Analysis of Allostery in the Tryptophan Synthase Complex by Horizontal and Vertical Approaches

The tryptophan synthase (TS) is a linear complex that catalyzes the last two steps of tryptophan biosynthesis in primary metabolism. It is a model system for subunit interactions in multi-enzyme complexes with a long-standing history of research. The -subunit TrpA and the -subunit TrpB display a rather low catalytic activity in isolation but strongly stimulate each other in the TS complex in an allosteric manner. In this thesis, allostery in the TS was studied by means of two different approaches. In the first manuscript, the TrpA subunit from Zea mays, which is dependent on activation by the respective TrpB subunit, was compared to a homologue of TrpA, called BX1. BX1 catalyzes the same reaction in secondary metabolism as does TrpA in primary metabolism but is highly active in the absence of a TrpB-like partner protein. The TrpA - BX1 comparison identified two differing amino acids within a loop region known for its role in allosteric activation of TrpA. The transfer of the corresponding BX1 loop residues into TrpA yielded variants with turnover numbers that were increased by at least one order of magnitude and up to 520-fold. This corresponds to the activation of TrpA by TrpB in the wild-type TS complex. At the same time substrate affinity was reduced drastically, an effect that could be reversed by binding of wild-type TrpB to the TrpA variants. These findings contribute to the understanding of the allosteric activation of the -subunit by the -subunit of TS and suggest an evolutionary trajectory that describes the transition from a primary metabolic enzyme regulated by an interaction partner to a self-reliant stand-alone secondary metabolic enzyme. In the second manuscript, the allosteric network of TS was analyzed by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. In previous work, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) were computed by means of ASR. The corresponding primordial proteins were produced in Escherichia coli, purified, and characterized. The results showed that LBCA-TS is reminiscent of modern TS by forming a complex with indole channeling taking place. However, LBCA-TrpA decreases the activity of LBCA-TrpB by a factor of 5 whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis increases the activity of ncTrpB by a factor of 30. In order to identify those amino acid residues that are responsible for this large difference, all six evolutionary TrpA and TrpB intermediates that stepwise link LBCA TS with N. caesariensis TS were produced and characterized. Remarkably, the switching from TrpB-inhibition to TrpB-activation by TrpA occurred between two successive TS intermediates. The comparison of these intermediates and the mutual exchange of residues by iterative rounds of site-directed mutagenesis allowed for the identification of four (out of 413) residues from TrpB that are necessary and sufficient for its allosteric activation by TrpA. These findings demonstrate that ancestral sequence reconstruction can efficiently identify residues essential for allosteric communication and contribute to our understanding of signal propagation in TS

The ICON Earth System Model Version 1.0

This work documents ICON-ESM 1.0, the first version of a coupled model based 19 on the ICON framework 20 • Performance of ICON-ESM is assessed by means of CMIP6 DECK experiments 21 at standard CMIP-type resolution 22 • ICON-ESM reproduces the observed temperature evolution. Biases in clouds, winds, 23 sea-ice, and ocean properties are larger than in MPI-ESM. Abstract 25 This work documents the ICON-Earth System Model (ICON-ESM V1.0), the first cou-26 pled model based on the ICON (ICOsahedral Non-hydrostatic) framework with its un-27 structured, icosahedral grid concept. The ICON-A atmosphere uses a nonhydrostatic dy-28 namical core and the ocean model ICON-O builds on the same ICON infrastructure, but 29 applies the Boussinesq and hydrostatic approximation and includes a sea-ice model. The 30 ICON-Land module provides a new framework for the modelling of land processes and 31 the terrestrial carbon cycle. The oceanic carbon cycle and biogeochemistry are repre-32 sented by the Hamburg Ocean Carbon Cycle module. We describe the tuning and spin-33 up of a base-line version at a resolution typical for models participating in the Coupled 34 Model Intercomparison Project (CMIP). The performance of ICON-ESM is assessed by 35 means of a set of standard CMIP6 simulations. Achievements are well-balanced top-of-36 atmosphere radiation, stable key climate quantities in the control simulation, and a good 37 representation of the historical surface temperature evolution. The model has overall bi-38 ases, which are comparable to those of other CMIP models, but ICON-ESM performs 39 less well than its predecessor, the Max Planck Institute Earth System Model. Problem-40 atic biases are diagnosed in ICON-ESM in the vertical cloud distribution and the mean 41 zonal wind field. In the ocean, sub-surface temperature and salinity biases are of con-42 cern as is a too strong seasonal cycle of the sea-ice cover in both hemispheres. ICON-43 ESM V1.0 serves as a basis for further developments that will take advantage of ICON-44 specific properties such as spatially varying resolution, and configurations at very high 45 resolution. 46 Plain Language Summary 47 ICON-ESM is a completely new coupled climate and earth system model that ap-48 plies novel design principles and numerical techniques. The atmosphere model applies 49 a non-hydrostatic dynamical core, both atmosphere and ocean models apply unstruc-50 tured meshes, and the model is adapted for high-performance computing systems. This 51 article describes how the component models for atmosphere, land, and ocean are cou-52 pled together and how we achieve a stable climate by setting certain tuning parameters 53 and performing sensitivity experiments. We evaluate the performance of our new model 54 by running a set of experiments under pre-industrial and historical climate conditions 55 as well as a set of idealized greenhouse-gas-increase experiments. These experiments were 56 designed by the Coupled Model Intercomparison Project (CMIP) and allow us to com-57 pare the results to those from other CMIP models and the predecessor of our model, the 58 Max Planck Institute for Meteorology Earth System Model. While we diagnose overall 59 satisfactory performance, we find that ICON-ESM features somewhat larger biases in 60 several quantities compared to its predecessor at comparable grid resolution. We empha-61 size that the present configuration serves as a basis from where future development steps 62 will open up new perspectives in earth system modellin

Recent trends in radioanalytical purification procedures and application to decommissioning of nuclear facilities

The decommissioning of nuclear facilities demands an optimisation of analytical procedures to determine a variety of radionuclides in a variety of different materials on a wide activity scale ranging from very low to very high activities. Severe analytical problems arise from disadvantageous decay properties of a number of radionuclides. In the following the opportunities and limitations of recently developed analytical and nuclear detection methods are presented which has been applied successfully determining radionuclides like Fe-55, Ni-59, Ni-63, Sr-90, Th, U, Pu, Am, Cm. The applied analytical methods have to fulfil high quality standards including very low lower limits of detection, very high selectivity, sufficient precision and accuracy even in samples containing enhanced amounts of interfering elements. A new powerful tool is the application of collimated in-situ-gamma-spectrometry. A recently developed calibration phantom named K-RISK is presented

Development of a cell culture-based assay for the evaluation of putative compounds against Cryptosporidium parvum

Cryptosporidiose ist eine sowohl bei Menschen als auch bei Tieren weltweit auftretende gastrointestinale Infektion, die durch das zoonotische Protozoon C. parvum und weitere Vertreter derselben Gattung verursacht wird. Der klinische Verlauf einer durch C. parvum induzierten Darminfektion ist eng mit dem Immunstatus des Wirtes assoziiert. Bei immunkompetenten Personen tritt als Folge einer Infektion mit diesem Erreger in der Regel eine akute, selbstlimitierende Durchfallerkrankung auf, die sich im Gegensatz dazu bei immundefizienten Personen zu einer chronischen und lebensbedrohlichen Erkrankungen mit unter Umständen (zusätzlichen) extraintestinalen Manifestationen entwickeln kann. Aber nicht nur in der Humanmedizin, sondern auch in der Veterinärmedizin zählt C. parvum zu den relevantesten parasitären Enteritiserregern. Durch ihr noch unausgereiftes Immunsystem sind in besonderem Maße Neonaten für eine Infektion mit diesem Erreger empfänglich und infizieren sich regelmäßig durch eine orale Aufnahme der übiquitär vorkommenden und gegenüber Umwelteinflüssen und Chemikalien sehr resistenten Dauerstadien (Oocysten). Als einer der Hauptverursacher der neonatalen Diarrhoe beim Kalb ist C. parvum verantwortlich für Verluste in der Kälberaufzucht, ein mit einem retardierten Wachstum einhergehendes vermindertes Schlachtgewicht und folglich Verursacher von erheblichen wirtschaftlichen Verlusten in der Landwirtschaft. Sowohl in der Human- als auch in der Veterinärmedizin existieren zugelassene Wirkstoffe zur Behandlung bzw. Pro- und Metaphylaxe einer humanen bzw. bovinen Cryptosporidiose. Leider erweist sich deren Einsatz nicht in allen Fällen einer Cryptosporidiose als effektiv, so dass uneingeschränkt wirksame Chemotherapeutika bis heute nicht verfügbar sind, aber dringend benötigt werden. In der vorliegenden Arbeit wurde eine Methode zur Evaluierung einer anticryptosporidialen Wirksamkeit potentieller bis dato noch nicht überprüfter Wirkstoffe etabliert. Bei dem als „C. parvum Inhibitionsassay“ bezeichneten Testverfahren handelt es sich um eine Kombination aus einer in vitro Kultivierung von C. parvum in HCT-8 Zellen und einer quantitativen real-time PCR (qPCR). Die qPCR ermöglichte eine relative Quantifizierung der Erregerentwicklung unter Wirkstoffeinfluss, so dass eine Aussage zu einer potentiell anticryptosporidialen Wirksamkeit der untersuchten Substanzen getroffen werden konnte. Als Zielsequenz fungierte hierbei ein 255 bp umfassender Abschnitt der cryptosporidialen 18S rDNA, dessen Gehalt in jeder einzelnen Probe anhand einer internen Kontrolle normiert wurde. Als interne Kontrolle wurde eine 189 bp umfassende Sequenz innerhalb des zellulären (humanen) 18S rDNA Gens verwendet, deren Gehalt in den einzelnen Proben anhand einer zweiten qPCR ermittelt wurde. Insgesamt wurden 51 Wirkstoffe in mindestens drei verschiedenen Konzentrationen überprüft. Zwei vielversprechend erscheinende Wirkstoffe, ein heterozyklisch substituiertes 1,2,4-Triazin (BAY-AB24992) und ein substituiertes Benzimidazol (BAY-AF76184), wurden eingehender untersucht. Ihr mittels des C. parvum Inhibitionsassays überprüftes Konzentrationsspektrum umfasste insgesamt acht Verdünnungsstufen und selbige Konzentrationen wurden ebenfalls mittels eines Lactatdehydrogenase (LDH)-Assays und eines WST-1-Assays auf zytotoxische und zytostatische Effekte auf die gewählte Wirtszelllinie untersucht. Für die Substanz BAY-AF76184 konnte eine gute konzentrationsabhängige Wirksamkeit gegen C. parvum mit Inhibitionen des cryptosporidialen Wachstums im Größenbereich von maximal 97 % gezeigt werden. Der ermittelte EC50-Wert belief sich auf 2,37 μM. Obgleich für diesen Wirkstoff auch konzentrationsabhängige antiproliferative und zytotoxische Effekte auf die HCT-8 Zelllinie nachgewiesen werden konnten, ist eine weitere Überprüfung in niedrigen Dosierungen in Tiermodellen angeraten. Selbst sehr niedrige Wirkstoffkonzentrationen in Höhe von 4,46 μM, die nicht mehr mit zytostatischen Effekten und einer nur sehr geringen Zytotoxizität assoziiert waren, erzielten in vitro noch Wachstumsinhibitionen von C. parvum im Größenbereich von ca. 78 %, weshalb diese Substanz trotz seiner unerwünschten Eigenschaften durchaus als vielsprechend gelten kann. Auch für BAY-AB24992 scheint eine nachfolgende in vivo Überprüfung empfehlenswert. Zwar war für diese Substanz keine Konzentrationsabhängigkeit nachweisbar und in vitro bewirkten nur die vier höchsten Konzentrationen eine über 60%ige Wachstumsinhibition von C. parvum (ca. 71 bis 88 %), allerdings konnten für diesen Wirkstoff keinerlei antiproliferative Effekte und auch nur eine sehr geringe Zytotoxizität nachgewiesen werden. Insgesamt betrachtet stellt das hier etablierte C. parvum Inhibitionsassay eine Methode dar, die eine schnelle und zuverlässige parallele Überprüfung mehrerer Substanzen auf ihre Wirksamkeit gegen das Protozoon C. parvum ermöglicht. Es ist folglich dazu geeignet, ethisch bedenkliche, kostenintensive und arbeitsaufwendige Tierversuche zumindest partiell zu ersetzten. Obgleich von einer attestierten Wirksamkeit in vitro nicht zwangsläufig auf eine Wirksamkeit in vivo geschlossen werden kann, so eignet sich ein Zellkultur-basiertes Testverfahren dennoch zur Vorselektion solcher Wirkstoffe, die in nachgeschalteten Tierversuchen mit einer größeren Wahrscheinlichkeit positive Ergebnisse erzielen könnten.Cryptosporidiosis is a common gastro-intestinal infection in both, humans and animals, worldwide caused by the zoonotic protozoan C. parvum and other members of the same genus. The clinical course of C. parvum infections is highly dependent on the immune status of the individual host. While in immunocompetent individuals Cryptosporidium infections most commonly result in acute but self-limiting gastroenteritis, in immunodeficient or immunosupressed individuals cryptosporidiosis can become a chronic and life-threatening diarrhoeal disease and also (additional) extraintestinal infections may occur. Not only in human medicine but also in veterinary medicine C. parvum ranges among the most relevant parasitic enteritis pathogens. Due to their immature immune system neonates are highly susceptible for infections with this parasite and routinely get infected by oral uptake of the ubiquitary oocysts which are very resistant against environmental changes and chemicals. As one of the major pathogens causing neonatal diarrhoea in calves, C. parvum is responsible for fatalities and retarded weight gain in calf rearing and therefore the cause of significant economical losses in agriculture. Even though approved agents for the treatment of cryptosporidiosis and for pro- and metaphylactic use are available in human as well as in veterinary medicine, fully effective drugs in all cases of cryptosporidiosis are still missing but urgently needed. In the present work a method was established to evaluate the efficacy of putative anticryptosporidial compounds which had not been tested yet. The so-called ”C. parvum inhibition assay“ is a combination of in vitro cultivation of C. parvum in HCT-8 cells and quantitative real-time PCR (qPCR). The qPCR-based method used here allowed the relative quantification of the development of C. parvum under drug exposure, therefore the efficacy of the tested compound could be determined. For the detection of the parasite DNA a target sequence of 255 bp within the cryptosporidial 18S rDNA gene was used. The amount of this target sequence was normalised to an internal control for each sample. As internal control a sequence with a size of 189 bp from the human host cell 18S rDNA gene was amplified in a second qPCR. Overall 51 compounds were tested in at least three different concentrations. Two promising compounds, a heterocyclic substituted 1,2,4-triazinedione (BAY- AB24992) and a substituted benzimidazole (BAY-AF76184), were examined in more detail. Both compounds were tested in eight different concentrations for their ability to interfere with C. parvum development in vitro. Furthermore the same concentrations were also used in a lactate dehydrogenase and a WST-1 assay to analyse potential cytotoxic and anti-proliferative effects on the chosen host cell line, respectively. The drug BAY-AF76184 displayed a good concentration- dependent inhibition of C. parvum growth with a maximum inhibition of 97%. The concentration response curve had an EC50 value of 2.37 μM. Although this compound also showed concentration-dependent antiproliferative and cytotoxic effects on HCT-8 cells, further in vivo tests at low dosages are advisable. Even a concentration of 4.46 μM, which was associated with low cytotoxicity and no cytostatic effect, reached an inhibition of the growth of C. parvum in vitro of approximately 78%. Therefore BAY-AF76184 can be considered a promising/putative compound despite its unwanted side effects. Future in vivo testing of BAY-AB24992 also appears to be recommendable. This compound did not inhibit C. parvum growth in a concentration-dependant manner but also showed good in vitro efficacy against C. parvum. At least the four highest concentrations yielded an inhibition of more than 60% (approx. 71-88%). Additionally this drug had neither antiproliferative nor cytotoxic effects. Overall the established C. parvum inhibition assay displays a fast and reliable method for assessing drug efficacy against the protozoan C. parvum. Consequently it is suitable to at least partially replace expensive and labour-intensive animal experiments which are also of ethical concern. Although there is of course no guarantee that drugs with good in vitro activity will also perform in vivo, a cell culture-based assay nevertheless improves prediction for overall success of consecutive in vivo tests

