TY  - JOUR
A1  - Rietjens, Ivonne M. C. M.
A1  - Dussort, P.
A1  - Günther, Helmut
A1  - Hanlon, Paul
A1  - Honda, Hiroshi
A1  - Mally, Angela
A1  - O'Hagan, Sue
A1  - Scholz, Gabriele
A1  - Seidel, Albrecht
A1  - Swenberg, James
A1  - Teeguarden, Justin
A1  - Eisenbrand, Gerhard
T1  - Exposure assessment of process-related contaminants in food by biomarker monitoring
JF  - Archives of Toxicology
N2  - Exposure assessment is a fundamental part of the risk assessment paradigm, but can often present a number of challenges and uncertainties. This is especially the case for process contaminants formed during the processing, e.g. heating of food, since they are in part highly reactive and/or volatile, thus making exposure assessment by analysing contents in food unreliable. New approaches are therefore required to accurately assess consumer exposure and thus better inform the risk assessment. Such novel approaches may include the use of biomarkers, physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry, and/or duplicate diet studies. This review focuses on the state of the art with respect to the use of biomarkers of exposure for the process contaminants acrylamide, 3-MCPD esters, glycidyl esters, furan and acrolein. From the overview presented, it becomes clear that the field of assessing human exposure to process-related contaminants in food by biomarker monitoring is promising and strongly developing. The current state of the art as well as the existing data gaps and challenges for the future were defined. They include (1) using PBK modelling and duplicate diet studies to establish, preferably in humans, correlations between external exposure and biomarkers; (2) elucidation of the possible endogenous formation of the process-related contaminants and the resulting biomarker levels; (3) the influence of inter-individual variations and how to include that in the biomarker-based exposure predictions; (4) the correction for confounding factors; (5) the value of the different biomarkers in relation to exposure scenario's and risk assessment, and (6) the possibilities of novel methodologies. In spite of these challenges it can be concluded that biomarker-based exposure assessment provides a unique opportunity to more accurately assess consumer exposure to process-related contaminants in food and thus to better inform risk assessment.
KW  - Dietary process-related contaminants
KW  - Biomarkers
KW  - External exposure assessment
KW  - Physiologically based kinetic models
KW  - Risk assessment
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226268
VL  - 92
IS  - 1
ER  - 
TY  - JOUR
A1  - Salinger, Tim
A1  - Hu, Kai
A1  - Liu, Dan
A1  - Taleh, Scharoch
A1  - Herrmann, Sebastian
A1  - Oder, Daniel
A1  - Gensler, Daniel
A1  - Müntze, Jonas
A1  - Ertl, Georg
A1  - Lorenz, Kristina
A1  - Frantz, Stefan
A1  - Weidemann, Frank
A1  - Nordbeck, Peter
T1  - Association between Comorbidities and Progression of Transvalvular Pressure Gradients in Patients with Moderate and Severe Aortic Valve Stenosis
JF  - Cardiology Research and Practice
N2  - Background. Fast progression of the transaortic mean gradient (P-mean) is relevant for clinical decision making of valve replacement in patients with moderate and severe aortic stenosis (AS) patients. However, there is currently little knowledge regarding the determinants affecting progression of transvalvular gradient in AS patients. Methods. This monocentric retrospective study included consecutive patients presenting with at least two transthoracic echocardiography examinations covering a time interval of one year or more between April 2006 and February 2016 and diagnosed as moderate or severe aortic stenosis at the final echocardiographic examination. Laboratory parameters, medication, and prevalence of eight known cardiac comorbidities and risk factors (hypertension, diabetes, coronary heart disease, peripheral artery occlusive disease, cerebrovascular disease, renal dysfunction, body mass index >= 30 Kg/m(2), and history of smoking) were analyzed. Patients were divided into slow (P-mean < 5 mmHg/year) or fast (P-mean >= 5 mmHg/year) progression groups. Results. A total of 402 patients (mean age 78 +/- 9.4 years, 58% males) were included in the study. Mean follow-up duration was 3.4 +/- 1.9 years. The average number of cardiac comorbidities and risk factors was 3.1 +/- 1.6. Average number of cardiac comorbidities and risk factors was higher in patients in slow progression group than in fast progression group (3.3 +/- 1.5 vs 2.9 +/- 1.7; P = 0.036). Patients in slow progression group had more often coronary heart disease (49.2% vs 33.6%; P = 0.003) compared to patients in fast progression group. LDL-cholesterol values were lower in the slow progression group (100 +/- 32.6 mg/dl vs 110.8 +/- 36.6 mg/dl; P = 0.005). Conclusion. These findings suggest that disease progression of aortic valve stenosis is faster in patients with fewer cardiac comorbidities and risk factors, especially if they do not have coronary heart disease. Further prospective studies are warranted to investigate the outcome of patients with slow versus fast progression of transvalvular gradient with regards to comorbidities and risk factors.
KW  - Valvular heart-desease
KW  - Prognostic impact
KW  - Risk-factors
KW  - Chronic heart-failure
KW  - Prevalence
KW  - mild
KW  - statins
KW  - therapy
KW  - mortality
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227291
ER  - 
TY  - JOUR
A1  - Weigand, Isabel
A1  - Ronchi, Cristina L.
A1  - Rizk-Rabin, Marthe
A1  - Dalmazi, Guido Di
A1  - Wild, Vanessa
A1  - Bathon, Kerstin
A1  - Rubin, Beatrice
A1  - Calebiro, Davide
A1  - Beuschlein, Felix
A1  - Bertherat, Jérôme
A1  - Fassnacht, Martin
A1  - Sbiera, Silviu
T1  - Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas
JF  - Scientific Reports
N2  - Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.
KW  - kinases
KW  - immunohistochemistry
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157952
VL  - 7
IS  - 49
ER  - 
TY  - THES
A1  - Bathe-Peters, Marc
T1  - Spectroscopic approaches for the localization and dynamics of β\(_1\)- and β\(_2\)-adrenergic receptors in cardiomyocytes
T1  - Spektroskopieansätze zur Bestimmung der Lokalisation und Dynamiken von β\(_1\)- und β\(_2\)-Adrenozeptoren in Kardiomyozyten
N2  - 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.
N2  - Im Herzen werden der β\(_1\)-adrenerge Rezeptor (AR) und der β\(_2\)-AR, zwei prototypische GPCR, durch die Hormone Adrenalin und Noradrenalin aktiviert. Dabei interagieren beide Rezeptoren mit dem stimulatorischen G\(_s\) Protein, bewirken eine Erhöhung des cyclischen Adenosinmonophosphates (cAMP) und beeinflussen die Kontraktionskraft und Frequenz des Herzens nach einem Stimulus. Jedoch hat die Aktivierung des β\(_1\)-ARs, nicht des β\(_2\)-ARs, auch weitere Effekte, wie z.B. Veränderungen in der Transkription von Genen. Dies wiederum führt zu Spekulationen, wie solch unterschiedliche Effekte von Rezeptoren hervorgerufen werden können, die gleiche Signalwege bedienen.
In dieser Arbeit wird untersucht, ob dieses unterschiedliche Verhalten durch eine ungleiche Verteilung dieser beiden Rezeptoren in adulten Kardiomyozyten hervorgerufen werden könnte. Dazu wird die Lokalisation und die Dynamik dieser endogenen Rezeptoren in der Plasmamembran sowie im T-tubulären Netzwerk von intakten adulten Kardiomyozyten, unter Entwicklung und Verwendung hochsensitiver Fluoreszenzspektroskopiemethoden, bestimmt. Dies ermöglicht die örtliche und dynamische Eingrenzung des β\(_2\)-adrenergen Rezeptors unter endogener Expression ausschließlich auf das T-tubuläre Netzwerk. Dementgegen stellt sich heraus, dass sich der β\(_1\)-adrenerge Rezeptor ubiquitär auf der äußeren Membran und den T-Tubuli befindet und diffundiert. In β\(_2\)-AR überexprimierenden transgenen Kardiomyozyten hingegen werden diese Kompartments nicht beibehalten und es findet eine Umverteilung der Rezeptoren, auch unter Einbezug der Zelloberfläche, statt. Diese Daten können stärker darauf hindeuten, dass einige Rezeptorsubtypen sich gezielt und spezifisch bestimmte Zelloberflächen aussuchen, um somit ihre verschiedenen Signale und funktionären Effekte erzeugen zu können.
Zu den Techniken, die in dieser Arbeit die Bestimmung der Lokalisation und der Dynamiken der niedrig exprimierten adrenergen Rezeptoren zulassen, gehört die Anwendung von Fluoreszenzspektroskopiemethoden in Kombination mit einem fluoreszierenden β-adrenergen Antagonisten. Weitere Techniken, die im Rahmen dieser Arbeit entwickelt wurden und in weiterführenden Studien aufschlussreiche Erkenntnisse liefern könnten, umfassen die Entwicklung eines Setups aus einer Kombination aus Weitfeld- und Konfokalmikroskopie und die Implementierung eines stabilen Autofokus mit Hilfe einer elektrisch veränderbaren Linse.
KW  - G-Protein gekoppelte Rezeptoren
KW  - Beta-Adrenozeptor
KW  - Kardiomyozyt
KW  - Fluoreszenzmikroskopie
KW  - Fluoreszenzkorrelationsspektroskopie
KW  - Fluorescence
KW  - Fluorescence Microscopy
KW  - G Protein-Coupled Receptor
KW  - Autofocus
KW  - Microscopy
KW  - Beta-Adrenergic Receptor
KW  - Cardiomyocyte
KW  - Fluorescence Correlation Spectroscopy
KW  - FCS
KW  - GPCR
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-258126
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  - Sedaghat-Hamedani, Farbod
A1  - Rebs, Sabine
A1  - Kayvanpour, Elham
A1  - Zhu, Chenchen
A1  - Amr, Ali
A1  - Müller, Marion
A1  - Haas, Jan
A1  - Wu, Jingyan
A1  - Steinmetz, Lars M.
A1  - Ehlermann, Philipp
A1  - Streckfuss-Bömeke, Katrin
A1  - Frey, Norbert
A1  - Meder, Benjamin
T1  - Genotype complements the phenotype: identification of the pathogenicity of an LMNA splice variant by nanopore long-read sequencing in a large DCM family
JF  - International Journal of Molecular Sciences
N2  - Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.
KW  - familial DCM
KW  - laminopathy
KW  - long-read sequencing
KW  - nanopore
KW  - induced pluripotent stem cell cardiomyocytes
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290415
SN  - 1422-0067
VL  - 23
IS  - 20
ER  - 
TY  - THES
A1  - Nemec, Katarina
T1  - Modulation of parathyroid hormone 1 receptor (PTH1R) signaling by receptor activity-modifying proteins (RAMPs)
T1  - Regulierung der Signalübertragung des Parathormon 1-Rezeptors (PTH1R) durch Rezeptoraktivitäts-modifizierende Proteine (RAMPs)
N2  - The receptor activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that interact with several G protein-coupled receptors (GPCRs), the largest and pharmacologically most important family of cell surface receptors. RAMPs can regulate GPCR function in terms of ligand-binding, G-protein coupling, downstream signaling, trafficking, and recycling. The integrity of their interactions translates to many physiological functions or pathological conditions.
Regardless of numerous reports on its essential importance for cell biology and pivotal role in (patho-)physiology, the molecular mechanism of how RAMPs modulate GPCR activation remained largely elusive. 
This work presents new insights that add to the common understanding of the allosteric regulation of receptor activation and will help interpret how accessory proteins - RAMPs - modulate activation dynamics and how this affects the fundamental aspects of cellular signaling. Using a prototypical class B GPCR, the parathyroid hormone 1 receptor (PTH1R) in the form of advanced genetically encoded optical biosensors, I examined RAMP's impact on the PTH1R activation and signaling in intact cells. A panel of single-cell FRET and confocal microscopy experiments as well canonical and non-canonical functional assays were performed to get a holistic picture of the signaling initiation and transduction of that clinically and therapeutically relevant GPCR. Finally, structural modeling was performed to add molecular mechanistic details to that novel art of modulation. 
I describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity and kinetics of cAMP accumulation. Additionally, RAMP2 increases PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R and modulates cytosolic ERK1/2 phosphorylation. Structural homology modeling shows that structural motifs governing GPCR-RAMP interaction originate in allosteric hotspots and rationalize functional modulation. Moreover, to interpret the broader role of RAMP's modulation in GPCRs pharmacology, different fluorescent tools to investigate RAMP's spatial organization were developed, and novel conformational biosensors for class B GPCRs were engineered. Lastly, a high throughput assay is proposed and prototyped to expand the repertoire of RAMPs or other membrane protein interactors.
These data uncover the critical role of RAMPs in GPCR activation and signaling and set up a novel platform for studying GPCR modulation. Furthermore, these insights may provide a new venue for precise modulation of GPCR 
function and advanced drug design.
N2  - G Protein-gekoppelte Rezeptoren (GPCRs) bilden die größte und pharmakologisch wichtigste Familie von
Zelloberflächenrezeptoren, die zahlreiche (patho-)physiologische Prozesse im menschlichen Körper steuern. GPCRs
übertragen während des Rezeptoraktivierungsprozesses extrazelluläre Signale in das Zellinnere, wo durch die
extrazelluläre Stimulation Konformationsänderungen des Rezeptorkerns auslöst und die Bindung intrazellulärer
Bindungspartner – G Proteine, G Protein-gekoppelte Rezeptorkinase und Arrestine - ermöglicht. Es handelt sich
also um einen kritischen Prozess in der Signaltransduktion, der durch einige endogene Moleküle wie Ionen, Lipide
oder andere Proteine moduliert werden kann und Auswirkungen auf nachgeschaltete Signalkaskaden hat.
GPCRs bilden gewebeabhängige Oligomere mit ihren interagierenden Partnern, Rezeptor-Aktivitäts-modifizierende
Proteinen (RAMPs), ubiquitär exprimierten Membranproteinen. Bekannt ist, dass sie die Ligandenbindung, die G-
Protein-Kopplung, die nachgeschaltete Signalisierung, das Trafficking und das Recycling einiger GPCRs modulieren.
Ihre Rolle im kritischsten Prozess der Signaltransduktion - der Rezeptoraktivierung - wurde jedoch nur begrenzt
erforscht.
Anhand des physiologisch und therapeutisch wichtigen Parathormon-Rezeptors (PTH1R), einem GPCR der Klasse B,
wurden die Modulationseffekte von RAMPs auf den Prozess der Rezeptoraktivierung und ihre Folgen für die
nachgeschaltete Signalübertragung analysiert. Hierzu wurden verschiedene optische Biosensoren zur Messung der
Aktivierung des PTH1R und seiner Signalkaskade entwickelt und in verschiedenen Versuchsanordnungen
eingesetzt, mit dem Ziel einen holistischen Blick auf die Interaktion zwischen PTH1R und RAMPs und ihre
funktionellen Auswirkungen zu erhalten.
Die Interaktion zwischen PTH1R und RAMPs erwies sich als besonders ausgeprägt für RAMP2, und RAMP2 zeigte
eine spezifische allosterische Modulation der PTH1R-Konformation, sowohl im basalen als auch im Liganden-
aktivierten Zustand. Ein einzigartiger voraktivierter oder (meta-stabiler) Zustand ermöglichte eine schnellere
Rezeptoraktivierung auf Liganden-spezifische Weise. Außerdem beeinflusste RAMP2 die G Protein- und Nicht-G
Protein-vermittelte Signalübertragung indem es die PTH-vermittelte Gi3-Signalempfindlichkeit und die Kinetik der
cAMP-Akkumulation modulierte. Weiterhin erhöhte RAMP2 die Menge der β-Arrestin2-Rekrutierung an PTH1R auf
Liganden-spezifische Weise. Dies könnte mit einer erhöhten zytosolischen ERK-Menge zusammenhängen, die hat
sich von der nukleären ERK-Phosphorylierung unterscheidet. Um einen molekularen Mechanismus für die
vorgestellten Ergebnisse vorzuschlagen, wurden mehrere strukturelle Modelle entwickelt und analysiert.
