Genetic and pharmacological manipulation of the tryptophan hydroxylases

Titelblatt und Inhaltsverzeichnis Zusammenfassung/Abstract Abkürzungsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Literaturverzeichnis AnhangSerotonin (5-HT) ist ein Neurotransmitter, der bei Regulierung von affektivem Verhalten wie Stimmung, Angst und Aggression beteiligt ist, aber auch vegetative Funktionen wie Schlaf, Appetit und Temperaturkontrolle beeinflusst. Dysfunktionen des serotonergen Systems spielen deshalb eine wichtige Rolle bei vielen psychiatrischen Erkrankungen wie Depression und Suizidalität, Angst- und Zwangserkrankungen, Impulskontrollstörungen und Störungen des Essverhaltens. 5-HT kommt aber auch in vielen peripheren Geweben vor und steuert dort unter anderem die primäre Hämostase, den Blutdruck, die Darmmotilität und die zellvermittelte Immunantwort. Die 5-HT-Biosynthese wird im geschwindigkeitsbestimmenden Schritt von der Tryptophan-hydroxylase (TPH) katalysiert. Das Gen einer zweiten TPH-Isoform (TPH2), die in neuronalen Geweben exprimiert wird, wurde in der vorliegenden Arbeit für die Maus, die Ratte und den Menschen zum ersten Mal kloniert, sequenziert und die Enzymeigenschaften der rekombinanten Enzyme charakterisiert. Die ermittelten biochemischen Charakteristika der TPH2 stimmen mit den Parametern überein, die in früheren Studien mit TPH aus neuronaler Quelle ermittelt wurden. Die durchgeführten Sequenzanalysen zeigen auch, dass die TPH2 phylogenetisch die jüngste Aromatische-Aminosäure-Hydroxylase neben der Phenylalanin- und Tyrosinhydroxylase ist. Der erstellte phylogenetische Stammbaum erlaubt einen interessanten Überblick auf die Evolution der Hydroxylasen, die demnach vor etwa drei Milliarden Jahren begann. Zum genaueren Verständnis der physiologischen Bedeutung des serotonergen Systems, wurde im Tierversuch an Mäusen der 5-HT-Spiegel pharmakologisch und molekularbiologisch manipuliert. Durch die Behandlung der Mäuse mit 1-Methyltryptophan (1-MeTrp) als Inhibitor des Tryptophan abbauenden Enzyms Indolamin-2,3-dioxygenase (IDO) konnte der 5 -HT-Spiegel peripher im Blutplasma um 800 % und zentral im Gehirn um 30 % erhöht werden. Um die 5-HT-Gehalte pharmakologisch zu vermindern, wurde das Tryptophananalogon 7-Hydroxytryptophan (7-HTP) synthetisiert und in Zellkultur und Tierversuchen getestet. Diese bisher noch nicht verwendete Substanz wird durch die TPH zum toxischen 5,7-Dihydroxytryptamin (5,7-DHT) metabolisiert und tötet so gezielt serotonerge Zellen ab. In Zellkulturexperimenten erwies sich 7-HTP als geeignet, um spezifisch 5-HT produzierende kleinzellige Lungenkrebs- und Karzinoidzellen abzutöten. Damit besitzt 7-HTP ein Potential als Zytostatikum gegen 5-HT produzierende Tumore. In Tierversuchen an Drosophila und Mäusen zeigte 7-HTP keine offensichtlichen toxischen Auswirkungen. An Mäusen konnte aber sechs Tage nach oraler Verabreichung von 7-HTP eine Verminderung der 5-HT-Spiegel im Blut um 37 % festgestellt werden. Molekularbiologisch erfolgte die spezifische Manipulation der TPH-Expression mit Hilfe von Ribozymen. Dazu wurden drei gegen TPH1 gerichtete Mini- Hammerheadribozyme konstruiert deren Spalteffizienzen in vitro von den Substrat/Ribozym-Verhältnissen und der Reaktionsdauer abhängig waren und mit den berechneten freien Energien der Spaltreaktionen korrelierten. Nach der Klonierung in tRNA-Expressionskonstrukte kehrte sich die Rangfolge der in vitro Spalteffizienzen der drei Ribozyme um. Das beste tRNA-Ribozymkonstrukt zeigte auch in Zellkulturexperimenten mit 5-HT produzierenden Mastzellen eine deutliche Reduktion der TPH1-Expression um 79 %. Das bedeutet, dass empirische Tests immer noch notwendig sind, um die beste Zielsequenz eines Ribozyms zu ermitteln. Die mit diesem Ribozym erzeugten heterozygot transgenen Tiere zeigten im Duodenum eine leichte Herabregulation der TPH1 um 34 % und auch eine Absenkung der 5-HT-Konzentration um 17 %. Diese Ribozyme können also neue Möglichkeiten zur Beeinflussung der Hämostase, und damit zu neuen Therapien von thrombotisch bedingten Erkrankungen bieten. Zur Untersuchung verminderter TPH2-Expression im Tiermodell wurden transgene Mäuse mit retroviralen Vektoren generiert, die Hammerheadribozyme gegen TPH2 exprimieren. Anhand dieser Mäuse konnte die Anwendung des retroviralen Gentransfers am Beispiel eines Ribozym vermittelten TPH2-Knockdowns demonstriert werden. Diese Mäuse zeigen eine verminderte 5-HT-Syntheserate im Gehirn