Zur Chemie und Biogenese der herzwirksamen Glykoside von Convallaria majalis L.

Im ersten Teil der vorliegenden Dissertation wird die Identifizierung von sechs aus Convallaria majalis L. erstmals isolierten Cardenoliden beschrieben. Bei den Substanzen a, b und c handelt es sich um Periplogenin, Strophanthidin und Strophanthidol, die bis dahin nur in gebundener Form (als Glykoside) in Convallaria bekannt waren. Substanz b bzw. Strophanthidin konnte durch direkten Vergleich mit einem authentischen Präparat mit Vallarotoxin identifiziert werden. Vallarotoxin war zwar schon 1954 von Tschesche und Seehofer in sehr kleiner Menge aus Convallaria isoliert, in seiner Struktur aber nicht erkannt worden. Die Glykoside c1 , B1 und X2 wurden als die Allomethyloside Peripallosid , Strophallosid und Strophanolosid ( = Periplogenin-, Strophanthidin- und Strophanthidol-6-desoxy- ßrD-allosid) identifiziert. Außer den bisher nachgewiesenen Zuckern Glucose , Rhamnose und Gulomethylose konnte damit erstmals Allomethylose als Zuckerkomponente von Convallaria-Glykosiden aufgefunden werden. Auf Grund der großen s trukturellen Ähnlichkeit der einzelnen Convallaria-Cardenolide erwiesen sich zur Sicherung der Identität neben herkömmlichen Verfahren (Hydrolyse, chromatographischer Vergleich der Spaltprodukte ) vor allem spektroskopische Methoden (UV-, IR- und NMRSpektroskopie, Massenspektrometrie) als unentbehrlich. Der zweite Teil der Arbeit befaßt sich mit den Fragen des Transuortes und Syntheseortes der herzwirksamen Substanzen sowie mit deren Biosynthese. Da bis dahin keine näheren Angaben über die Inhaltsstoffe in den unterirdischen Organen der Maiglöckchenpflanze vorlagen, war zuerst eine genauere Analyse dieser Pflanzenteile erforderlich: Der Gesamtcardenolidgehalt im Rhizom von 0,26% entspricht dem einer guten Blattdroge, der Gehalt in den Wurzeln liegt etwa um die Hälfte niedriger (0 , 145%). In der Glykosidzusammensetzung unterscheiden sich die unterirdischen Organe nur quantitativ, nicht aber qualitativ von den oberirdischen Anteilen. Bei der Untersuchung von Rhizom und Wurzel wurde auch auf in freier Form vorkommende Zucker geprüft: in beiden Organen sind Saccharose (3 % ), Fructose und Glucose ( je 0,7%, bezogen auf Trockensubstanz) frei enthalten. Die Bestimmung des Cardenolidgehaltes in den einzelnen Organen von Convallaria majalis während einer Vegetationsperiode ergab, daß in den oberirdischen Teilen der Pflanze zu Beginn der Blütenbildung eine vermehrte Cardenolidsynthese einsetzt; ab der Hochblüte erfolgt hingegen eine stete Abnahme der herzaktiven Inhaltsstoffe. Ein Ansteigen der Glykosidmenge in den unterirdischen Anteilen erfolgt in zeitlicher Obereinstimmung mit der Neubildung sowohl ober- als auch unterirdischer Organe (Blüten, Ausläufer, Knospen). An eine solche Phase schließt jeweils ein " Glykosidabbau" an. Nach den erhaltenen Befunden kann ein Ansteigen der Cardenolidmenge in den unterirdischen Organen auf Grund eines Transportes aus dem Sproß ausgeschlossen werden. Die Glykosidzusammensetzung erfährt während einer Vegetationsperiode in den einzeInen Organen keine tiefgreifenden Veränderungen. Einzig in den oberirdischen Anteilen ist nach der Blütezeit eine verstärkte Abnahme von Convallatoxin zugunsten von Convallatoxol und vor allem von Convallosid zu beobachten. Die Glucosidierung stellt somit einen wesentlichen Vorgang in der Endausgestaltung des Glykosidkomplexes in Blatt und Blüte dar. Um festzustellen, ob in der Maiglöckchenpflanze eine Translokation von Cardenoliden stattfindet, applizierten wir tritiiertes Convallatoxol einmal über das Wurzelsystem, einmal über die Blattspreite. Dabei zeigte sich, daß ein Transport von Cardenoliden aus den Wurzeln in den Sproß, nicht jedoch in umgekehrter Richtung erfolgt. Weiters gelang der Nachweis, daß sowohl Wurzeln als auch Rhizome und Blätter zur Biosynthese von herzwirksamen Stoffen befähigt sind: Nach Verabreichung von Mevalonsäure-14C an die isolierten Pflanzenteile ließ sich in jedem Fall Periplorhamnosid- 140 isolieren. Für die Verfolgung einzelner Biogeneseschritte auf der Monoglykosidstufe in Convallaria majalis, wurden Cardenolide (Periplorhamnosid, Convallatoxol, Convallatoxin und Rhodexin A) mit Tritium markiert. Nach Applikation der radioaktiven Glykoside an Maiglöckchenblätter konnten wir die entstandenen Folgeprodukte isolieren und identifizieren. Es gelang zu zeigen, daß Periplorhamnosid eine Art Schlüsselstellung in der Biogenese der Convallaria Glykoside einnimmt. Durch schrittweise Oxydation wird aus dieser Verbindung zuerst Convallatoxol und dann Convallatoxin gebildet; die Fortsetzung des Biosyntheseweges liegt in der Glucos idierung von Convallatoxin zu Convallosid. Anderseits entsteht durch Hydroxylierung von Periplorhamnosid am C-Atom 11 Lokundjosid. Die Bildung dieses Cardenolides erfolgt allerdings auch über einen zweiten Biogeneseweg, nämlich durch Hydroxylierung von Rhodexin A am C-Atom 5. Die Isolierung von Lokundjosid-3H als Folgeprodukt von Rhodexin A beweist, daß die Einführung der 5 β OH-Gruppe - entgegen früheren Annahmen - auch auf der Glykosidstufe möglich ist und zum Einbau dieses Substituenten eine Δ5-Doppelbindung nicht unbedingt Voraussetzung ist. Durch den Nachweis dieser einzelnen Biosyntheseschritte sind nun die wichtigsten auf der Monoglykosidstufe ablaufenden Umsetzungen bei Convallaria majalis bekannt