Diese Arbeit liefert den Beweis, dass RAMP die GPCR-Aktivierung mit funktionellen Auswirkungen auf die zelluläre
Signalübertragung reguliert. Die Ergebnisse sollten im Zusammenhang mit zellspezifischen Koexpressionsmustern
interpretiert werden und können zur Entwicklung von fortschrittlichen Therapeutika positiv beitragen. Da GPCRs
praktisch alle Zellfunktionen koordinieren und seit jeher wichtigen Angriffspunkten für Medikamente sind, tragen
die vorgestellten Erkenntnisse zum universellen Verständnis der molekularen Mechanismen bei, die den
menschlichen Körper orchestrieren.
KW  - G-Protein gekoppelter Rezeptor
KW  - GPCR
KW  - RAMP
KW  - PTH1R
KW  - FRET
KW  - BRET
KW  - pharmacology
KW  - Fluoreszenz-Resonanz-Energie-Transfer
KW  - Förster Resonanz Energie Transfer
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288588
ER  - 
TY  - JOUR
A1  - Hartmann, Nico
A1  - Knierim, Maria
A1  - Maurer, Wiebke
A1  - Dybkova, Nataliya
A1  - Hasenfuß, Gerd
A1  - Sossalla, Samuel
A1  - Streckfuss-Bömeke, Katrin
T1  - Molecular and functional relevance of Na\(_V\)1.8-induced atrial arrhythmogenic triggers in a human SCN10A knock-out stem cell model
JF  - International Journal of Molecular Sciences
N2  - In heart failure and atrial fibrillation, a persistent Na\(^+\) current (I\(_{NaL}\)) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that Na\(_V\)1.8 contributes to arrhythmogenesis by inducing a I\(_{NaL}\). Genome-wide association studies indicate that mutations in the SCN10A gene (Na\(_V\)1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these Na\(_V\)1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the I\(_{NaL}\) and action potential duration. Ca\(^{2+}\) measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca\(^{2+}\) leak. The I\(_{NaL}\) was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of Na\(_V\)1.8. No effects on atrial APD\(_{90}\) were detected in any groups. Both SCN10A KO and specific blockers of Na\(_V\)1.8 led to decreased Ca\(^{2+}\) spark frequency and a significant reduction of arrhythmogenic Ca\(^{2+}\) waves. Our experiments demonstrate that Na\(_V\)1.8 contributes to I\(_{NaL}\) formation in human atrial CMs and that Na\(_V\)1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore Na\(_V\)1.8 could be a new target for antiarrhythmic strategies.
KW  - Na\(_V\)1.8
KW  - iPSC-cardiomyocytes
KW  - late Na\(^+\) current (I\(_{NaL}\))
KW  - CRISPR Cas9
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-362708
SN  - 1422-0067
VL  - 24
IS  - 12
ER  - 
TY  - JOUR
A1  - Lorenz, Kristina
A1  - Rosner, Marsha Rich
T1  - Harnessing RKIP to combat heart disease and cancer
JF  - Cancers
N2  - Cancer and heart disease are leading causes of morbidity and mortality worldwide. These diseases have common risk factors, common molecular signaling pathways that are central to their pathogenesis, and even some disease phenotypes that are interdependent. Thus, a detailed understanding of common regulators is critical for the development of new and synergistic therapeutic strategies. The Raf kinase inhibitory protein (RKIP) is a regulator of the cellular kinome that functions to maintain cellular robustness and prevent the progression of diseases including heart disease and cancer. Two of the key signaling pathways controlled by RKIP are the β-adrenergic receptor (βAR) signaling to protein kinase A (PKA), particularly in the heart, and the MAP kinase cascade Raf/MEK/ERK1/2 that regulates multiple diseases. The goal of this review is to discuss how we can leverage RKIP to suppress cancer without incurring deleterious effects on the heart. Specifically, we discuss: (1) How RKIP functions to either suppress or activate βAR (PKA) and ERK1/2 signaling; (2) How we can prevent cancer-promoting kinase signaling while at the same time avoiding cardiotoxicity.
KW  - RKIP
KW  - ERK1/2
KW  - PKA
KW  - βAR
KW  - heart failure
KW  - cancer
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262185
SN  - 2072-6694
VL  - 14
IS  - 4
ER  - 
TY  - JOUR
A1  - Obidiegwu, Jude E.
A1  - Lyons, Jessica B.
A1  - Chilaka, Cynthia A.
T1  - The Dioscorea genus (yam) — an appraisal of nutritional and therapeutic potentials
JF  - Foods
N2  - The quest for a food secure and safe world has led to continuous effort toward improvements of global food and health systems. While the developed countries seem to have these systems stabilized, some parts of the world still face enormous challenges. Yam (Dioscorea species) is an orphan crop, widely distributed globally; and has contributed enormously to food security especially in sub-Saharan Africa because of its role in providing nutritional benefits and income. Additionally, yam has non-nutritional components called bioactive compounds, which offer numerous health benefits ranging from prevention to treatment of degenerative diseases. Pharmaceutical application of diosgenin and dioscorin, among other compounds isolated from yam, has shown more prospects recently. Despite the benefits embedded in yam, reports on the nutritional and therapeutic potentials of yam have been fragmented and the diversity within the genus has led to much confusion. An overview of the nutritional and health importance of yam will harness the crop to meet its potential towards combating hunger and malnutrition, while improving global health. This review makes a conscious attempt to provide an overview regarding the nutritional, bioactive compositions and therapeutic potentials of yam diversity. Insights on how to increase its utilization for a greater impact are elucidated.
KW  - yam
KW  - Dioscorea
KW  - nutritional composition
KW  - bioactive compounds
KW  - therapeutic potential
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213102
SN  - 2304-8158
VL  - 9
IS  - 9
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  - THES
A1  - Raab, Annette
T1  - The role of Rgs2 in animal models of affective disorders
T1  - Über die Bedeutung von Rgs2 in Tiermodellen affektiver Störungen
N2  - Anxiety and depressive disorders result from a complex interplay of genetic and environmental factors and are common mutual comorbidities. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety as well as rodent depression. Rgs2 negatively regulates G protein-coupled receptor (GPCR) signaling by acting as a GTPase accelerating protein towards the Gα subunit. 
The present study investigates, whether mice with a homozygous Rgs2 deletion (Rgs2-/-) show behavioral alterations as well as an increased susceptibility to stressful life events related to human anxiety and depressive disorders and tries to elucidate molecular underlying’s of these changes. 
To this end, Rgs2-/- mice were characterized in an aversive-associative learning paradigm to evaluate learned fear as a model for the etiology of human anxiety disorders. Spatial learning and reward motivated spatial learning were evaluated to control for learning in non-aversive paradigms. Rgs2 deletion enhanced learning in all three paradigms, rendering increased learning upon deletion of Rgs2 not specific for aversive learning. These data support reports indicating increased long-term potentiation in Rgs2-/- mice and may predict treatment response to conditioning based behavior therapy in patients with polymorphisms associated with reduced RGS2 expression. Previous reports of increased innate anxiety were corroborated in three tests based on the approach-avoidance conflict. Interestingly, Rgs2-/- mice showed novelty-induced hypo-locomotion suggesting neophobia, which may translate to the clinical picture of agoraphobia in humans and reduced RGS2 expression in humans was associated with a higher incidence of panic disorder with agoraphobia. Depression-like behavior was more distinctive in female Rgs2-/- mice. Stress resilience, tested in an acute and a chronic stress paradigm, was also more distinctive in female Rgs2-/- mice, suggesting Rgs2 to contribute to sex specific effects of anxiety disorders and depression.
Rgs2 deletion was associated with GPCR expression changes of the adrenergic, serotonergic, dopaminergic and neuropeptide Y systems in the brain and heart as well as reduced monoaminergic neurotransmitter levels. Furthermore, the expression of two stress-related microRNAs was increased upon Rgs2 deletion. The aversive-associative learning paradigm induced a dynamic Rgs2 expression change. The observed molecular changes may contribute to the anxious and depressed phenotype as well as promote altered stress reactivity, while reflecting an alter basal stress level and a disrupted sympathetic tone. Dynamic Rgs2 expression may mediate changes in GPCR signaling duration during memory formation.
Taken together, Rgs2 deletion promotes increased anxiety-like and depression-like behavior, altered stress reactivity as well as increased cognitive function.
N2  - Angststörungen sowie Depressionserkrankungen entstehen in der Regel aus der Interaktion genetischer Faktoren mit Umwelteinflüssen und sind häufig gegenseitige Begleiterkrankungen. Das Protein, Regulator of G protein signaling 2 (Rgs2), wurde mit dem vermehrten Auftreten von Angststörungen im Menschen, sowie mit angstähnlichem sowie depressionsähnlichem Verhalten im Mausmodell assoziiert. Rgs2 beeinflusst auf zellulärer Ebene G Protein gekoppelte Signalwege, indem es die GTPase Aktivität der Gα Untereinheit beschleunigt.
In der vorliegenden Arbeit wurden die Folgen einer homozygoten Rgs2-Defizienz im Mausmodell untersucht. In Anlehnung an die humanen Krankheitsbilder wurde angst- und depressions-ähnliches Verhalten, Stress Reaktivität und den phänotypischen Veränderungen zugrundeliegende molekulare Ursachen evaluiert.
Erlernte Furcht gilt als Model der Ätiologie humaner Angsterkrankungen. Aus diesem Grund, wurden Rgs2-/- Mäuse in einem aversiv-assoziativen Lernmodell, der sogenannten Furcht-Konditionierung, untersucht. Dabei zeigte sich erhöhtes Furchtlernen und Furchtgedächtnis in Rgs2-/- Mäusen. Um zu zeigen, dass die erhöhte kognitive Fähigkeit spezifisch für erlernte Furcht sei, wurde räumliches Lernen in zwei Modellen getestet. Rgs2-Defizienz verbesserte auch in diesen Modellen die Lernfähigkeit. Somit konnte gezeigt werden, dass verbesserte kognitive Fähigkeit nicht spezifisch für emotionales Lernen war. Diese Daten auf Verhaltensebene unterstützen bisherige Befunde von erhöhter Langzeit Potenzierung im Hippocampus von Rgs2-/- Mäusen. Im Menschen könnte eine durch Polymorphismen vermittelte reduzierte Rgs2 Expression das Therapieansprechen auf konditionierungsbasierte Verhaltenstherapien verbessern. Bisherige Befunde von erhöhter, angeborener Angst in Rgs2-/- Mäusen konnten in drei Tests, basierend auf dem Annäherungs-Vermeidungs-Konflikt, bestätigt werden. Interessanterweise, zeigten Rgs2-/- Mäuse in allen Tests verminderte Lokomotion in neuen, ungewohnten Umgebungen. Dies könnte auf Neophobie und somit auf das Krankheitsbild der Agoraphobie im Menschen hindeuten. Tatsächlich wurden RGS2 Polymorphismen bereits mit einer erhöhten Inzidenz von Panikstörung mit Agoraphobie assoziiert. Rgs2-/- Mäuse zeigten zudem depressionsähnliches Verhalten, welches in weiblichen Mäusen ausgeprägter war. Des Weiteren zeigten, insbesondere weibliche Rgs2-/- Mäuse, erhöhte Stress Resilienz nach akuter und chronischer Stressexposition. Rgs2 könnte somit ein Faktor der Geschlechtsspezifität von Angst und Depressionserkrankungen sein. 
Rgs2-Defizienz konnte mit Expressionsänderungen von G Protein gekoppelten Rezeptoren des adrenergen, serotonergen, dopaminergen und Neuropeptid Y Systems in Gehirn und Herz, sowie mit verminderten Spiegeln monoaminerger Neurotransmitter assoziiert werden. Diese Veränderungen könnten zu dem beobachteten ängstlichen sowie depressiven Phänotyp und der veränderten Stress Reaktivität beitragen. Des Weiteren war die Expression zweier, in der Stressreaktion involvierten, microRNAs erhöht. Dies könnte auf einen veränderten basalen Stress Level hindeuten. Furcht-Konditionierung löste dynamische Expressionsänderungen der Rgs2 mRNA aus. Somit könnte die GPCR Signaldauer während der Gedächtnisbildung durch Rgs2 moduliert werden. 
Zusammengefasst, führt Rgs2-Defizienz im Mausmodell zu erhöhtem angst- und depressions-ähnlichem Verhalten, veränderter Stress Reaktivität sowie erhöhter kognitiver Leistung.
KW  - Angst
KW  - Depression
KW  - Tiermodell
KW  - Rgs2
KW  - Regulator of G protein signaling 2
KW  - Animal model
KW  - Anxiety
KW  - Depression
KW  - Stress
KW  - Knockout
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-152550
ER  - 
TY  - THES
A1  - Wilde, Sabrina
T1  - Einsatz von mechanistischen Biomarkern zur Charakterisierung und Bewertung von \(in\) \(vitro\) Genotoxinen
T1  - Use of mechanistic biomarkers for the characterization and evaluation of \(in\) \(vitro\) genotoxins
N2  - Die verfügbaren in vitro Genotoxizitätstests weisen hinsichtlich ihrer Spezifität und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschränkungen auf. Um diese Mängel zu überwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Prüfung auf Genotoxizität in der Arzneimittelentwicklung beitragen.
1.	Etablierung und Bewertung einer neuen in vitro Genotoxizitätsmethode (MultiFlow Methode)
Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbrüche), phosphorylierten H3 (S10) (mitotische Zellen), nukleären Protein p53 (Genotoxizität) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und ergänzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre Fähigkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen“ gegenüber der „alten“ Methode führte zu einer verbesserten Sensitivität von 95% gegenüber 90%, Spezifität von 90% gegenüber 72% und einer MoA-Klassifizierungsrate von 85% gegenüber 45% (Aneugen vs. Klastogen).
2.	Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen
Die Analyse 67 ausgewählter  DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen können. Die Kombination der höchstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 ermöglichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivität, Spezifität und Prädiktivität von 86%, 83% und 85%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizität mit TP53I3, Klastogenität mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann.
Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen für BubR1 (S670) und pH3 (S28) mit Aneugenität, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenität, p53 (K373) mit Genotoxizität und Nrf2 (S40) mit oxidativem Stress identifiziert.
Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinveränderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivitäten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden können und daher eine bessere Risikobewertung von Wirkstoffkandidaten ermöglichen.
N2  - Available in vitro genotoxicity tests have limitations regarding their specificity and mode of action (MoA) information. To overcome these shortages, two objectives were pursued in this work to develop and establish new in vitro tools for genotoxicity testing.
1.	Establishment and evaluation of a novel in vitro genotoxicity method (MultiFlow  method)
The MultiFlow method is based on DNA damage-related protein responses of γH2AX (DNA double-strand breaks), phosphorylated H3 (S10) (mitotic cells), nuclear protein p53 (genotoxicity) and cleaved PARP1 (apoptosis) in TK6 cells. In total, 31 model substances were studied flow cytometrically in the MultiFlow assay - and also with the well-established micronucleus test (MicroFlow MNT) - for their ability to classify across MoA groups: aneugens, clastogens and non-genotoxicants. The performance of the new method resulted in an improved sensitivity of 95% to 90%, specificity of 90% to 72% and a MoA classification rate of 85% to 45% (aneugen vs. clastogen). 