und als Folge ein anxiolytisches Verhalten. Es ist damit gelungen, ein zweites unabhängig reguliertes serotonerges System im ZNS genauer zu charakterisieren und spezifisch zu manipulieren und daraus entscheidende Erkenntnisse für das Verständnis der 5 -HT-Biosynthese zu gewinnen.The neurotransmitter Serotonin (5-HT) is involved in the regulation of affective behavior, such as mood, anxiety, and aggression and influences vegetative functions like regulation of sleep, appetite, and body temperature. Therefore, dysfunctions of the serotonergic system play an important role in numerous psychiatric disorders such as depression and suicidality, obsessive- compulsive disorder, impulsive behavior and eating disorders. 5-HT also occurs in many peripheral tissues and regulates amongst others primary haemostasis, blood pressure, gut motility, and cell-mediated immune response. The rate- limiting step in the biosynthesis of 5-HT is catalysed by the tryptophan hydroxylase (TPH). The gene of a newly discovered second TPH-isoform (TPH2), which is expressed in neuronal tissues, was cloned and sequenced from mouse, rat, and human and the enzymatic properties of the recombinant enzymes were characterized for the first time. The biochemical characteristics of the TPH2 were found in agreement with the parameters, which were determined in earlier studies with TPH from neuronal sources. Sequence analyses also show that TPH2 is phylogenetically the youngest aromatic amino acid hydroxylase beside the phenylalanine and tyrosine hydroxylases. The constructed phylogenetic pedigree gives an interesting overview on the evolution of hydroxylases, which thus began approximately three billion years ago. In order to better understand the physiological impact of the serotonergic system the 5-HT-levels were pharmacologically and genetically manipulated in vivo. Treatment of mice with 1-methyl-tryptophan (1-MeTrp), an inhibitor of the tryptophan metabolising enzyme indolamine-2,3-dioxygenase (IDO), resulted in increased levels of 5-HT in blood by 800 % and in the central nervous system by 30 %. To deplete the 5 -HT-levels pharmacologically the tryptophan analogue 7-hydroxytryptophan (7-HTP) was synthesised and tested in cell culture and animal experiments. It could be shown that this novel substance is metabolised by TPH to its toxic derivative 5,7-dihydroxytryptamine (5,7-DHT) which is able to kill serotonergic cells specifically. In cell culture experiments 7-HTP was proven to be suitable to kill 5-HT producing small cell lung cancer cells and carcinoid tumor cells specifically showing its potential as a specific chemotherapeutic compound against 5-HT-producing tumors. Animal experiments with Drosophila and mice revealed no obvious toxic effects of 7-HTP, but oral administration of 7-HTP to mice diminished 5-HT blood levels by 37 % after six days. The expression of TPH was targeted genetically using ribozymes. Therefore, three mini-hammerhead ribozymes against the Tph1-mRNA were designed and their efficacies were tested in vitro. Their determined cleavage efficiencies were dependent on the substrate/ribozyme ratios and the duration of the cleavage reactions and were correlated with their calculated free energies. After cloning these ribozymes into tRNA-expression constructs, different cleavage rates were detected in vitro. The most effective one of these tRNA-miniribozyme chimeras reduced the expression of TPH1 in 5-HT producing mastocytoma cells by 79 %. This shows that empirical tests are still necessary to find the best target sequence of ribozymes. Heterozygous transgenic animals expressing this tRNA-miniribozyme chimera showed a down regulation of the TPH1 in the duodenum by 34 % and of the 5-HT-levels by 17 %. New pharmacological possibilities to influence haemostasis using this ribozyme may provide novel therapies of thrombotic diseases. To investigate a decreased expression of TPH2 in vivo, transgenic mice were generated using retro-viral vectors, which express hammerhead ribozymes against the TPH2. With these mice the generation of a ribozyme-mediated TPH2-knock-down using retro-viral gene transfer could be demonstrated. These mice show a decreased 5-HT synthesis in the brain, which entails anxiolytic behavior. Thus, a second independently regulated serotonergic system in the CNS was better characterized and manipulated specifically, and fundamental insights for the understanding of the 5-HT biosynthesis were gained

