis erfolgreich angewendet. Genauere Kenntnisse {\"u}ber die zu Grunde liegenden toxikologischen in-vitro Mechanismen k{\"o}nnten zu neuen M{\"o}glichkeiten in der klinischen Anwendung f{\"u}hren. In unserer Arbeit untersuchten wir verschiedenen Tumorzelllinien (HT29,CaCo-2,HCT116,HaCaT) in Kombination mit Cisplatin und Hyperthermie mit verschiedenen Methoden, wie zum Beispiel Mikrokerntest, Comet-Assay, Durchflusszytometrie, Vitalit{\"a}tstest und mikroskopischen Analysen. Unsere Ergebnisse f{\"u}hrten uns zu der Hypothese, dass Hyperthermie alleine zu einer sogenannte mitotic catastrophe f{\"u}hrt und zum Absterben der Tumorzellen. Im Gegensatz dazu zeigten Tumorzellen, welche mit Cisplatin alleine oder auch in Kombination mit Hyperthermie nicht in die Mitose eintreten und daher nicht durch Apoptose in den Zelltod gehen.}, subject = {Hyperthermie}, language = {de} } @phdthesis{Deiss2012, author = {Deiß, Katharina}, title = {Die Regulation des Kinasemodulators Raf Kinase Inhibitor Protein (RKIP): Einfluss von Phosphorylierung und Dimerisierung auf die Interaktion mit Raf1 und G Protein-gekoppelter Rezeptorkinase 2 (GRK2)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73884}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {RKIP reguliert Proteinkinasen der Signaltransduktionskaskaden von G Protein-gekoppelten Rezeptoren, der Raf/MEK/ERK-MAPK, des Transkriptionsfaktors NFκB und von GSK3β. Unklar war bisher, wie die spezifische Interaktion von RKIP mit seinen mannigfaltigen Interaktionspartnern erm{\"o}glicht und reguliert wird. Raf1 und GRK2 sind die einzigen bekannten direkten Interaktionspartner von RKIP und wurden deshalb gew{\"a}hlt, um die zugrundeliegenden molekularen Mechanismen dieser Interaktion genauer zu untersuchen. In dieser Arbeit wurde gezeigt, dass RKIP nach PKC-vermittelter Phosphorylierung von Serin153 dimerisiert und dass diese Dimerisierung f{\"u}r die RKIP/Raf1-Dissoziation und die RKIP/GRK2-Interaktion essentiell ist. Co-Immunpr{\"a}zipitationsexperimente mit einer phosphorylierungsdefizienten Mutante zeigten, dass f{\"u}r diese Dimerisierung die Phosphorylierung von beiden RKIP-Molek{\"u}len notwendig ist. Als Dimerinteraktionsfl{\"a}che wurden die Aminos{\"a}uren 127-146 von RKIP identifiziert, da das Peptid RKIP127-146 die Dimerisierung von RKIP spezifisch und effizient hemmte. Um die Bedeutung dieser phosphorylierungsinduzierten Dimerisierung von RKIP f{\"u}r seine Interaktion mit Raf1 und GRK2 zu untersuchen, wurden eine phosphomimetische Mutante (RKIPSK153/7EE) und eine Mutante von RKIP generiert, welche bereits unphosphoryliert dimerisiert (RKIP∆143-6). Folgende Ergebnisse legen nahe, dass die Dimerisierung von RKIP f{\"u}r die spezifische Interaktion mit Raf1 bzw. GRK2 entscheidend ist: (i) Die Dimerisierung von phosphoryliertem RKIP ging mit der Dissoziation von RKIP und Raf1 und der Assoziation von RKIP und GRK2 einher; (ii) die Mutanten RKIPSK153/7EE und RKIP∆143-6, die bereits in unstimulierten Zellen eine starke Dimerisierung zeigten, hatten eine h{\"o}here Affinit{\"a}t zu GRK2 als zu Raf1; (iii) die Hemmung der RKIP-Dimerisierung interferierte nur mit der RKIP/GKR2- aber nicht mit der RKIP/Raf1-Interaktion; (iv) in vitro und in Mausherzen konnte ein RKIP- und GRK2-immunreaktiver Komplex nachgewiesen werden; (v) Untersuchungen zur RKIP-vermittelten Hemmung der Kinaseaktivit{\"a}t von GRK2 und Raf implizierten, dass dimerisiertes RKIP nur die Aktivit{\"a}t von GRK2, nicht aber von Raf hemmt. Diese Arbeit zeigt, dass die phosphorylierungsinduzierte Dimerisierung von RKIP die spezifische Interaktion von RKIP mit Raf1 und GRK2 koordiniert. Die Aufkl{\"a}rung dieses Mechanismus erweitert unser Verst{\"a}ndnis der spezifischen Interaktion von Kinasen mit ihren Regulatorproteinen.}, subject = {Signaltransduktion}, language = {de} } @article{DekantLangerLuppetal.2021, author = {Dekant, Raphael and Langer, Michael and Lupp, Maria and Adaku Chilaka, Cynthia and Mally, Angela}, title = {In vitro and in vivo analysis of ochratoxin A-derived glucuronides and mercapturic acids as biomarkers of exposure}, series = {Toxins}, volume = {13}, journal = {Toxins}, number = {8}, issn = {2072-6651}, doi = {10.3390/toxins13080587}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-245146}, year = {2021}, abstract = {Ochratoxin A (OTA) is a widespread food contaminant, with exposure estimated to range from 0.64 to 17.79 ng/kg body weight (bw) for average consumers and from 2.40 to 51.69 ng/kg bw per day for high consumers. Current exposure estimates are, however, associated with considerable uncertainty. While biomarker-based approaches may contribute to improved exposure assessment, there is yet insufficient data on urinary metabolites of OTA and their relation to external dose to allow reliable estimates of daily intake. This study was designed to assess potential species differences in phase II biotransformation in vitro and to establish a correlation between urinary OTA-derived glucuronides and mercapturic acids and external exposure in rats in vivo. In vitro analyses of OTA metabolism using the liver S9 of rats, humans, rabbits and minipigs confirmed formation of an OTA glucuronide but provided no evidence for the formation of OTA-derived mercapturic acids to support their use as biomarkers. Similarly, OTA-derived mercapturic acids were not detected in urine of rats repeatedly dosed with OTA, while indirect analysis using enzymatic hydrolysis of the urine samples prior to LC-MS/MS established a linear relationship between urinary glucuronide excretion and OTA exposure. These results support OTA-derived glucuronides but not mercapturic acids as metabolites suitable for biomonitoring.}, language = {en} } @article{DekantBridges2016, author = {Dekant, Wolfgang and Bridges, James}, title = {Assessment of reproductive and developmental effects of DINP, DnHP and DCHP using quantitative weight of evidence}, series = {Regulatory Toxicology and Pharmacology}, volume = {81}, journal = {Regulatory Toxicology and Pharmacology}, doi = {10.1016/j.yrtph.2016.09.032}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186750}, pages = {397-406}, year = {2016}, abstract = {Quantitative weight of evidence (QWoE) methodology utilizes detailed scoring sheets to assess the quality/reliability of each publication on toxicity of a chemical and gives numerical scores for quality and observed toxicity. This QWoE-methodology was applied to the reproductive toxicity data on diisononylphthalate (DINP), di-n-hexylphthalate (DnHP), and dicyclohexylphthalate (DCHP) to determine if the scientific evidence for adverse effects meets the requirements for classification as reproductive toxicants. The scores for DINP were compared to those when applying the methodology DCHP and DnHP that have harmonized classifications. Based on the quality/reliability scores, application of the QWoE shows that the three databases are of similar quality; but effect scores differ widely. Application of QWoE to DINP studies resulted in an overall score well below the benchmark required to trigger classification. For DCHP, the QWoE also results in low scores. The high scores from the application of the QWoE methodology to the toxicological data for DnHP represent clear evidence for adverse effects and justify a classification of DnHP as category 1B for both development and fertility. The conclusions on classification based on the QWoE are well supported using a narrative assessment of consistency and biological plausibility.}, language = {en} } @article{DekantKlaunig2016, author = {Dekant, Wolfgang and Klaunig, James E.}, title = {Toxicology of decamethylcyclopentasiloxane (D5)}, series = {Regulatory Toxicology and Pharmacology}, volume = {74}, journal = {Regulatory Toxicology and Pharmacology}, number = {Supplement}, doi = {10.1016/j.yrtph.2015.06.011}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-190914}, pages = {S67-S76}, year = {2016}, abstract = {Decamethylcyclopentasiloxane (D5) is a cyclic siloxane used in the formulation of consumer products as well as an industrial intermediate. A summary of the previous studies on the toxicology of D5 is provided. Toxicokinetic studies with D5 after dermal administration demonstrate a very low uptake of due to rapid evaporation. Following inhalation exposure, exhalation of unchanged D5 and excretion of metabolites with urine are major pathways for clearance in mammals. Due to this rapid clearance by exhalation, the potential for bioaccumulation of D5 is considered unlikely. The available toxicity data on D5 adequately cover the relevant endpoints regarding potential human health hazards. D5 was not DNA reactive or mutagenic in standard in vitro and in vivo test systems. D5 also did not induce developmental and reproductive toxicity in appropriately performed studies. In repeated studies in rats with subacute, subchronic and chronic inhalation exposure, mild effects on the respiratory tract typically seen after inhalation of irritating materials, increases in liver weight (28- and 90-day inhalation studies), and a small increase in the incidence of uterine adenocarcinoma (uterine tumor) in female rats (two-year inhalation chronic bioassay) were observed. The liver effects induced by D5 were consistent with D5 as a weak "phenobarbital-like" inducer of xenobiotic metabolizing enzymes and these effects are considered to be an adaptive response. Mechanistic studies to elucidate the mode-of-action for uterine tumor induction suggest an interaction of D5 with dopamine signal transduction pathways altering the pituitary control of the estrus cycle. The resulting estrogen imbalance may cause the small increase in uterine tumor incidence at the highest D5-exposure concentration over that seen in control rats. A genotoxic mechanism or a direct endocrine activity of D5 is not supported as a mode-of-action to account for the induction of uterine tumors by the available data.}, language = {en} } @article{DjelićBorozanDimitrijevićSrećkovićetal.2022, author = {Djelić, Ninoslav and Borozan, Sunčica and Dimitrijević-Srećković, Vesna and Pajović, Nevena and Mirilović, Milorad and Stopper, Helga and Stanimirović, Zoran}, title = {Oxidative stress and DNA damage in peripheral blood mononuclear cells from normal, obese, prediabetic and diabetic persons exposed to thyroid hormone in vitro}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {16}, issn = {1422-0067}, doi = {10.3390/ijms23169072}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285988}, year = {2022}, abstract = {Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM.}, language = {en} } @phdthesis{Dorsch2009, author = {Dorsch, Sandra}, title = {Rezeptor-Rezeptor-Interaktion ß-adrenerger Rezeptoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39712}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Viele Membranrezeptoren liegen als {\"u}ber Disulfidbr{\"u}cken-verbundene Dimere vor. Ein Nachweis der Dimerisierung ist in diesen F{\"a}llen methodisch klar und einfach zu erbringen. F{\"u}r die meisten G-Protein-gekoppelten Rezeptoren dagegen ist weder die Existenz von Di- oder Oligomeren noch deren Funktion eindeutig belegt. Meist wurden Methoden wie Coimmunopr{\"a}zipitation und Resonanz-Energie-Transfer-Verfahren wie BRET oder FRET verwendet, um Protein-Protein-Interaktionen zu untersuchen. Trotz ihrer hohen Sensitivit{\"a}t besitzen diese Methoden einige Grenzen und k{\"o}nnen je nach experimentellem Ansatz und Verwendung verschiedener Kontrollen, unterschiedliche Ergebnisse hinsichtlich des Vorliegens einer Protein-Protein-Interaktion liefern. Weder die Stabilit{\"a}t der Interaktion, noch die Fraktion der interagierenden Proteine kann mittels Resonanz-Energie-Transfer-Assays zuverl{\"a}ssig ermittelt werden. Auch die Gr{\"o}ße der Komplexe ist nicht oder nur technisch aufwendig bestimmbar. Deshalb wurde in dieser Arbeit eine neue, unabh{\"a}ngige Methode entwickelt, um Rezeptor-Rezeptor-Interaktionen in lebenden Zellen genauer untersuchen zu k{\"o}nnen. Diese auf „Fluorescence Recovery after Photobleaching" basierende Mikroskopie-Methode erlaubt die Mobilit{\"a}t von Proteinen zu bestimmen. Um Homointeraktionen zwischen Proteinen messen zu k{\"o}nnen, m{\"u}ssen zwei Protein-Fraktionen mit unterschiedlicher Mobilit{\"a}t vorliegen. Deshalb wurde eine Rezeptor-Fraktion extrazellul{\"a}r mit YFP markiert und mit Hilfe polyklonaler Antik{\"o}rper gegen YFP spezifisch immobilisiert. Die andere Rezeptorfraktion wurde intrazellul{\"a}r mit CFP oder Cerulean markiert und wurde deshalb nicht von extrazellul{\"a}ren Antik{\"o}rpern erkannt. So konnten mittels Zwei-Farben-FRAP potenzielle Interaktionen zwischen den immobilisierten extrazellul{\"a}r-markierten Rezeptoren und den intrazellul{\"a}r-markierten Rezeptoren durch eine Mobilit{\"a}ts{\"a}nderung letzterer detektiert werden. Diese Methode wurde mittels eines monomeren (CD86) und kovalent dimeren (CD28) Rezeptors validiert. Es zeigte sich, dass eine spezifische Immobilisierung extrazellul{\"a}r-markierter Proteine nur durch polyklonale, nicht aber durch monoklonale Antik{\"o}rper gegen YFP erreicht werden konnte. Intrazellul{\"a}r-markierte Proteine wurden hierbei in ihrer Mobilit{\"a}t nicht durch die extrazellul{\"a}ren Antik{\"o}rper beeinflusst. Bei Immobilisierung des extrazellul{\"a}r-markierten CD86 war das coexprimierte, intrazellul{\"a}r-markierte CD86-CFP weiterhin voll mobil. Außerdem zeigte das Monomer CD86 eine vom relativen CFP-YFP-Expressionsverh{\"a}ltnis unabh{\"a}ngige Mobilit{\"a}t. Dieses Ergebnis ließ den Schluss zu, dass extra- und intrazellul{\"a}r-markiertes CD86 nicht miteinander interagieren und als Monomer vorliegen. Die Mobilit{\"a}t des kovalenten Dimers CD28 war dagegen abh{\"a}ngig vom CFP-YFP-Expressionsverh{\"a}ltnis und stimmte gut mit theoretisch erwarteten Werten f{\"u}r ein Dimer {\"u}berein. Die Anwendung der Zwei-Farben-Methode zur Untersuchung von Interaktionen zwischen ß1- und ß2-adrenergen Rezeptoren zeigte Unterschiede zwischen beiden Rezeptor-Subtypen. ß1-AR zeigte eine spezifische transiente Interaktion, ß2-AR dagegen lagen als stabile Oligomere h{\"o}herer Ordnung vor. Die transiente Interaktion zwischen ß1-AR und die stabile Oligomerisierung von ß2-AR wurde nicht nur in HEK 293T-Zellen sondern auch in neonatalen Rattenkardiomyozyten und bei 37 °C beobachtet. Ferner hatte der Aktivierungszustand des jeweiligen Rezeptors keinen Einfluß auf das Ausmaß der Interaktion. Zwischen ß1- und ß2-AR wurde nur eine sehr schwache und instabile Heterointeraktion mittels der Zwei-Farben-FRAP-Methode beobachtet. Um zu {\"u}berpr{\"u}fen, ob eine direkte Interaktion zwischen den adrenergen Rezeptoren vorliegt, wurde die BRET-Methode verwendet. Mittels BRET wurde eine direkte Interaktion zwischen ß2-AR festgestellt, jedoch konnte nicht zwischen Dimeren und Oligomeren h{\"o}herer Ordnung unterschieden werden. Bei ß1-AR fand bei h{\"o}heren YFP-Rluc-Expressionsverh{\"a}ltnissen ein spezifischer Energietransfer statt. Bei niedrigeren Expressionsverh{\"a}ltnissen lag das Signal jedoch im unspezifischen Bereich. Auch bei Untersuchung der Heterointeraktion zwischen ß1- und ß2-AR konnte keine klare Aussage {\"u}ber eine spezifische Interaktion zwischen beiden Rezeptor-Subtypen getroffen werden.}, subject = {Dimerisierung}, language = {de} } @phdthesis{Duraphe2010, author = {Duraphe, Prashant}, title = {Identification and characterization of AUM, a novel human tyrosine phosphatase}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-44256}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Protein Phosphatasen werden aufgrund der Aminos{\"a}uresequenzen ihrer aktiven Zentren in drei große Familien unterteilt. In einer neu entdeckten Familie von Phosphatasen ist das aktive Zentrum durch die Sequenz DXDX(T/V) charakterisiert. Diese Aspartat-abh{\"a}ngigen Phosphatasen geh{\"o}ren zu der Superfamilie der Hydrolasen vom Haloazid Dehalogenase(HAD)-Typ, einer evolution{\"a}r konservierten und ubiquit{\"a}r verbreiteten Enzymfamilie. Bislang konnten 58 menschliche HAD Enzyme durch Datenbankanalysen identifiziert werden. Ihre Funktionen sind jedoch nach wie vor nur rudiment{\"a}r verstanden. Im Rahmen dieser Arbeit wurde zun{\"a}chst das Komplement aller menschlichen HAD Phosphatasen durch Datenbank-Recherchen erfasst. Zusammen mit phylogenetischen Analysen gelang es, eine zum damaligen Zeitpunkt unbekannte, putative Phosphatase zu identifizieren, die eine vergleichsweise hohe Sequenz-Homologie zu der Zytoskelettregulierenden HAD Phosphatase Chronophin aufweist. Dieses neuartige Enzym wurde kloniert und mit biochemischen und zellbiologischen Methoden charakterisiert. Auf der Basis dieser Befunde bezeichnen wir dieses neuartige Protein als AUM (actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase).Mittels Northern blot, real-time PCR und Western blot Analysen konnte gezeigt werden, dass AUM in allen untersuchten menschlichen und murinen Geweben exprimiert wird. Die h{\"o}chste Expression konnte in Hodengewebe nachgewiesen werden. Durch immunohistochemische Untersuchungen konnte gezeigt werden, dass AUM spezifisch in reifenden Keimzellen mit einem Expressionsmaximum zum Zeitpunkt der Spermiogenese exprimiert wird. Um die Substratpr{\"a}ferenz von AUM zu charakterisieren, wurde zun{\"a}chst ein peptidbasierter in vitro Phosphatase-Substrat-Screen durchgef{\"u}hrt. Hierbei wurden 720 aus menschlichen Phosphoproteinen abgeleitete Phosphopeptide untersucht. Interessanterweise dephosphorylierte AUM ausschließlich Phosphotyrosin (pTyr)-enthaltende Peptide. Nur 17 pTyr-Peptide (~2\% aller untersuchten Peptide) fungierten als AUM-Substrate. Diese Daten legen eine hohe Substratspezifit{\"a}t von AUM nahe. Zu den putativen AUM Substraten geh{\"o}ren Proteine, die in die Dynamik der Zytoskelett-Reorganisation sowie in Tyrosin Kinasevermittelte Signalwege eingebunden sind. In {\"U}bereinstimmung mit den Ergebnissen dieses Phosphopeptid-Screens konnte mittels Phosphatase overlay assays sowie in Zellextrakten aus Pervanadat-behandelten HeLa Zellen demonstriert werden, dass AUM eine begrenzte Anzahl Tyrosin-phosphorylierter Proteinen dephosphorylieren kann.In zellul{\"a}ren Untersuchungen wurde die m{\"o}gliche Rolle von AUM im Rahmen der durch den epidermalen Wachstumsfaktor (EGF) ausgel{\"o}sten Tyrosin-Phosphorylierung in einer Spermatogonien Zelllinie (GC-1 spg-Zellen) analysiert. So konnte nachgewiesen werden, dass die {\"U}berexpression von AUM zu einer moderaten Abnahme Tyrosin phosphorylierter Proteine nach EGF-Stimulation f{\"u}hrte. Im Gegensatz dazu l{\"o}ste jedoch die durch RNAInterferenz vermittelte Depletion von endogenem AUM einen robusten Anstieg Tyrosinphosphorylierter Proteine aus, zu denen auch der EGF-Rezeptor selbst z{\"a}hlt. Zus{\"a}tzlich zu dem EGF-Rezeptor wurde die Src-Kinase im Zuge des Phosphopeptid- Screens als m{\"o}gliches AUM Substrat identifiziert. Daher wurden in vitro Kinase/Phosphatase-Assays mit gereinigtem Src und AUM durchgef{\"u}hrt. Mit diesem Ansatz konnte erstmals gezeigt werden, dass AUM in der Lage ist, die Src-Kinase zu aktivieren, w{\"a}hrend Src AUM phosphoryliert und die AUM Phosphatase-Aktivit{\"a}t blockiert. Diese Ergebnisse deuten auf eine gekoppelte, wechselseitige Regulation von AUM und Src hin. Obwohl die Details dieser Regulation derzeit noch unklar sind, zeigen unsere initialen Ergebnisse, dass AUM die Src-Aktivit{\"a}t unabh{\"a}ngig von seiner Phosphatase Aktivit{\"a}t steigert, w{\"a}hrend Src die AUM Phosphatase-Aktivit{\"a}t Kinase-abh{\"a}ngig vermindert. Auf zellul{\"a}rer Ebene sind AUM-depletierte Zellen durch Ver{\"a}nderungen der Aktin- Zytoskelett-Dynamik und der Zelladh{\"a}sion charakterisiert. So weisen AUM-defiziente Zellen stabilisierte Aktin Streßfasern und vergr{\"o}ßerte fokale Adh{\"a}sionen auf. Weiterhin sind AUMdepletierte Zellen durch ein beschleunigtes spreading auf Fibronektin gekennzeichnet. Wir haben mit AUM ein bisher nicht beschriebenes Mitglied der Familie Aspartat-abh{\"a}ngiger Phosphatasen entdeckt. In dieser Arbeit ist es gelungen, AUM phylogenetisch, biochemisch und zellbiologisch zu charakterisieren. Unsere Ergebnisse legen nahe, dass AUM einen wichtigen, neuartigen Regulator der Src-vermittelten Zytoskelett-Dynamik im Rahmen der Zelladh{\"a}sion und Migration darstellt.}, subject = {Tyrosin}, language = {en} } @article{DaenikenFriederichLutzetal.1981, author = {D{\"a}niken, A. von and Friederich, U. and Lutz, Werner K. and Schlatter, C.