TY - THES A1 - Kramer, Sofia T1 - Hemmung pathologischer kardialer Hypertrophie über das Dimer-Interface von ERK1/2 T1 - Inhibition of pathological cardiac hypertrophy by the dimer interface of ERK1/2 N2 - Die extrazellulär Signal-regulierten Kinasen 1 und 2 (ERK1/2) spielen eine zentrale Rolle bei der Vermittlung kardialer Hypertrophie und dem Zellüberleben. Hypertrophe Stimuli aktivieren ERK1/2, triggern deren Dimerisierung und in der Folge die ERK188-Autophosphorylierung. Diese neu entdeckte Autophosphorylierung ist eine Voraussetzung für den nukleären Import von ERK1/2 und führt zum Entstehen pathologischer kardialer Hypertrophie. Da das Dimer Interface von ERK eine mögliche Zielstruktur darstellt, um selektiv die nukleären Signalwege von ERK zu unterbrechen, wurde untersucht, ob man mit Hemmung der ERK-Dimerisierung eine therapeutische Möglichkeit hat, um pathologische kardiale Hypertrophie zu verhindern. Dazu wurden verschiedene ERK2 Mutanten und Peptide generiert, um die ERK-Dimerisierung zu verhindern. Die Effekte dieser Konstrukte auf die ERK-Dimerisierung und den Kernimport wurden in verschiedenen Zelltypen mittels Fluoreszenzmikroskopie, Co-Immunopräzipitationen und Duolink proximity ligation assays getestet. Es konnte gezeigt werden, dass die Peptide effektiv die ERK-ERK Interaktion nach Stimulation mit Phenylephrin und/oder Carbachol verhindern. Zusätzlich reduzierten die Peptide ERKT188-Phosphorylierung und in der Folge den ERK-Import in den Nukleus und Kardiomyozytenhypertrophie. Normale ERK-Aktivierung wurde jedoch durch die Peptide nicht verhindert. Insgesamt konnte gezeigt werden, dass das ERK-Dimer Interface eine wertvolle Zielstruktur ist, mit dem man nukleäre ERK1/2 Signalwege selektiv unterbrechen und damit effektiv Kardiomyozytenwachstum reduzieren kann, ohne gleichzeitig das Zellüberleben zu gefährden. N2 - The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have a central role in cardiac hypertrophy and cell survival. Hypertrophic stimuli activate ERK1/2, trigger ERK dimerization and subsequently ERKT188-autophosphorylation. This newly discovered autophosphorylation is a prerequisite for nuclear ERK1/2 translocation and leads to the development of pathological cardiac hypertrophy. As the ERK dimer interface is a potential target to selectively interfere with nuclear ERK1/2 signaling, we investigated whether the interference with ERK dimerization may serve as a therapeutic strategy to prevent pathological cardiac hypertrophy. For this, we generated ERK2 mutants and peptide constructs that interfere with ERK dimerization. These constructs were evaluated in various cell types by their effects on nuclear ERK translocation and dimerization by fluorescence microcopy, co-immunoprecipitation and Duolink proximity ligation assays. We showed that the peptides indeed prevented ERK-ERK interaction in response to phenylephrine and/or carbachol stimulation. In addition, the peptides prevented ERKT188-phosphorylation and interfered with all ERK1/2 effects that are associated with ERKT188-phosphorylation e.g. nuclear ERK translocation and cardiomyocyte hypertrophy. Interestingly, the peptide did not inhibit the general activation of ERK1/2. These data indicate that the ERK dimer interface is a valuable target for selective inhibition of nuclear ERK1/2 signaling, that is able to effectively attenuate ERK1/2 mediated cardiomyocyte growth but without impairing cell survival. KW - Dimerisierung KW - Herzhypertrophie KW - Interferenz KW - MAP-Kinase KW - ERK1/2 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233739 ER - TY - THES A1 - Claßen, Alexandra T1 - The ERK-cascade in the pathophysiology of cardiac hypertrophy T1 - Die ERK-Kaskade in der Pathophysiologie der Herzhypertrophie N2 - 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. N2 - ERK1/2 sind bekannte Schlüsselfiguren bei der Entstehung der Herzinsuffizienz. Weitere Komponenten der ERK-Kaskade, insbesondere Raf1, können das Herz jedoch vor Zelltod und ischämischem Schaden schützen. Eine zusätzliche Autophosphorylierung von ERK1 an Thr208 bzw. von ERK2 an Thr188 ermöglicht ERK1/2 die Translokation zum Zellkern und befähigt ERK dort zur Phosphorylierung von nukleosolischen Zielproteinen, welche eine Herzmuskelhypertrophie auslösen. Daher erscheint diese zusätzliche Autophosphorylierung als eine vielversprechende pharmakologische Zielstruktur. In dieser Arbeit wird ein in-silico Modell der ERK-Kaskade im Kardiomyozyten präsentiert. Das Modell ist ein semi-quantitatives Modell und wurde mit den Programmen SQUAD und CellNetAnalyzer getestet. Verschiedene Phosphorylierungs-Zustände von ERK1/2 als auch verschiedene Stimuli (hypertrophe als auch nicht-hypertrophe) können mit dem Modell reproduziert werden. Mit dem präsentierten in-silico Modell können sowohl zeitliche Abläufe als auch synergistische und antagonistische Effekte vorhergesagt werden. Die simulierten zeitlichen Abläufe wurden durch in-vitro Experimente validiert. SQUAD wurde hauptsächlich für die Modellierung von zeitlichen Abläufen und Schwellenwerte genutzt, wohingegen CellNetAnalyzer vor allen Dingen zur Analyse von Fließgleichgewichten und Rückkopplungs-Mechanismen genutzt wurde. Darüberhinaus wurden Zielstrukturen von ERK1/2, welche zusätzlich an der Entstehung der Herzhypertrophie mitwirken, identifiziert. Diese umfassen unter anderem Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 und SPIN90. Gen-Expressions-Datensätze von Kardiomyozyten nach TAC (transverse aortic constriction) wurden analysiert. Diese wurden mit den im Model vorhandenen Strukturen verglichen und signifikant veränderte Expressionslevel wurden identifiziert. Veränderungen der Ultrastruktur des Kardiomyozyten sind das Ergebnis der induzierten Hypertrophie. KW - Herzhypertrophie KW - Systembiologie KW - ERK-cascade KW - ERK-Kaskade KW - cardiac hypertrophy KW - in-silico model KW - In-silico Modell Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229664 ER - TY - JOUR A1 - Cheng, Cheng A1 - Othman, Eman M. A1 - Stopper, Helga A1 - Edrada-Ebel, RuAngelie A1 - Hentschel, Ute A1 - Abdelmohsen, Usama Ramadan T1 - Isolation of petrocidin A, a new cytotoxic cyclic dipeptide from the marine sponge-derived bacterium \(Streptomyces\) sp. SBT348 JF - Marine Drugs N2 - 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. KW - biology KW - sponges KW - actinomycetes KW - streptomyces KW - cyclic dipeptide KW - cytotoxic Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172644 VL - 15 IS - 12 ER - TY - JOUR A1 - Segerer, Gabriela A1 - Hadamek, Kerstin A1 - Zundler, Matthias A1 - Fekete, Agnes A1 - Seifried, Annegrit A1 - Mueller, Martin J. A1 - Koentgen, Frank A1 - Gessler, Manfred A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation JF - Scientific Reports N2 - Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive Pgp\(^{D34N}\) mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation. KW - cell proliferation KW - DNA metabolism KW - lipidomics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181094 VL - 6 ER - TY - JOUR A1 - Maurer, Jana A1 - Hupp, Sabrina A1 - Bischoff, Carolin A1 - Foertsch, Christina A1 - Mitchell, Timothy J. A1 - Chakraborty, Trinad A1 - Iliev, Asparouh I. T1 - Distinct neurotoxicity profile of listeriolysin O from \(Listeria\) \(monocytogenes\) JF - Toxins N2 - Cholesterol-dependent cytolysins (CDCs) are protein toxins that originate from Gram-positive bacteria and contribute substantially to their pathogenicity. CDCs bind membrane cholesterol and build prepores and lytic pores. Some effects of the toxins are observed in non-lytic concentrations. Two pathogens, \(Streptococcus\) \(pneumoniae\) and \(Listeria\) \(monocytogenes\), cause fatal bacterial meningitis, and both produce toxins of the CDC family—pneumolysin and listeriolysin O, respectively. It has been demonstrated that pneumolysin produces dendritic varicosities (dendrite swellings) and dendritic spine collapse in the mouse neocortex, followed by synaptic loss and astrocyte cell shape remodeling without elevated cell death. We utilized primary glial cultures and acute mouse brain slices to examine the neuropathological effects of listeriolysin O and to compare it to pneumolysin with identical hemolytic activity. In cultures, listeriolysin O permeabilized cells slower than pneumolysin did but still initiated non-lytic astrocytic cell shape changes, just as pneumolysin did. In an acute brain slice culture system, listeriolysin O produced dendritic varicosities in an NMDA-dependent manner but failed to cause dendritic spine collapse and cortical astrocyte reorganization. Thus, listeriolysin O demonstrated slower cell permeabilization and milder glial cell remodeling ability than did pneumolysin and lacked dendritic spine collapse capacity but exhibited equivalent dendritic pathology. KW - medicine KW - listeriolysin O KW - meningitis KW - acute slices KW - variocosities KW - dendritic spines Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172130 VL - 9 IS - 1 ER - TY - JOUR A1 - Schönegge, Anne-Marie A1 - Gallion, Jonathan A1 - Picard, Louis-Philippe A1 - Wilkins, Angela D. A1 - Le Gouill, Christian A1 - Audet, Martin A1 - Stallaert, Wayne A1 - Lohse, Martin J. A1 - Kimmel, Marek A1 - Lichtarge, Olivier A1 - Bouvier, Michel T1 - Evolutionary action and structural basis of the