Young people's trauma-related cognitions before and after cognitive processing therapy for post-traumatic stress disorder

Objectives: Cognitive processing therapy (CPT) is a psychotherapy for post‐traumatic stress disorder (PTSD) with a broad evidence base. Change in trauma‐related cognitions is considered its primary working mechanism. When trying to integrate a traumatic event into existing cognitive schemas, the adaptive mechanism is changing the schema (accommodation). However, PTSD patients frequently either change their schemas too much (over‐accommodation), or cognitively distort the event (assimilation). We aimed to test the hypothesized connections between the three types of cognition and symptom load. Design: This study adds to the literature using ‘impact statements’, essays on their trauma‐related thoughts written by patients at the beginning and end of CPT, to investigate cognitive change and its relationship to symptomatic outcome. Methods: We analysed statements written by 31 adolescents and young adults who received developmentally adapted CPT (a longer treatment where CPT is the core component) in a randomized controlled trial. Results: As expected, post‐CPT statements contained more accommodated and fewer over‐accommodated and assimilated clauses than pre‐CPT statements. Correlations between cognition frequencies and concurrent symptom load were as expected for assimilation, and, in part, over‐accommodation and accommodation. Decreased PTSD and depressive symptoms were correlated with increased accommodated thoughts. For over‐accommodation and assimilation, however, expected correlations could not be shown. Conclusions: Our results support the notion that cognitive change is an important mechanism of change in CPT in a sample of younger, non‐English‐speaking clients

Towards Modernization of the Formulation of the Traditional Uighur Medicine Herbal Preparation Abnormal Savda Munziq

