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Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy, which is associated with life-threatening ventricular arrhythmias. Approximately 60% of patients carry a putative disease-causing genetic variant, but interpretation of genetic test results can be challenging. The aims of this study were to systematically reclassify genetic variants in patients with ARVC and to assess the impact on ARVC diagnosis. Methods: This study included patients from the Multicenter Zurich ARVC Registry who hosted a genetic variant deemed to be associated with the disease. Reclassification of pathogenicity was performed according to the modified 2015 American College of Medical Genetics criteria. ARVC diagnosis (categories: definite, borderline, possible) based on the 2010 Task Force Criteria was reclassified after genetic readjudication. Results: In 79 patients bearing 80 unique genetic variants, n=47 (58.8%) genetic variants were reclassified, and reclassification was judged to be clinically relevant in n=33 (41.3%). Variants in plakophilin-2 ( PKP2 ) were shown to reclassify less frequently as compared with other genes ( PKP2 , n=1, 8.3%; desmosomal non- PKP2 , n=20, 66.7%; nondesmosomal, n=26, 68.4%; P =0.001for overall comparison; PKP2 versus desmosomal non- PKP2, P =0.001; PKP2 versus nondesmosomal, P Conclusions: Given that approximately half of genetic variants were reclassified, with 10.1% of patients losing their definite disease status, accurate determination of variant pathogenicity is of utmost importance in the diagnosis of ARVC.

Background Cancer cachexia as a metabolic syndrome can lead to at least 25% of cancer deaths. The inhibition of muscle atrophy is a main strategy to treat cancer cachexia. In this process, myostatin (MSTN) can exert a dual effect on protein metabolism, including inhibition of protein biosynthesis and enhancement of protein degradation. In this study, we will test the effect on muscle atrophy induced by cancer cachexia of IMB0901, a MSTN inhibitor. Methods Two high-throughput screening models against MSTN were developed. By screening, IMB0901, 2-((1-(3,4-dichlorophenyl)-1H-pyrazolo [3,4-d] pyrimidin-4-yl) amino) butan-1-ol, was picked out from the compound library. The in vitro cell model and the C26 animal model of muscle atrophy induced by cancer cachexia were used to determine the pharmacological activity of IMB0901. Whether IMB0901 could inhibit the aggravating effect of doxorubicin on muscle wasting was examined in vitro and in vivo. Results IMB0901 inhibited the MSTN promoter activity, the MSTN signaling pathway, and the MSTN positive feedback regulation. In atrophied C2C12 myotubes, IMB0901 had a potent efficiency of decreasing MSTN expression and modulating MSTN signaling pathway which was activated by C26-conditioned medium (CM). In C2C12 myotubes, the expressions of three common myotube markers, myosin heavy chain (MyHC), myogenic differentiation 1 (MyoD), and myogenin (MyoG), were downregulated by CM, which could be efficiently reversed by IMB0901 via reduction of ubiquitin-mediated proteolysis and enhancement of AKT/mTOR-mediated protein synthesis. In the C26 animal model, IMB0901 mitigated the weight loss of body, quadricep and liver, and protected the quadriceps cell morphology. Furthermore, IMB0901 decreased the expression of two E3 ligases Atrogin-1 and MuRF-1 in the quadriceps in vivo. At the cellular level, IMB0901 had no influence on anti-tumor effect of three chemotherapeutic agents (cisplatin, doxorubicin, and gemcitabine) and lowered doxorubicin-induced upregulation of MSTN in C2C12 myotubes. IMB0901 did not affect the inhibitory effect of doxorubicin on C26 tumor and delayed the weight loss of muscle and adipose tissue caused by C26 tumor and doxorubicin. Conclusions IMB0901 inhibits muscle atrophy induced by cancer cachexia by suppressing ubiquitin-mediated proteolysis and promoting protein synthesis. These findings collectively suggest that IMB0901 is a promising leading compound for the management of muscle atrophy induced by cancer cachexia. Electronic supplementary material The online version of this article (10.1186/s13395-019-0193-2) contains supplementary material, which is available to authorized users.