}, title = {Tests for mutagenicity in Salmonella and covalent binding to DNA and protein in the rat of the riot control agent o-chlorobenzylidene malononitrile (CS)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61073}, year = {1981}, abstract = {The aim of this study was to determine whether o-chlorobenzylidene malononitrile ( CS) exhibits any genotoxic activity towards Salmonella or mammalian DNA in vivo. CS was synthesized with a [\(^{14}\)C]-label at the benzylic carbon atom. It was administered i. p. at a dose level of 13 mg/kg (1 mCi/kg) to young adult male rats. Liverand kidney DNA was isolated after 8, 25, and 75 h. The radioactivity was at (liver, 8 and 75 h) or below (all other samples) the limit of detection of 3 dpm. Therefore, a possible binding of CS to DNA is at least 10\(^5\) times lower than that of the strong hepatocarcinogen aflatoxin B1, and 4,000 times lower than that of vinyl chloride. In contrast to this lack of DNA binding, but in agreement with the chemical reactivity of CS, a binding to nuclear proteins could be detected with specific activities ranging between 50 and 121 dpm/mg for liver and between 3 and 41 dpm/mg for kidney. Protein binding could well be responsible for its pronounced cytotoxic effects. Cs was also tested in the Ames Salmonella/microsome assay. Strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were used with or without pre-incubation. Only with strain TA 100 and only without pre-incubation, a doubling of the number of revertants was detectable at the highest dose Ievels used, 1,000 and 2,000 !lg CS per plate. With pre-incubation of TA 100 with CS, a slight increase of the number of revertants was seen at 100 and 500 !lg per plate, and a subsequent fall below control values at 1,000 J.tg. A check for the number of surviving bacteria revealed a strong bacteriotoxicity of the higher doses of es so that the calculated mutation frequencies, i.e., the oumber of revertants per number of surviving bacteria, increased with doses up to 500 !J.g. This toxicity could be counteracted in part by the addition of increasing amounts of rat liver microsomes. In the view of these results, and taking into account the rare and low exposure of man, it is concluded that CS will not create a risk for the induction of point mutations or of carcinogenic processes mediated by DNA binding.}, subject = {Toxikologie}, language = {en} } @article{DaenikenLutzJaeckhetal.1984, author = {D{\"a}niken, A. von and Lutz, Werner K. and J{\"a}ckh, R. and Schlatter, C.}, title = {Investigation of the potential for binding of Di(2-ethylhexyl) phthalate (DEHP) and Di(2-ethylhexyl) adipate (DEHA) to liver DNA in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61004}, year = {1984}, abstract = {Investigation of the Potential for Binding of Di(2-ethylhexyl) Phthalate (DEHP) and Di(2- ethylhexyl) Adipate (DEHA) to Liver DNA in Vivo. VON D{\"A}NIKEN, A., LUTZ, W. K., J{\"A}CKH, R., AND ScHLATTER, C. (1984). Toxico/. App/. Pharmaco/. 73, 373-387. It was the aim oftbis investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or witbout pretreatment for 4 weeks with 1\% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [\(^{14}\)H]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The Iimit of detection, CBI < 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [\(^{14}\)C]- and [\(^{3}\)H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where tbe carcinogenmodified nucleoside or base adducts are expected. The respective Iimits of detection were at 0.07 and 0.04 CBI units for the \(^{14}\)C and \(^{3}\)H Iabels, respectively. The experiments with [\(^{14}\)C]- and [\(^{3}\)H]DEHA, labeled in the alcobol moiety and administered to mice, revealed aminute radioactivity of <50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which bad been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for \(^{14}\)C and 0.01 to 0.12 for \(^{3}\)H resulted. Determination of the level· of \(^{14}\)C02 expiration revealed a linear correlation with the speciftc activity of DNA. Experiments with 2-ethyl[ 1-\(^{14}\)C]hexanol perfonned with both rats and mice allowed the conclusion tbat most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01. Pretreatment of the animals witb unlabeled compound bad no effect on the DNA radioactivities in either species. The present negative data, in conjunction witb other negative short-term tests for mutagenicity, strongly indicate that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP and DEHA in rodents.}, subject = {Toxikologie}, language = {en} } @article{DaenikenLutzSchlatter1981, author = {D{\"a}niken, A. von and Lutz, Werner K. and Schlatter, C.}, title = {Lack of covalent binding to rat liver DNA of the hypolipidemic drugs clofibrate and fenofibrate}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61087}, year = {1981}, abstract = {\(^{14}\)C-Labelled clofibric acid and fenofibric acid were administered p.o. to 200 g male and female rats. After 10 h, liver nuclear DNA and protein were isolated and the radioactivity was determined. Binding to protein was clearly measurable whereas no binding to DNA could be detected from any drug. A comparison of the Iimit of detection of such DNA binding with well-known chemical carcinogens revealed that the known hepatocarcinogenicity of clofibrate cannot be based upon an initiating, DNA damaging, mode of action but must be due to other, nongenotoxic, mechanisms such as peroxisome proliferation, hepatomegaly, or cytotoxicity due to protein binding. The risk assessment in man and the interpretation of the carcinogenicity data for rodents are discussed.}, subject = {Toxikologie}, language = {en} } @article{EberlRebsHoppeetal.2024, author = {Eberl, Hanna and Rebs, Sabine and Hoppe, Stefanie and Sedaghat-Hamedani, Farbod and Kayvanpour, Elham and Meder, Benjamin and Streckfuss-B{\"o}meke, Katrin}, title = {Generation of an RBM20-mutation-associated left-ventricular non-compaction cardiomyopathy iPSC line (UMGi255-A) into a DCM genetic background to investigate monogenetic cardiomyopathies}, series = {Stem Cell Research}, volume = {74}, journal = {Stem Cell Research}, issn = {1873-5061}, doi = {10.1016/j.scr.2023.103290}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350565}, year = {2024}, abstract = {RBM20 mutations account for 3 \% of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient.}, language = {en} } @phdthesis{EmamiNemini2012, author = {Emami-Nemini, Alexander Darius}, title = {Differential parathyroid hormone receptor signaling directed by adaptor proteins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72369}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {The superfamily of G protein-coupled receptors (GPCR) regulates numerous physiological and pathophysiological processes. Hence GPCRs are of significant interest for pharmacological therapy. Embedded into cytoplasmic membranes, GPCRs represent the core of large signaling complexes, which are critical for transduction of exogenous stimuli towards activation of downstream signaling pathways. As a member of the GPCR family B, the parathyroid hormone receptor (PTHR) activates adenylyl cyclases, phospholipases C β as well as mitogen-activated protein kinase-dependent signaling pathways, thereby mediating endocrine and paracrine effects of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. This regulates, calcium homeostasis, bone metabolism and bone development. Paradoxically, PTH is able to induce both catabolic and anabolic bone metabolism. The anabolic effect of PTH is successfully applied in the therapy of severe osteoporosis. Domination of anabolic or catabolic bone-metabolism is entailed by temporal and cell-type specific determinants. The molecular bases are presumably differential arrangements of adaptor proteins within large signaling complexes that may lead to differential activation of signaling pathways, thereby regulating physiological effects. The molecular mechanisms are largely unclear; thus, there is significant interest in revealing a better understanding of PTHR-related adaptor proteins. To identify novel adaptor proteins which direct PTHR signaling pathways, a proteomic screening approach was developed. In this screening, vav2, a guanine-nucleotide exchange factor (GEF) for small GTPases which regulates cytoskeleton reorganization, was found to interact with intracellular domains of PTHR. Evidence is provided that vav2 impairs PTH-mediated phospholipase C β (PLCβ) signaling pathways by competitive interactions with G protein αq subunits. Vice versa, PTH was shown to regulate phosphorylation and subsequent GEF activity of vav2. These findings may thus shed new light on the molecular mechanisms underlying the effects of PTH on bone metabolism by PLC-signaling, cell migration and cytoskeleton organization. In addition to the understanding of intracellular molecular signaling processes, screening for ligands is a fundamental and demanding prerequisite for modern drug development. To this end, ligand binding assays represent a fundamental technique. As a substitution for expensive and potentially harmful radioligand binding, fluorescence-based ligand-binding assays for PTHR were developed in this work. Based on time-resolved fluorescence, several assay variants were established to facilitate drug development for the PTHR.}, subject = {G-Protein gekoppelte Rezeptoren}, language = {en} } @phdthesis{Eman2013, author = {Eman, Maher Othman Sholkamy}, title = {In Vitro and In Vivo Analysis of Insulin-Induced Oxidative Stress and DNA Damage}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69274}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Hyperinsulinemia, a condition with excessively high insulin blood levels, is related to an increased cancer incidence. Diabetes mellitus, metabolic syndrome, obesity and polycystic ovarian syndrome are the most common of several diseases accompanied by hyperinsulinemia. Since an elevated cancer risk especially for colon and kidney cancers, was reported for those patients, we investigated for the first time the induction of genomic damage by insulin mainly in HT29 (human colon cells), LLC-PK1 (pig kidney cells), HK2 (human kidney cells) and peripheral lymphocytes, and to confirm the genotoxicity of insulin in other cells from different tissues. To ascertain that the insulin effects were not only limited to permanent cell lines, rat primary colon, kidney, liver and fatty tissue cells were also studied. To connect the study and the findings to in vivo conditions, two in vivo models for hyperinsulinemia were used; Zucker diabetic fatty rats in a lean and diabetic state infused with different insulin concentrations and peripheral lymphocytes from type 2 diabetes mellitus patients. First, the human colon adenocarcinoma cells (HT29) showed significant elevation of DNA damage using comet assay and micronucleus frequency analysis upon treatment with 5 nM insulin in standard protocols. Extension of the treatment to 6 days lowered the concentration needed to reach significance to 0.5-1 nM. Insulin enhanced the cellular ROS production as examined by the oxidation of the dyes 2´,7´-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium (DHE). The FPG modified comet assay and the reduction of damage by the radical scavenger tempol connected the insulin-mediatedDNA damage to ROS production. To investigate the sources of ROS upon insulin stimulation, apocynin and VAS2870 as NADPH oxidase inhibitors and rotenone as mitochondrial inhibitor were applied in combination with insulin and all of them led to a reduction of the genomic damage. Investigation of the signaling pathway started by evaluation of the binding of insulin to its receptor and to the IGF-1 receptor. The results showed the involvement of both receptors in the signaling mechanism. Following the activation of both receptors, PI3K