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The "Candidatus Synechococcus spongiarum" group includes different clades of cyanobacteria with high 16S rRNA sequence identity (~99%) and is the most abundant and widespread cyanobacterial symbiont of marine sponges. The first draft genome of a "Ca. Synechococcus spongiarum" group member was recently published, providing evidence of genome reduction by loss of genes involved in several nonessential functions. However, "Ca. Synechococcus spongiarum" includes a variety of clades that may differ widely in genomic repertoire and consequently in physiology and symbiotic function. Here, we present three additional draft genomes of "Ca. Synechococcus spongiarum," each from a different clade. By comparing all four symbiont genomes to those of free-living cyanobacteria, we revealed general adaptations to life inside sponges and specific adaptations of each phylotype. Symbiont genomes shared about half of their total number of coding genes. Common traits of "Ca. Synechococcus spongiarum" members were a high abundance of DNA modification and recombination genes and a reduction in genes involved in inorganic ion transport and metabolism, cell wall biogenesis, and signal transduction mechanisms. Moreover, these symbionts were characterized by a reduced number of antioxidant enzymes and low-weight peptides of photosystem II compared to their free-living relatives. Variability within the "Ca. Synechococcus spongiarum" group was mostly related to immune system features, potential for siderophore-mediated iron transport, and dependency on methionine from external sources. The common absence of genes involved in synthesis of residues, typical of the O antigen of free-living Synechococcus species, suggests a novel mechanism utilized by these symbionts to avoid sponge predation and phage attack.
IMPORTANCE
While the Synechococcus/Prochlorococcus-type cyanobacteria are widely distributed in the world's oceans, a subgroup has established its niche within marine sponge tissues. Recently, the first genome of sponge-associated cyanobacteria, " Candidatus Synechococcus spongiarum," was described. The sequencing of three representatives of different clades within this cyanobacterial group has enabled us to investigate intraspecies diversity, as well as to give a more comprehensive understanding of the common symbiotic features that adapt "Ca. Synechococcus spongiarum" to its life within the sponge host.
Bariatric operations in obese patients with type 2 diabetes often improve diabetes before weight loss is observed. In patients mainly Roux-en-Y-gastric bypass with partial stomach resection is performed. Duodenojejunal bypass (DJB) and ileal interposition (IIP) are employed in animal experiments. Due to increased glucose exposition of L-cells located in distal ileum, all bariatric surgery procedures lead to higher secretion of antidiabetic glucagon like peptide-1 (GLP-1) after glucose gavage. After DJB also downregulation of Na\(^{+}\)-D-glucose cotransporter SGLT1 was observed. This suggested a direct contribution of decreased glucose absorption to the antidiabetic effect of bariatric surgery. To investigate whether glucose absorption is also decreased after IIP, we induced diabetes with decreased glucose tolerance and insulin sensitivity in male rats and investigated effects of IIP on diabetes and SGLT1. After IIP, we observed weight-independent improvement of glucose tolerance, increased insulin sensitivity, and increased plasma GLP-1 after glucose gavage. The interposed ileum was increased in diameter and showed increased length of villi, hyperplasia of the epithelial layer, and increased number of L-cells. The amount of SGLT1-mediated glucose uptake in interposed ileum was increased 2-fold reaching the same level as in jejunum. Thus, improvement of glycemic control by bariatric surgery does not require decreased glucose absorption.
Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing
(2015)
The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program “Predict-IV”; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of “omics” technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling.
This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day.
Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors.
Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling.
Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions.
Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo.
Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound’s distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.
The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for \(H^+\) efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit \(H^+\) efflux (\(H^+\)-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H+-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (\(H^+\) efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to \(K^+\) efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the \(H^+\)-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of (\(H^+\)-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of (\(H^+\)-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.
Der Natrium-D-Glukose Kotransporter 1 (SGLT1) spielt eine wichtige Rolle bei der Aufnahme von Glukose aus dem Darmlumen in die Enterozyten des Darms. Anhand von Untersuchungen an Xenopus laevis-Oozyten konnte in unserem Labor das Protein RS1 als posttranslationales Regulatorprotein für SGLT1 und diverse andere Transporter ermittelt werden. Es wurde eine regulatorische Domäne aus RS1 mit vielen potentiellen Phosphorylierungsstellen isoliert (RS1-Reg) und gezeigt dass RS1-Reg die Abschnürung von Transporter enthaltenen Vesikeln vom Transgolgi-Netzwerk hemmt. Neben SGLT1 reguliert RS1 auch die konzentrierenden Nukleosidtransporter (CNTs) am TGN. Die Regulation der Transporter ist vom Phosphorylierungszustand von RS1-Reg abhängig. So wurde durch Versuche an Oozyten von Xenopus laevis und Injektion von RS1-Reg Mutanten gezeigt, dass die Phosphorylierung von RS1-Reg an einigen Stellen zu einer Inhibition von SGLT1 führte, während der Nukleosidtransporter CNT1 durch die dephosphorylierte Mutante herunterreguliert wurden. Neben der phosphorylierungsabhängigen Regulation konnte für SGLT1 auch gezeigt werden, dass die Herunterregulation nur unter Niedrigzucker-Bedingungen erfolgte, nicht jedoch bei hohen Glukosekonzentrationen. Für die CNTs war eine derartige Zuckerabhängigkeit nicht zu beobachten.