Background MiR-9 is a small non-coding RNA that is highly conserved between species and primarily expressed in the central nervous system (CNS). It is known to influence proliferation and neuronal differentiation in the brain and spinal cord of different vertebrates. Different studies have pointed to regional and species-specific differences in the response of neural progenitors to miR-9. Methods In ovo and ex ovo electroporation was used to overexpress or reduce miR-9 followed by mRNA in situ hybridisation and immunofluorescent stainings to evaluate miR- expression and the effect of changed miR-9 expression. Results We have investigated the expression and function of miR-9 during early development of the mid-hindbrain region (MH) in chick. Our analysis reveals a closer relationship of chick miR-9 to mammalian miR-9 than to fish and a dynamic expression pattern in the chick neural tube. Early in development, miR-9 is diffusely expressed in the entire brain, bar the forebrain, and it becomes more restricted to specific areas of the CNS at later stages. MiR-9 overexpression at HH9–10 results in a reduction of FGF8 expression and premature neuronal differentiation in the mid-hindbrain boundary (MHB). Within the midbrain miR-9 does not cause premature neuronal differentiation it rather reduces proliferation in the midbrain. Conclusion Our findings indicate that miR-9 has regional specific effects in the developing mid-hindbrain region with a divergence of response of regional progenitors. Electronic supplementary material The online version of this article (10.1186/s12861-017-0159-8) contains supplementary material, which is available to authorized users.

Programmed death-ligand 1 (PD-L1) has been speculated to play a critical role in suppression of the immune system and it can be upregulated in cancer cells, which may allow cancers to evade the host immune system. MicroRNAs (miRNAs) are small non-coding RNA molecules (containing about 22 nucleotides), that function in RNA silencing and post-transcriptional regulation of gene expression. MiRNAs were found deregulated (upregulated or downregulated) and implicated in cancer development with various roles which depend on their gene target. Using targetscan web server prediction algorithm, we concluded that miR-140-3p is a targeting mirRNA with conserved consequential pairing of target region for PD-L1. Moreover, by reviewing all the available cancer studies in Pub/Medline about miR-140-3p, was found permanently down regulated. Furthermore, in recent immunotherapy related clinical trials in most cancers, evaluated PD-L1, it is found overexpressed. In the near future, in vitro or in vivo studies need to validate whether there is direct correlation between PD-L1 overexpression and miR-140-3p downregulation as targetscan performed algorithm predicted.

Demonstration of downregulation of luteinizing hormone (LH) using the gonadotropin-releasing hormone (GnRH)-agonist buserelin as the active ingredient in the form of a slow-release implant.To eliminate any negative feedback mechanisms of endogenous sex steroids, nine bitches were ovariohysterectomized prior to treatment. The applied drug was the slow-release implant Profact DepotNo dose dependency was observed, leading to the conclusion that maximum efficiency could already be achieved using the lowest dose of 3.3 mg. Therefore, for further evaluation, the dogs were combined as a single group. An increasing downregulatory effect was observed from weeks 2 to 26, with the pharmacodynamic activity lasting approximately 34 weeks. There was a significant increase 1 hour after implantation to almost twice the pre-treatment value; elevated but continuously declining LH concentrations lasted a further 8 hours.Similar as in male dogs, in the bitch buserelin in the form of a slow-release implant also led to downregulation of LH secretion with a preceding initial increase lasting for several hours. This increase must be seen in relation to the unwanted side effects when using this type of drug for downregulation of ovarian function in the bitch.Inhibition of the initial LH increase appears to be an important factor to allow for routine clinical use of slow-release GnRH-agonist implants in the bitch.

The accumulation of hepatocellular lipid droplets, referred to as steatosis, is the most frequent liver pathology in western industrial countries. The surface of lipid droplets is stabilized by amphiphilic proteins of the PAT family (perilipin, adipophilin, TIP47). We recently showed that PAT proteins are differentially expressed in liver.The effects of lipid droplet induction with oleate and of siRNA-mediated downregulation of PAT proteins were evaluated using immunofluorescence microscopy, immunoblot and functional assays.Whereas perilipin, adipophilin and (in only minor amounts) also TIP47 coat lipid droplets in steatotic hepatocytes, adipophilin and TIP47, but not perilipin, are detectable in hepatocellular cell cultures. Likewise, perilipin is not induced after downregulation of adipophilin and TIP47.Common cell culture models show specific differences to human hepatocyte steatosis in vivo. Perilipin may play a role in the long-term storage of fat, which may be only partially reflected by cell culture models.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