activation occurs leading to phosphorylation of AKT which in turn activates two pathways for ROS production, the first related to mitochondria and the second through activation of Rac1 , resulting in the activation of Nox1. Both pathways could be activated through AKT or through the mitochondrial ROS which in turn could activates Nox1. Studying another human colon cancer cell line, Caco-2 and rat primary colon cells in vitro confirmed the effect of insulin on cellular chromatin. We conclude that pathophysiological levels of insulin can cause DNA damage in colon cells, which may contribute to the induction or progression of colon cancer. Second, in kidney cells, insulin at a concentration of 5 nM caused a significant increase in DNA damage in vitro. This was associated with the formation of reactive oxygen species (ROS). In the presence of antioxidants, blockers of the insulin and IGF-1 receptors, and a phosphatidylinositol 3-kinases (PI3K) inhibitor, the insulin mediated DNA damage was reduced. Phosphorylation of AKT was increased and p53 accumulated. Inhibition of the mitochondrial and NADPH oxidase related ROS production reduced the insulin mediated damage. In primary rat cells insulin also induced genomic damage. HK2 cells were used to investigate the mechanistic pathway in the kidney The signaling is identical to the one in the colon cells untill the activation of the mitochondrial ROS production, because after the activation of PI3K activation of Nox4 occurs at the same time across talk between mitochondria and Nox4 activation has been suggested and might play a role in the observed effects. In the in vivo model, kidneys from healthy, lean ZDF rats, which were infused with insulin to yield normal or high blood insulin levels, while keeping blood glucose levels constant, the amounts of ROS and p53 were elevated in the high insulin group compared to the control level group. ROS and p53 were also elevated in diabetic obese ZDF rats. The treatment of the diabetic rats with metformin reduced the DNA oxidation measured as 8-oxodG as well as the ROS production in that group. HL60 the human premyelocytic cells and cultured lymphocytes as models for the hemopoietic system cells showed a significant induction for DNA damage upon treatment with insulin. The diabetic patients also exhibited an increase in the micronucleus formation over the healthy individuals. In the present study, we showed for the first time that insulin induced oxidative stress resulting in genomic damage in different tissues, and that the source of the produced ROS differs between the tissues. If the same mechanisms are active in patients, hyperinsulinemia might cause genomic damage through the induction of ROS contributing to the increased cancer risk, against which the use of antioxidants as well as mitochondrial and NADPH oxidase inhibitors might exert protective effects with cancer preventive potential under certain conditions. Normal healthy human plasma insulin concentrations are in the order of 0.04 nM after overnight fasting and increase to less than about 0.2 nM after a meal. Pathophysiological levels can reach 1 nM and can stay above 0.2 nM for the majority of the daytime yielding condictions close to the insulin concentrations determined in the present study. Whether the observed effects also occur in vivo and whether they actually initiate or promote tumor formation remains to be determined. However, if proof of that can be obtained, our experiments with inhibitors indicate chances for pharmacological intervention applying antioxidants or enzyme inhibitors. It will not be the aim to reduce ROS in any case or as much as possible because ROS have now been recognized as important signaling molecules and participatants in immune defense, but a reduction to physiological levels instead of pathophysiological levels in the context of a disease associated with ROS overproduction might be beneficial.}, subject = {Insulin}, language = {en} } @phdthesis{Engelhardt2001, author = {Engelhardt, Stefan}, title = {Transgene Mausmodelle zur Charakterisierung der Funktion kardialer beta-adrenerger Rezeptoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-1181950}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2001}, abstract = {In der vorliegenden Arbeit wurde die Funktion kardialer beta-adrenerger Rezeptoren mit Hilfe einer Kombination aus transgenen Mausmodellen und physiologischen und molekularbiologischen Methoden untersucht. Durch gezielte {\"U}berexpression des humanen beta1-adrenergen Rezeptors im Herzen transgener M{\"a}use konnte gezeigt werden, daß die chronische Aktivierung dieses Rezeptors eine trophische Wirkung auf die Herzmuskelzellen hat. {\"U}ber einen Zeitraum von mehreren Monaten f{\"u}hrte dies zur Entwicklung einer Herzinsuffizienz. In der menschlichen Herzinsuffizienz kommt es zu einem {\"a}hnlichen Ph{\"a}nomen: Durch deutlich erh{\"o}hte Freisetzung von endogenen Katecholaminen kommt es zu einer chronischen Dauerstimulation kardialer beta1-adrenerger Rezeptoren. Daß diese sch{\"a}dlich ist belegen das hier beschriebene Mausmodell und zudem einige neuere klinische Studien, die zeigen daß eine pharmakologische Blockade beta-adrenerger Rezeptoren zu einer Verminderung der Herzinsuffizienzmortalit{\"a}t f{\"u}hrt. Dieses Mausmodell erlaubte es erstmals den beta1-adrenergen Rezeptor hinsichtlich seiner spontanen Rezeptoraktivit{\"a}t in einem physiologischen Modell zu untersuchen. Dabei zeigte sich, daß der humane beta1-adrenerge Rezeptor spontane Aktivit{\"a}t aufweist, jedoch in einem deutlich geringeren Ausmaß als der beta2-adrenerge Rezeptor. Dies k{\"o}nnte klinisch relevant sein, da klinisch verwendete beta-Rezeptor-Antagonisten die spontane Aktivit{\"a}t des beta1-adrenergen Rezeptors in unserem Modell unterschiedlich stark unterdr{\"u}ckten. In der vorliegenden Arbeit wurde zudem untersucht, ob sich die beiden kardial exprimierten Beta-Rezeptor-Subtypen Beta1 und Beta2 hinsichtlich ihrer Signaltransduktion unterscheiden. Ausgehend von dem Befund, daß die chronische Aktivierung der beiden Subtypen in transgenen Mausmodellen zu deutlich unterschiedlichen Ph{\"a}notypen f{\"u}hrt, wurden verschiedene intrazellul{\"a}re Signalwege auf ihre Aktivierung hin {\"u}berpr{\"u}ft. Abweichend von publizierten, in vitro nach kurzzeitiger Rezeptorstimulation erhobenen Daten zeigte sich, daß die chronische Aktivierung der Rezeptorsubtypen zu einer unterschiedlichen Aktivierung der kardialen MAP-kinasen (ERK) f{\"u}hrt. Die beta1-spezifische Aktivierung dieser Kinasen k{\"o}nnte die beobachtete unterschiedliche Hypertrophieentwicklung in diesen beiden Mausmodellen erkl{\"a}ren. Einen weiteren Schwerpunkt bei der Aufkl{\"a}rung des Mechanismus beta-adrenerg induzierter Hypertrophie bildete die Untersuchung der zellul{\"a}ren Calcium-hom{\"o}ostase. Als fr{\"u}heste funktionelle Ver{\"a}nderung in der Entwicklung einer beta-adrenerg induzierten Herzhypertrophie und -insuffizienz trat dabei eine St{\"o}rung des intrazellul{\"a}ren Calciumtransienten auf. Als m{\"o}glicher Mechanismus f{\"u}r die St{\"o}rung des Calciumhaushalts konnte eine zeitgleich auftretende ver{\"a}nderte Expression des Calcium-regulierenden Proteins Junctin beschrieben werden. Einen neuen therapeutischen Ansatz f{\"u}r die Therapie der Herzinsuffizienz k{\"o}nnten schließlich vielleicht die Untersuchungen zum kardialen Na/H-austauscher ergeben: Es konnte erstmals gezeigt werden, daß der kardiale Na/H-Austauscher maßgeblich an der beta-adrenerg induzierten Herzhypertrophie- und Fibrose-entstehung beteiligt ist und daß die pharmakologische Inhibition dieses Proteins sowohl Hypertrophie als auch die Fibrose wirksam unterdr{\"u}cken kann.}, subject = {Beta-Rezeptor}, language = {de} } @article{EpeHarttigStopperetal.1990, author = {Epe, B. and Harttig, U. and Stopper, Helga and Metzler, M.}, title = {Covalent binding of reactive estrogen metabolites to microtubular protein as a possible mechanism of aneuploidy induction and neoplastic cell transformation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-63478}, year = {1990}, abstract = {Neoplastic cell transfonnation induced by estrogens and some other carcinogen\& such as benzene appears to involve the induction of mitotic aneuploidy rather than DNA damage and point mutations. As metabolic activation may also play an important roJe in the mechanism of carcinogenesis of these nongenotoxic compounds, we have studied the Interaction of reactive quinone metabolites of various estrogens and of benzene with the major microtubular protein, tubulin, in a cell-free system. Covalent binding of the radioactively labeled metabolites to the a- and 13-subunit of tubulin was found to depend on the structure of the metabolite. When the adducted tubulins were tested in vitro for their ability to polymerize to microtubules, Inhibition of microtubule assembly was obsened in every case, although to varying extents. It is proposed that the fonnation of covalent tubulin adducts may impair the formation of mitotic spindies and thus contribute to chromosomal nondisjunction and aneuploidy induction.}, subject = {Toxikologie}, language = {en} } @article{EpeHaeringRamaiahetal.1993, author = {Epe, Bernd and H{\"a}ring, Martin and Ramaiah, Danaboyina and Stopper, Helga and Abou-Elzahab, Mohamed M. and Adam, Waldemar and Saha-M{\"o}ller, Chantu R.}, title = {DNA damage induced by furocoumarin hydroperoxides plus UV (360 nm)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86870}, year = {1993}, abstract = {Wben irradiated at 360 nm, furocoumarins with a hydroperoxide group in a side chain effciently give rise to a type of DNA damage that can best be explained by a photoinduced generation of hydroxyl radicals from the excited pbotosensitizers. The observed DNA damage profiles, i.e. the ratios of single-strand breaks, sites of base loss (AP sites) and base modifications sensitive to fonnamidopyrimidine-DNA glycosylase (FPG protein) and endonuclease m, are similar to the DNA damage profile produced by hydroxyl radicals generated by lonizing radiation or by xanthine and xanthine oxidase in the presence of Fe(III)-EDTA. No such damage is observed with the corresponding furocoumarin alcohols or in the absence of near-UV radiation. The damage caused by the photo-excited hydroperoxides is not influenced by superoxide dismutase (SOD) or catalase or by D2O as solvent. The presence of t-butanol, however, reduces both the formation of single-strand breaks and of base odifications sensitive to FPG protein. The cytotoxicity caused by one of the hydroperoxides in L5178Y mome lymphoma cells is found to be dependent on the near-UV irradiation and to be much higher than that of the corresponding alcohol. Therefore the new type of photoinduced damage occurs inside cells. Intercalating photosensitizers with an attached hydroperoxide group might represent a novel and versatile class of DNA damaging agents, e.g. for phototherapy.