Monitoring the equilibration of 228Th and decay products over time after extraction from ivory

The worldwide population of elephants shows a rapid decline due to trafficking of ivory. Hence, a method is needed for the age determination to distinguish legal from illegal ivory. The analysis of Th-228 and Th-232 in ivory can be used for this purpose. This study provides a technique for the analysis of Th-228 in ivory. The thorium was extracted from ivory and measured after various time periods applying -spectrometry until a secular radioactive equilibrium between Th-228 and its decay products was established. An equilibration period of 32days was found. The uncertainty of the determination of the activity of Th-228 could be reduced by integrating the counts of the equilibrated decay products into the determination process

Aneurysma der Vena poplitea als Ursache rezidivierender Lungenarterienembolien mit Reanimationspflichtigkeit

History and admission findings: A previously healthy 40-year-old varnisher was admitted because of increasing dyspnoea. His clinical status rapidliy deteriorated. He was referred to a cardiology intensive care unit but had to be resuscitated during transport. His condition became stable under controlled ventilation and analgesics. There were no other contributory abnormal findings. Investigations: The concentration of D-dimers was raised. Pulmonary angiography demonstrated multiple bilateral occlusions of the segmental arteries. Treatment and course: Extubation became possible after thrombolysis with recombinant tissue plasminogen activator (rTPA). There was no evidence of leg or pelvic vein thrombosis. But a hard mass was palpated in the left popliteal fossa and extensively thrombosed saccular aneurysm of the popliteal vein was found. The aneurysm was resected and a venous graft was interposed. There were no further thromboemboli under oral anticoagulation. Two years later the venous graft was occluded with adequate collateral circulation. Conclusion: Aneurysm of the popliteal vein is a rare vascular anomaly of unknown pathogenesis. In patients with repetitive episodes of lung embolism peripheral aneurysms must be taken into consideration