Im Rahmen der vorliegenden Studie wurde untersucht, ob die Ergebnisse aus den Oozytenmessungen auch in vivo in einem Säugetier gezeigt werden können. Hierzu wurden Mutanten der regulatorischen Domäne (RS1-Reg) des Maus-Proteins, welche den phosphorylierten Zustand simulierten (RS1-Reg (S19E)), oder die Phosphorylierung verhinderten (RS1-Reg (S19A)) eingesetzt. Diese wurden an ein Nanohydrogel gekoppelt, um eine Aufnahme in die Enterozyten im Darm zu gewährleisten. Es wurde in der RS1KO-Mausohne funktionelles RS1 gezeigt, dass auch im in vivo-System eine Herunterregulation von SGLT1 durch mRS1-Reg (S19E), nicht jedoch durch mRS1-Reg (S19A) erfolgte, während die CNTs nur durch mRS1-Reg (S19A) inhibiert wurden. Des Weiteren führte mRS1-Reg (S19A) in der Wildtypmaus bei niedrigen Zuckerkonzentrationen zu einer Stimulation von SGLT1, was für eine Kompetition mit dem endogenen RS1-Proteins spricht. Es konnte indirekt der Beweis erbracht werden, dass über Nanohydrogele längere Proteine in die Zelle gebracht werden können und dort funktionell freigesetzt werden.
Die Venusfliegenfalle, Dionaea muscipula, weckte aufgrund ihrer karnivoren Lebensweise schon sehr früh das Interesse vieler Wissenschaftler. Für karnivore Pflanzen, die auf Nährstoff-armen Böden wachsen, spielen Insekten als Beute und somit als Nährstofflieferant eine entscheidende Rolle. So können die Pflanzen durch die Verdauung der Beute mit wichtigen Makro- und Mikronährstoffen, wie Stickstoff, Phosphat, Kalium oder Natrium versorgt werden. Aus diesem Grund sollte im Rahmen meiner Arbeit ein besonderes Augenmerk auf die molekularen Mechanismen der Kationenaufnahme während der Nährstoffresorption gerichtet werden. Insbesondere die aus dem Insekt stammenden Nährstoffe Kalium und Natrium waren dabei von großem Interesse.
Im Allgemeinen sind Kaliumionen für Pflanzen eine essentielle anorganische Substanz und von großer physiologischer Bedeutung für die Entwicklung, den Metabolismus, die Osmoregulation, das Membranpotential und viele zelluläre Prozesse. Analysen der Kaliumaufnahme an Wurzeln von Modellpflanzen wie Arabidopsis thaliana und Reis zeigten, dass die Aufnahme von K+ ein Zusammenspiel von hoch-affinen K+-Transportern der HAK5-Familie und nieder-affinen Kaliumkanälen (AKT1/AtKC1) erfordert, die in ein komplexes
(De-)Phosphorylierungsnetzwerk eingebunden sind. In der vorliegenden Arbeit war es mir möglich das Netzwerk zur Kaliumaufnahme in den Drüsen der Venusfliegenfalle zu entschlüsseln. Es konnten Orthologe zum Kaliumtransporter HAK5 aus Arabidopsis (DmHAK5) und zum Kaliumkanal AKT1 (DmKT1) identifiziert und im heterologen Expressionssystem der Xenopus laevis Oozyten elektrophysiologisch charakterisiert werden. Dabei zeigte sich, das DmKT1 durch einen Ca2+-Sensor/Kinase-Komplex aus der CBL/CIPK-Familie phosphoryliert und somit aktiviert wird. Phylogenetische Analysen von DmKT1 bestätigten die Eingruppierung dieses Kaliumkanals in die Gruppe der pflanzlichen Shaker-Kaliumkanäle des AKT1-Typs. Die Transporteigenschaften zeigten zudem, dass DmKT1 bei hyperpolarisierenden Membranpotentialen aktiviert wird und einen K+-selektiven Einwärtsstrom vermittelt. In Oozyten konnte eine Kaliumaufnahme bis zu einer externen Konzentration von ≥1 mM beobachtet werden. DmKT1 repräsentiert also einen Kaliumkanal mit einer hohen Transportkapazität, der die nieder-affine Kaliumaufnahme in die Drüsenzellen der Venusfliegenfalle vermitteln kann.
Unterhalb einer externen Kaliumkonzentration von 1 mM würde der anliegende elektrochemische Kaliumgradient einen Kaliumausstrom und somit einen Verlust von Kalium favorisieren. Hoch-affine K+/H+-Symporter können durch die Ausnutzung des Protonengradienten eine Kaliumaufnahme im mikromolaren Bereich gewährleisten. In Wurzelhaaren von Arabidopsis vermittelt der Transporter AtHAK5 die Kaliumaufnahme unter Kaliummangelbedingungen. DmHAK5, ein Ortholog zu AtHAK5, ist in Dionaea Drüsen exprimiert und konnte zum ersten Mal im heterologen Expressionssystem der Xenopus Oozyten im Detail charakterisiert werden. Interessanterweise zeigte sich, dass DmHAK5 wie der K+-Kanal DmKT1 durch denselben CBL/CIPK-Komplex posttranslational reguliert und aktiviert wird. Die Transporteigenschaften von DmHAK5 wiesen auf einen Transporter mit einer breiten Substratspezifität hin, sodass sich DmHAK5 neben Kalium auch für Ammonium permeabel zeigte. Affinitätsuntersuchungen von DmHAK5 zu seinem Substrat Kalium klassifizierten das Protein als einen hoch-affinen Kaliumtransporter, der im Symport mit Protonen die Kaliumaufnahme im mikromolaren Konzentrationsbereich vermitteln kann.
Das Kaliumtransportmodul besteht also aus dem K+-selektiven Kanal DmKT1 und dem
K+/H+-Symporter DmHAK5, die die hoch- und nieder-affine Kaliumaufnahme in den Drüsenzellen während der Beuteverdauung in Dionaea muscipula Fallen ermöglichen. Beide Transportmodule werden Kalzium-abhängig durch die Kinase CIPK23 und den Ca2+-Sensor CBL9 auf posttranslationaler Ebene reguliert.
Zusammenfassend gelang es in dieser Arbeit Einblicke in die Kationenaufnahme während der Nährstoffresorptionsphase der Venusfliegenfalle, Dionaea muscipula, zu gewinnen. Dabei wurde klar, dass Dionaea muscipula im Laufe ihrer Evolution zu einer karnivoren Pflanze, nicht neue Transportmodule zur Nährstoffresorption aus der Beute entwickelte, sondern bekannte aus Wurzeln stammende Transportmodule umfunktionierte. Auf molekularer Ebene konnten die biophysikalischen Charakteristika der K+- und Na+-Transportproteine, sowie ihre Regulation entschlüsselt werden. Diese Erkenntnisse wurden schließlich in den Kontext des Beutefangs der Venusfliegenfalle gebracht und diskutiert.
Marine sponge–associated actinomycetes are considered as promising sources for the discovery of novel biologically active compounds. In the present study, a total of 64 actinomycetes were isolated from 12 different marine sponge species that had been collected offshore the islands of Milos and Crete, Greece, eastern Mediterranean. The isolates were affiliated to 23 genera representing 8 different suborders based on nearly full length 16S rRNA gene sequencing. Four putatively novel species belonging to genera Geodermatophilus, Microlunatus, Rhodococcus and Actinomycetospora were identified based on a 16S rRNA gene sequence similarity of < 98.5% to currently described strains. Eight actinomycete isolates showed bioactivities against Trypanosma brucei brucei TC221 with half maximal inhibitory concentration (IC50) values <20 μg/mL. Thirty four isolates from the Milos collection and 12 isolates from the Crete collection were subjected to metabolomic analysis using high resolution LC-MS and NMR for dereplication purposes. Two isolates belonging to the genera Streptomyces (SBT348) and Micromonospora (SBT687) were prioritized based on their distinct chemistry profiles as well as their anti-trypanosomal activities. These findings demonstrated the feasibility and efficacy of utilizing metabolomics tools to prioritize chemically unique strains from microorganism collections and further highlight sponges as rich source for novel and bioactive actinomycetes.
Mining Genomes of Three Marine Sponge-Associated Actinobacterial Isolates for Secondary Metabolism
(2015)
Here, we report the draft genome sequences of three actinobacterial isolates, Micromonospora sp. RV43, Rubrobacter sp. RV113, and Nocardiopsis sp. RV163 that had previously been isolated from Mediterranean sponges. The draft genomes were analyzed for the presence of gene clusters indicative of secondary metabolism using antiSMASH 3.0 and NapDos pipelines. Our findings demonstrated the chemical richness of sponge-associated actinomycetes and the efficacy of genome mining in exploring the genomic potential of sponge-derived actinomycetes.
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS.
Background
Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor.
Results
Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites.
Conclusions
Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.