2. Identification of mechanistic biomarkers for the characterization of genotoxicants
The analysis of 67 selected DNA-damage associated genes using the QuantiGene Plex method showed that a combinaten of genes can contribute to MoA classification. The combination of the highest-ranked markers (BIK, KIF20A, TP53I3, DDB2 and OGG1) highlighted the best identification rate of model substances. The synergistic statistic tool correctly categorized 16 of 16 substances into aneugens, clastogens and non-genotoxicants. By using leave-one out cross validation, the model was evaluated and achieved a sensitivity, specificity and predictivity of 86%, 83%, 85% respectively. 
Follow-up with qPCR was conducted and revealed associations with TP53I3 for genotoxicity, ATR and RAD17 for clastogenicity and NFE2L2 for oxidative stress. 
By investigating posttranslational modifications using high-content imaging, associations for BubR1 (S670) and pH3 (S28) with aneugenicity, 53BP1 (S1778) and FANCD2 (S1404) with clastogenicity, p53 (K373) with genotoxicity and Nrf2 (S40) with oxidative stress were found to be further useful for MoA identification.
This work demonstrates that genotoxicants and non-genotoxicants induce different gene- and protein expression changes in the TK6 cells that can be used to classify the MoA groups (aneugen/clastogen/non-genotoxicant/reactive oxygen species), thus enabling better risk assessment of potential drug candidates.
KW  - Genotoxizität
KW  - Genotoxicitiy
KW  - Klastogene
KW  - Aneugene
KW  - Biomarker
KW  - Klassifizierung
KW  - clastogens
KW  - aneugens
KW  - biomarker
KW  - classification
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-182782
ER  - 
TY  - JOUR
A1  - Vazquez-Rodriguez, Saleta
A1  - Vilar, Santiago
A1  - Kachler, Sonja
A1  - Klotz, Karl-Norbert
A1  - Uriarte, Eugenio
A1  - Borges, Fernanda
A1  - Matos, Maria João
T1  - Adenosine receptor ligands: coumarin−chalcone hybrids as modulating agents on the activity of hARs
JF  - Molecules
N2  - Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin–chalcone hybrids were synthesized (compounds 1–8) and screened in radioligand binding (hA\(_1\), hA\(_{2A}\) and hA\(_3\)) and adenylyl cyclase (hA\(_{2B}\)) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin–chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA\(_1\) or hA\(_3\) subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA\(_1\), while the methoxy counterparts were selective for the hA\(_3\). The most potent hA\(_1\) ligand was compound 7 (K\(_i\) = 17.7 µM), whereas compound 4 was the most potent ligand for hA\(_3\) (K\(_i\) = 2.49 µM). In addition, docking studies with hA\(_1\) and hA\(_3\) homology models were established to analyze the structure–function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.
KW  - coumarin
KW  - chalcone
KW  - neurodegenerative diseases
KW  - adenosine receptors
KW  - binding affinity
KW  - docking
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213165
SN  - 1420-3049
VL  - 25
IS  - 18
ER  - 
TY  - THES
A1  - Anton, Selma
T1  - Characterization of cAMP nanodomains surrounding the human Glucagon-like peptide 1 receptor using FRET-based reporters
T1  - Charakterisierung der Rezeptor-assoziierten cAMP Nanodomänen des humanen Glucagon-like peptide 1 Rezeptors mittels FRET-basierter Sensoren
N2  - 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 Förster 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.
N2  - G Protein gekoppelte Rezeptoren (GPCRs) stellen eine große und sehr vielfältige Familie an Membranproteinen dar, deren primäre Funktion die Signalübertragung von extrazellulären Stimuli in intrazelluläre Signale ist. Dank ihrer breiten Expression im gesamten menschlichen Körper regulieren sie unterschiedliche zelluläre Prozesse und damit deren physiologische Funktion, unter anderem die Sinnesempfindung, zelluläre Kommunikation und Neurotransmission. GPCRs stehen im Zusammenhang mit unterschiedlichen Erkrankungen wie Herzinsuffizienz, Krebs, neurologischen Funktionsstörungen und diverser metabolischer Krankheiten, weswegen sie als Ziele („Targets“) zur Behandlung verschiedener Erkrankungen erforscht und genutzt werden. Aufgrund ihrer Expression auf der Zelloberfläche sind sie leicht zugänglich, und die Diversität ihrer Liganden begünstigt zusätzlich ihre Nutzung als pharmakologische Targets. Heutzutage vermitteln bereits 30% aller weltweit zugelassenen Arzneistoffe ihre Wirkung an GPCRs.
GPCRs üben ihre Funktion aus, indem sie hauptsächlich an G Proteine binden, welche wiederum die Produktion sogenannter second messenger in Gang setzen. cAMP ist das Hauptsignalmolekül der Rezeptoren, welche an das stimulatorische GS Protein koppeln. cAMP überträgt hunderte ankommende Signale in einer hochspezifischen Weise, indem es an unterschiedliche Effektorproteine bindet, welche sich in bestimmten zellulären Regionen befinden. Dadurch koordiniert dieses Signalmolekül eine Vielzahl zellulärer Prozesse, angefangen bei der Regulierung von Ionenkanalaktivität über die Kontraktilität glatter- und quergestreifter Muskulatur bis hin zur Genexpression, Zellproliferation und Apoptose. Durch die pleiotropen Effekte, welche durch cAMP reguliert werden, stellt sich die Frage, wie GS-gekoppelte Rezeptoren Signalspezifität erreichen, obwohl sie ihre Funktion durch dieses eine Signalmolekül ausführen.
Ursprünglich ging man von einer uneingeschränkten Diffusion und dadurch homogenen Verteilung von cAMP in der Zelle aus. Diese Vorstellung ist jedoch nicht mit der Signalisierungsspezifität von GPCRs vereinbar, da unter diesen Umständen cAMP unselektiv all seine Effektorproteine in der gesamten Zelle aktivieren könnte. Daher entstand die Hypothese der cAMP-Kompartimentierung, wobei die Zelle lokal begrenzte Bereiche mit hohen oder niedrigen cAMP Konzentrationen umfassen würde. Jedoch gab es bisher keinerlei Beweise für die Existenz und die molekulare Zusammensetzung mutmaßlicher Domänen. Folglich setzten wir uns als Ziel, hochkonzentrierte cAMP-Kompartimente in der Zelle zu lokalisieren, ihre räumliche Dimension aufzuklären und ihre Rolle zur Realisierung zellulärer Signalisierungsspezifität zu ermitteln. Im Rahmen der vorliegenden Studie setzten wir einen Förster resonance energy transfer (FRET)-basierten cAMP Sensor ein, fusionierten ihn mit dem humanen glucagone-like peptide 1 Rezeptor (hGLP1R) als Prototyp eines GS-koppelnden Rezeptors, um cAMP am Ursprung des Signals zu messen. Mittels dieser Sensoren weisen wir eine Rezeptor-umgebende begrenzte cAMP Domäne nach, welche eine erhöhte cAMP Konzenztration aufweist (Figure ‎3.10). Bei Stimulation des Rezeptors mit GLP1 Konzenztrationen beginnend bei 10 fM entsteht eine Rezeptordomäne mit lokal erhöhten cAMP Konzentrationen, welche getrennt von Plasmamembran und Cytosol ist. Wir zeigen, dass das hGLP1R-Kompartiment geschützt ist vor cAMP Signalen, welche an weiteren, unabhängigen GS-gekoppelten Rezeptoren ihren Ursprung haben (Figure ‎3.11). Um die räumliche Dimension dieser Domäne zu untersuchen, verwendeten wir Nanolinker der Länge 30- und 60 nm als Abstandhalter zwischen Rezeptor und Sensor (Figure ‎3.12) und zeigen dabei, dass sich die Domäne über eine Länge von 60 Nanometern erstreckt, wobei ein abnehmender cAMP-Gradient erkennbar ist. Weiterhin beweisen wir, dass Phosphodiesterasen (PDEs) Schlüsselfaktoren für die Bildung des cAMP-Gradienten um den Rezeptor herum sind, indem sie die Diffusion ins Cytosol beschränken (Figure ‎3.13). Darüber hinaus zeigen wir (Figure ‎3.15), dass Rezeptor-spezifische cAMP Signale PKA-Phosphorylierung in der Rezeptordomäne auslösen und, dass AKAPs elementar für nanodomänen PKA-Aktivität sind, wohingegen die cytosolische PKA-Phosphorylierung unabhängig von AKAP-Targeting der PKA ist (Figure ‎3.16). 
Zusammenfassend beweisen unsere Ergebnisse die Existenz einer Rezeptor-umgebenden Nanodomäne mit erhöhten cAMP Spiegeln eines GS-gekoppelten Rezeptors. Zeitgleiche Studien in unserer Gruppe zeigen, dass cAMP in der Zelle weitgehend gebunden vorliegt und diffusionslimitiert ist. Dies stellt den Nachweis für eine eingeschränkte Diffusion als molekulare Voraussetzung für die Bildung von Signalkompartimenten dar. Wir gehen davon aus, dass unsere Ergebnisse ein Ausgangspunkt für die Aufklärung von Rezeptoren als Quelle für Signalkompartimente darstellen, jedoch bedarf es weiterer Studien, um die präzise molekulare Zusammensetzung und die beteiligten Proteine dieser Signaldomäne zu untersuchen. Das Grundverständnis der Signalisierungskaskaden auf molekularer Ebene könnte es uns ermöglichen, die zellulären Reaktionen zu manipulieren, um eine Fehlfunktion der Signalisierung in erkrankten Zellen wiederherzustellen. Da der hGLP1R entscheidend für Aufrechterhaltung ausgeglichener Blutglucosespiegel ist, würde die Erfassung der molekularen Details der kompartimentalisierten Signalübertragung die Feinabstimmung der Rezeptorsignale ermöglichen, um ihn als spezifisches Target zur Behandlung von Diabetes Mellitus einzusetzen.
KW  - FRET
KW  - cAMP
KW  - compartments
KW  - GPCR
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190695
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  - Reimann, Hauke
A1  - Stopper, Helga
A1  - Polak, Thomas
A1  - Lauer, Martin
A1  - Herrmann, Martin J.
A1  - Deckert, Jürgen
A1  - Hintzsche, Henning
T1  - Micronucleus frequency in buccal mucosa cells of patients with neurodegenerative diseases
JF  - Scientific Reports
N2  - Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer's disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome.
KW  - peripheral-blood lymphocytes
KW  - Alzheimers disease
KW  - DNA damage
KW  - cognitive impairment
KW  - cytome biomarkers
KW  - diagnosis
KW  - association
KW  - assay
KW  - life
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231430
VL  - 10
ER  - 
TY  - JOUR
A1  - Wagner, Michael
A1  - Sadek, Mirna S.
A1  - Dybkova, Nataliya
A1  - Mason, Fleur E.
A1  - Klehr, Johann
A1  - Firneburg, Rebecca
A1  - Cachorro, Eleder
A1  - Richter, Kurt
A1  - Klapproth, Erik
A1  - Kuenzel, Stephan R.
A1  - Lorenz, Kristina
A1  - Heijman, Jordi
A1  - Dobrev, Dobromir
A1  - El-Armouche, Ali
A1  - Sossalla, Samuel
A1  - Kämmerer, Susanne
T1  - Cellular mechanisms of the anti-arrhythmic effect of cardiac PDE2 overexpression
JF  - International Journal of Molecular Sciences
N2  - Background: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to β-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. Methods: Cellular arrhythmia, ion currents, and Ca\(^{2+}\)-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates. Results: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in I\(_{CaL}\) seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in I\(_{NaL}\) and Ca\(^{2+}\)-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. Conclusion: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.
KW  - PDE2
KW  - arrhythmia
KW  - CaMKII
KW  - heart failure
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285888
SN  - 1422-0067
VL  - 22
IS  - 9
ER  - 
TY  - JOUR
A1  - Schanbacher, Constanze
A1  - Bieber, Michael
A1  - Reinders, Yvonne
A1  - Cherpokova, Deya
A1  - Teichert, Christina
A1  - Nieswandt, Bernhard
A1  - Sickmann, Albert
A1  - Kleinschnitz, Christoph
A1  - Langhauser, Friederike
A1  - Lorenz, Kristina
T1  - ERK1/2 activity is critical for the outcome of ischemic stroke
JF  - International Journal of Molecular Sciences
N2  - Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.
KW  - ERK1/2
KW  - tMCAO
KW  - ischemic stroke
KW  - RKIP
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-283991
SN  - 1422-0067
VL  - 23
IS  - 2
ER  - 
TY  - THES
A1  - Kauk, Michael
T1  - Investigating the Molecular Mechanism of Receptor Activation at Muscarinic Receptors by Means of Pathway-Specific Dualsteric Ligands and Partial Agonists
T1  - Molekulare Grundlagen der Rezeptoraktivierung von muskarinergen Acetylcholin Rezeptoren durch dualstere Liganden und Partialagonisten
N2  - G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology.
Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored.
In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 % could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful.
N2  - G Protein gekoppelte Rezeptoren (GPCRs) bilden die größte Proteinfamilie, die im humanen Genom verschlüsselt ist. Sie sind nicht nur die Zielstruktur für eine Vielzahl von derzeit gebräuchlichen Medikamenten, sondern gehören auch zu den vielversprechendsten Therapieansätzen für die moderne Medikamentenentwicklung. Muskarinerge Acetylcholin Rezeptoren (mAChRs) gehören zu den aminergen Klasse A GPCRs und sind bereits heute von klinischer Relevanz. Die muskarinerge Rezeptorfamilie wird von fünf Subtypen gebildet, die sich besonders durch eine hohe Sequenzidentität in der endogenen Ligandenbindestelle (orthostere Bindestelle) auszeichnen. Aus diesem Grund ist es mit den herkömmlich verwendeten Medikamenten nicht möglich, einen ganz bestimmten Subtyp zu therapieren, ohne auch andere Subtypen zu beeinflussen und so unerwünschte Nebenwirkungen zu erhalten. Eine Möglichkeit Ligandenbindung, dynamische Rezeptoraktivierung oder Signalweiterleitung von GPCRs nach pharmakologischen Gesichtspunkten zu charakterisieren, stellt der Floreszenz Resonanz Energietransfer (FRET) dar. Mit Hilfe dieser Methode kann über kleine Entfernungen (<10 nm) die relative Orientierung von zwei Fluorophoren mit überlappenden Spektralbereichen mit hoher zeitlicher Auflösung verfolgt werden. Integriert man das Fluorophorpaar mit Hilfe gentechnischer Methoden in die Molekülstruktur des Rezeptors, kann man dessen Konformationsänderung bzw. Aktivierung infolge einer Ligandenbindung aufzeichnen.
Dualstere Liganden sind eine Substanzklasse von hohem zukünftigen klinischen Potential und zeichnen sich durch die Verknüpfung mehrerer pharmakologisch aktiver Untereinheiten aus. Der orthostere Molekülteil interagiert mit der endogenen Ligandenbindestelle und der allostere Molekülteil interagiert mit einem zweiten Rezeptorabschnitt, der häufig in den extrazellulären Schlaufen des Rezeptors zu finden ist. Diese allosteren Bindestellen zeichnet sich durch eine vergleichsweise geringe Sequenzidentität aus, weswegen allostere Modulatoren auch selektiv an Subtypen binden können. Aufgrund des Aufbaus können dualstere Liganden auf vielfältige Weise mit dem Rezeptor interagieren und dieser Bindemechanismus wurde als dynamische Ligandenbindung beschrieben. Zum einen können beide Molekülteile gleichzeitig mit dem Rezeptor interagieren und ihn aktivieren (dualsterer Bindemodus) und zum anderen findet man einen rein allosteren Bindemodus, der den Rezeptor nicht aktiviert. Der orthostere Molekülteil ist vor allem für die Rezeptoraktivierung zuständig, die sich durch eine hohe Affinität auszeichnet und der allostere Molekülteil kann selektive Rezeptorinteraktionen vermitteln. Da dualstere Moleküle immer Eigenschaften beider Untereinheiten besitzen, werden dualstere Liganden als sehr vielversprechend erachtet, zukünftig subtypselektive Medikamente darzustellen.
In dieser Arbeit wurde der erste Rezeptor FRET Sensor für den muskarinergen Subtyp 1 (M1) beschrieben und es konnte gezeigt werden, dass sich dieser Rezeptorsensor in seiner physiologischen Funktion nicht von dem wild Typ unterscheidet. Des Weiteren können mit Hilfe dieses Sensors liganden- und konzentrationsabhängige Rezeptorantworten aufgezeichnet werden. Der M1-I3N-CFP wurde dazu genutzt verschiedene Reihen dualsterer Liganden zu charakterisieren und auf ihre aktivierenden Eigenschaften bezüglich des M1 zu testen. Es wurde gezeigt, dass die Kombination aus dem synthetischen und hochpotenten Agonisten Iperoxo als Orthoster und dem in der Literatur als M1 selektiven positiven allosteren Modulator beschriebenen BQCA als Alloster sehr vielversprechend ist. Es konnte gezeigt werden, dass die rezeptoraktivierenden Eigenschaften sowohl von orthosteren wie auch von dualsteren Liganden stark von der Linkerlänge abhängig sind. Für dualstere Liganden konnte so ein glockenförmiger Zusammenhang zwischen Linkerlänge und Rezeptoraktivierung herausgearbeitet werden. Des Weiteren wurde gezeigt, dass bestimmte Hybride, die den M1 aktivieren, an anderen Subtypen keine Effekte hervorrufen und somit als subtypselektiv beschrieben werden können. Im Anschluss wurde mit Hilfe des gewonnenen Wissens über Iperoxo/BQCA Hybride, das Moleküldesign der dualsteren Liganden weiterentwickelt. So wurden in dieser Arbeit die ersten photo-schaltbaren bzw. photo-dimmbaren dualsteren Liganden beschrieben und charakterisiert. Des Weiteren wurde in dieser Arbeit die herkömmliche Charakterisierung von dualsteren Liganden weiterentwickelt. Es konnte zum ersten Mal gezeigt werden, dass es möglich ist, die Aktivierung eines Rezeptors durch einen dualsteren Liganden nachzustellen, indem die einzelnen Fragmente des ursprünglichen Liganden gleichzeitig appliziert werden. Diese auf Fragmenten basierende Charakterisierung ist die erste Anwendung dieser Art und birgt großes Potential für die zukünftige Suche nach neuen Wirkstoffen. Neben der Untersuchung von pharmakologischen Schwerpunkten wurde auch die Weiterentwicklung der Rezeptor FRET Methodik beschrieben. Die herkömmliche Anwendung der Rezeptor FRET Sensoren geschieht auf Objektträgern aus Quarzglas. In dieser Arbeit wurde diese Anwendung dahingehend weiterentwickelt, dass die Objektträger mit einer 20 nm dicken Goldschicht beschichtet wurden, um den Einfluss von Plasmonoberflächen auf physiologisch relevante FRET Messungen zu untersuchen. Es konnte gezeigt werden, dass mit Hilfe der Goldbeschichtung und in Abhängigkeit des Versuchsaufbaus die Energietransfereffizienz um bis zu 60 % gesteigert werden konnte. Diese Entdeckung zeigt Potential zukünftig die FRET-Reichweite zu erhöhen und so bisher nicht charakterisierbare Sachverhalte aufklären zu können.
KW  - G-Protein gekoppelte Rezeptoren
KW  - Muscarinrezeptor
KW  - Dualsteric Ligands
KW  - Partial Agonists
KW  - Dualstere Liganden
KW  - Partialagonismus
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173729
N1  - Online-Version enthält nicht den Appendix (Volltexte der Originalveröffentlichungen der Zeitschriftenaufsätze)
ER  - 
TY  - JOUR
A1  - Sedaghat-Hamedani, Farbod
A1  - Rebs, Sabine
A1  - El-Battrawy, Ibrahim
A1  - Chasan, Safak
A1  - Krause, Tobias
A1  - Haas, Jan
A1  - Zhong, Rujia
A1  - Liao, Zhenxing
A1  - Xu, Qiang
A1  - Zhou, Xiaobo
A1  - Akin, Ibrahim
A1  - Zitron, Edgar
A1  - Frey, Norbert
A1  - Streckfuss-Bömeke, Katrin
A1  - Kayvanpour, Elham
T1  - Identification of SCN5a p.C335R variant in a large family with dilated cardiomyopathy and conduction disease
JF  - International Journal of Molecular Sciences
N2  - Introduction: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. Methods: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. Results: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na\(^+\) channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. Conclusion: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.
KW  - familial DCM
KW  - conduction disease
KW  - SCN5a
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284442
SN  - 1422-0067
VL  - 22
IS  - 23
ER  - 
TY  - THES
A1  - İşbilir, Ali
T1  - Localization and Trafficking of CXCR4 and CXCR7
T1  - Lokalisation und Verteilung von CXCR4 und CXCR7
N2  - G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins, and are the master components that translate extracellular stimulus into intracellular signaling, which in turn modulates key physiological and pathophysiological processes. Research within the last three decades suggests that many GPCRs can form complexes with each other via mechanisms that are yet unexplored. Despite a number of functional evidence in favor of GPCR dimers and oligomers, the existence of such complexes remains controversial, as different methods suggest diverse quaternary organizations for individual receptors. Among various methods, high resolution fluorescence microscopy and imagebased fluorescence spectroscopy are state-of-the-art tools to quantify membrane protein oligomerization with high precision. This thesis work describes the use of single molecule fluorescence microscopy and implementation of two confocal microscopy based fluorescence fluctuation spectroscopy based methods for characterizing the quaternary organization of two class A GPCRs that are important clinical targets: the C-X-C type chemokine receptor 4 (CXCR4) and 7 (CXCR7), or recently named as the atypical chemokine receptor 3 (ACKR3). The first part of the results describe that CXCR4 protomers are mainly organized as monomeric entities that can form transient dimers at very low expression levels allowing single molecule resolution. The second part describes the establishment and use of spatial and temporal brightness methods that are based on fluorescence fluctuation spectroscopy. Results from this part suggests that ACKR3 forms clusters and surface localized monomers, while CXCR4 forms increasing amount of dimers as a function of receptor density in cells. Moreover, CXCR4 dimerization can be modulated by its ligands as well as receptor conformations in distinct manners. Further results suggest that antagonists of CXCR4 display distinct binding modes, and the binding mode influences the oligomerization and the basal activity of the receptor: While the ligands that bind to a “minor” subpocket suppress both dimerization and constitutive activity, ligands that bind to a distinct, “major” subpocket only act as neutral antagonists on the receptor, and do not modulate neither the quaternary organization nor the basal signaling of CXCR4. Together, these results link CXCR4 dimerization to its density and to its activity, which may represent a new strategy to target CXCR4.
N2  - G protein-gekoppelte Rezeptoren (GPCRs) bilden die größte Klasse der Membranproteine und sind entscheidend an der Übersetzung extrazellulärer Reize in intrazelluläre Signale beteiligt, welche wiederum unzählige physiologische und pathophysiologische Prozesse regulieren. Die Forschungsergebnisse der letzten drei Jahrzehnte deutet darauf hin, dass viele GPCRs mittels noch weitgehend unbekannter Mechanismen miteinander Komplexe bilden können. Trotz vielfältiger Beobachtungen, die für die funktionelle Relevanz von GPCR-Dimeren und -Oligomeren sprechen, ist deren Existenz dennoch weiterhin umstritten, vor allem da verschiedene Methoden auf unterschiedliche quaternäre Anordnungen derselben Rezeptoren hinweisen. Von den derzeit verfügbaren Methoden zur genauen Untersuchung der GPCR Dimerisierung/-Oligomerisierung, stellen die hochauflösende Fluoreszenzmikroskopie sowie die bildbasierte Fluoreszenzspektroskopie die Techniken der Wahl dar. Die hier vorliegende Arbeit beschreibt die Anwendung der Einzelmolekül Fluoreszenzmikroskopie sowie zweier konfokalmikroskopischer
Methoden zur Messung der Fluoreszenzfluktuation, mit deren Hilfe die quaternäre Anordnung zweier klinisch hochattraktiver Klasse A GPCRs untersucht wurde: der C-X-C Typ Chemokinrezeptoren 4 (CXCR4) und 7 (CXCR7), letzterer auch bekannt als atypischer Chemokinrezeptor 3 (ACKR3). Der erste Teil der Ergebnisse legt anhand Untersuchungen an einzelnen Molekülen dar, dass CXCR4 überwiegend in Form monomerer Einheiten auftritt, die bei sehr geringen Expressionsleveln kurzlebige Dimere bilden können. Der zweite Teil beschreibt die Etablierung und Anwendung räumlicher und zeitlicher Brillanzmethoden, die auf der spektroskopischen Untersuchung der Fluoreszenzfluktuation beruhen. Die Ergebnisse dieses Abschnitts deuten darauf hin, dass ACKR3 sowohl in Form beständiger Rezeptor-Cluster, und monomere Einheit an der Oberfläche lebender Zellen auftritt. CXCR4 ist bei zunehmender Rezeptordichte hingegen vermehrt in Form von Dimeren zu finden. Zudem kann die Dimerisierung von CXCR4 von dessen Liganden, als auch von der drei dimensionalen Anordnung der Rezeptorteilstrukturen (Rezeptorkonformation)auf unterschiedliche Weise reguliert werden. Die weiteren Ergebnisse legen nahe, dass Antagonisten auf unterschiedliche Weise an CXCR4 binden können und dass der jeweilige Bindungsmodus entscheidend für den Einfluss des Liganden auf Oligomerisierung und basale Aktivität von CXCR4 ist: Während Liganden, die an eine kleinere Untertasche des Rezeptors binden, sowohl die Dimerisierung als auch die Basalaktivität unterdrücken, fungieren Verbindungen, die an eine andere, größere Untertasche binden, lediglich als neutrale Antagonisten und zeigen keinerlei Einfluss auf die quaternäre Anordnung und basale Aktivität von CXCR4. Zusammenfassend verknüpfen diese Ergebnisse CXCR4-Dimerisierung mit der Rezeptordichte in Zellen und seiner Aktivität, was die Grundlage für neue Strategien zur phamakologischen Modulation von CXCR4 darstellen könnte.
KW  - G-Protein gekoppelter Rezeptor
KW  - GPCR
KW  - Receptor
KW  - Chemokine
KW  - oligomerization
KW  - CXCR4
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249378
ER  - 
TY  - JOUR
A1  - Spinaci, Andrea
A1  - Lambertucci, Catia
A1  - Buccioni, Michela
A1  - Dal Ben, Diego
A1  - Graiff, Claudia
A1  - Barbalace, Maria Cristina
A1  - Hrelia, Silvana
A1  - Angeloni, Cristina
A1  - Tayebati, Seyed Khosrow
A1  - Ubaldi, Massimo
A1  - Masi, Alessio
A1  - Klotz, Karl-Norbert
A1  - Volpini, Rosaria
A1  - Marucci, Gabriella
T1  - A\(_{2A}\) adenosine receptor antagonists: are triazolotriazine and purine scaffolds interchangeable?
JF  - Molecules
N2  - The A\(_{2A}\) adenosine receptor (A\(_{2A}\)AR) is one of the four subtypes activated by nucleoside adenosine, and the molecules able to selectively counteract its action are attractive tools for neurodegenerative disorders. In order to find novel A\(_{2A}\)AR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A\(_{2A}\)AR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted.
KW  - A\(_{2A}\) adenosine receptor antagonist
KW  - purine derivatives
KW  - triazolotriazine derivatives
KW  - anti-Parkinson agents
KW  - anti-inflammatory agents
KW  - molecular modeling
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270618
SN  - 1420-3049
VL  - 27
IS  - 8
ER  - 
TY  - JOUR
A1  - Jarzina, Sebastian
A1  - Di Fiore, Stefano
A1  - Ellinger, Bernhard
A1  - Reiser, Pia
A1  - Frank, Sabrina
A1  - Glaser, Markus
A1  - Wu, Jiaqing
A1  - Taverne, Femke J.
A1  - Kramer, Nynke I.
A1  - Mally, Angela
T1  - Application of the adverse outcome pathway concept to in vitro nephrotoxicity assessment: kidney injury due to receptor-mediated endocytosis and lysosomal overload as a case study
JF  - Frontiers in Toxicology
N2  - Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.
KW  - adverse outcome pathway (AOP)
KW  - nephrotoxicity
KW  - QIVIVE
KW  - risk assessment
KW  - key event relationship
KW  - In vitro toxicity testing
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284796
SN  - 2673-3080
VL  - 4
ER  - 
TY  - THES
A1  - Ramge, Vanessa Magali
T1  - Untersuchung der Genotoxizität von Pyrrolizidinalkaloiden \(in\) \(vitro\) am Beispiel von Riddelliin und Lasiocarpin
T1  - Investigating the genotoxic potential of pyrrolizidine alkaloids \(in\) \(vitro\) using the example of riddelliin and lasiocarpin
N2  - PA sind natürliche Pflanzeninhaltsstoffe, die wegen ihres genotoxischen Potentials bekannt sind. Nach Applikation mikromolarer Konzentrationen können bei in vitro Untersuchungen von Leberzellen chromosomale Schäden detektiert werden. PA stehen im Verdacht nach Aufnahme bei Menschen hepatotoxische und kanzerogene Wirkungen nach sich zu ziehen. In dieser Studie wurden Lasiocarpin und Riddelliin an der humanen Leberkarzinomzelllinie Huh6 auf Genotoxizität getestet. Die ausgewählten Methoden waren der MK-Test, der alkalische und der FPG Comet Assay und die γ-H2AX-Färbung. In den Vorversuchen mit BaP und CPA wurde gezeigt, dass die Zellen durch Prodrugs genotoxisch geschädigt werden. Zusammenfassend kann gesagt werden, dass Riddelliin und Lasiocarpin im MK-Test eine dosisabhängige, genotoxische Wirkung auf die Huh6 Zellen haben. Der Einfluss von Lasiocarpin war im MK-Test im Vergleich zum Einfluss von Riddelliin bei geringerer Konzentration detektierbar. Nach einer simultanen Behandlung der Huh6 Zellen mit verschiedenen PA kann konkludiert werden, dass keine signifikante Erhöhung an DNA-Schäden im Vergleich zu Behandlungen mit den Einzelsubstanzen festgestellt werden konnte, was möglicherweise auf eine Erschöpfung der metabolischen Kapazität der Zellen zurückzuführen ist. Insgesamt ist es den Ergebnissen zufolge wahrscheinlich, dass die Entstehung von Crosslinks durch Lasiocarpin und Riddelliin eher eine Rolle in der Genotoxizitätsinduktion auf Huh6 Zellen spielen als oxidativer Stress. Doppelstrangbrüche konnten nicht als sicherer Induktor von Genotoxizität identifiziert werden. Die Besonderheiten der Stoffwechselwege einzelner PA und die Spezifizierung einzelner, für die Metabolisierung relevanter Enzyme sollte in Zukunft Gegenstand der Forschung sein, um die kumulativen Wirkungen von PA besser nachzuvollziehen und die für den Menschen entstehenden Risiken durch die Aufnahme von PA konkretisieren zu können.
N2  - PA are naturally occurring secondary plant metabolites which are known for their genotoxic potential. In vitro studies can detect chromosomal damage after application of micromolar doses. Notoriously, some PA are suspected to cause hepatotoxicity and carcinogenicity in human beings as well as in animals. In this study the genotoxic effects of Lasiocarpin and Riddelliin were tested on the human hepatoma cell line Huh6. Therefore, the micronucleus test, the alkaline and the FPG Comet Assay and γ-H2AX Assay were performed. The genotoxic potential of these prodrugs on Huh6 cells was proven in preliminary tests with BaP and CPA. In conclusion, the selected PA Riddelliin and Lasiocarpin induced micronuclei in Huh6 cells in a dose-dependent manner. In comparison to Riddelliin, the influence of Lasiocarpin was detectable at lower concentrations in micronucleus test. After the simultaneous treatment with different structure types of PA it can be observed that there is no significant elevation of DNA damage compared to the single substance tests. Possibly the reason for this is a depletion of the metabolic capacity of the Huh6 cells. Overall, according to the findings of the performed toxicological tests with Lasiocarpin and Riddelliin, it is likely that the formation of crosslinks plays a more important role in the induction of genotoxicity on Huh6 cells than oxidative stress. Double-strand breaks could not be identified as a reliable inducer of genotoxicity in this study. The peculiarities of the metabolic pathways of individual PA and the specification of relevant enzymes for metabolization should be subject of future research to create a better understanding of the cumulative effects and the resulting risks to humans from the ingestion of PA.
KW  - Pyrrolizidinalkaloide
KW  - Lasiocarpin
KW  - Riddelliin
KW  - Huh6
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319793
ER  - 
TY  - JOUR
A1  - Jochmann, Svenja
A1  - Elkenani, Manar
A1  - Mohamed, Belal A.
A1  - Buchholz, Eric
A1  - Lbik, Dawid
A1  - Binder, Lutz
A1  - Lorenz, Kristina
A1  - Shah, Ajay M.
A1  - Hasenfuß, Gerd
A1  - Toischer, Karl
A1  - Schnelle, Moritz
T1  - Assessing the role of extracellular signal‐regulated kinases 1 and 2 in volume overload‐induced cardiac remodelling
JF  - ESC Heart Failure
N2  - Aims
Volume overload (VO) and pressure overload (PO) induce differential cardiac remodelling responses including distinct signalling pathways. Extracellular signal‐regulated kinases 1 and 2 (ERK1/2), key signalling components in the mitogen‐activated protein kinase (MAPK) pathways, modulate cardiac remodelling during pressure overload (PO). This study aimed to assess their role in VO‐induced cardiac remodelling as this was unknown.

Methods and results
Aortocaval fistula (Shunt) surgery was performed in mice to induce cardiac VO. Two weeks of Shunt caused a significant reduction of cardiac ERK1/2 activation in wild type (WT) mice as indicated by decreased phosphorylation of the TEY (Thr‐Glu‐Tyr) motif (−28% as compared with Sham controls, P < 0.05). Phosphorylation of other MAPKs was unaffected. For further assessment, transgenic mice with cardiomyocyte‐specific ERK2 overexpression (ERK2tg) were studied. At baseline, cardiac ERK1/2 phosphorylation in ERK2tg mice remained unchanged compared with WT littermates, and no overt cardiac phenotype was observed; however, cardiac expression of the atrial natriuretic peptide was increased on messenger RNA (3.6‐fold, P < 0.05) and protein level (3.1‐fold, P < 0.05). Following Shunt, left ventricular dilation and hypertrophy were similar in ERK2tg mice and WT littermates. Left ventricular function was maintained, and changes in gene expression indicated reactivation of the foetal gene program in both genotypes. No differences in cardiac fibrosis and kinase activation was found amongst all experimental groups, whereas apoptosis was similarly increased through Shunt in ERK2tg and WT mice.

Conclusions
VO‐induced eccentric hypertrophy is associated with reduced cardiac ERK1/2 activation in vivo. Cardiomyocyte‐specific overexpression of ERK2, however, does not alter cardiac remodelling during VO. Future studies need to define the pathophysiological relevance of decreased ERK1/2 signalling during VO.
KW  - ERK1/2
KW  - volume overload
KW  - aortocaval fistula model
KW  - cardiac remodelling
KW  - eccentric hypertrophy
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212735
VL  - 6
IS  - 5
SP  - 1015
EP  - 1026
ER  - 
TY  - THES
A1  - Schihada, Hannes
T1  - Novel optical methods to monitor G-protein-coupled receptor activation in microtiter plates
T1  - Neue optische Methoden zur Messung der Aktivierung von G-Protein-gekoppelten Rezeptoren in Mikrotiter-Platten
N2  - G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function.
N2  - Die Klasse der G-protein-gekoppelten Rezeptoren (GPCRs) stellt die größte Familie membranständiger Proteine dar. GPCRs regulieren eine Vielzahl diverser physiologischer Prozesse in eukaryotischen Zellen und kontrollieren so unterschiedliche Zellfunktionen im menschlichen Organismus. Sie stellen die Zelloberflächenrezeptoren für verschiedenartige extrazelluläre Stimuli, wie zum Beispiel Photonen, niedermolekulare chemische Verbindungen, Peptide und Lipide dar. Die Wechselwirkung mit diesen sogenannten Liganden stabilisiert spezifische GPCR-Konformationen. Diese dienen wiederum als Ausgangspunkt für nachgeschaltete intrazelluläre Signalkaskaden, die beispielweise über membranverankerte G-Proteine vermittelt werden können. Während endogene GPCR-Agonisten diese Signalweiterleitung verstärken, können andere Biomoleküle wie Lipide, Ionen oder andersartige Membranproteine die Funktion, und damit die Signalweiterleitung der GPCRs modulieren.  	
Aufgrund ihrer Einbindung in eine Vielzahl physiologischer und pathophysiologischer Prozesse, wurden GPCRs schon früh als Angriffspunkte („Targets“) zur Behandlung verschiedener Erkrankungen erforscht und genutzt. Heutzutage vermitteln etwa 30% aller zugelassenen Arzneistoffe ihre Wirkung über G-protein-gekoppelte Rezeptoren. Dennoch wird das große Potential dieser Rezeptorfamilie als Targets für medikamentöse Behandlungen noch nicht in vollem Umfang ausgeschöpft. Tatsächlich gibt es für mehr als 200 GPCRs, die nicht der olfaktorischen Wahrnehmung dienen, noch keine Arzneistoffe, da wenig über deren Pharmakologie und physiologische Bedeutung bekannt ist. Zudem wird die Entwicklung neuartiger GPCR-Liganden erheblich durch das eingeschränkte Methodenrepertoire beeinträchtigt. Alle derzeit etablierten Techniken zur Identifizierung neuer GPCR-Liganden erfassen entweder den Ligand-GPCR-Bindungsprozess, der keine Informationen über die tatsächliche Aktivität der Verbindung liefert, oder messen weit-nachgeschaltete Signale, wie Änderungen sogenannter „Second-Messenger“-Konzentrationen (meist cAMP oder Calcium) und Reporter-Gen-Expressionslevel. Aufgrund ihrer Entfernung vom eigentlichen Rezeptor-Aktivierungsprozess haben diese Methoden allerdings bedeutende Nachteile und produzieren so häufig Falsch-Positive und Falsch-Negative Ergebnisse.	
Seit den frühen 2000er wurden GPCR-Konformationssensoren auf Basis von Fluoreszenz-Resonanz-Energie-Transfer (FRET) zur Messung der Ligand-induzierten Rezeptordynamik genutzt. Jedoch wies keiner der bisher entwickelten FRET- oder BRET- (Biolumineszenz-Resonanz-Energie-Transfer) Sensoren ausreichende Signalstärke auf, um im Hochdurchsatz-Screening (HTS) angewendet werden zu können. 
Die vorliegende Studie beschreibt das erste GPCR-Sensordesign, das aufgrund seiner exzellenten Signalstärke im Hochdurchsatz-Verfahren verwendet werden kann. Wir haben 21 unterschiedliche FRET- und BRET-Sensoren des α2A-adrenergen Rezeptors (α2AAR) getestet und dabei die Kombination der kleinen und hellen Luziferase NanoLuciferase (Nluc) mit dem rot-fluoreszierenden HaloTag-Farbstoff 618 als sensitivstes RET-Paar identifiziert. Der α2AARNluc/Halo(618) Biosensor ermöglicht die Messung der Aktivität und Wirkstärke von α2AAR-Liganden im Mikrotiterplattenformat. Um die universelle Anwendbarkeit dieses Sensordesigns zu prüfen, wurden fünf weitere Nluc/Halo(618)-basierende Sensoren für GPCRs unterschiedlicher Unterfamilien entwickelt. Zudem konnten wir zeigen, dass diese GPCRNluc/Halo(618)-Fusionsproteine weiterhin ihre natürlichen Signalkaskaden in Gang setzen können und damit die biologische Funktionalität dieser Rezeptoren erhalten ist. Außerdem belegt die vorlegende Arbeit, dass diese neue Sensor-Generation zur Messung Ligand-vermittelter Rezeptordynamiken im Hochdurchsatz-Format und zur Untersuchung der GPCR-Regulation durch endogene Modulatoren genutzt werden kann.
Zusammenfassend kann gesagt werden, dass wir den ersten HTS-kompatiblen Assay zur Messung der GPCR-Konformationsänderungen entwickelt haben. Diese Biosensoren erlauben die Charakterisierung neuartiger GPCR-Liganden direkt auf der Rezeptorebene und funktionieren damit unabhängig von nachgeschalteter Signalamplifikation oder Überlagerung verschiedener Signalwege, welche die Aussagekraft traditioneller GPCR-Screening-Verfahren häufig beeinträchtigen. Diese Technik kann zur Entdeckung neuartiger GPCR-Arzneistoffe genutzt werden, zu einem besseren Verständnis bisher kaum erforschter Rezeptoren beitragen und der Identifizierung und Charakterisierung potentieller GPCR-Modulatoren dienen.
KW  - G-Protein gekoppelte Rezeptoren
KW  - Hochdurchsatz-Screening
KW  - Förster Resonanz Energie Transfer
KW  - High-thropughput screening
KW  - Bioluminescence resonance energy transfer
KW  - G-protein-coupled receptors
KW  - Receptor dynamics
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175415
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  - 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  - 
TY  - JOUR
A1  - Klenk, Christoph
A1  - Hommers, Leif
A1  - Lohse, Martin J.
T1  - Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor
JF  - Frontiers in Endocrinology
N2  - Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.
KW  - GPCRs
KW  - parathyroid hormone 1 receptor
KW  - matrix metalloproteinase
KW  - ectodomain cleavage
KW  - biased signaling
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262055
SN  - 1664-2392
VL  - 13
ER  - 
TY  - JOUR
A1  - Reimann, Hauke
A1  - Stopper, Helga
A1  - Hintzsche, Henning
T1  - Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells
JF  - Archives of Toxicology
N2  - Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.
KW  - micronuclei
KW  - cell fate
KW  - etoposide
KW  - live imaging
KW  - DNA damage
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235039
SN  - 0340-5761
VL  - 94
ER  - 
TY  - THES
A1  - Seier, Kerstin
T1  - Investigation of dynamic processes of prototypical class A GPCRs by single-molecule microscopy
T1  - Untersuchung von dynamischen Prozessen von prototypischen Klasse A GPCR's durch Einzelmolekülmikroskopie
N2  - In this work, two projects were pursued. 

In the first project, I investigated two different subtypes of opioid receptors, which play a key role as target for analgesia. A set of subtype specific fluorescent ligands for μ opioid receptor (MOR) and δ opioid receptor (DOR) was characterised and used to gain insights into the diffusion behaviour of those receptors. It was shown that the novel ligands hold photophysical and pharmacological properties making them suitable for single-molecule microscopy. Applying them to wild-type receptors expressed in living cells revealed that both sub-types possess a heterogeneous diffusion behaviour. Further- more, the fluorescent ligands for the MOR were used to investigate homodomerisation, a highly debated topic. The results reveal that only ≈ 5 % of the receptors are present as homodimers, and thus the majority is monomeric. G-protein coupled receptors (GPCRs) play a major role as drug targets. Accordingly, understanding the activation process is very important. For a long time GPCRs have been believed to be either active or inactive. In recent years several studies have shown, that the reality is more complex, involving more substates. [1, 2, 3, 4] In this work the α 2A AR was chosen to investigate the activation process on a single-molecule level, thus being able to distinguish also rare or short-lived events that are hidden in ensemble mea- surements. With this aim, the receptor was labelled intracellular with two fluorophores using supported membranes. Thus it was possible to acquire movies showing qualita- tively smFRET events. Unfortunately, the functionality of the used construct could not be demonstrated. To recover the functionality the CLIP-tag in the third intracellular loop was replaced successfully with an amber codon. This stop codon was used to insert an unnatural amino acid. Five different mutants were created and tested and the most promising candidate could be identified. First ensemble FRET measurements indicated that the functionality might be recovered but further improvements would be needed. Overall, I could show that single-molecule microscopy is a versatile tool to investigate the behaviour of typical class A GPCRs. I was able to show that MOR are mostly monomeric under physiological expression levels. Furthermore, I could establish intra- cellular labelling with supported membranes and acquire qualitative smFRET events.
N2  - In dieser Arbeit wurden zwei Projekte verfolgt. 

Im ersten Projekt wurden zwei Subtypen der Opioidrezeptoren untersucht, die eine wichtige Rolle für die Wirksamkeit von Analgetika spielen. Ein Set von subtypspezifischen fluoreszierenden Liganden für den MOR und den DOR wurde charakterisiert und eingesetzt, um Einblicke in das Diffuionsverhalten der Rezeptoren zu gewinnen. Es konnte gezeigt werden, dass die neuartigen Liganden sowohl photophysikalische als auch pharmakologische Eigenschaften besitzen, die sie für die Einzelmolekülmikroskopie interessant machen. Versuche mit Opioidrezeptoren, die in lebenden Zellen exprimiert werden, zeigten, dass beide Subtypen heterogenes Diffuionsverhalten aufweisen. Des Weiteren wurden die fluoreszierenden Liganden für den MOR genutzt um Homodimerisierung zu untersuchen, da dies ein kontrovers diskutiertes Thema ist. Die Ergebnisse zeigen, dass lediglich ≈ 5% der Rezeptoren als Homodimere vorliegen und der Großteil monomerisch ist. 
GPCRs sind besonderem Interesse, weil sie Angriffspunkt vieler Medikamente sind. Deshalb ist es wichtig ihren Aktivierungsmechanismus besser zu verstehen. Lange Zeit wurde angenommen, dass GPCRs entweder aktiv oder inaktiv sind. Neuere Studien zeigten jedoch, dass die Realität komplexer ist und der Prozess Zwischenschritte involviert. [1, 2, 3, 4] In dieser Arbeit wurde der α 2A Adrenorezeptor als prototypischer Klasse A GPCR gewählt, um den Aktivierungsprozess auf Einzelmoleküllevel zu untersuchen. Durch die Betrachtung einzelner Rezeptoren ist es möglich auch seltene oder sehr kurzlebige Ereignisse zu unterscheiden, die in Kollektivmessungen untergehen. Um dies zu erreichen wurde der Rezeptor erfolgreich intrazellulär mit zwei Fluorophoren markiert. Dies gelang durch die Herstellung von „supported membranes", also Zellmembranen die auf einem Objektträger fixiert wurden. Dadurch war es möglich Videos aufzunehmen, die Einzelmolekül-FRET-Ereignisse zeigen. Jedoch gelang es nicht zu zeigen, dass der Rezeptor als Ganzes noch funktional war. Um einen funktionalen Rezeptor zu erhalten, wurde das CLIP-Tag in der dritten intrazellulären Schleife erfolgreich durch ein Stopcodon ersetzt, welches für eine nicht kanonische Aminosäure kodierte. Fünf verschiedene Mutanten wurden kloniert und getestet, wobei der vielversprechendste Mutant identifiziert werden konnte. Erste FRET-Kollektivmessungen deuten darauf hin, dass dieser Mutant funktional sein könnte. Jedoch sind weitere Verbesserungen nötig. 
Insgesamt konnte ich zeigen, dass Einzelmolekülmikroskopie vielseitige Möglichkeiten bietet um das Verhalten von GPCRs zu untersuchen. Ich konnte nachweisen, dass MOR unter physiologischen Bedingungen hauptsächlich als Monomere vorliegen. Des Weiteren konnte ich Dank supported membranes die Markierung durch Farbstoffe im Intrazellularbereich etablieren und qualitative smFRET Ereignisse aufnehmen.
KW  - PhD thesis pharmacology
KW  - GPCR dimerisation
KW  - single-molecule imaging
KW  - opioid receptor
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199739
ER  - 
TY  - JOUR
A1  - Bankoglu, Ezgi Eyluel
A1  - Schuele, Carolin
A1  - Stopper, Helga
T1  - Cell survival after DNA damage in the comet assay
JF  - Archives of Toxicology
N2  - 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.
KW  - Cell death and comet assay
KW  - DNA damage
KW  - DNA repair
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265339
VL  - 95
IS  - 12
ER  - 
TY  - JOUR
A1  - Jeanclos, Elisabeth
A1  - Knobloch, Gunnar
A1  - Hoffmann, Axel
A1  - Fedorchenko, Oleg
A1  - Odersky, Andrea
A1  - Lamprecht, Anna‐Karina
A1  - Schindelin, Hermann
A1  - Gohla, Antje
T1  - Ca\(^{2+}\) functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin
JF  - FEBS Letters
N2  - Pyridoxal 5′‐phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ‐aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)‐ and integrin‐binding protein 1 (CIB1) as a PDXP interactor by yeast two‐hybrid screening and find a calmodulin (CaM)‐binding motif that overlaps with the PDXP‐CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP‐CIB1 complex may functionally differ from the PDXP‐Ca\(^{2+}\)‐CaM complex.
KW  - calmodulin
KW  - chronophin
KW  - CIB1
KW  - haloacid dehalogenase
KW  - pyridoxal phosphatase
KW  - vitamin B6
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217963
VL  - 594
IS  - 13
SP  - 2099
EP  - 2115
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  - THES
A1  - Bertelsmann, Dietmar
T1  - Analysis of the Frequency of Kidney Toxicity in Preclinical  Safety Studies using the eTOX Database
T1  - Analyse der Häufigkeit von Nierentoxizität in präklinischen Sicherheitsstudien unter Verwendung der eTOX-Datenbank
N2  - 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.
N2  - Diese Arbeit zielte darauf ab, verlässliche Daten über die Häufigkeit verschiedener Arten von Nierentoxizitätsbefunden in 28-tägigen oralen Sondenstudien an Wistar-Ratten zu erhalten. Untersucht wurde weiterhin die Konsistenz 
der Toxizitätsbefunde unterschiedlicher Spezies und Studiendauer sowie die 
Korrelation zwischen histopathologischen Endpunkten und routinemäßig verwendeten klinisch-chemischen Parametern, die auf eine Nierenschädigung hinweisen. Die Analyse der histopathologischen Nierenbefunde wurde durch Extraktion von Informationen aus der IMI eTOX-Datenbank durchgeführt.
Spontane renale histopathologische Befunde in 28-tägigen oralen Sondenstudien an Wistar-Ratten und Beagles bestätigten tubuläre Basophilie und 
renale Dilatation als häufigste Nebenbefunde bei den Kontrolltieren, während 
Nekrose und Glomerulosklerose gar nicht oder nur selten als Hintergrundläsion 
identifiziert wurden. 
Der histopathologische Nachweis von Nekrose und Glomerulosklerose 
war mit Änderungen der klinisch-chemischen Parameter in 28-tägigen Wistar-Rattenstudien mit oraler Sonde verbunden. Nekrose ging häufig mit einem statistisch signifikanten Anstieg von Serumkreatinin und Serumharnstoff einher, 
während Serumalbumin in Behandlungsgruppen, in denen Nekrose aufgezeichnet wurde, häufig statistisch signifikant abnahm. Im Gegensatz zur Nekrose war 
Glomerulosklerose in keiner der 28-tägigen Wistar-Ratten-Behandlungsgruppen 
mit oraler Sonde mit statistisch signifikanten Veränderungen von Serumkreatinin 
und Harnstoff assoziiert, sondern scheint sich am besten in einem Muster von 
statistisch signifikant erniedrigtem Serumalbumin und Serumprotein zusammen 
mit einem statistisch signifikanten Anstieg des Serumcholesterins widerzuspiegeln. Wie aufgrund der hohen Hintergrundinzidenzen von tubulärer Basophilie 
und Dilatation zu erwarten war, waren bei Tieren, bei denen Nierenläsionen auf 
renale tubuläre Basophilie oder Dilatation beschränkt waren, keine konsistenten 
Änderungen der klinisch-chemischen Parameter erkennbar. Zusammenfassend 
sind die routinemäßig bereitgestellten klinisch-chemischen Parameter eher unempfindlich - neuartige Nieren-Biomarker wie „Cystatin C“, „β-trace protein“ und „Kidney injury molecule 1“ sollten weiter evaluiert und in die routinemäßige 
präklinische und klinische Praxis integriert werden. Die Auswertung der Daten 
zur klinischen Chemie war jedoch durch das Fehlen individueller Tierdaten begrenzt.
Trotz der umfangreichen Anzahl an präklinischen Studien in der eTOX-Datenbank wurde der zeitliche Vergleich der Konsistenz durch die begrenzte 
Anzahl von Kurz- und Langzeitstudien eingeschränkt, welche mit denselben
Substanzen durchgeführt wurden, die innerhalb einer 28-Tage-Studie an Ratten 
als Verursacher von renalen histopathologischen Veränderungen identifiziert 
wurden. Eine hohe zeitliche Konsistenz sowohl für die behandlungsbedingte tubuläre Basophilie und Dilatation kann für keinen der beiden Effekte bestätigt 
werden, da diese beiden Befunde nur selten in Studien über eine andere Behandlungsdauer als 28 Tage nach Verabreichung derselben Substanzen, die den 
jeweiligen Effekt in einer 28-Tage-Studie hervorriefen, induziert wurden. Für den 
Befund der Nekrose war die zeitliche Konsistenz gering. Eine Ausnahme stellte
Substanz "AZ_GGA_200002321" dar, bei der über verschiedene Behandlungsdauern (2, 4, 26, 104 Wochen) hinweg konstant renale papilläre Nekrose festgestellt wurde. Für die Substanzen, die in einer 28-Tage-Studie an Ratten als glomeruloskleroseauslösend identifiziert wurden, waren keine Kurz- und Langzeitstudien verfügbar.
In keiner der korrespondierenden 28-Tage-Studien an Beagles mit oraler 
Sonde wurden konsistente Befunde der ausgewählten histopathologischen Endpunkte nach Behandlung mit den identischen Verbindungen, die den jeweiligen 
Effekt nach 28-tägiger Behandlung in Ratten verursachten, festgestellt. In der 
überwiegenden Mehrheit der Fälle wurden den Beagles in diesen Studien im 
Vergleich zu den entsprechenden 28-Tage-Wistar-Rattenstudien niedrigere Dosen verabreicht.
In der eTOX-Datenbank konnten keine 28-tägigen oralen Sondenstudien
an Wistar- und Wistar-Han-Ratten gefunden werden, in denen eine Akkumulation von hyalinen Tröpfchen, tubuläre Atrophie oder Hyperplasie aufgezeichet wurde. Nur eine 28-tägige Wistar-Rattenstudie wurde mit dem histopathologischen Ergebnis einer neutrophilen Entzündung identifiziert. Folglich kann eine 
Bewertung dieser vier Nierenbefunde in Bezug auf klinische Chemie und Konsistenz über Zeit und Spezies nicht vorgenommen werden.
Insgesamt zeigt dieser Arbeit, dass eine gezielte statistische Auswertung 
von in vivo-Daten im Rahmen von Forschungsverbünden wie dem eTOX-Projekt 
eine enorme Chance bietet, die präklinische Forschung in Zukunft auf dem Weg 
zu einer effizienteren Forschung in der präklinischen Phase der Arzneimittelentwicklung zu unterstützen. Dies könnte außerdem durch die Erweiterung methodischer Strategien und möglicherweise neuartiger Software-Tools erreicht werden, um die In-vivo-Toxikologie neuer molekularer Entitäten mit Hilfe von Informationen vorherzusagen, die bereits vor Beginn der Arzneimittelentwicklungspipeline verfügbar sind.
KW  - renal toxicity
KW  - etox database
KW  - rats
KW  - toxicity
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257104
ER  - 
TY  - JOUR
A1  - Hintzsche, Henning
A1  - Montag, Gracia
A1  - Stopper, Helga
T1  - Induction of micronuclei by four cytostatic compounds in human hematopoietic stem cells and human lymphoblastoid TK6 cells
JF  - Scientific Reports
N2  - For mutagenicity testing, primary lymphocytes or mammalian cell lines are employed. However, the true target for carcinogenic action of mutagenic chemicals may be stem cells. Since hematopoietic cancers induced by chemical agents originate at the hematopoietic stem cell (HSC) stage and since one of the side effects of chemotherapeutic cancer treatment is the induction of secondary tumors, often leukemias, HSC may be a suitable cell system. We compared the sensitivity of HSC with the genotoxicity testing cell line TK6 for chromosomal mutations. HSC were less sensitive than TK6 cells for the genotoxic effects of the model genotoxins and chemotherapeutic agents doxorubicin, vinblastine, methyl methanesulfonate (MMS) and equally sensitive for mitomycin C (MMC). However, loss of viability after mitomycin C treatment was higher in HSC than in TK6 cells. Among the factors that may influence sensitivity for genomic damage, the generation or response to reactive oxygen species (ROS) and the effectiveness of DNA damage response can be discussed. Here we show that HSC can be used in a standard micronucleus test protocol for chromosomal mutations and that their sensitivity was not higher than that of a classical testing cell line.
KW  - apoptosis
KW  - haematopoietic stem cells
KW  - TK6 cells
KW  - micronuclei
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176210
VL  - 8
IS  - 3371
ER  - 
TY  - JOUR
A1  - Chilaka, Cynthia Adaku
A1  - Obidiegwu, Jude Ejikeme
A1  - Chilaka, Augusta Chinenye
A1  - Atanda, Olusegun Oladimeji
A1  - Mally, Angela
T1  - Mycotoxin regulatory status in Africa: a decade of weak institutional efforts
JF  - Toxins
N2  - 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.
KW  - fungi
KW  - mycotoxin
KW  - legislation
KW  - food safety
KW  - food security
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-278941
SN  - 2072-6651
VL  - 14
IS  - 7
ER  - 
TY  - JOUR
A1  - Winkelbeiner, Nicola
A1  - Wandt, Viktoria K.
A1  - Ebert, Franziska
A1  - Lossow, Kristina
A1  - Bankoglu, Ezgi E.
A1  - Martin, Maximilian
A1  - Mangerich, Aswin
A1  - Stopper, Helga
A1  - Bornhorst, Julia
A1  - Kipp, Anna P.
A1  - Schwerdtle, Tanja
T1  - A multi-endpoint approach to base excision repair incision activity augmented by PARylation and DNA damage levels in mice: impact of sex and age
JF  - International Journal of Molecular Sciences
N2  - Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
KW  - maintenance of genomic integrity
KW  - ageing
KW  - sex
KW  - DNA damage
KW  - base excision repair (incision activity)
KW  - DNA damage response
KW  - poly(ADP-ribosyl)ation
KW  - liver
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285706
SN  - 1422-0067
VL  - 21
IS  - 18
ER  - 
TY  - JOUR
A1  - Tolstik, Elen
A1  - Ali, Nairveen
A1  - Guo, Shuxia
A1  - Ebersbach, Paul
A1  - Möllmann, Dorothe
A1  - Arias-Loza, Paula
A1  - Dierks, Johann
A1  - Schuler, Irina
A1  - Freier, Erik
A1  - Debus, Jörg
A1  - Baba, Hideo A.
A1  - Nordbeck, Peter
A1  - Bocklitz, Thomas
A1  - Lorenz, Kristina
T1  - CARS imaging advances early diagnosis of cardiac manifestation of Fabry disease
JF  - International Journal of Molecular Sciences
N2  - Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient’s prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90–96%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD.
KW  - coherent anti-Stokes Raman scattering (CARS) microscopy
KW  - Raman micro-spectroscopy
KW  - cardiovascular diseases
KW  - Fabry Disease (FD)
KW  - Gb3 and lyso-Gb3 biomarkers
KW  - multivariate data analysis
KW  - immunohistochemistry
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284427
SN  - 1422-0067
VL  - 23
IS  - 10
ER  - 
TY  - THES
A1  - Kodandaraman, Geema
T1  - Influence of insulin-induced oxidative stress in genotoxicity and disease
T1  - Einfluss von insulininduziertem oxidativem Stress auf Genotoxitität und Krankheit
N2  - Hormones are essential components in the body and their imbalance leads to pathological consequences. T2DM, insulin resistance and obesity are the most commonly occurring lifestyle diseases in the past decade. Also, an increased cancer incidence has been strongly associated with obese and T2DM patients.
Therefore, our aim was to study the influence of high insulin levels in accumulating DNA damage in in vitro models and patients, through the induction of oxidative stress. The primary goal of this study was to analyze the genotoxicity induced by the combined action of two endogenous hormones (insulin and adrenaline) with in vitro models, through the induction of micronuclei and to see if they cause an additive increase in genomic damage. This is important for multifactorial diseases having high levels of more than one hormone, such as metabolic syndrome and conditions with multiple pathologies (e.g., T2DM along with high stress levels).
Furthermore, the combination of insulin and the pharmacological inhibition of the tumor suppressor gene: PTEN, was to be tested in in vitro models for their genotoxic effect and oxidative stress inducing potential. As the tumor suppressor gene: PTEN is downregulated in PTEN associated syndromes and when presented along with T2DM and insulin resistance, this may increase the potential to accumulate genomic damage.
The consequences of insulin action were to be further elucidated by following GFP-expressing cells in live cell-imaging to observe the ability of insulin, to induce micronuclei and replicative stress. Finally, the detrimental potential of high insulin levels in obese patients with hyperinsulinemia and pre-diabetes was to be studied by analyzing markers of oxidative stress and genomic damage. In summary, the intention of this work was to understand the effects of high insulin levels in in vitro and in patients to understand its relevance for the development of genomic instability and thus an elevated cancer risk.
N2  - In-vitro-Genotoxizitätsstudien mit hohen Konzentrationen von Insulin und die Kombination mit Adrenalin zeigten keinen additiven Anstieg der Mikrokernzahl. Der Insulinrezeptor und der AKT-Signalweg waren in den insulinvermittelten Genomschaden involviert. Die endogenen ROS-Quellen, Mitochondrien und NOX, waren an dem insulinvermittelten DNA-Schaden beteiligt. Hohe Konzentrationen von mitochondrialen ROS alleine, verursacht durch einen Komplex III Mitochondrien-Inhibitor, führten zu Zytotoxizität, aber nicht zu einer Zunahme des Genomschadens. Daher ist die durch das NOX-Enzym vermittelte ROS-Produktion wahrscheinlich der gemeinsame Faktor des genotoxischen Signalweges von Insulin und Adrenalin. Die Überstimulation des NOX-Enzyms führte zu einer Sättigung der zellulären biologischen Effekte und fehlender Additivität bei der Induktion von Genomschaden. Dies könnte jedoch unter physiologischen Bedingungen anders sein, da die Hormonspiegel niedriger sind und die ROS-Quellen nicht durch jedes einzelne der Hormone bereits maximal genutzt und daher erschöpft werden. Damit könnte die Möglichkeit eines additiven Genomschadens in vivo bestehen.  
Die Rolle des AKT-Signalwegs bei der Insulin-vermittelten genomischen Schädigung ist bereits etabliert und hier wurde nun die Funktion des negativen Regulatorproteins PTEN untersucht. Die Ergebnisse zeigten, dass die PTEN Inhibierung nicht nur zu einer erhöhten Genotoxizität durch MN-Induktion führte, sondern auch zur Beeinträchtigung der mitochondrialen Funktion. Obwohl kein Anstieg von ROS nach PTEN-Inhibierung beobachtet wurde, könnte die mitochondriale Dysfunktion zur metabolischen Imbalance sowie zur Zunahme des Genomschadens führen. Dies könnte insbesondere bei Patienten mit bestimmten PTEN-assoziierten Syndromen und Krebserkrankungen, die eine defekte PTEN-vermittelte Tumorsuppressorfunktion, DNA-Reparaturdefekte und kompromittierte antioxidative Abwehrmechanismen aufweisen, eine wichtige Rolle spielen. Wenn diese Patienten zusätzlich von Hyperinsulinämie betroffen sind, könnte eine Akkumulation von Genomschaden erfolgen und das Risiko zur Krebsentstehung wäre erhöht. 
Der Mechanismus der Genomschadensinduktion durch Insulin wurde bisher mit einer ROS-vermittelten DNA-Oxidation in Verbindung gebracht, aber noch nicht mit der mitogenen Signalgebung. Bei dieser beschleunigte das mitogene Potential des Insulins die Zellteilung und verursachte einen leichten replikativen Stress. Der milde replikative Stress könnte der Kontrolle durch die mitotischen Checkpoint-Proteine entgehen und zu Chromosomen-Fehlverteilungen und Chromosomenbrüchen führen. Dieser Effekt wurde in der Krebszelllinie Hela in Form von multipolaren Spindeln und Mikronuklei beobachtet und es ist nicht klar ob normale Zellen mit effizienterer Kontrolle dies verhindern könnten. Insgesamt könnte ein durch hohe Insulinspiegel vermittelter Schaden im Kontext anderer Komorbiditäten wie etwa PTEN Syndromen, metabolischem Syndrom oder Adipositas zu einer Akkumulation von DNA-Schäden führen.  
Schließlich zeigte die Analyse von Proben adipöser Patienten eine Zunahme von DNA-Schaden und oxidativem Stress im Vergleich zu den gesunden Kontrollen. Der Anstieg des DNA-Schadens war am höchsten in der Untergruppe der Patienten mit Insulinresistenz. Hoher Insulinspiegel bedeutet somit ein Risiko vom erhöhten oxidativen Stress und Genomschaden, insbesondere in Kombination mit Komorbiditäten.
Erschwert wird das Verständnis dieser multifaktoriellen Zusammenhänge durch das komplexe Zusammenspiel von oxidativem Stress und seiner zellulären Regulation in vielen physiologischen sowie pathophysiologischen Prozessen. Daneben ist es eine Herausforderung, Genomschäden bei den geringen Wirkspiegeln hormoneller Effekte zu detektieren. Weitere Untersuchungen der komplexen Insulin-vermittelten Genomschadenswege werden notwendig sein, um mögliche Risiken der Hyperinsulinämie bei Erkrankungen wie Stoffwechselkrankheiten, Diabetes Typ 2 und Adipositas besser zu charakterisieren.
KW  - Insulin
KW  - Genotoxicity
KW  - Micronucleus
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242005
ER  - 
TY  - JOUR
A1  - Bankoglu, Ezgi Eyluel
A1  - Arnold, Charlotte
A1  - Hering, Ilona
A1  - Hankir, Mohammed
A1  - Seyfried, Florian
A1  - Stopper, Helga
T1  - Decreased chromosomal damage in lymphocytes of obese patients after bariatric surgery
JF  - Scientific Reports
N2  - 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.
KW  - obesity
KW  - bariatric surgery
KW  - cancer risk
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177090
VL  - 8
IS  - 11195
ER  - 
TY  - JOUR
A1  - Tan, Aaron
A1  - Babak, Maria V.
A1  - Venkatesan, Gopalakrishnan
A1  - Lim, Clarissa
A1  - Klotz, Karl-Norbert
A1  - Herr, Deron Raymond
A1  - Cheong, Siew Lee
A1  - Federico, Stephanie
A1  - Spalluto, Giampiero
A1  - Ong, Wei-Yi
A1  - Chen, Yu Zong
A1  - Loo, Jason Siau Ee
A1  - Pastorin, Giorgia
T1  - Design, Synthesis and Evaluation of New Indolylpyrimidylpiperazines for Gastrointestinal Cancer Therapy
JF  - Molecules
N2  - Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors’ ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3–6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 μM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.
KW  - gastrointestinal cancer
KW  - hA<sub>3</sub>AR
KW  - partial agonists
KW  - indolylpyrimidylpiperazines
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193271
SN  - 1420-3049
VL  - 24
IS  - 20
ER  - 
TY  - JOUR
A1  - Harnoš, Jakub
A1  - Cañizal, Maria Consuelo Alonso
A1  - Jurásek, Miroslav
A1  - Kumar, Jitender
A1  - Holler, Cornelia
A1  - Schambony, Alexandra
A1  - Hanáková, Kateřina
A1  - Bernatík, Ondřej
A1  - Zdráhal, Zbynêk
A1  - Gömöryová, Kristína
A1  - Gybeľ, Tomáš
A1  - Radaszkiewicz, Tomasz Witold
A1  - Kravec, Marek
A1  - Trantírek, Lukáš
A1  - Ryneš, Jan
A1  - Dave, Zankruti
A1  - Fernández-Llamazares, Ana Iris
A1  - Vácha, Robert
A1  - Tripsianes, Konstantinos
A1  - Hoffmann, Carsten
A1  - Bryja, Vítězslav
T1  - Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1
JF  - Nature Communications
N2  - Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.
KW  - biological techniques
KW  - cell signalling
KW  - phosphorylation
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227837
VL  - 10
ER  - 
TY  - JOUR
A1  - Joos, J. P.
A1  - Saadatmand, A. R.
A1  - Schnabel, C.
A1  - Viktorinová, I.
A1  - Brand, T.
A1  - Kramer, M.
A1  - Nattel, S.
A1  - Dobrev, D.
A1  - Tomancak, P.
A1  - Backs, J.
A1  - Kleinbongard, P.
A1  - Heusch, G.
A1  - Lorenz, K.
A1  - Koch, E.
A1  - Weber, S.
A1  - El-Armouche, A.
T1  - Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila
JF  - Scientific Reports
N2  - Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.
KW  - cardiac hypertrophy
KW  - epigenetics
KW  - heart failure
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323637
VL  - 8
ER  - 
TY  - JOUR
A1  - Hommers, L. G.
A1  - Richter, J.
A1  - Yang, Y.
A1  - Raab, A.
A1  - Baumann, C.
A1  - Lang, K.
A1  - Schiele, M. A.
A1  - Weber, H.
A1  - Wittmann, A.
A1  - Wolf, C.
A1  - Alpers, G. W.
A1  - Arolt, V.
A1  - Domschke, K.
A1  - Fehm, L.
A1  - Fydrich, T.
A1  - Gerlach, A.
A1  - Gloster, A. T.
A1  - Hamm, A. O.
A1  - Helbig-Lang, S.
A1  - Kircher, T.
A1  - Lang, T.
A1  - Pané-Farré, C. A.
A1  - Pauli, P.
A1  - Pfleiderer, B.
A1  - Reif, A.
A1  - Romanos, M.
A1  - Straube, B.
A1  - Ströhle, A.
A1  - Wittchen, H.-U.
A1  - Frantz, S.
A1  - Ertl, G.
A1  - Lohse, M. J.
A1  - Lueken, U.
A1  - Deckert, J.
T1  - A functional genetic variation of SLC6A2 repressor hsa-miR-579-3p upregulates sympathetic noradrenergic processes of fear and anxiety
JF  - Translational Psychiatry
N2  - Increased sympathetic noradrenergic signaling is crucially involved in fear and anxiety as defensive states. MicroRNAs regulate dynamic gene expression during synaptic plasticity and genetic variation of microRNAs modulating noradrenaline transporter gene (SLC6A2) expression may thus lead to altered central and peripheral processing of fear and anxiety. In silico prediction of microRNA regulation of SLC6A2 was confirmed by luciferase reporter assays and identified hsa-miR-579-3p as a regulating microRNA. The minor (T)-allele of rs2910931 (MAFcases = 0.431, MAFcontrols = 0.368) upstream of MIR579 was associated with panic disorder in patients (pallelic = 0.004, ncases = 506, ncontrols = 506) and with higher trait anxiety in healthy individuals (pASI = 0.029, pACQ = 0.047, n = 3112). Compared to the major (A)-allele, increased promoter activity was observed in luciferase reporter assays in vitro suggesting more effective MIR579 expression and SLC6A2 repression in vivo (p = 0.041). Healthy individuals carrying at least one (T)-allele showed a brain activation pattern suggesting increased defensive responding and sympathetic noradrenergic activation in midbrain and limbic areas during the extinction of conditioned fear. Panic disorder patients carrying two (T)-alleles showed elevated heart rates in an anxiety-provoking behavioral avoidance test (F(2, 270) = 5.47, p = 0.005). Fine-tuning of noradrenaline homeostasis by a MIR579 genetic variation modulated central and peripheral sympathetic noradrenergic activation during fear processing and anxiety. This study opens new perspectives on the role of microRNAs in the etiopathogenesis of anxiety disorders, particularly their cardiovascular symptoms and comorbidities.
KW  - clinical genetics
KW  - psychiatric disorders
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-322497
VL  - 8
ER  - 
TY  - THES
A1  - Hadi, Naji Said Aboud
T1  - In vitro Studies on the Genotoxicity of Selected Pyrrolizidine Alkaloids
T1  - In-vitro-Studien zur Genotoxizität ausgewählter Pyrrolizidinalkaloide
N2  - Cancer is one of the leading causes of death worldwide. Toxic contaminants in human food or medicinal products, such as substances like pyrrolizidine alkaloids (PAs), have been thought to contribute to cancer incidence. PAs are found in many plant species as secondary metabolites, and they may affect humans through contaminated food sources, herbal medicines, and dietary supplements. Hundreds of compounds belonging to PAs have been identified, differing in their chemical structures, either in their necine base moiety or esterification at their necic acid moiety. PAs undergo hepatic metabolism, and after this process, they can induce hepatotoxicity, genotoxicity, and carcinogenicity. However, the mechanism of inducing genotoxicity and carcinogenicity is still unclear and warrants further investigation.
Therefore, the present study aims to investigate the mechanism of genotoxicity induced by selected PAs with different chemical structures in in vitro systems. Primarily, human hepatoma HepG2 cells were utilized, and in co-culture, metabolically active HepG2 cells were combined with non-metabolically active human cervical HeLa H2B-GFP cells.

First, the genotoxicity of the PAs europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine, and lasiocarpine was investigated in the cytokinesis-block micronucleus (CBMN) assay. All seven selected PAs caused the formation of micronuclei in a dose-dependent manner, with the maximal increase of micronucleus formation ranging from 1.64 to 2.0 fold. The lowest concentrations at which significant induction of micronuclei was found were 3.2 µM for lasiocarpine and riddelliine, 32 µM for retrorsine and echimidine, and 100 µM for seneciphylline, europine, and lycopsamine. These results confirmed previously published potency rankings in the micronucleus assay.
The same PAs, with the exception of seneciphylline, were also investigated in a crosslink-modified comet assay, and reduced tail formation after hydrogen peroxide treatment was found in all diester-type PAs. Meanwhile, an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration. Thus, the crosslinking activity was related to the ester type.

Next, the role of metabolic enzymes and membrane transporters in PA-induced genotoxicity was assessed. Ketoconazole (CYP 450-3A4 inhibitor) prevented lasiocarpine-induced micronucleus formation completely, while furafylline (CYP 450-1A2 inhibitor) reduced lasiocarpine-induced micronucleus formation, but did not abolish it completely. This implies that the CYP 450 enzymes play an important role in PA-induced genotoxicity. 
Carboxylesterase 2 enzyme (CES 2) is commonly known to be involved in the detoxification of xenobiotics. Loperamide (CES 2 inhibitor) yielded an increased formation of lasiocarpine-induced micronuclei, revealing a possible role of CES-mediated detoxification in the genotoxicity of lasiocarpine. Also, intracellular glutathione (GSH) plays an important role in the detoxification of xenobiotics or toxins in the cells. Cells which had been pretreated with L-buthionine sulfoximine (BSO) to reduce GSH content were significantly more sensitive for the induction of micronucleus formation by lasiocarpine revealing the importance of GSH in PA-induced genotoxicity.
 
Quinidine (Q) and nelfinavir (NFR) are OCT1 and OATP1B1 influx transporter inhibitors, respectively, which reduced micronucleus induction by lasiocarpine (only quinidine significantly), but not completely, pointing to a relevance of OCT1 for PA uptake in HepG2 cells. Verapamil (V) and benzbromarone (Bz) are MDR1 and MRP2 efflux transporter inhibitors, respectively, and they caused a slightly increased micronucleus induction by lasiocarpine (significant only for benzbromarone) thus, revealing the role of efflux transporters in PA-induced genotoxicity. 

The mechanistic approach to PA-induced genotoxicity was further studied based on oxidative stress via the formation of reactive oxygen species (ROS) in HepG2 cells. Overproduction of ROS can cross-link cellular macromolecules such as DNA, leading to genomic damage. An equimolar concentration of 10 µM of lasiocarpine (open-diester PA), riddelliine (cyclic-diester PA), and europine (monoester) significantly induced ROS production, with the highest ROS generation observed after lasiocarpine treatment, followed by riddelliine and then europine. No significant increase in ROS production was found with lycopsamine (10 µM; monoester PA), even at a higher concentration (320 µM). The generation of ROS by these PAs was further analyzed for confirmation by using 5 mM of the thiol radical scavenger antioxidant N-acetyl cysteine (NAC) combined with lasiocarpine, riddelliine, or europine. This analysis yielded a significant decrease in ROS after combining NAC with lasiocarpine, riddelliine, and europine. In addition, lasiocarpine, riddelliine, and europine induced a loss of mitochondrial membrane potential, pointing to mitochondria as the source of ROS generation.

In vivo, hepatic sinusoidal epithelial cells (HSECs) are known to be damaged first by PAs after hepatic metabolization, but HSECs themselves do not express the required metabolic enzymes for activation of PAs. To mimic this situation, HepG2 cells were used to metabolically activate PA in a co-culture with HeLa H2B-GFP cells as non-metabolically active neighbours. Due to the green fluorescent GFP label the HeLa cells could be identified easily based in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronucleus formation in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of CYP 450 enzymes with ketoconazole abrogated micronucleus formation induced by the same PAs tested in the co-culture. The efflux transporter inhibitors verapamil and benzbromarone reduced the micronucleus formation in the co-culture. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured non-metabolically active green HeLa cells.

Finally, in HepG2 cells as well as the co-culture, combinations of PAs lasiocarpine and riddelliine favoured an additive effect rather than synergism. Thus, this study therefore provides support that the assumption of dose-addition can be applied in the characterization of the genotoxicity risk of PAs present in a mixture.
N2  - Krebs ist eine der häufigsten Todesursachen weltweit. Toxische Verunreinigungen in Lebensmitteln oder pflanzlichen Arzneimitteln, wie Pyrrolizidinalkaloide (PAs), können zur Krebsinzidenz beitragen. PAs kommen in vielen Pflanzenarten als Sekundärmetabolite vor. Menschen können diese über kontaminierte Nahrungsquellen, pflanzliche Arzneimittel und Nahrungsergänzungsmittel aufnehmen. Eine Vielzahl von Verbindungen, die zu pyrrolizidinalkaloidhaltigen Substanzen (PAs) gehören, wurden identifiziert. Diese unterscheiden sich in ihrer chemischen Struktur entweder durch ihre Necinbaseneinheit oder ihre Veresterung an der Necicsäureeinheit. Nach metabolischer Aktivierung in der Leber können PAs Hepatotoxizität, Genotoxizität und Karzinogenität induzieren. Jedoch ist der Genotoxizitätsmechanismus nicht vollständig aufgeklärt und erfordert weitere Untersuchungen.
Das Ziel dieser Studie liegt in der Untersuchung des Mechanismus der Genotoxizität, die in vitro durch bestimmte PAs mit unterschiedlicher chemischer Struktur induziert wird. Hierbei wurden primär humane Hepatom-HepG2-Zellen verwendet sowie in Co-Kultur metabolisch aktive HepG2-Zellen und nicht-metabolisch aktive humane zervikale HeLa H2B-GFP-Zellen.
Zunächst wurde die Genotoxizität der PAs Europin, Lycopsamin, Retrorsin, Riddelliin, Seneciphyllin, Echimidin und Lasiocarpin im Zytokinese-Block-Mikronukleus-Assay (CBMN) untersucht. Die sieben (7) ausgewählten PAs führten dosisabhängig zur Bildung von Mikrokernen. Der maximale Anstieg der Mikronukleusbildung lag für alle PAs im Bereich des 1,64- bis 2,0-fachen des Ausgangswertes. Die niedrigsten Konzentrationen, bei denen eine signifikante Induktion von Mikrokernen gefunden wurde, waren 3,2 μM für Lasiocarpin und Riddelliin, 32 μM für Retrorsin und Echimidin sowie 100 μM für Seneciphyllin, Europin und Lycopsamin. Diese Ergebnisse bestätigen zuvor veröffentlichte Potenz-Rankings im Mikronukleus-Assay.

Die Genotoxizität der gleichen PAs, mit Ausnahme von Seneciphyllin, wurde zusätzlich mittels eines Crosslink-modifizierten Comet-Assay untersucht. Es wurde eine reduzierte Schweifbildung nach der Behandlung mit Wasserstoffperoxid in allen PAs des Diestertyps gefunden, während eine äquimolare Konzentration der Monoester Europin und Lycopsamin die DNA-Migration nicht signifikant reduzierte. Dies deutet darauf hin, dass die Vernetzungsaktivität von PAs auf der Ester-Einheit beruht.
Als nächstes wurde die Rolle von Stoffwechselenzymen und Membrantransportern in der PA-induzierten Genotoxizität untersucht. Ketoconazol (CYP 450-3A4-Inhibitor) verhinderte die Lasiocarpin-induzierte Mikronukleusbildung vollständig, während Furafyllin (CYP 450-1A2-Inhibitor) die Lasiocarpin-induzierte Mikronukleusbildung reduzierte, aber nicht vollständig beseitigte. Dies deutet darauf hin, dass CYP 450-Enzyme eine wichtige Rolle bei der PA-induzierten Genotoxizität spielen.

Es ist allgemein bekannt, dass das Enzym Carboxylesterase 2 (CES-2) an der Entgiftung von Xenobiotika beteiligt ist. Loperamid (CES-2-Inhibitor) führte zu einer erhöhten Bildung von Lasiocarpin-induzierten Mikrokernen, was auf eine mögliche Rolle der CES-vermittelten Entgiftung bei der Genotoxizität von Lasiocarpin hindeutet. Auch intrazelluläres Glutathion (GSH) spielt eine wichtige Rolle bei der Entgiftung von Xenobiotika oder Toxinen. Zellen, die mit L-Buthioninsulfoximin (BSO) vorbehandelt worden waren, um den GSH-Gehalt zu reduzieren, waren signifikant empfindlicher für die Induktion der Mikronukleusbildung durch Lasiocarpin, was die Bedeutung von GSH für die PA-induzierte Genotoxizität zeigt.
Chinidin (Q) und Nelfinavir (NFR) sind OCT1- bzw. OATP1B1-Influx-Transporter-Inhibitoren, die die Mikronukleus-Induktion durch Lasiocarpin reduzierten (nur Chinidin signifikant), aber nicht vollständig, was auf eine Relevanz von OCT1 für die PA-Aufnahme in HepG2-Zellen hindeutet.Verapamil (V) und Benzbromaron (Bz) sind MDR1- bzw. MRP2-Efflux-Transporter-Inhibitoren und verursachten eine leicht erhöhte Mikronukleus-Induktion durch Lasiocarpin (signifikant nur für Benzbromaron), was die Rolle von Efflux-Transportern bei der PA-induzierten Genotoxizität aufzeigt.
Der Mechanismus der PA-induzierten Genotoxizität wurde auf der Grundlage von oxidativem Stress durch die Bildung von reaktiven Sauerstoffspezies (ROS) in HepG2-Zellen weiter untersucht. Eine Überproduktion von ROS kann zelluläre Makromoleküle wie DNA vernetzen, was zu genomischen Schäden führt. Eine äquimolare Konzentration von 10 μM von Lasiocarpin (Open-Diester PA), Riddelliin (Cyclic-Diester PA) und Europin (Monoester) induzierte signifikant die ROS-Produktion, wobei die höchste ROS-Erzeugung nach Lasiocarpin-Behandlung beobachtet wurde, gefolgt von Riddelliin und Europin. Mit Lycopsamin (10 μM; Monoester PA) wurde auch bei höherer Konzentration (320 μM) keine signifikante Steigerung der ROS-Produktion gefunden.
Um die Beteiligung von ROS am Mechanismus der Genotoxizität einzelner PAs genauer zu betrachten und die bisherigen Ergebnisse zu bestätigen, wurden weitere Untersuchungen in Anwesenheit des Sauerstoffradikalfängers N-Acetylcysteine (NAC) in Kombination mit Lasiocarpin, Riddelliin oder Europin durchgeführt. Diese Analyse ergab eine signifikante Abnahme der ROS-Produktion nach der Kombination von NAC mit Lasiocarpin, Riddelliin und Europin. Darüber hinaus induzierten Lasiocarpin, Riddelliin und Europin Veränderungen im mitochondrialen Membranpotenzial. Dies deutet darauf hin, dass ROS vermehrt in den Mitochondrien der Zellen gebildet werden.
Aus in vivo Daten ist bekannt, dass hepatische sinusoidale Epithelzellen (HSECs) die Zelltypen innerhalb der Leber sind, die nach der metabolischen Aktivierung von PAs zuerst geschädigt werden. Jedoch exprimieren HSECs nicht die erforderlichen Stoffwechselenzyme für die Aktivierung von PAs. Um diese Situation nachzuahmen, wurden HepG2-Zellen verwendet, um PAs in einer Kokultur mit HeLa H2B-GFP-Zellen als nicht-metabolisch aktive Nachbarn metabolisch zu aktivieren. Durch die grün fluoreszierende GFP-Markierung konnten die HeLa-Zellen in der Co-Kultur leicht identifiziert werden. Die PAs Europine, Riddelliin und Lasiocarpin induzierten die Bildung von Mikrokernen in HepG2-Zellen und in HeLa H2B-GFP-Zellen, die mit HepG2-Zellen kokultiviert wurden, jedoch nicht in HeLa H2B-GFP-Zellen, die allein kultiviert wurden. Die metabolische Hemmung von CYP 450-Enzymen mit Ketoconazol hob die Mikronukleusbildung, welche durch die zuvor getesteten PAs induziert wurde, auf. Die Efflux-Transporter-Inhibitoren Verapamil und Benzbromaron reduzierten die Mikronukleusbildung in der Kokultur. Darüber hinaus wurden mitotische Störungen als zusätzlicher genotoxischer Wirkmechanismus in der Co-Kultur aus HepG2-Zellen und in HeLa H2B-GFP-Zellen beobachtet, jedoch nicht in HeLa H2B-GFP-Zellen, die allein kultiviert wurden. Zusammengefasst deuten diese Ergebnisse darauf hin, dass PAs durch HepG2-Zellen bioaktiviert werden können und aus PAs gebildete Metabolite genomische Schäden in kokultivierten, nicht-metabolisch aktiven HeLa-Zellen induzierten.

Abschließend zeigen Kombinationen der PAs Lasiocarpin und Riddelliin sowohl in HepG2-Zellen als auch in der Co-Kultur eher einen additiven Effekt als einen Synergismus. Diese Studie liefert daher Unterstützung für die Annahme, dass die Dosisaddition zur Charakterisierung des genotoxischen Risikos von in einem Gemisch vorhandenen PAs angewendet werden kann.
KW  - Pyrrolizidine alkaloids
KW  - HeLa H2B-GFP-Zellen
KW  - Pyrrolizidinalkaloide
KW  - Kleinkern
KW  - Mutagenität
KW  - Genotoxizität
KW  - DNA-Vernetzung
KW  - mitotische Störung
KW  - Co-culture
KW  - metabolische Aktivierung
KW  - Membrantransporter
KW  - metabolische Enzyme
KW  - HepG2-Zellen
KW  - Genotoxicity
KW  - Micronuclei
KW  - DNA crosslink
KW  - Mitotic disturbance
KW  - Metabolic activation
KW  - Membrane transporters
KW  - Metabolic enzymes
KW  - HepG2 cells
KW  - HeLa H2B-GFP cells
KW  - micronucleus
Y1  - 2024
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-370376
ER  - 
TY  - JOUR
A1  - Gohla, Antje
T1  - Do metabolic HAD phosphatases moonlight as protein phosphatases?
JF  - BBA - Molecular Cell Research
N2  - Mammalian haloacid dehalogenase (HAD)-type phosphatases have evolved to dephosphorylate a wide range of small metabolites, but can also target macromolecules such as serine/threonine, tyrosine-, and histidine-phosphorylated proteins. To accomplish these tasks, HAD phosphatases are equipped with cap domains that control access to the active site and provide substrate specificity determinants. A number of capped HAD phosphatases impact protein phosphorylation, although structural data are consistent with small metabolite substrates rather than protein substrates. This review discusses the structures, functions and disease implications of the three closely related, capped HAD phosphatases pyridoxal phosphatase (PDXP or chronophin), phosphoglycolate phosphatase (PGP, also termed AUM or glycerol phosphatase) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP or HDHD2B). Evidence in support of small metabolite and protein phosphatase activity is discussed in the context of the diversity of their biological functions.
KW  - actin cytoskeleton
KW  - cancer
KW  - haloacid dehalogenase-type phosphatase
KW  - major depression
KW  - metabolism
KW  - vitamin B6
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233168
VL  - 1866
ER  - 
TY  - JOUR
A1  - Jeanclos, Elisabeth
A1  - Albersen, Monique
A1  - Ramos, Rúben J. J.
A1  - Raab, Annette
A1  - Wilhelm, Christian
A1  - Hommers, Leif
A1  - Lesch, Klaus-Peter
A1  - Verhoeven-Duif, Nanda M.
A1  - Gohla, Antje
T1  - Improved cognition, mild anxiety-like behavior and decreased motor performance in pyridoxal phosphatase-deficient mice
JF  - BBA - Molecular Basis of Disease
N2  - Pyridoxal 5′-phosphate (PLP) is an essential cofactor in the catalysis of ~140 different enzymatic reactions. A pharmacological elevation of cellular PLP concentrations is of interest in neuropsychiatric diseases, but whole-body consequences of higher intracellular PLP levels are unknown. To address this question, we have generated mice allowing a conditional ablation of the PLP phosphatase PDXP. Ubiquitous PDXP deletion increased PLP levels in brain, skeletal muscle and red blood cells up to 3-fold compared to control mice, demonstrating that PDXP acts as a major regulator of cellular PLP concentrations in vivo. Neurotransmitter analysis revealed that the concentrations of dopamine, serotonin, epinephrine and glutamate were unchanged in the brains of PDXP knockout mice. However, the levels of γ-aminobutyric acid (GABA) increased by ~20%, demonstrating that elevated PLP levels can drive additional GABA production. Behavioral phenotyping of PDXP knockout mice revealed improved spatial learning and memory, and a mild anxiety-like behavior. Consistent with elevated GABA levels in the brain, PDXP loss in neural cells decreased performance in motor tests, whereas PDXP-deficiency in skeletal muscle increased grip strength. Our findings suggest that PDXP is involved in the fine-tuning of GABA biosynthesis. Pharmacological inhibition of PDXP might correct the excitatory/inhibitory imbalance in some neuropsychiatric diseases.
KW  - pyridoxal phosphatase
KW  - vitamin B6
KW  - γ-Aminobutyric acid (GABA)
KW  - motor performance
KW  - neuropsychiatric diseases
KW  - neurotransmitter biosynthesis
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323396
VL  - 1865
ER  - 
TY  - JOUR
A1  - Meral, Derya
A1  - Provasi, Davide
A1  - Prada-Gracia, Diego
A1  - Möller, Jan
A1  - Marino, Kristen
A1  - Lohse, Martin J.
A1  - Filizola, Marta
T1  - Molecular details of dimerization kinetics reveal negligible populations of transient µ-opioid receptor homodimers at physiological concentrations
JF  - Scientific Reports
N2  - Various experimental and computational techniques have been employed over the past decade to provide structural and thermodynamic insights into G Protein-Coupled Receptor (GPCR) dimerization. Here, we use multiple microsecond-long, coarse-grained, biased and unbiased molecular dynamics simulations (a total of ~4 milliseconds) combined with multi-ensemble Markov state models to elucidate the kinetics of homodimerization of a prototypic GPCR, the µ-opioid receptor (MOR), embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol lipid bilayer. Analysis of these computations identifies kinetically distinct macrostates comprising several different short-lived dimeric configurations of either inactive or activated MOR. Calculated kinetic rates and fractions of dimers at different MOR concentrations suggest a negligible population of MOR homodimers at physiological concentrations, which is supported by acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments. This study provides a rigorous, quantitative explanation for some conflicting experimental data on GPCR oligomerization.
KW  - computational biophysics
KW  - fluorescence resonance energy transfer
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223995
VL  - 8
ER  -