allosteric switch controlling β\(_2\)AR functional selectivity JF - Nature Communications N2 - Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the β\(_2\)-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or βarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity. KW - toxicology KW - functional clustering KW - molecular modelling KW - protein design KW - receptor pharmacology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172268 VL - 8 ER - TY - JOUR A1 - Adaku Chilaka, Cynthia A1 - Mally, Angela T1 - Mycotoxin Occurrence, Exposure and Health Implications in Infants and Young Children in Sub-Saharan Africa: A Review JF - Foods N2 - 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. KW - mycotoxin KW - occurrence KW - exposure KW - child health KW - sub-Saharan Africa Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219250 SN - 2304-8158 VL - 9 IS - 11 ER - TY - JOUR A1 - Klaunig, James E. A1 - Dekant, Wolfgang A1 - Plotzke, Kathy A1 - Scialli, Anthony R. T1 - Biological relevance of decamethylcyclopentasiloxane (D5) induced rat uterine endometrial adenocarcinoma tumorigenesis: Mode of action and relevance to humans JF - Regulatory Toxicology and Pharmacology N2 - Decamethylcyclopentasiloxane (D5) is a cyclic siloxane used in the production and formulation of consumer products with potential exposure to manufacturing workers, consumer, and the general public. Following a combined 2-year inhalation chronic bioassay performed in Fischer 344 (F344) rats, an increase in uterine endometrial adenocarcinomas was noted at the highest concentration to which animals were exposed. No other neoplasms were detected. In this study, a dose of 160 ppm produced an incidence of 8% endometrial adenocarcinomas. Based on a number of experimental studies with D5, the current manuscript examines the biological relevance and possible modes of action for the uterine endometrial adenocarcinomas observed in the rat following chronic exposure to D5. Variable rates of spontaneous uterine endometrial adenocarcinomas have been reported for untreated F344 CrIBr rats. As such, we concluded that the slight increase in uterine endometrial adenocarcinomas observed in the D5 chronic bioassay might not be the result of D5 exposure but may be related to variability of the spontaneous tumor incidence in this strain of rat. However, if the uterine endometrial adenocarcinomas are related to D5-exposure, alteration in the estrous cycle in the aging F344 rat is the most likely mode of action. D5 is not genotoxic or estrogenic. The alteration in the estrous cycle is caused by a decrease in progesterone with an increase in the estrogen:progesterone ratio most likely induced by a decrease in prolactin concentration. Available data support that exposure to D5 influences prolactin concentration. Although the effects on prolactin concentrations in a number of experiments were not always consistent, the available data support the conclusion that D5 is acting via a dopamine receptor agonist-like mechanism to alter the pituitary control of the estrous cycle. In further support of this mode of action, studies in F344 aged animals showed that the effects of D5 on estrous cyclicity produced a response consistent with a dopamine-like effect and further suggest that D5 is accelerating the aging of the reproductive endocrine system in the F344 rat utilized in this study. This mode of action for uterine endometrial adenocarcinoma tumorigenesis is not relevant for humans. KW - Reproductive toxicity KW - Carcinogenicity KW - Silicones KW - Enzyme induction KW - Uterine tumors KW - Rat Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190952 VL - 74 IS - Supplement ER - TY - JOUR A1 - Balasubramanian, Srikkanth A1 - Othman, Eman M. A1 - Kampik, Daniel A1 - Stopper, Helga A1 - Hentschel, Ute A1 - Ziebuhr, Wilma A1 - Oelschlaeger, Tobias A. A1 - Abdelmohsen, Usama R. T1 - Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation JF - Frontiers in Microbiology N2 - 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. KW - medicine KW - marine sponges KW - actinomycetes KW - Streptomyces KW - staphilococci KW - biofilms KW - contact lens Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171844 VL - 8 ER - TY - JOUR A1 - Dekant, Wolfgang A1 - Klaunig, James E. T1 - Toxicology of decamethylcyclopentasiloxane (D5) JF - Regulatory Toxicology and Pharmacology N2 - 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. KW - Prolactin KW - Fischer 344 rats KW - MMQ cells KW - Reproductive toxicity KW - Carcinogenicity KW - Silicones KW - Enzyme induction Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190914 VL - 74 IS - Supplement ER -