Down-regulation of the IAP antagonistis XAF1, Smac/DIABLO and HtrA2, has been related to the onset and progression of various malignancies. We examined the mRNA-expression of these pro-apoptotic parameters in testicular germ cell tumors (TGCT) and normal testicular tissue and correlated their expression levels to clinicopathological tumour features.Real-time RT-PCR was used to quantify the mRNA-expression of XAF1, Smac/DIABLO and HtrA2 in normal testicular tissue (n = 18), carcinoma in situ (n = 4), seminomas (n = 64), and non-seminomatous germ cell tumors (n = 35).Compared to normal testicular tissue, the expression levels of XAF1 were increased in TGCT (p0.001), whereas those of Smac/DIABLO and HtrA2 were decreased (p0.001 and p0.001). Smac/DIABLO expression levels showed a significant trend towards a gradual decrease from normal testicular tissue to CIS and seminomas and finally to NSGCT (p0.001). Moreover, XAF1 and HtrA2 expression levels gradually increased with progression of clinical tumour stage in seminoma patients (p = 0.001 and p = 0.018), their expression levels being strongly intercorrelated (Spearman rho correlation coefficient: 0.674; p0.001).These data suggest that a down-regulation of Smac/DIABLO and HtrA2 is implicated in the development and progression of TGCT, whereas overexpression of XAF1 in TGCT might contribute to their extraordinary sensitivity to chemotherapy. Regarding the additional correlation of XAF1 and HtrA2 expression with clinical tumour stage in seminoma patients, it appears reasonable to further evaluate these three IAP antagonists as molecular parameters for the prediction of treatment response and prognosis of TGCT patients.

Conventional therapeutic approaches to treat advanced renal cell carcinoma (RCC) are of limited benefit. Receptor tyrosine kinase inhibitors (RTKI) may open up novel treatment options. In the present study, the effects of the RTKI AEE788 on the growth and adhesion capacity of RCC cell lines were evaluated in vitro.RCC cells were treated with AEE788, and alterations of tumor growth and tumor cell interaction with vascular endothelium or extracellular matrix proteins were analyzed. Furthermore, the addition of interferon alpha (IFNalpha) was investigated to see whether it may enhance the anti-tumoral potential of AEE788.AEE788 significantly blocked RCC cell growth and adhesion. Analysis of alpha- and beta-integrins revealed distinct alterations of the receptor expression profile and downregulation of integrin-dependent signaling. Growth-blocking effects were further enhanced when the AEE788-IFNalpha combination protocol was applied. In addition, downregulation of integrin-dependent signaling was more intense in the presence of a combination of AEE788 and IFNalpha than with AEE788 monotherapy.AEE788 exerts significant anti-tumoral properties, particularly when combined with IFNalpha. AEE788 may therefore be an encouraging compound to treat advanced RCC.

In the recent years, a tremendous amount has been learned about the pathophysiology of atrial fibrillation (AF). AF induces electrophysiological changes in the atria causing a perpetuation of the arrhythmia ("electrical remodeling"). Besides such AF-induced electrophysiological changes, which involve the downregulation of L-type calcium channels and thereby the calcium inward current, AF induces structural and ultrastructural changes in atrial tissue ("structural remodeling"). Calcium-dependent tissue alterations are induced by proteases and phosphatases like calpain and calcineurin. Furthermore, cardiac diseases like hypertension, heart failure, etc. activate the atrial angiotensin II system, and thereby, a progressive pro-arrhythmogenic atrial fibrosis is induced. Besides first clinical trials assessing the antiarrhythmic effects of angiotensin II receptor blockers in patients with AF, experimental data suggest that viral gene transfer can be used to transform fibroblasts to electrically conducting cardiomyocytes. This highly interesting methodology may be helpful to restore electrical conduction in fibrotic cardiac tissue.

Angiogenesis and tumor-associated immunosuppression are two of the hallmarks of carcinogenesis. In previous studies we demonstrated in vitro that HNSCC tumor cells attract monocytes via monocyte chemotactic protein-1 (MCP-1) and activate them via transforming growth factor-beta 1(TGF-beta1) to secrete interleukin (IL)-1alpha, which in turn stimulates tumor cells to secrete increased levels of the angiogenic and immunosuppressive vascular endothelial growth factor (VEGF). These findings suggest that interaction between the immune system and VEGF-mediated angiogenesis is important for progression of HNSCC. Recent studies in vitro show that retinoic acid (RA) downregulates the release of MCP-1 and TGF-beta1 by tumor cells. Therefore, we investigated the ability of RA to modulate the ability of tumor cells to recruit and activate monocytes for participation in VEGF-mediated angiogenesis and immunosuppression in vivo.Mice (ten/group) were injected daily with RA (160 microg/kg) for 3 weeks. After that time mice were sacrificed, and paraffin sections of tumors were obtained and stained for VEGF-A, CD68, and PECAM (CD31) by immunohistochemistry. The lungs, liver, and myocardium were analyzed for macro- and micrometastases. The plasma protein levels of VEGF-A and MCP-1 were determined by ELISA.In RA-treated mice tumors regressed completely and RA prevented metastases (p=0.00) and macrophage infiltration (p=0.007). Treated mice downregulated VEGF-A (0 pg/ml) and MCP-1 (12 pg/ml) in peripheral blood (p=0.001).Our findings suggest a new therapeutic possibility: the development of treatment protocols that can block each of the ways in which tumors induce new blood vessel growth and immunosuppression of the host.

Duisburg, Essen, Univ., Diss., 2003 Extrazelluläre Signalmoleküle, wie Hormone, Neurotransmitter und Mediatoren binden in der Regel an membranständige Rezeptoren und lösen eine Kaskade von zellulären Reaktionen aus, die zu einer spezifischen Zellantwort führen. Die größte Gruppe der membranständigen Rezeptoren sind die sog. G-Protein-gekoppelten Rezeptoren (GPCRen), die nach Bindung von spezifischen Agonisten nachgeschaltete heterotrimere G-Proteine aktivieren, worüber unterschiedliche Signaleffektoren, wie die Adenylylcyclase, die Phospholipase C oder Ionenkanäle aktiviert oder gehemmt werden. Die muskarinischen Acetylcholin-Rezeptoren (mAChRen) mit ihren bekannten Subtypen M1 - M5 gehören zur GPCRSuperfamilie. Ähnlich wie andere GPCRen unterliegen auch die mAChRen selbst einer Regulation. Dies betrifft einmal die Abschwächung der Signalweiterleitung (Desensibilisierung) sowie die Internalisierung und den Abbau (Down-Regulation) der Rezeptoren. Für die beiden ersten Prozesse ist es anscheinend erforderlich, daß die Rezeptoren durch Rezeptorkinasen phosphoryliert werden, an die dann Adapter-Proteine wie ?-Arrestin binden, was dann die Desensibilisierung und Internalisierung der Rezeptoren auslöst. Frühere Untersuchungen an den mAChR-Subtypen hatten ergeben, daß die M1, M3 und M4 mAChRen unter Beteiligung von ?-Arrestin über sog. clathrin-coated vesikel (CCV) in HEK293-Zellen internalisieren und daß sie anschließend wieder an die Zelloberfläche zurückkehren. Im Gegensatz dazu internalisiert der M2 mAChR nicht über CCV, sondern wird über andere, bisher noch nicht bekannte Vesikel in das Zellinnere transportiert und anschließend in der Zelle abgebaut. Für die Vesikel-Abschnürung bei der mAChR-Internalisierung ist Dynamin für alle vier mAChR-Subtypen essentiell. Ziel dieser Arbeit war es, die Rolle der beiden Rezeptorkinasen Caseinkinase 1? (CK1?) und G-Protein-gekoppelte Rezeptorkinase 2 (GRK2) sowie der Proteine Intersectin, Endophilin und Ubiquitin bei der Desensibilisierung, Internalisierung und Down-Regulation der mAChR-Subtypen M1 - M4 zu charakterisieren. Die Untersuchungen, die mittels transienter oder stabiler Expression der M1 - M4 mAChRen und der genannten Proteine sowie von spezifischen Mutanten dieser Proteine in HEK293- und HEK293 tsA201-Zellen durchgeführt wurden, zeigten hinsichtlich der beiden Rezeptorkinasen, daß die GRK2, aber nicht die CK1? für die Desensibilisierung und Internalisierung des M2 mAChRs sowie für die Internalisierung des M4 mAChRs verantwortlich ist. Dagegen kann die Internalisierung des M1 mAChRs sowohl durch CK1? als auch GRK2 reguliert werden, während die Internalisierung des M3 mAChRs weder durch CK1? noch durch GRK2 wesentlich gesteuert wird. Weitergehende Untersuchungen zur CK1?, die die M1 und M3 mAChRen in einer Agonist-abhängigen Weise phosphoryliert, ergaben, daß die CK1? weder bei der akuten Signaltransduktion (Stimulation von Phospholipase C und D, Calcium-Freisetzung und Aktivierung von Mitogen-aktivierten Proteinkinasen) über diese mAChR-Subtypen noch ihrer Desensibilisierung beteiligt ist. Studien mit heterologer Expression von Wildtyp Intersectin und der fünf isolierten SH3-Domänen (SH3A - E) dieses Proteins ergaben, daß Intersectin wahrscheinlich über seine SH3E- und SH3D-Domänen die Internalisierung des M2 mAChRs negativ reguliert. Die Wirkung der SH3E-Domäne erfolgt offensichtlich über eine Interaktion mit Dynamin, während die SH3D-Domäne mit einem bisher nicht identifizierten Protein interagiert. Interessanterweise wurde die Geschwindigkeit der Internalisierung des M1 mAChRs durch Expression der SH3A-, SH3D- und SH3E-Domänen von Intersectin gesteigert. Hiermit konnte erstmalig eine Beteiligung von Intersectin bei der Internalisierung von GPCRen gezeigt werden. Endophilin ist ein für die Invagination und Endozytose von synaptischen Vesikeln essentielles Protein, das über seine SH3-Domäne mit verschiedenen anderen Proteinen, wie Dynamin, Synaptojanin oder auch Rezeptoren interagiert. Untersuchungen mit Wildtyp Endophilin und seiner isolierten SH3-Domäne ergaben, daß die Internalisierung der M2 und M4 mAChRen, aber nicht die der M1 und M3 mAChRen durch Endophilin reguliert wird. Diese Regulation erfolgt wahrscheinlich über eine Interaktion von Endophilin mit Dynamin, während keine Hinweise für eine Beteiligung der Inositol-5-Phosphatase Synaptojanin oder eine direkte Interaktion von Endophilin mit mAChRen gefunden wurden. Da im Gegensatz zu den anderen mAChR-Subtypen internalisierte M2 mAChRen nicht mehr an die Plasmamembran zurückkehren, sondern intrazellulär abgebaut werden, wurde geprüft, ob eine Ubiquitinylierungsreaktion bei der Internalisierung und Down-Regulation des M2 mAChRs beteiligt ist. Untersuchungen mit einer Ubiquitin-Mutante, die eine Multi-Ubiquitinylierung von Proteinen verhindert, ergaben, daß die Internalisierung und Down-Regulation des M2 mAChRs, aber nicht die der M1 und M4 mAChRen, von einer Multi-Ubiquitinylierungsreaktion abhängig sind. Weiterhin konnte die Down-Regulation des M2 mAChRs sowohl durch spezifische proteasomale als auch durch lysosomale Hemmstoffe unterbunden werden. Auch wenn das hierbei beteiligte Ubiquitin-Target bisher nicht identifiziert wurde, so lassen diese Ergebnisse den Schluß zu, daß das oben erwähnte besondere Verhalten der internalisierten M2 mAChRen möglicherweise darauf beruht, daß die Internalisierung dieses Rezeptors von einer Multi-Ubiquitinylierungsreaktion begleitet ist, die ihn zum proteasomalen und lysosomalen Abbau hinführt und damit seine Rückkehr an die Plasmamembran verhindert.