Measurements of Transverse Transport and the Effect of Suspended Load

Source: ICHE Conference Archive - https://mdi-de.baw.de/icheArchiv

Investigation of the Degradation of Chelate Complexes in Liquid Redox Desulfurization Processes

Metal complexes such as Fe‐EDTA, which are used as pseudo‐catalysts or oxygen carriers in wet oxidative desulfurization processes, are subject to a degradation mechanism that significantly influences the economics of such processes. Therefore, this study presents a methodology for determining the degree of degradation during the reactive hydrogen sulfide absorption in a Fe‐EDTA solution within a continuously operating semi‐batch reactor system. For this purpose, the reactive conversion of H2S in the liquid phase was used as a reference, and a clear dependence of the degradation on the pH could be shown. In addition, indicators are introduced that evaluate the observed pH dependency of the degradation and distinguish pH‐induced effects such as the pH‐dependent absorption performance of H2S.TU Berlin, Open-Access-Mittel - 202

study protocol for a randomized controlled trial

Background For the surgical treatment of recurrent primary spontaneous pneumothoraces (rPSP) different operative therapies are applied to achieve permanent freedom from recurrence. Methods/design This multicenter clinical trial evaluates the long-term results of two commonly applied surgical techniques for the treatment of rPSP. Based on the inclusion and exclusion criteria, and after obtaining the patients’ informed consent, participants are randomized into the two surgical treatment arms: pulmonary wedge resection plus parietal pleurectomy (WRPP) or parietal pleurectomy alone (PP). Consecutively, all study participants will be followed up for two years to evaluate the surgical long-term effect. The primary efficacy endpoint is the recurrence rate of pneumothorax within 24 months after surgery. The calculated sample size is 360 patients (n = 180 per treatment arm) to prove superiority of one of the two treatments. So far, 22 surgical sites have submitted their declaration of commitment, giving the estimated number of participating patients. Discussion A prospective randomized clinical trial has been started to compare two established surgical therapies to evaluate the long-term results regarding recurrence rates. Furthermore, cost of treatment, and influence on the perioperative morbidity and mortality as well as on quality of life are analyzed. If the study reveals equivalence for both surgical techniques, unnecessary pulmonary resections could be avoided

Effect of fullerene C60 thermal and tribomechanical loading on Raman signals

Fullerene C60 powder was loaded by 1 N normal force and exposed to sliding under different frequencies for 15 min. It is shown that the velocity of the sliding movement determines the stability of the fullerene C60 powder. At slow velocity of movement with a frequency of 1 Hz under 1 N normal force, the fullerene C60 structure remains undamaged after 15 min sliding. On the contrary, high sliding velocities of 10 Hz and 50 Hz affected fragmentation of the fullerene C60, which resulted in a reduction of the coefficient of friction (COF). During sliding with 1 Hz, the friction reached the highest level with an average COF of 0.59 ± 0.03. The faster relative motion under 1 N normal force gave a lower average COF with 0.39 ± 0.03. The initial fullerene C60 powder formed a thick compressed layer in the tribomechanical loaded zone. As proven by Raman spectroscopy, operating the tribomechanical sliding test at 50 Hz stimulated the re-attraction of fresh C60 fullerene island onto the fragmented layer from outside of the loaded powder regions. The COF was increasing again up to 0.44 ± 0.04 for 1 N normal force and 50 Hz frequency. The fragmentation and decomposition of fullerene C60 with increasing sliding velocity is attributed to thermal heating up during fast relative movement. Raman spectra of the tribomechanical loaded fullerene C60 are compared with Raman spectra from slowly heated up C60 in air and with Raman spectra of laser irradiated fullerene C60

Antiferromagnetic structure and electronic properties of BaCr2As2 and BaCrFeAs2

The chromium arsenides BaCr2As2 and BaCrFeAs2 with ThCr2Si2 type structure (space group I4/mmm; also adopted by '122' iron arsenide superconductors) have been suggested as mother compounds for possible new superconductors. DFT-based calculations of the electronic structure evidence metallic antiferromagnetic ground states for both compounds. By powder neutron diffraction we confirm for BaCr2As2 a robust ordering in the antiferromagnetic G-type structure at T_N = 580 K with mu_Cr = 1.9 mu_B at T = 2K. Anomalies in the lattice parameters point to magneto-structural coupling effects. In BaCrFeAs2 the Cr and Fe atoms randomly occupy the transition-metal site and G-type order is found below 265 K with mu_Cr/Fe = 1.1 mu_B. 57Fe Moessbauer spectroscopy demonstrates that only a small ordered moment is associated with the Fe atoms, in agreement with electronic structure calculations with mu_Fe ~ 0. The temperature dependence of the hyperfine field does not follow that of the total moments. Both compounds are metallic but show large enhancements of the linear specific heat coefficient gamma with respect to the band structure values. The metallic state and the electrical transport in BaCrFeAs2 is dominated by the atomic disorder of Cr and Fe and partial magnetic disorder of Fe. Our results indicate that Neel-type order is unfavorable for the Fe moments and thus it is destabilized with increasing iron content.Comment: 14 pages, 14 figures, submitted to Physical Review