}, subject = {DNS-Sch{\"a}digung}, language = {en} } @phdthesis{Eppli2023, author = {Eppli, Nenad}, title = {Untersuchung des Einflusses der ERK1/2-Autophosphorylierung an Threonin 188 auf Mausherzen mittels transgener M{\"a}use mit ubiquit{\"a}rer {\"U}berexpression von ERK2\(^{T188D}\)}, doi = {10.25972/OPUS-21655}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216558}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Die ERK2Thr188-Autophosphoylierung stellt einen regulatorischen Signalweg dar, der infolge einer hypertrophen Stimulation die kardiale Hypertrophie beg{\"u}nstigt. Eine Hemmung dieser Phosphorylierung in Kardiomyozyten verhindert die Ausbildung der kardialen Hypertrophie ohne Beeinflussung der kardioprotektiven Funktionen von ERK1/2. Demgegen{\"u}ber f{\"u}hrt die dauerhafte Simulation zu einem gain-of-function-Ph{\"a}notypen mit ausgepr{\"a}gter Hypertophie, Fibrose und einer reduzierten Herzfunktion. In dieser Arbeit wurde die dauerhafte Simulation ERK2Thr188-Phosphorylierung (T188D) in einem Mausmodell mit ubiquit{\"a}rer Expression dieser Mutation untersucht. Dabei konnte gezeigt werden, dass sich nach Stimulation durch TAC in diesen Tieren ein etwas st{\"a}rkerer hypertropher Ph{\"a}notyp mit vergr{\"o}ßerten Kardiomyozyten, gesteigerter interstitieller Fibrosierung und reduzierter Herzfunktion ausbildet als in M{\"a}usen mit kardiomyozyten-spezifischer {\"U}berexpression diese Mutante. In Fibroblasten- und VSMC-Zelllinien wurde eine gesteigerte Proliferation der T188D-{\"u}berexprimierenden Zellen im Vergleich zu Kontrollen festgestellt. Somit scheint die ERK2Thr188-Phosphorylierung auch in kardialen Nicht-Myozyten einen maladaptiven Einfluss auf das Herz auszu{\"u}ben.}, subject = {Herzhypertrophie}, language = {de} } @article{FathyFawzyHintzscheetal.2019, author = {Fathy, Moustafa and Fawzy, Michael Atef and Hintzsche, Henning and Nikaido, Toshio and Dandekar, Thomas and Othman, Eman M.}, title = {Eugenol exerts apoptotic effect and modulates the sensitivity of HeLa cells to cisplatin and radiation}, series = {Molecules}, volume = {24}, journal = {Molecules}, number = {21}, issn = {1420-3049}, doi = {10.3390/molecules24213979}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193227}, pages = {3979}, year = {2019}, abstract = {Eugenol is a phytochemical present in different plant products, e.g., clove oil. Traditionally, it is used against a number of different disorders and it was suggested to have anticancer activity. In this study, the activity of eugenol was evaluated in a human cervical cancer (HeLa) cell line and cell proliferation was examined after treatment with various concentrations of eugenol and different treatment durations. Cytotoxicity was tested using lactate dehydrogenase (LDH) enzyme leakage. In order to assess eugenol's potential to act synergistically with chemotherapy and radiotherapy, cell survival was calculated after eugenol treatment in combination with cisplatin and X-rays. To elucidate its mechanism of action, caspase-3 activity was analyzed and the expression of various genes and proteins was checked by RT-PCR and western blot analyses. Eugenol clearly decreased the proliferation rate and increased LDH release in a concentration- and time-dependent manner. It showed synergistic effects with cisplatin and X-rays. Eugenol increased caspase-3 activity and the expression of Bax, cytochrome c (Cyt-c), caspase-3, and caspase-9 and decreased the expression of B-cell lymphoma (Bcl)-2, cyclooxygenase-2 (Cox-2), and interleukin-1 beta (IL-1β) indicating that eugenol mainly induced cell death by apoptosis. In conclusion, eugenol showed antiproliferative and cytotoxic effects via apoptosis and also synergism with cisplatin and ionizing radiation in the human cervical cancer cell line.}, language = {en} } @phdthesis{Fazeli2010, author = {Fazeli, Gholamreza}, title = {Signaling in the induction of genomic damage by endogenous compounds}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-55634}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Reactive oxygen species (ROS) are continuously generated in cells and are involved in physiological processes including signal transduction but also their damaging effects on biological molecules have been well described. A number of reports in the literature implicate excessive oxidative stress and/or inadequate antioxidant defense in the pathogenesis of cancer, atherosclerosis, chronic and age related disorders. Several studies have indicated that activation of the renin-angiotensin-aldosterone-system can lead to the formation of ROS. Epidemiological studies have revealed higher renal cell cancer incidences and also higher cancer mortalities in hypertensive individuals. Recently, our group has shown that perfusion of the isolated mouse kidney with Ang II or treatment of several cell lines with Ang II leads to formation of DNA damage and oxidative base modifications. Here, we tried to scrutinize the pathway involved in genotoxicity of Ang II. We confirmed the genotoxicity of Ang II in two kidney cell lines of human origin. Ang II treatment led to the production of superoxide anions which we could hinder when we used the membrane permeable superoxide dismutase (SOD) mimetic TEMPOL. One of the enzymes which is activated in the cells after Ang II treatment and is able to produce ROS is NADPH oxidase. We demonstrated the activation of NADPH oxidase in response to Ang II by upregulation of its p47 subunit using RT-PCR. Also, pPhosphorylation of p47 subunit of NADPH oxidase after Ang II treatment was enhanced. Using two inhibitors we showed that NADPH oxidase inhibition completely prevents DNA damage by Ang II treatment. To differentiate between Nox2 and Nox4 isoforms of NADPH oxidase subunits in the genotoxicity of Ang II, we performed siRNA inhibition and found a role only for Nox4, while Nox2 was not involved. Next, we investigated PKC as a potential activator of NADPH oxidase. We showed that PKC becomes phosphorylated after Ang II treatment and also that inhibition of PKC hinders Ang II from damaging the cells. Our results from using several inhibitors of different parts of the pathway revealed that PKC activation in this pathway is dependent on the action of PLC on membrane phospholipids and production of IP3. IP3 binds to its receptor at endoplasmic reticulum (ER), opening a channel which allows calcium efflux into the cytoplasm. In this manner, both ER calcium stores and extracellular calcium cooperate so that Ang II can exert its genotoxic effect. PLC is activated by AT1R stimulation. We could also show that the genotoxicity of Ang II is mediated via AT1R signaling using the AT1R antagonist candesartan. In conclusion, here we have shown that Ang II is able to damage genomic damage in cell lines of kidney origin. The observed damage is associated with production of ROS. A decrease in Ang II-induced DNA damage was observed after inhibition of G-proteins, PLC, PKC and NADPH oxidase and interfering with intra- as well as extracellular calcium signaling. This leads to the following preliminary model of signaling in Ang II-induced DNA damage: binding of Ang II to the AT1 receptor activates PLC via stimulation of G-proteins, resulting in the activation of PKC in a calcium dependent manner which in turn, activates NADPH oxidase. NADPH oxidase with involvement of its Nox4 subunit then produces reactive oxygen species which cause DNA damage. Dopamine content and metabolism in the peripheral lymphocytes of PD patients are influenced by L-Dopa administration. The PD patients receiving a high dose of L-Dopa show a significantly higher content of dopamine in their lymphocytes compared to PD patients who received a low dose of L-Dopa or the healthy control. Central to many of the processes involved in oxidative stress and oxidative damage in PD are the actions of monoamine oxidase (MAO), the enzyme which is responsible for the enzymatic oxidation of dopamine which leadsing to production of H2O2 as a by-product. We investigated whether dopamine oxidation can cause genotoxicity in lymphocytes of PD patents who were under high dose L-Dopa therapy and afterward questioned the occurrence of DNA damage after dopamine treatment in vitro and tried to reveal the mechanism by which dopamine exerts its genotoxic effect. The frequency of micronuclei in peripheral blood lymphocytes of the PD patients was not elevated compared to healthy age-matched individuals, although the formation of micronuclei revealed a positive correlation with the daily dose of L-Dopa administration in patients who received L-Dopa therapy together with dopamine receptor agonists. In vitro, we describe an induction of genomic damage detected as micronucleus formation by low micromolar concentrations in cell lines with of different tissue origins. The genotoxic effect of dopamine was reduced by addition of the antioxidants TEMPOL and dimethylthiourea which proved the involvement of ROS production in dopamine-induced DNA damage. To determine whether oxidation of dopamine by MAO is relevant in its genotoxicity, we inhibited MAO with two inhibitors, trans-2-phenylcyclopropylamine hydrochloride (PCPA) and Ro 16-6491 which both reduced the formation of micronuclei in PC-12 cells. We also studied the role of the dopamine transporter (DAT) and dopamine type 2 receptor (D2R) signaling in the genotoxicity of dopamine. Inhibitors of the DAT, GBR-12909 and nomifensine, hindered dopamine-induced genotoxicity. These results were confirmed by treatment of MDCK and MDCK-DAT cells, the latter containing the human DAT gene, with dopamine. Only MDCK-DAT cells showed elevated chromosomal damage and dopamine uptake. Although stimulation of D2R with quinpirole in the absence of dopamine did not induce genotoxicity in PC-12 cells, interference with D2R signaling using D2R antagonist and inhibition of G-proteins, phosphoinositide 3 kinase and extracellular signal-regulated kinases reduced dopamine-induced genotoxicity and affected the ability of DAT to take up dopamine. Furthermore, the D2R antagonist sulpiride inhibited the dopamine-induced migration of DAT from cytosol to cell membrane. Overall, the neurotransmitter dopamine causes DNA damage and oxidative stress in vitro. There are also indications that high dose L-Dopa therapy might lead to oxidative stress. Dopamine exerts its genotoxicity in vitro upon transport into the cells and oxidization oxidation by MAO. Transport of dopamine by DAT has the central role in this process. D2R signaling is involved in the genotoxicity of dopamine by affecting activation and cell surface expression of DAT and hence modulating dopamine uptake. We provided evidences for receptor-mediated genotoxicity of two compounds with different mechanism of actions. The involvement of these receptors in many human complications urges more investigations to reveal whether abnormalities in the endogenous compounds-mediated signaling can play a role in the initiation of new conditions like carcinogenesis.}, subject = {Angiotensin II}, language = {en} } @article{FedericoRedentiSturleseetal.2015, author = {Federico, Stephanie and Redenti, Sara and Sturlese, Mattia and Ciancetta, Antonella and Kachler, Sonja and Klotz, Karl-Norbert and Cacciari, Barbara and Moro, Stefano and Spalluto, Giampiero}, title = {The Influence of the 1-(3-Trifluoromethyl-Benzyl)-1H-Pyrazole-4-yl Moiety on the Adenosine Receptors Affinity Profile of Pyrazolo[4,3-e][1,2,4]Triazolo[1,5-c]Pyrimidine Derivatives}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0143504}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137133}, pages = {e0143504}, year = {2015}, abstract = {A new series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) derivatives has been developed in order to explore their affinity and selectivity profile at the four adenosine receptor subtypes. In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A\(_{2B}\) adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl) benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6(3H, 7H)-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA\(_{2B}\) AR but they displayed affinity at the hA\(_3\) AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA\(_3\) AR K\(_i\) = 11 nM) and selectivity (A\(_1\)/A\(_3\) and A\(_{2A}\)/A\(_3\) > 9090; A\(_{2B}\)/A\(_3\) > 909) at the hA\(_3\) AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs.}, language = {en} } @phdthesis{Fink2008, author = {Fink, Kristin}, title = {Toxins in Renal Disease and Dialysis Therapy : Genotoxic Potential and Mechanisms}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-31082}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {In patients suffering from end-stage renal disease who are treated by hemodialysis genomic damage as well as cancer incidence is elevated. One possible cause for the increased genomic damage could be the accumulation of genotoxic substances in the blood of patients. Two possible sources for those toxins have to be considered. The first possibility is that substances from dialysers, the blood tubing system or even contaminated dialysis solutions may leach into the blood of the patients during dialysis. Secondly, the loss of renal filtration leads to an accumulation of substances which are normally excreted by the kidney. If those substances possess toxic potential, they are called uremic toxins. Several of these uremic toxins are potentially genotoxic. Within this thesis several exemplary uremic toxins have been tested for genotoxic effects (homocysteine, homocysteine-thiolactone,leptine, advanced glycated end-products). Additionally, it was analysed whether substances are leaching from dialysers or blood tubing and whether they cause effects in in vitrotoxicity testing. The focus of chemical analytisis was on bisphenol A (BPA), the main component of plastics used in dialysers and dialyser membranes.}, subject = {Bisphenol A}, language = {en} } @phdthesis{Fischer2010, author = {Fischer, Thomas Horst}, title = {Die transkriptionelle Regulation der microRNA-21 im Herzen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-50702}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {MicroRNAs sind kleine, nicht kodierende RNA-Molek{\"u}le, die posttranskriptionell die Genexpression regulieren. Sie binden hierf{\"u}r spezifisch an 3'-UTRs von messenger-RNAs und f{\"u}hren entweder direkt zu deren Abbau oder inhibieren deren Translation. {\"U}ber die Mechanismen, die die Expression von microRNAs regulieren, ist jedoch noch wenig bekannt. Die Tatsache, dass sie als lange Vorl{\"a}ufermolek{\"u}le (pri-microRNAs) durch die RNA-Polymerase-II transkribiert werden, legt die Existenz eines Promotorbereiches nahe, der dem proteinkodierender Gene {\"a}hnelt. Mit Hilfe von microRNA-Arrays konnten wir im linksventrikul{\"a}ren Myokard mehrere bei Herzinsuffizienz deutlich ver{\"a}ndert exprimierte microRNAs identifizieren. Die microRNA-21 ist dabei bereits im Fr{\"u}hstadium der Herzinsuffizienz verst{\"a}rkt exprimiert (Northern Blot). Auch in prim{\"a}ren, kardialen Zellen (Fibroblasten, Kardiomyozyten) wird die microRNA-21 nach Induktion einer Hypertrophie verst{\"a}rkt exprimiert. Weiterf{\"u}hrendes Ziel dieser Arbeit war es nun, diejenigen Mechanismen aufzukl{\"a}ren, die der starken Induktion der microRNA-21 im erkrankten Myokard zu Grunde liegen. Durch bioinformatische Analyse des zugeh{\"o}rigen Promotorbereiches (Trans-Spezies-Konservierung) und Klonierung danach ausgerichteter Fragmente in Luciferase-basierte Reporter-Plasmide konnte ein 118 Basen langer Bereich identifiziert werden, der maßgeblich die Expression der microRNA-21 im Herzen bedingt. Durch Deaktivierung einzelner cis-Elemente konnte die kardiale Expression auf zwei essentielle Transkriptionsfaktorbindungsstellen zur{\"u}ckgef{\"u}hrt werden. Es handelt sich dabei um Erkennungssequenzen f{\"u}r die im Herz bedeutsamen Transkriptionsfaktoren CREB und SRF. Sie liegen in enger r{\"a}umlicher Nachbarschaft ungef{\"a}hr 1150 bp vor der Transkriptionsstartstelle. Die Suppression der Expression dieser beiden Transkriptionsfaktoren mittels geeigneter siRNAs f{\"u}hrte jeweils zu einer signifikanten Aktivit{\"a}tsminderung des microRNA-21-Promotors und konnte somit die vorangehenden Ergebnisse validieren. Durch Generierung einer transgenen Tierlinie, die lacZ unter der Kontrolle des microRNA-21-Promotors exprimiert, werden in naher Zukunft n{\"a}here Aufschl{\"u}sse {\"u}ber die gewebsspezifische Verteilung der microRNA-21-Expresssion in vivo m{\"o}glich sein. Zusammenfassend beschreiben wir hier erstmals den Mechanismus der transkriptionellen Regulation der microRNA-21 im Herzen. Dieser Mechanismus bedingt wahrscheinlich die starke Induktion dieser microRNA bei kardialer Hypertrophie und Herzinsuffizienz.}, subject = {Small RNA}, language = {de} } @article{FischerBelandLutz1993, author = {Fischer, W. H. and Beland, P. E. and Lutz, Werner K.}, title = {DNA adducts, cell proliferation and papilloma latency time in mouse skin after repeated dermal application of DMBA and TPA}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60673}, year = {1993}, abstract = {'lbe mouse skin tumor model was used to investigate whether the Ievel of DNA 8dducts and/or the rate of cell division in the epidermis are indicators of the risk of cancer formation for an individual in an outbred animal popul8tion. A high risk was considered to be reftected by 8 short latency period for the 8ppearance of 8 papilloma. Fernale NMRI mice were treated twice weekly with 2.5 nmol 7 ,12-dimethylbenz[a]antbracene (DMBA) and 3 nmoi12-0-tetradecanoylphorbol-13- 8cetate (TPA) and the appearance of papillomas was registered. The first papilloma 8ppeared after 7.5 weeks. After 17 weeks, when 12 of 14 mice bad 8t least one papilloma, an osmotic minipump deliverlog 5-bromo-2'deoxyuridine (BrdU) was implanted into eacb mouse for 24 h. The mice were killed after 24 h ~d the epidermis was analyzed for D:MBA-nucleotide 8dducts by 32p.postlabeling, for the cell number per unit skin length, and for the labeling index for DNA synthesls. Unexpectedly, D:MBA-nucleotide 8dduct Ievels were highest in those anima1s wbich showed the Iongest latency periods. Adduct Ievels were negatively correlated with the 18beling index, indicating that dilution of adducts by cell division was a predominant factor in determining average adduct concentrations. Individual tumor-latency time was not corTelated with either cell ntunber or labeling index. This could be due to the fact that the measurements only provided 8veraged data and gave no infonnation on the specific situation in clones of premalignant cells. Under the conditions of tbis assay, therefore, neither DNA adduct Ievels nor information on the average kinetics of cell division bad a predidive value for the individual amcer risk withln a group of outbred animals receiving the same treatment}, subject = {Toxikologie}, language = {en} } @article{FischerLutz1994, author = {Fischer, W. H. and Lutz, Werner K.}, title = {Short communication : Mouse skin papilloma formation by chronic dermal application of 7,12-dimethylbenz[a]anthracene is not reduced by diet restriction}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60644}, year = {1994}, abstract = {No abstract available}, subject = {Toxikologie}, language = {en} } @article{FreyGassenmaierHofmannetal.2020, author = {Frey, Anna and Gassenmaier, Tobias and Hofmann, Ulrich and Schmitt, Dominik and Fette, Georg and Marx, Almuth and Heterich, Sabine and Boivin-Jahns, Val{\´e}rie and Ertl, Georg and Bley, Thorsten and Frantz, Stefan and Jahns, Roland and St{\"o}rk, Stefan}, title = {Coagulation factor XIII activity predicts left ventricular remodelling after acute myocardial infarction}, series = {ESC Heart Failure}, volume = {7}, journal = {ESC Heart Failure}, number = {5}, doi = {10.1002/ehf2.12774}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236013}, pages = {2354-2364}, year = {2020}, abstract = {Aims Acute myocardial infarction (MI) is the major cause of chronic heart failure. The activity of blood coagulation factor XIII (FXIIIa) plays an important role in rodents as a healing factor after MI, whereas its role in healing and remodelling processes in humans remains unclear. We prospectively evaluated the relevance of FXIIIa after acute MI as a potential early prognostic marker for adequate healing. Methods and results This monocentric prospective cohort study investigated cardiac remodelling in patients with ST-elevation MI and followed them up for 1 year. Serum FXIIIa was serially assessed during the first 9 days after MI and after 2, 6, and 12 months. Cardiac magnetic resonance imaging was performed within 4 days after MI (Scan 1), after 7 to 9 days (Scan 2), and after 12 months (Scan 3). The FXIII valine-to-leucine (V34L) single-nucleotide polymorphism rs5985 was genotyped. One hundred forty-six patients were investigated (mean age 58 ± 11 years, 13\% women). Median FXIIIa was 118 \% (quartiles, 102-132\%) and dropped to a trough on the second day after MI: 109\%(98-109\%; P < 0.001). FXIIIa recovered slowly over time, reaching the baseline level after 2 to 6 months and surpassed baseline levels only after 12 months: 124 \% (110-142\%). The development of FXIIIa after MI was independent of the genotype. FXIIIa on Day 2 was strongly and inversely associated with the relative size of MI in Scan 1 (Spearman's ρ = -0.31; P = 0.01) and Scan 3 (ρ = -0.39; P < 0.01) and positively associated with left ventricular ejection fraction: ρ = 0.32 (P < 0.01) and ρ = 0.24 (P = 0.04), respectively. Conclusions FXIII activity after MI is highly dynamic, exhibiting a significant decline in the early healing period, with reconstitution 6 months later. Depressed FXIIIa early after MI predicted a greater size of MI and lower left ventricular ejection fraction after 1 year. The clinical relevance of these findings awaits to be tested in a randomized trial.}, language = {en} } @phdthesis{Froelich2012, author = {Fr{\"o}lich, Nadine}, title = {Analyse der µ-Opiatrezeptoraktivierung und Signaltransduktion in lebenden Zellen mittels FRET-Mikroskopie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71009}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Der Fluoreszenz-Resonanz-Energie-Transfer ist ein Ph{\"a}nomen, welches erstmals 1948 von Theodor F{\"o}rster beschrieben wurde. Mit der Entwicklung von Fluoreszenzproteinen konnten in Kombination mit Mikroskopietechniken Einblicke in zellbiologische Vorg{\"a}nge gewonnen werden, die durch biochemische oder physiologische Experimente nicht m{\"o}glich sind. Dabei spielt die hohe zeitliche und r{\"a}umliche Aufl{\"o}sung eine wichtige Rolle. Auf dem Forschungsgebiet der GPCR, welche die gr{\"o}ßte Gruppe von Membranproteinen bei den S{\"a}ugetieren darstellen, wurden insbesondere Erkenntnisse {\"u}ber Konformations{\"a}nderungen der Rezeptoren, die Kinetik der Rezeptoraktivierung und die Interaktion mit intrazellul{\"a}ren Signalproteinen gewonnen. Der µ-Opioidrezeptor geh{\"o}rt zur Familie der GPCR und stellt aufgrund seiner analgetischen Wirkungen eine wichtige pharmakologische Zielstruktur dar. Das Ziel dieser Arbeit war sowohl den Rezeptor als auch seine Signalwege mittels FRET-Mikroskopie zu untersuchen. Zun{\"a}chst sollte ein intramolekularer FRET-Sensor des µ-Opioidrezeptors entwickelt werden, dazu wurden basierend auf den Kenntnissen {\"u}ber die Terti{\"a}rstruktur und dem Aufbau bereits bekannter GPCR-Sensoren verschiedene Rezeptorkonstrukte kloniert. Bei den Konstrukten wurden entweder zwei Fluoreszenzproteine oder ein Fluoreszenzprotein und ein Fluorophor-bindendes Tetracysteinmotiv kombiniert. Auch die Positionen der eingef{\"u}gten Sequenzen wurden in den intrazellul{\"a}ren Dom{\"a}nen variiert, da der Rezeptor auf die Modifikationen mit beeintr{\"a}chtigter Membranlokalisation reagierte. Durch die Optimierung wurden Rezeptoren konstruiert, die an der Zellmembran lokalisiert waren. Jedoch zeigte keines der Rezeptorkonstrukte Funktionalit{\"a}t im Hinblick auf die Rezeptoraktivierung. Im zweiten Teil wurden die pharmakologischen Effekte der Metabolite von Morphin am humanen µ-Opioidrezeptor systematisch analysiert. Dazu wurde die F{\"a}higkeit der Metabolite, Gi-Proteine zu aktivieren und β-Arrestin2 zu rekrutieren, mittels FRET-basierter Messungen an lebenden Zellen untersucht. Außerdem wurde die Affinit{\"a}t der Metabolite zum humanen µ Opioidrezeptor anhand der Verdr{\"a}ngung eines radioaktiven Liganden analysiert. Meine Experimente identifizierten eine Gruppe mit stark agonistischen und eine mit schwach agonistischen Eigenschaften. Die starken Partialagonisten aktivieren den Rezeptor bereits bei nanomolaren Konzentrationen, w{\"a}hrend die schwachen Metabolite den Rezeptor erst bei Konzentrationen im mikromolaren Bereich aktivieren. Die Metabolite Normorphin, Morphin-6-Glucuronid und 6-Acetylmorphin zeigen geringere Potenz als Morphin bei der Gi-Aktivierung aber {\"u}berraschenderweise h{\"o}here Potenz und Effizienz f{\"u}r die β-Arrestin-Rekrutierung. Dies deutet auf eine bevorzugte Aktivierung von β-Arrestin2 hin. Die aus diesen Studien gewonnenen Ergebnisse liefern Hinweise darauf, welche Metabolite bei der Signalverarbeitung am µ Opioidrezeptor in vivo beteiligt sind.}, subject = {Opiatrezeptor}, language = {de} } @phdthesis{Foertsch2012, author = {F{\"o}rtsch, Christina}, title = {Pneumolysin: the state of pore-formation in context to cell trafficking and inflammatory responses of astrocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70892}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Pneumolysin, a protein toxin, represents one of the major virulence factors of Streptococcus pneumoniae. This pathogen causes bacterial meningitis with especially high disease rates in young children, elderly people and immunosuppressed patients. The protein toxin belongs to the family of cholesterol-dependent cytolysins, which require membrane cholesterol in order to bind and to be activated. Upon activation, monomers assemble in a circle and undergo conformational change. This conformational change leads to the formation of a pore, which eventually leads to cell lysis. This knowledge was obtained by studies that used a higher concentration compared to the concentration of pneumolysin found in the cerebrospinal fluid of meningitis patients. Thus, a much lower concentration of pneumolysin was used in this work in order to investigate effects of this toxin on primary mouse astrocytes. Previously, a small GTPase activation, possibly leading to cytoskeletal changes, was found in a human neuroblastoma cell line. This led to the hypothesis that pneumolysin can lead to similar cytoskeletal changes in primary cells. The aim of this work was to investigate and characterise the effects of pneumolysin on primary mouse astrocytes in terms of a possible pore formation, cellular trafficking and immunological responses. Firstly, the importance of pore-formation on cytoskeletal changes was to be investigated. In order to tackle this question, wild-type pneumolysin and two mutant variants were used. One variant was generated by exchanging one amino acid in the cholesterol recognising region, the second variant was generated by deleting two amino acids in a protein domain that is essential for oligomerisation. These variants should be incapable of forming a pore and were compared to the wild-type in terms of lytic capacities, membrane binding, membrane depolarisation, pore-formation in artificial membranes (planar lipid bilayer) and effects on the cytoskeleton. These investigations resulted in the finding that the pore-formation is required for inducing cell lysis, membrane depolarisation and cytoskeletal changes in astrocytes. The variants were not able to form a pore in planar lipid bilayer and did not cause cell lysis and membrane depolarisation. However, they bound to the cell membrane to the same extent as the wild-type toxin. Thus, the pore-formation, but not the membrane binding was the cause for these changes. Secondly, the effect of pneumolysin on cellular trafficking was investigated. Here, the variants showed no effect, but the wild-type led to an increase in overall endocytotic events and was itself internalised into the cell. In order to characterise a possible mechanism for internalisation, a GFP-tagged version of pneumolysin was used. Several fluorescence-labelled markers for different endocytotic pathways were used in a co-staining approach with pneumolysin. Furthermore, inhibitors for two key-players in classical endocytotic pathways, dynamin and myosin II, were used in order to investigate classical endocytotic pathways and their possible involvement in toxin internalisation. The second finding of this work is that pneumolysin is taken up into the cell via dynamin- and caveolin-independent pinocytosis, which could transfer the toxin to caveosomes. From there, the fate of the toxin remains unknown. Additionally, pneumolysin leads to an overall increase in endocytotic events. This observation led to the third aim of this work. If the toxin increases the overall rate of endocytosis, the question arises whether toxin internalisation favours bacterial tissue penetration of the host or whether it serves as a defence mechanism of the cell in order to degrade the protein. Thus, several proinflammatory cytokines were investigated, as previous studies describe an effect of pneumolysin on cytokine production. Surprisingly, only interleukin 6-production was increased after toxin-treatment and no effect of endocytotic inhibitors on the interleukin 6-production was observed. The conclusion from this finding is that pneumolysin leads to an increase of interleukin 6, which would not depend on the endocytotic uptake of pneumolysin. The production of interleukin 6 would enhance the production of acute phase proteins, T-cell activation, growth and differentiation. On the one hand, this activation could serve pathogen clearance from infected tissue. On the other hand, the production of interleukin 6 could promote a further penetration of pathogen into host tissue. This question should be further investigated.}, subject = {Streptococcus pneumoniae}, language = {en} } @article{FoertschHuppMaetal.2011, author = {F{\"o}rtsch, Christina and Hupp, Sabrina and Ma, Jiangtao and Mitchell, Timothy J. and Maier, Elke and Benz, Roland and Iliev, Asparouh I.}, title = {Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69084}, year = {2011}, abstract = {Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin's pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20-40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin's lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.}, subject = {Toxikologie}, language = {en} } @article{GimplGerstbergerMaussetal.1990, author = {Gimpl, G. and Gerstberger, R. and Mauss, U. and Klotz, Karl-Norbert and Lang, R. E.}, title = {Solubilization and characterization of active neuropeptide-Y receptors from rabbit kidney}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60375}, year = {1990}, abstract = {Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[ (3-cholamidopropy l)dimethylammonio ]- 1-propanesulfonic acid (CHAPS). In membrane fragmentsandsoluble extracts neuropeptide Y bindingwas time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective Kn and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y bindingwas specifically inhibited by the nonhydrolyzable GTP analog guanosine 5' -0- (3-thiotripbosphate) in a concentration-dependent manner, with IC\(_{50}\) values of 28 and 0.14 \(\mu\)M for membrane- bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. CrossHoking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.}, subject = {Toxikologie}, language = {en} } @phdthesis{Glaser2012, author = {Glaser, Nina}, title = {Influence of natural food compounds on DNA stability}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72872}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Cancer is one of the leading causes of death all over the world. Malnutrition and toxic contaminations of food with substances such as mycotoxins have been thought to account for a high percentage of cancers. However, human diet can deliver both mutagens and components that decrease the cancer risk. Genomic damage could be reduced by food components through different mechanisms such as scavenging of reactive oxygen species. In the first part of this study we tried to investigate the effects of patulin and resveratrol on DNA stability in V79 cells. Patulin is a mycotoxin, which is frequently found in spoiled apples and other fruits. The WHO has established a safety level of 50 µg/L, which is indeed not observed by all manufacturers. The acute toxicity of patulin in high concentrations is well known, however its potential carcinogenicity is still a matter of debate. Therefore we wanted to investigate further steps in the mechanism of patulin-induced genotoxicity. Patulin caused the formation of micronuclei and nucleoplasmic bridges in a dose-dependent manner. Further analysis revealed that patulin induced both kinetochore-negative and positive micronuclei. Time course of incubation indicate a new mechanism for patulin-induced nucleoplasmic bridge formation. We hypothized a mechanism via cross-linking of DNA, which was confirmed by a modified version of comet assay. Incubations of cells with patulin led to an increased number of multinucleated cells and multipolar mitoses. Cell cytometry revealed a G2 arrest by patulin, which might explain the amplification of centrosomes and patulin-induced aneuploidy. Patulin cause a dose-dependent DNA damage in comet assay which was influenced by the cellular GSH content. However, an induction of oxidative stress was just seen with higher concentrations of patulin. Levels of cellular glutathione were increased after 24 h incubation indicating an adaptive response to patulin-induced stress. There is growing interest in polyphenols such as resveratrol which have shown many positive effects on human health. The beneficial properties are partially attributed to their ability to scavenge reactive oxygen species. Co-incubation of V79 cells with patulin and 10 µM of the antioxidant resveratrol led to a slight reduction of micronucleus frequency compared to cells which were just treated with patulin. However, in higher concentrations resveratrol themselves caused the formation of micronuclei in V79 cells. Kinetochore analysis indicated only clastogenic properties for resveratrol but no disturbance of mitosis. The antioxidant properties of resveratrol were shown in ferric reducing antioxidant power (FRAP) assay. However, in cellular system resveratrol in higher concentrations revealed also prooxidative properties, as shown in 2,7-dichlordihydrofluorescein (DCF) assay. The increased level of glutathione after resveratrol treatment might reflect an adaptive response to resveratrol-induced oxidative stress. For the second part of this thesis we investigated the effects of an anthocyanin-rich grape extract on hypertensive Ren-2 rats. Ren-2 rats are an accepted genetically modified rat model for the investigation of hypertension and increased oxidative stress. We divided 23 female Ren-2 rats into three groups. One group was fed with an anthocyanin-rich Dacapo grape extract, one group was treated with the angiotensin converting enzyme (ACE) inhibitor ramipril and the third group was kept without medication during the experiment. After one week untreated group showed a clear increase in systolic and diastolic blood pressure compared to the ramipril treated rats. This was in part attenuated in the animals fed with anthocyanin-rich Dacapo grape extract. Effects on blood pressure were also reflected in an increased thirst of untreated and extract fed animals. Comet assay with cells of kidney and liver revealed a slight protective impact of Dacapo extract on DNA damage compared to the other groups. Similar results were obtained after evaluation of ɣ-H2AX-staining of kidney and heart sections. However, in the small intestine oppositional effects were seen, indicating an increased number of double strand breaks probably due to the high local concentration of polyphenols after oral ingestion. Antioxidative properties of the extract were shown in FRAP assay. However, this effect was not reflected in an increased antioxidative capacity in serum or a protective impact in the dihydroethidium (DHE) assay. The extract showed protective effects on DNA damage in comet assay and ɣ-H2AX-staining, but was not able to reduce hypertension back to the control level of ramipril treated animals. High local concentrations could also result in an increased damage of the affected tissue. Therefore, the administration of such concentrated compounds should be handled with care.}, subject = {Patulin}, language = {en} } @phdthesis{Glaeser2017, author = {Gl{\"a}ser, Katharina}, title = {Einfluss hochfrequenter Felder des Mobilfunks auf das blutbildende System in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145733}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Elektromagnetische Felder (EMF) sind in der Umwelt des Menschen allgegenw{\"a}rtig. Unter Verwendung unterschiedlicher Frequenzen bilden sie die Grundlage zahlreicher Technologien und begegnen uns im Alltag in einer Vielzahl von Anwendungen. Eine sehr wichtige Anwendung von EMF ist die mobile Kommunikation. Die hierf{\"u}r verwendeten Frequenzen liegen im hochfrequenten Bereich und variieren mit dem Mobilfunkstandard. Weit verbreitet ist die GSM- und UMTS-Modulation der zweiten (2G) und dritten Generation (3G). Zum neuesten Mobilfunkstandard z{\"a}hlt LTE (4G). Aus statistischen Daten geht hervor, dass derzeit weltweit mehr als sieben Milliarden Mobilfunk-Endger{\"a}te existieren. Die weitverbreitete und stetig ansteigende Verwendung dieser Technologien verdeutlicht, dass viele Menschen, darunter auch zunehmend Kinder und Jugendliche, regelm{\"a}ßig einer Exposition gegen{\"u}ber EMF ausgesetzt sind. Die wichtigste Expositionsquelle stellt dabei das Mobiltelefon dar, da sich in diesem Szenario die Quelle sehr nah am menschlichen K{\"o}rper befindet. In der Vergangenheit wurden zahlreiche in-vitro- und in-vivo-Untersuchungen sowie epidemiologische Studien durchgef{\"u}hrt, um potentielle, nicht-thermische Effekte von Mobilfunkstrahlung auf biologische Systeme beurteilen zu k{\"o}nnen. Ein vollst{\"a}ndiger Konsens konnte auf der Basis der erhaltenen Ergebnisse jedoch nicht erzielt werden, sodass weiterhin Bedenken zum sch{\"a}dlichen Potential dieser nichtionisierenden Strahlung bestehen. Insbesondere wurden Fragestellungen zu Langzeiteffekten sowie zu Effekten, die speziell bei Kindern eine besondere Rolle spielen, bisher nicht ausreichend adressiert. Kinder k{\"o}nnen empfindlicher auf Umwelteinfl{\"u}sse reagieren und sind im Vergleich zu Erwachsenen teilweise h{\"o}her gegen{\"u}ber EMF exponiert. Dies gilt vor allem f{\"u}r Kopfregionen, in denen sich das aktive, f{\"u}r die H{\"a}matopoese verantwortliche Knochenmark befindet. Vor diesem Hintergrund war es das Ziel der vorliegenden Arbeit, den Einfluss von Mobilfunkstrahlung auf das humane blutbildende System zu untersuchen. Im Fokus standen dabei humane h{\"a}matopoetische Stammzellen, die mit Frequenzen der Mobilfunkstandards GSM (900 MHz), UMTS (1.950 MHz) und LTE (2.535 MHz) jeweils {\"u}ber einen kurzen (4 h) und einen langen (20 h) Zeitraum und mit unterschiedlichen Intensit{\"a}ten (0 W/kg, 0,5 W/kg, 1 W/kg, 2 W/kg und 4 W/kg) exponiert wurden. Vergleichende Experimente erfolgten mit Zellen der Promyelozyten-Zelllinie HL-60. M{\"o}gliche Effekte wurden mit den Endpunkten Apoptose, oxidativer Stress, Zellzyklus, DNA-Schaden und -Reparatur sowie Differenzierung und Epigenetik in Form von Histonacetylierung bewertet. In keinem der genannten Endpunkte konnten klare Effekte durch Mobilfunkstrahlung ausgemacht werden, weder f{\"u}r die h{\"a}matopoetischen Stammzellen, noch f{\"u}r die Zelllinie HL-60. Die einzige Ver{\"a}nderung wurde bei der Quantifizierung von DNA-Sch{\"a}den beobachtet. Hier zeigte sich nach der Kurzzeitexposition der Stammzellen mit der Modulation GSM eine kleine, aber statistisch signifikante Abnahme der DNA-Sch{\"a}den verglichen mit der Scheinexposition. Diese Beobachtung ließ sich in weiteren Replikaten jedoch nicht reproduzieren und wurde daher als nicht biologisch relevant eingestuft. Insgesamt konnte mit dieser Arbeit gezeigt werden, dass durch Mobilfunkstrahlung mit Frequenzen der verbreiteten Modulationen GSM, UMTS und LTE sowie SAR-Werten, die unterhalb und oberhalb des empfohlenen Sicherheitsstandards liegen und typischerweise bei Handytelefonaten auftreten, keine Effekte in Zellen des blutbildenden Systems unter den gegebenen Versuchsbedingungen induziert wurden. Ein besonderer Fokus lag hierbei auf der Reproduzierbarkeit der Ergebnisse. Weiterhin wurden zum ersten Mal humane h{\"a}matopoetische Stammzellen f{\"u}r derartige Untersuchungen eingesetzt. Dies hat insofern eine besondere Bedeutung, als h{\"a}matopoetische Stammzellen aufgrund ihrer multipotenten Eigenschaften eine breitere Analyse mit Hinblick auf die Kanzerogenese und auf das Immunsystem erm{\"o}glichen. Um {\"u}ber die Mobilfunk-Untersuchungen hinaus die h{\"a}matopoetischen Stammzellen besser charakterisieren zu k{\"o}nnen, sowie die Sensitivit{\"a}t von Blutzellen mit unterschiedlichem Differenzierungsstatus zu analysieren, wurden sie anderen Zellen des blutbildenden Systems (undifferenzierte und differenzierte HL-60-Zellen und TK6-Zellen) gegen{\"u}bergestellt. Eine Behandlung der verschiedenen Zelltypen mit mutagenen Substanzen zeigte, dass sich die h{\"a}matopoetischen Stammzellen in den meisten der untersuchten Endpunkte von den Zelllinien unterschieden. Deutliche Abweichungen zeigten sich beim oxidativen Stress, der DNA-Reparatur und der Histonacetylierung; kein Unterschied konnte dagegen bei den DNA-Sch{\"a}den beobachtet werden. Eine erste Interpretation der erhaltenen Ergebnisse ist auf der Grundlage der unterschiedlichen Eigenschaften von Zellen mit abweichendem Differenzierungsstatus m{\"o}glich. Um jedoch eine eindeutige Aussage treffen zu k{\"o}nnen, m{\"u}ssten noch weitere Untersuchungen durchgef{\"u}hrt werden.}, subject = {Mobilfunk}, language = {de} } @article{GmachBathePetersTeluguetal.2022, author = {Gmach, Philipp and Bathe-Peters, Marc and Telugu, Narasimha and Miller, Duncan C. and Annibale, Paolo}, title = {Fluorescence spectroscopy of low-level endogenous β-adrenergic receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {18}, issn = {1422-0067}, doi = {10.3390/ijms231810405}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288277}, year = {2022}, abstract = {The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β\(_1\)- and β\(_2\)-adrenergic receptors (β\(_{1/2}\)-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs.}, language = {en} }