It is not clear how initial therapy with CsA effects indefinite allograft acceptance after small bowel transplantation in the rat. Our study of post-transplant alterations of immune parameters within donor-type mesenteric lymph nodes suggests CsA-dependent, Th2-mediated induction of graft-specific T-cell tolerance.

0\. Titelblatt und Inhaltsverzeichnis 1\. Einleitung 11 1.1. G-Protein-gekoppelte Rezeptoren 11 1.2. Die Endotheline und ihre Rezeptoren 12 1.3. Physiologische und pathophysiologische Bedeutung des Endothelins 15 1.4. Signalkaskaden der Endothelin-Rezeptoren 16 1.5. Endothelin-induzierte Vasokonstriktion 18 1.6. Rezeptor-vermittelte Endozytose 19 1.7. Transportprozesse in polarisierten Zellen 22 1.8. Visualisierung von intrazellulären Transportprozessen 23 1.9. Fragestellung 25 2\. Material und Methode 26 2.1. Material 26 2.1.1 Chemikalien und Reagenzien 26 2.1.2 Geräte und Rechner 28 2.1.3 Bakterienstämme und Zellinien 30 2.1.4 Desoxyribonucleotide 31 2.1.5 Medien und Agarplatten für E.coli 32 2.2. Methoden 33 2.2.1 Methode zur Aufreinigung von DNS 33 2.2.2 Amplifizierung von DNS durch Polymerase Kettenreaktion (PCR) 34 2.2.3 Spezifische Spaltung von DNS durch Verdau mit Restriktionsendonukleasen 35 2.2.4 DNS-Sequenzierung nach der Dideoxymethode 35 2.2.5 Horizontale Gelelektrophorese 36 2.2.6 Eluierung von DNS Fragmenten aus Agarose-Gelen mit dem Gene Clean Kit 37 2.2.7 Ligation von DNS Fragmenten 37 2.2.8 Herstellung kompetenter Zellen und Transformation 38 2.2.9 Zellkultur 38 2.2.10 125Iod-Endothelin1 Bindung und Verdrängung an Gesamtmembranen 40 2.2.11 Internalisierungsexperimente 41 2.2.12 Inhibition der Clathrin-abhängigen Internalisierung 42 2.2.13 Kolokalisation von Fluo-ET1 mit DiI-LDL oder TRITC-Transferrin 42 2.2.14 Konfokale- bzw. Epifluoreszenz-Mikroskopie 43 2.2.15 Biotinilierung von Oberflächenproteinen 43 2.2.16 Inositolphosphat Assay 44 2.2.17 cAMP-Radioimmunoassay (RIA) 45 3\. Ergebnisse 46 3.1. Pharmakologische Eigenschaften der Endothelin-Rezeptoren, im Vergleich mit ihren GFP-Fusionsproteinen 47 3.2. Internalisierung und intrazellulärer Transport 48 3.2.1 Visualisierung der Endozytose von Endothelin-Rezeptoren 48 3.2.2 Wiedererscheinen des ETB Rezeptors an der Zelloberfläche nach erfolgter Internalisierung 52 3.2.3 Kolokalisation mit Fluorochrom-markiertem Low-Density-Lipoprotein (LDL) 54 3.2.4 Sucrose inhibiert die Clathrin-abhängige Endozytose des ETB Rezeptors 57 3.3. Strukturelle Motive für die Oberflächenexpression des ETB Rezeptors in polarisierten MDCK-Zellen 57 3.3.1 Verteilung der ETB/GFP Konstrukte in MDCK-Zellen 59 3.3.2 Radioaktiver Ligand-Bindungs-Assay an MDCK-Zellen 59 3.3.3 Biotinilierungs-Assay zur Messung der apikalen und basolateralen Membranoberflächen 61 3.3.4 Signaltransduktion des ETB Rezeptors an polaren MDCK-Zellen 62 4\. Diskussion 65 4.1. Subtyp spezifische Desensitisierung der Endothelin-Rezeptoren 66 4.2. Differenzierte Internalisierung des Endothelin-Rezeptor Subtypen 67 4.3. Strukturelle Motive für die Oberflächenexpression in polarisierten MDCK-Zellen 70 4.4. Zusammenfassung und Ausblick 72 Literaturverzeichnis 73 Klonierung 86 Lebenslauf 87 Danksagung 88, Endotheline (ET1, ET2, ET3) sind Peptide von 21 Aminosäuren, die ihre Wirkung über zwei G-Protein-gekoppelte Rezeptoren entfalten: 1) Den v.a. auf vaskulären Muskelzellen exprimierten Endothelin A-Rezeptor (ETA) und 2) den in Endothelzellen exprimierten Endothelin B-Rezeptor (ETB). Der ETA vermittelt eine lang-anhaltende Vasokonstriktion, der ETB eine kurzfristige Vasodilatation. Die Ursache für die unterschiedliche Dauer der durch die Endothelin-Rezeptorsubtypen vermittelten Gefäßreaktionen ist bislang unklar. Unterschiede der Internalisierung und des intrazellulären Transports könnten hier eine mögliche Ursache sein. Während für den ETA ein Recycling in glatten Muskelzellen beschrieben wurde, lagen für den ETB bisher keine Untersuchungen vor. In der vorgelegten Arbeit wurde durch Verwendung von fluoreszierendem ET1 und Endothelin-Rezeptor/GFP-Fusionsproteinen gezeigt, daß der ETB nach Endothelin-1 (ET1) Bindung innerhalb weniger Minuten über einen Sucrose- sensitiven, d.h. Clathrin-vermittelten Weg internalisiert wird. Der Ligand verbleibt mit dem Rezeptor über mehrere Stunden in einem stabilen Komplex und wird in späte Endosomen/Lysosomen transportiert und schließlich abgebaut ("Downregulation"). Diese an transfizierten CHO-Zellen durchgeführten Analysen konnten auch an primär kultivierten Astrozyten bestätigt werden. Die Downregulation von Rezeptor und Ligand ist für G-Protein-gekoppelte Rezeptoren bislang einzigartig. Typischerweise dissoziiert der Ligand bei vielen anderen G-Protein-gekoppelten Rezeptoren in dem sauren Milieu der Endosomen. Ein spät endosomaler/lysosomaler Transport von aktivierten Rezeptoren war bislang nur für die Protease-aktivierten Rezeptoren eindeutig belegt, die eine konstitutive Aktivierung aufweisen. Für den ETA wurde hingegen an CHO-Zellen oder ETB-defizienten primär kultivierten Astrozyten keine wesentliche Internalisierung von Rezeptor und Ligand in endosomale Kompartimente beobachtet. Damit konnten für ETA und ETB unterschiedliche Internalisierungswege beschrieben werden, die auch zu Unterschieden der Oberflächenexpression beider Rezeptoren führen. Darüber hinaus zeigt die über Stunden nachweisbare Kolokalisation des ETB mit ET1, daß der ETB auch als Clearance-Rezeptor fungiert. Im zweiten Teil dieser Arbeit wurde eine mögliche polarisierte Oberflächenexpression des ETB an MDCK-Zellen untersucht. Für diese Zellen konnte gezeigt werden, daß der ETB gleichermaßen basolateral und apikal exprimiert wird. Ein potentielles tyrosinhaltiges basolaterales Sortiermotiv (GYXXF) ist für den Transport des Rezeptors ohne Bedeutung. Damit sind Epithelzellen sowohl von der apikalen als auch der basolateralen Seite für ET1 sensitiv. Dies trifft z.B. für Nierenepithelzellen zu, die den ETB exprimieren; da ET1 sowohl im Urin als auch Interstitium vorliegt ist eine Stimualtion der Zellen von beiden Seiten möglich. Weitere Arbeiten müssen nun klären, ob ETB auch in Endothelzellen auf apikalen (luminal) und basalen (abluminal) Plasmamembranen vorliegen. Diese Fragestellung ist von Bedeutung, da ET1 von den Endothelzellen v.a. nach basolateral sezerniert wird. Das Vorliegen basolateraler ETB würde damit auch eine autokrine/parakrine Stimulation der Endothelzellen bedeuten., Endohelins (ET1, ET2, ET3) are peptides consisting of 21 amino acids, which evolve their properties over G-protein coupled receptors: 1) particularly Endothelin A-receptors (ETA) expressed on vascular smooth muscle cells and 2) Endothelin B-receptors (ETB) on endothelial cells. ETA mediate a long lasting vasoconstriction, while ETB exhibit only a short vascular relaxation. The reason for these differences on vascular reactions among endothelin-receptor subtypes is not known at this time. Differences in internalisation and intracellular transport could be a possible reason. While ETA recycling in smooth muscle cells could be described, no analysis exist for the ETB. In this work, fluorescent ET1 and Endothelin-receptor/GFP-fusion protein was used to show, that after binding of ET1, ETB is internalised in a sucrose-sensitive, meaning clathrin-mediated pathway. The ligand remain associated in a stable complex with the receptor and is transported in late endosomes/lysosoms for degradation ("down-regulation"). These analyses on CHO-cells could also be validated on cultivated primary astrocytes. At this time the down-regulation of receptor and ligand is unique for G-protein coupled receptors. Typically in many cases ligand dissociates from G-protein coupled receptors in the acidic environment of endosomes. Only protease activated receptor, clearly show a constitutive transport in late endosoms/lysosoms. Whereas no internalisation could be observed for ETA, neither in CHO-cells nor in primary cultivated astrocytes. For this reason different internalisation pathways for ETA and ETB could be described leading to different surface expression. Furthermore colocalisation of ETB with ET1, detectable for hours, document the clearance function of ETB. In the second part of this work, a possible polarised surface expression of the ETB was analysed with MDCK-cells. For these cells it could be shown, that ETB is expressed in equal measure on apical and basolateral membrane. A potential basolateral sorting motive including tyrosin (GYXXF) has no effect on receptor transport. It can be concluded that epithelial cells are sensitive for ET1 also from basolateral side. This applies for epithelial kidney cells expressing ETB; because ET1 exist as well in urine as in interstitium, thus stimulation is possible from both sides. Further work should be done to clarify if ETB also exist in endothelial cells on apikal (luminal) and basal (abluminal) plasmamembrane. This question is important, because ET1 is released from endothelial cells mainly on basolateral side. The existence of ETB on this side, would signify an autocrine/paracrine stimulation of endothelial cells.

Transcription factors adjust the up- and downregulation of inflammatory genes. Within the bounds of the SIC-study (Selenium in Intensive Care) the authors will investigate the role of transcription factors NF-kappa B and AP-1 in S.I.R.S/septic patients. The goal of these investigations is the corroboration of therapy of septic patients with sodium selenite at the molecular biological level.

The dopaminergic reward system is activated by primary rewarding factors such as food, sexual activity and parental care. Its activation enhances the occurrence of behaviors which induced the stimulation of dopaminergic neurotransmission. Indications of a dysfunction of the dopaminergic reward system are found in major depression, schizophrenia, and addictive disorders. It has been hypothesized that dysfunction of the dopaminergic reward system is associated with anhedonia, the inability to experience pleasure. However, animal studies indicate that a reduction of central dopaminergic neurotransmission is associated with a decrease in incentive salience of reward-indicating stimuli and not with anhedonia per se. Sensitization of dopaminergic neurotransmission, on the other hand, seems to induce cue-dependent craving in addicted patients. In schizophrenia, phasic, stimulus-dependent dopamine release in the striatum may play a role in the abnormal attribution of salience to previously neutral stimuli.

Histopathological and clinical parameters are of limited value in characterization of the individual course of disease in bladder cancer patients. Down-regulation of E-cadherin, an intercellular adhesion molecule, and upregulation of autocrine motility factor receptor (AMFR) expression have been shown to play a part in tumour cell invasion and metastasis. In a retrospective analysis concerning 95 different bladder specimens and a prospective study in 100 bladder cancer patients, reduction in E-cadherin concomitantly with an increase in AMFR expression was associated with a poor prognosis. The dual use of these two antigens may improve early diagnosis of high-risk bladder cancer patients and influence treatment decisions.

So far, two angiotensin receptor subtypes, called AT1 and AT2, have been described in an animal model and in human. AT1 mediates almost all known effects of angiotensin II and its gene sequence and regulation is well studied. In contrast, only few data on function and regulation of AT2 are available. The complete mRNA sequence of AT2 has only recently been cloned and sequenced. The angiotensin receptors' receptor density and subtype distribution is organ specific. In the rat, lowest densities are found in the myocardium, followed by kidney, liver, adrenal medulla and cortex. The percentage of AT1 in the different organs amounts to 80, 85, 90, 57 and 10%. Angiotensin receptor subtypes have also been quantitated in human myocardium. There, the relatively unknown subtype AT2 dominates (67%). Myocardial receptor density is low, amounting to about 11 fmol/mg protein corresponding to 1/20-1/50 of the density of beta-adrenergic receptors. Angiotensin receptors in the human heart are present on cardiac fibroblasts and induce proliferation of these cells. Blockade of the renin angiotensin system by ACEI and AT1 antagonists in the rat downregulates angiotensin receptors in liver, kidney and adrenals to about 50% in an organ- and subtype specific manner, whereas cyclosporin A upregulates receptors twice. In end-stage human heart failure, but not in early stages, angiotensin receptors are downregulated to 1/3 of control values. Regulator mechanisms at transcriptional level have been elucidated by reporter gene assays; PMA, an activator of proteinkinase C, stimulates the transcription of the AT1 gene. The organ- and subtypespecific regulation of angiotensin receptors by pharmacological agents and/or cardiovascular diseases can contribute to the understanding of these drugs and of the pathophysiology of the corresponding diseases.

IL-8 is a chemotactic cytokine with proinflammatory and growth-promoting activities. Recently it has been shown to influence several functions of keratinocytes, including HLA-DR expression, chemotaxis, and proliferation by binding to a specific receptor. Because psoriasis vulgaris is characterized by epidermal hyperproliferation and infiltration of inflammatory cells, we investigated the expression of IL-8 and its receptor in normal and psoriatic epidermis using semiquantitative reverse-transcriptase-polymerase chain reaction. In addition the mRNA levels of the proto-oncogenes c-ras, c-raf, c-myc, and HER-2 were also investigated as potential growth-promoting stimuli in psoriatic epidermis. IL-8 mRNA was only detected in lesional psoriatic epidermis, and IL-8R-specific mRNA was found to be 10 times increased in lesional psoriatic epidermis. There was no significant difference in the protooncogene mRNA levels. In order to test the relevance of the massively increased IL-8R levels in psoriatic epidermis, we investigated the effect of the antipsoriatic drug FK-506 on specific IL-8 and IL-8R mRNA expression. FK-506 dose dependently inhibited IL-8R expression and function. Our data suggest that in psoriatic skin, elevated IL-8 levels and markedly increased IL-8R expression may act in concert to induce the cardinal signs of psoriasis--epidermal hyperproliferation and leukocyte infiltration. IL-8R may prove a molecular target for antipsoriatic drugs such as FK-506.

GnRH analogues are widely used for the reduction of uterine fibroids. This case report describes the therapy of a 27-year-old woman, whose uterus had a diameter of about 13 cm. After 7 injections of Zoladex Depot (ICI Pharma, Heidelberg, Germany), the uterus was reduced to normal size carrying dorsally a myoma of the same size. After only 7 weeks of medroxyprogesterone acetate (5 mg twice daily, Clinofem, Upjohn, Heppenheim, Germany) the uterus had grown again, so that therapy was changed to Zoladex Depot for another 3 months. On the 27th day of the first spontaneous cycle, the patient ovulated and conceived. Up to the 29th week of gestation (Aug '91), the foetal growth and development was normal, the uterus was normal in size, a childhead sized myoma being situated unproblematically behind the foetus.