Abnormal Savda Munziq (ASMq) is a herbal preparation used in Traditional Uighur Medicine for the treatment and prevention of diabetes, cardiovascular diseases, chronic asthma and cancer. The recommended dose of this decoction for cancer patients is 500 mL administered orally three times a day. Our approach aimed at reducing the high amount of fluid intake required by fractionation of ASMq guided by the antiproliferative activity on HL-60 cells. The fractionation of ASMq resulted in the preparation of an active extract, Extr-4. Using solid phase extraction, Extr-4 was further fractionated into five fractions (SPE-0, SPE-20, SPE-40, SPE-60 and SPE-80), with SPE-40 showing the strongest antiproliferative activity. Caffeic acid, rutin, isoquercitrin, isorhamnetin 3-O-rutinoside, apigenin 7-O-glucoside, rosmarinic acid, luteolin and formononetin were identified in Extr-4 and fractions thereof by means of TLC, HPLC-DAD and LC-MS. SPE-40 contained the main compounds responsible for the antiproliferative activity on HL-60 cells. Thus, a phenolic fraction with high antiproliferative activity on HL-60 cells was obtained from ASMq through the bioassay-guided fractionation process. This could provide a better pharmaceutical formulation that minimizes the administration inconveniencies of a high volume (1.5 L per day) of ASMq decoction for cancer patients

Microfluidic and nanotechnology based assays for the development of safe biopharmaceuticals

Protein stability towards aggregation represents a potential challenge for the production and administration of pharmaceuticals. In particular, aggregation can compromise the developability and shelf-life of the products, with consequences for yield and safety, respectively. In this work, we discuss two novel approaches for the analysis of the stability of protein formulations: (1) A microfluidic diffusion-sizing platform to analyze protein sizes and interactions at high protein concentration directly in the solution state with minimal perturbation of the sample. The limited dilution of the sample during the analysis and the possibility to characterize properties directly in the solution state make the technique suitable for the analysis of heterogeneous solutions of proteins under dynamic equilibrium. We show how the platform represents an attractive tool for the analysis of sizes and interactions of proteins in both diluted and high-concentration solutions during development, manufacturing, and formulation. (2) A highly controlled assay of surface-induced protein aggregation based on nanoparticles. Protein aggregation is often due to heterogeneous nucleation events occurring at interfaces, including air/water interface, impurities and leachable particles. However, the development of screening tools against surface aggregation has been hindered by the difficulty in generating a controlled amount of surface stress in the formulation as well as in decoupling the surface effect from the contribution of hydrodynamic flows. In our assay, we leverage the flexibility of polymer chemistry to finely tune the properties and amount of surfaces provided by the nanoparticles, inducing aggregation of soluble peptides and proteins, including antibodies, in a time scale of a few hours. This platform represents i) an attractive tool for fundamental studies of heterogeneous nucleation events under stagnant and flow conditions, and ii) a high-throughput screening assay of the effect of intrinsic and extrinsic variables on protein stability towards interface-induced aggregation. Please click Additional Files below to see the full abstract

Incremental, Adaptive and Interruptive Speech Realization for Fluent Conversation with ECAs

van Welbergen H, Baumann T, Kopp S, Schlangen D. Incremental, Adaptive and Interruptive Speech Realization for Fluent Conversation with ECAs. In: Aylett R, Krenn B, Pelachaud C, Shimodaira H, eds. Intelligent Virtual Agents. Lecture Notes in Computer Science. Vol 8108. Springer; 2013: 468-469

Fractionation of an Extract of Pluchea odorata Separates a Property Indicative for the Induction of Cell Plasticity from One That Inhibits a Neoplastic Phenotype

Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory

Application of a bacterial two-hybrid system for the analysis of protein-protein interactions between FemABX family proteins

Protein-protein interactions play an important role in all cellular processes. The development of two-hybrid systems in yeast and bacteria allows for in vivo assessment of such interactions. Using a recently developed bacterial two-hybrid system, the interactions of the Staphylococcus aureus proteins FemA, FemB and FmhB, members of the FemABX protein family, which is involved in peptidoglycan biosynthesis and beta-lactam resistance of numerous Gram-positive bacteria, were analysed. While FmhB is involved in the addition of glycine 1 of the pentaglycine interpeptide of S. aureus peptidoglycan, FemA and FemB are specific for glycines 2/3 and 4/5, respectively. FemA-FemA, FemA-FemB and FemB-FemB interactions were found, while FmhB exists solely as a monomer. Interactions detected by the bacterial two-hybrid system were confirmed using the glutathione S-transferase-pulldown assay and gel filtration

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements