TY - JOUR A1 - Stoof-Leichsenring, Kathleen Rosemarie A1 - Bernhardt, Nadine A1 - Pestryakova, Luidmila Agafyevna A1 - Epp, Laura Saskia A1 - Herzschuh, Ulrike A1 - Tiedemann, Ralph T1 - A combined paleolimnological/genetic analysis of diatoms reveals divergent evolutionary lineages of Staurosira and Staurosirella (Bacillariophyta) in Siberian lake sediments along a latitudinal transect JF - Journal of paleolimnolog N2 - Diatom diversity in lakes of northwest Yakutia (Siberia) was investigated by microscopic and genetic analysis of surface and cored lake sediments, to evaluate the use of sedimentary DNA for paleolimnological diatom studies and to identify obscure genetic diversity that cannot be detected by microscopic methods. Two short (76 and 73 bp) and one longer (577 bp) fragments of the ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) gene, encoding the large subunit of the rbcL, were used as genetic markers. Diverse morphological assemblages of diatoms, dominated by small benthic fragilarioid taxa, were retrieved from the sediments of each lake. These minute fragilarioid taxa were examined by scanning electron microscopy, revealing diverse morphotypes in Staurosira and Staurosirella from the different lakes. Genetic analyses indicated a dominance of haplotypes that were assigned to fragilarioid taxa and less genetic diversity in other diatom taxa. The long rbcL_577 amplicon identified considerable diversification among haplotypes clustering within the Staurosira/Staurosirella genera, revealing 19 different haplotypes whose spatial distribution appears to be primarily related to the latitude of the lakes, which corresponds to a vegetation and climate gradient. Our rbcL markers are valuable tools for tracking differences between diatom lineages that are not visible in their morphologies. These markers revealed putatively high genetic diversity within the Staurosira/Staurosirella species complex, at a finer scale than is possible to resolve by microscopic determination. The rbcL markers may provide additional reliable information on the diversity of barely distinguishable minute benthic fragilarioids. Environmental sequencing may thus allow the tracking of spatial and temporal diversification in Siberian lakes, especially in the context of diatom responses to recent environmental changes, which remains a matter of controversy. KW - Arctic lake sediments KW - Diatoms KW - Environmental DNA KW - Intraspecific variation KW - RbcL Y1 - 2014 U6 - https://doi.org/10.1007/s10933-014-9779-1 SN - 0921-2728 SN - 1573-0417 VL - 52 IS - 1-2 SP - 77 EP - 93 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Zor, K. A1 - Heiskanen, A. A1 - Caviglia, Claudia A1 - Vergani, M. A1 - Landini, E. A1 - Shah, F. A1 - Carminati, Marco A1 - Martinez-Serrano, A. A1 - Ramos Moreno, T. A1 - Kokaia, M. A1 - Benayahu, Dafna A1 - Keresztes, Zs. A1 - Papkovsky, D. A1 - Wollenberger, Ursula A1 - Svendsen, W. E. A1 - Dimaki, M. A1 - Ferrari, G. A1 - Raiteri, R. A1 - Sampietro, M. A1 - Dufva, M. A1 - Emneus, Jenny T1 - A compact multifunctional microfluidic platform for exploring cellular dynamics in real-time using electrochemical detection JF - RSC Advances N2 - Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivation and electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in both applications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring. Y1 - 2014 U6 - https://doi.org/10.1039/c4ra12632g SN - 2046-2069 VL - 4 IS - 109 SP - 63761 EP - 63771 PB - Royal Society of Chemistry CY - Cambridge ER - TY - GEN A1 - Zór, K. A1 - Heiskanen, A. A1 - Caviglia, Claudia A1 - Vergani, M. A1 - Landini, E. A1 - Shah, F. A1 - Carminati, Marco A1 - Martínez-Serrano, A. A1 - Ramos Moreno, T. A1 - Kokaia, M. A1 - Benayahu, Dafna A1 - Keresztes, Zs. A1 - Papkovsky, D. A1 - Wollenberger, Ursula A1 - Svendsen, W. E. A1 - Dimaki, M. A1 - Ferrari, G. A1 - Raiteri, R. A1 - Sampietro, M. A1 - Dufva, M. A1 - Emnéus, J. T1 - A compact multifunctional microfluidic platform for exploring cellular dynamics in real-time using electrochemical detection N2 - Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivation and electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in both applications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 289 Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-99492 ER - TY - JOUR A1 - Brothers, Soren M. A1 - Koehler, J. A1 - Attermeyer, Katrin A1 - Grossart, Hans-Peter A1 - Mehner, T. A1 - Meyer, N. A1 - Scharnweber, Inga Kristin A1 - Hilt, Sabine T1 - A feedback loop links brownification and anoxia in a temperate, shallow lake JF - Limnology and oceanography N2 - This study examines a natural, rapid, fivefold increase in dissolved organic carbon (DOC) concentrations in a temperate shallow lake, describing the processes by which increased DOC resulted in anoxic conditions and altered existing carbon cycling pathways. High precipitation for two consecutive years led to rising water levels and the flooding of adjacent degraded peatlands. Leaching from the flooded soils provided an initial increase in DOC concentrations (from a 2010 mean of 12 +/- 1 mg L-1 to a maximum concentration of 53 mg L-1 by June 2012). Increasing water levels, DOC, and phytoplankton concentrations reduced light reaching the sediment surface, eliminating most benthic primary production and promoting anoxia in the hypolimnion. From January to June 2012 there was a sudden increase in total phosphorus (from 57 mg L-1 to 216 mg L-1), DOC (from 24.6 mg L-1 to 53 mg L-1), and iron (from 0.12 mg L-1 to 1.07 mg L-1) concentrations, without any further large fluxes in water levels. We suggest that anoxic conditions at the sediment surface and flooded soils produced a dramatic release of these chemicals that exacerbated brownification and eutrophication, creating anoxic conditions that persisted roughly 6 months below a water depth of 1 m and extended periodically to the water surface. This brownification-anoxia feedback loop resulted in a near-complete loss of macroinvertebrate and fish populations, and increased surface carbon dioxide (CO2) emissions by an order of magnitude relative to previous years. Y1 - 2014 U6 - https://doi.org/10.4319/lo.2014.59.4.1388 SN - 0024-3590 SN - 1939-5590 VL - 59 IS - 4 SP - 1388 EP - 1398 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Baeten, Lander A1 - Warton, David I. A1 - Van Calster, Hans A1 - De Frenne, Pieter A1 - Verstraeten, Gorik A1 - Bonte, Dries A1 - Bernhardt-Römermann, Markus A1 - Cornelis, Johnny A1 - Decocq, Guillaume A1 - Eriksson, Ove A1 - Hedl, Radim A1 - Heinken, Thilo A1 - Hermy, Martin A1 - Hommel, Patrick A1 - Kirby, Keith J. A1 - Naaf, Tobias A1 - Petrik, Petr A1 - Walther, Gian-Reto A1 - Wulf, Monica A1 - Verheyen, Kris T1 - A model-based approach to studying changes in compositional heterogeneity JF - Methods in ecology and evolution : an official journal of the British Ecological Society Y1 - 2014 SN - 2041-210X SN - 2041-2096 VL - 5 IS - 2 SP - 156 EP - 164 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Shapiro, B. A1 - Hofreiter, Michael T1 - A paleogenomic perspective on evolution and gene function: new insights from ancient DNA JF - Science N2 - The publication of partial and complete paleogenomes within the last few years has reinvigorated research in ancient DNA. No longer limited to short fragments of mitochondrial DNA, inference of evolutionary processes through time can now be investigated from genome-wide data sampled as far back as 700,000 years. Tremendous insights have been made, in particular regarding the hominin lineage. With rare exception, however, a paleogenomic perspective has been mired by the quality and quantity of recoverable DNA. Though conceptually simple, extracting ancient DNA remains challenging, and sequencing ancient genomes to high coverage remains prohibitively expensive for most laboratories. Still, with improvements in DNA isolation and declining sequencing costs, the taxonomic and geographic purview of paleogenomics is expanding at a rapid pace. With improved capacity to screen large numbers of samples for those with high proportions of endogenous ancient DNA, paleogenomics is poised to become a key technology to better understand recent evolutionary events. Y1 - 2014 U6 - https://doi.org/10.1126/science.1236573 SN - 0036-8075 SN - 1095-9203 VL - 343 IS - 6169 PB - American Assoc. for the Advancement of Science CY - Washington ER - TY - JOUR A1 - Audisio, Paolo A1 - Cline, Andrew R. A1 - Solano, Emanuela A1 - Mancini, Emiliano A1 - Lamanna, Francesco A1 - Antonini, Gloria A1 - Trizzino, Marco T1 - A peculiar new genus and species of pollen-beetle (Coleoptera, Nitidulidae) from eastern Africa, with a molecular phylogeny of related Meligethinae JF - Systematics and biodiversity KW - new species KW - new genus KW - molecular analysis KW - pollen-beetles KW - host-plants KW - Asteraceae KW - Kenya KW - Tarchonanthopria freidbergi Y1 - 2014 U6 - https://doi.org/10.1080/14772000.2013.877539 SN - 1477-2000 SN - 1478-0933 VL - 12 IS - 1 SP - 77 EP - 91 PB - Routledge, Taylor & Francis Group CY - Abingdon ER - TY - JOUR A1 - Bianco, Pier Giorgio A1 - Ketmaier, Valerio T1 - A revision of the Rutilus complex from Mediterranean Europe with description of a new genus, Sarmarutilus, and a new species, Rutilus stoumboudae (Teleostei: Cyprinidae) JF - Zootaxa : an international journal of zootaxonomy ; a rapid international journal for animal taxonomists N2 - By combining morphology, ecology, biology, and biogeography with the available molecular (sequence variation of the entire mitochondrial cytochrome b gene; cyt-b) and karyology data, the taxonomy of several species of the Rutilus complex inhabiting southern Europe is revised. Rutilus stoumboudae, new species, is described from Lake Volvi, Greece. It differs from Rutilus rutilus in possessing more total GR and less branched rays in both dorsal and anal fins and in its placement in the cyt-b based phylogeny of the genus. The resurrected genus Leucos Heckel, 1843 (type species Leucos aula, Bonaparte, 1841), which according to molecular data diverged from Rutilus more than 5 million years ago, during the Messinian salinity crisis, includes five species of small size, without spinous tubercles on scales and head in reproductive males, pharyngeal teeth formula 5-5, and all show a preference for still waters. Leucos aula is the Italian species endemic in the Padany-Venetian district: L. basak is widespread in Croatia, Albania, Montenegro and former Yugoslav Republic of Macedonia (FYROM); L. albus, recently described from Lake Skadar, Montenegro, is also found in rivers Moraca and Zeta (Montenegro). L. albus differs from L. basak, its closest relative, in having more scales on the LL and less anal-fin rays; L. panosi is endemic to the western-Greece district, and L. ylikiensis is endemic to lakes Yliki and Paralimni in eastern Greece (introduced in Lake Volvi). Among the nominal species examined, Rutilus karamani, R. ohridanus, R. prespensis and R. prespensis vukovici are all junior synonyms of Leucos basak. Rutilus vegariticus is definitively regarded as junior synonym for R. rutilus. Sarmarutilus n.gen. is a monotypic genus, with Sarmarutilus rubilio as the type species. According to phylogenetic data, Sarmarutilus rubilio is basal to a cluster of species that includes Leucos basak, L. albus, L. aula, L. panosi and L. ylikiensis. Sarmarutilus possibly evolved in pre-Messinian time, in the Lago Mare, entered the Mediterranean area during the Messinian Lago Mare phase of the Mediterranean Sea and survived only in the Tuscany-Latium district. This genus differs from Leucos in having large pearl organs on the central part of head and body scales in mature males and for the habitat preference, being a riverine-adapted species. It differs from Rutilus in pharyngeal teeth formula (5-5 in Sarmarutilus and 6-5 in Rutilus), size (small in Sarmarutilus and large in Rutilus) and for the preferential habitat (riverine vs. still water). Finally, lectotypes for Leucos basak, Leucos aula, and Sarmarutilus rubilio are designated. KW - Freshwater fish KW - Mediterranean Europe KW - Cyprinidae KW - genera Rutilus KW - Leucos KW - Sarmarutilus new genus KW - new species Y1 - 2014 SN - 1175-5326 SN - 1175-5334 VL - 3841 IS - 3 SP - 379 EP - 402 PB - Magnolia Press CY - Auckland ER - TY - JOUR A1 - Omidbakhshfard, Mohammad Amin A1 - Winck, Flavia Vischi A1 - Arvidsson, Samuel Janne A1 - Riano-Pachon, Diego M. A1 - Müller-Röber, Bernd T1 - A step-by-step protocol for formaldehyde-assisted isolation of regulatory elements from Arabidopsis thaliana JF - Journal of integrative plant biology N2 - The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species. KW - Arabidopsis thaliana KW - chromatin KW - cis-regulatory elements KW - epigenomics KW - FAIRE-qPCR KW - FAIRE-seq KW - gene expression KW - gene regulatory network KW - transcription factor Y1 - 2014 U6 - https://doi.org/10.1111/jipb.12151 SN - 1672-9072 SN - 1744-7909 VL - 56 IS - 6 SP - 527 EP - 538 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Lukas, Marcus A1 - Wacker, Alexander T1 - Acclimation to dietary shifts impacts the carbon budgets of Daphnia magna JF - Journal of plankton research N2 - Daphnia responds to low availability of carbon (food quantity) or limiting concentrations of nutrients relative to carbon (C) in excess (food quality) by respectively saving or discharging C via different pathways. We investigated which kind of food limitation leads to a faster regulation in Daphnia C budgets, and whether the pre-assimilative C pathways, ingestion and faeces egestion and the post-assimilative C pathways, excretion and respiration, are regulated concurrently. Daphnia magna were exposed to dietary shifts in different food quantities or qualities; food quality was varied in terms of the essential component, cholesterol. After acclimation to the new diet ranging from 0 to 96 h, C budgets were measured by a radiotracer technique. Dietary shifts in quantity and quality caused Daphnia to quickly adjust their C budgets within 6 h, but different C pathways were affected. A shift to low food quantity reduced Daphnia respiration indicating C retention. In contrast, sudden low quality food caused increased faeces egestion to discharge excess C. Furthermore, we observed a delayed increase in excretion but no change in respiration within the time frame studied. Such time-shifted responses appear to be an appropriate means to keep the costs of physiological adjustments relatively low, which in turn would benefit Daphnia performance. KW - carbon pathway KW - cholesterol KW - zooplankton KW - food quality KW - food quantity Y1 - 2014 U6 - https://doi.org/10.1093/plankt/fbu018 SN - 0142-7873 SN - 1464-3774 VL - 36 IS - 3 SP - 848 EP - 858 PB - Oxford Univ. Press CY - Oxford ER - TY - INPR A1 - Lendlein, Andreas A1 - Neffe, Axel T. A1 - Jerome, Christine T1 - Advanced functional polymers for medicine T2 - Advanced healthcare materials Y1 - 2014 U6 - https://doi.org/10.1002/adhm.201400718 SN - 2192-2640 SN - 2192-2659 VL - 3 IS - 12 SP - 1939 EP - 1940 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Kind, Barbara A1 - Muster, Britta A1 - Staroske, Wolfgang A1 - Herce, Henry D. A1 - Sachse, Rene A1 - Rapp, Alexander A1 - Schmidt, Franziska A1 - Koss, Sarah A1 - Cardoso, M. Cristina A1 - Lee-Kirsch, Min Ae T1 - Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutieres syndrome JF - Human molecular genetics N2 - Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutieres syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy. We demonstrate that the RNase H2 complex is assembled in the cytosol and imported into the nucleus in an RNase H2B-dependent manner. RNase H2 is not only recruited to DNA replication foci, but also to sites of PCNA-dependent DNA repair. By fluorescence recovery after photobleaching, we demonstrate a high mobility and fast exchange of RNase H2 at sites of DNA repair and replication. We provide evidence that recruitment of RNase H2 is not only PCNA-dependent, mediated by an interaction of the B subunit with PCNA, but also PCNA-independent mediated via the catalytic domain of the A subunit. We found that AGS-associated mutations alter complex formation, recruitment efficiency and exchange kinetics at sites of DNA replication and repair suggesting that impaired ribonucleotide removal contributes to AGS pathogenesis. Y1 - 2014 U6 - https://doi.org/10.1093/hmg/ddu319 SN - 0964-6906 SN - 1460-2083 VL - 23 IS - 22 SP - 5950 EP - 5960 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Lecourieux, Fatma A1 - Kappel, Christian A1 - Lecourieux, David A1 - Serrano, Alejandra A1 - Torres, Elizabeth A1 - Arce-Johnson, Patricio A1 - Delrot, Serge T1 - An update on sugar transport and signalling in grapevine JF - Journal of experimental botany N2 - In addition to their role as a source of reduced carbon, sugars may directly or indirectly control a wide range of activities in plant cells, through transcriptional and post-translational regulation. This control has been studied in detail using Arabidopsis thaliana, where genetic analysis offers many possibilities. Much less is known about perennial woody species. For several years, various aspects of sugar sensing and signalling have been investigated in the grape (Vitis vinifera L.) berry, an organ that accumulates high concentrations of hexoses in the vacuoles of flesh cells. Here we review various aspects of this topic: the molecular basis of sugar transport and its regulation by sugars in grapevine; the functional analysis of several sugar-induced genes; the effects of some biotic and abiotic stresses on the sugar content of the berry; and finally the effects of exogenous sugar supply on the ripening process in field conditions. A picture of complex feedback and multiprocess regulation emerges from these data. KW - Fruit biology KW - grapevine KW - signalling KW - stress KW - sugar KW - transport Y1 - 2014 U6 - https://doi.org/10.1093/jxb/ert394 SN - 0022-0957 SN - 1460-2431 VL - 65 IS - 3 SP - 821 EP - 832 PB - Oxford Univ. Press CY - Oxford ER - TY - THES A1 - Girbig, Dorothee T1 - Analysing concerted criteria for local dynamic properties of metabolic systems T1 - Die Analyse koordinierter Kriterien für lokale dynamische Eigenschaften metabolischer Systeme N2 - Metabolic systems tend to exhibit steady states that can be measured in terms of their concentrations and fluxes. These measurements can be regarded as a phenotypic representation of all the complex interactions and regulatory mechanisms taking place in the underlying metabolic network. Such interactions determine the system's response to external perturbations and are responsible, for example, for its asymptotic stability or for oscillatory trajectories around the steady state. However, determining these perturbation responses in the absence of fully specified kinetic models remains an important challenge of computational systems biology. Structural kinetic modeling (SKM) is a framework to analyse whether a metabolic steady state remains stable under perturbation, without requiring detailed knowledge about individual rate equations. It provides a parameterised representation of the system's Jacobian matrix in which the model parameters encode information about the enzyme-metabolite interactions. Stability criteria can be derived by generating a large number of structural kinetic models (SK-models) with randomly sampled parameter sets and evaluating the resulting Jacobian matrices. The parameter space can be analysed statistically in order to detect network positions that contribute significantly to the perturbation response. Because the sampled parameters are equivalent to the elasticities used in metabolic control analysis (MCA), the results are easy to interpret biologically. In this project, the SKM framework was extended by several novel methodological improvements. These improvements were evaluated in a simulation study using a set of small example pathways with simple Michaelis Menten rate laws. Afterwards, a detailed analysis of the dynamic properties of the neuronal TCA cycle was performed in order to demonstrate how the new insights obtained in this work could be used for the study of complex metabolic systems. The first improvement was achieved by examining the biological feasibility of the elasticity combinations created during Monte Carlo sampling. Using a set of small example systems, the findings showed that the majority of sampled SK-models would yield negative kinetic parameters if they were translated back into kinetic models. To overcome this problem, a simple criterion was formulated that mitigates such infeasible models and the application of this criterion changed the conclusions of the SKM experiment. The second improvement of this work was the application of supervised machine-learning approaches in order to analyse SKM experiments. So far, SKM experiments have focused on the detection of individual enzymes to identify single reactions important for maintaining the stability or oscillatory trajectories. In this work, this approach was extended by demonstrating how SKM enables the detection of ensembles of enzymes or metabolites that act together in an orchestrated manner to coordinate the pathways response to perturbations. In doing so, stable and unstable states served as class labels, and classifiers were trained to detect elasticity regions associated with stability and instability. Classification was performed using decision trees and relevance vector machines (RVMs). The decision trees produced good classification accuracy in terms of model bias and generalizability. RVMs outperformed decision trees when applied to small models, but encountered severe problems when applied to larger systems because of their high runtime requirements. The decision tree rulesets were analysed statistically and individually in order to explore the role of individual enzymes or metabolites in controlling the system's trajectories around steady states. The third improvement of this work was the establishment of a relationship between the SKM framework and the related field of MCA. In particular, it was shown how the sampled elasticities could be converted to flux control coefficients, which were then investigated for their predictive information content in classifier training. After evaluation on the small example pathways, the methodology was used to study two steady states of the neuronal TCA cycle with respect to their intrinsic mechanisms responsible for stability or instability. The findings showed that several elasticities were jointly coordinated to control stability and that the main source for potential instabilities were mutations in the enzyme alpha-ketoglutarate dehydrogenase. N2 - Metabolische Systeme neigen zur Ausbildung von Fließgleichgewichten, deren Konzentrationen und Reaktionsflüsse experimentell charakterisierbar sind. Derartige Messungen bieten eine phänotypische Repräsentation der zahlreichen Interaktionen und regulatorischen Mechanismen des zugrundeliegenden metabolischen Netzwerks. Diese Interaktionen bestimmen die Reaktion des Systems auf externe Perturbationen, wie z.B. dessen asymptotische Stabilität und Oszillationen. Die Charakterisierung solcher Eigenschaften ist jedoch schwierig, wenn kein entsprechendes kinetisches Modell mit allen Ratengleichungen und kinetischen Parametern für das untersuchte System zur Verfügung steht. Die strukturelle kinetische Modellierung (SKM) ermöglicht die Untersuchung dynamischer Eigenschaften wie Stabilität oder Oszillationen, ohne die Ratengleichungen und zugehörigen Parameter im Detail zu kennen. Statt dessen liefert sie eine parametrisierte Repräsentation der Jacobimatrix, in welcher die einzelnen Parameter Informationen über die Sättigung der Enzyme des Systems mit ihren Substraten kodieren. Die Parameter entsprechen dabei den Elastizitäten aus der metabolischen Kontrollanalyse, was ihre biologische Interpretation vereinfacht. Stabilitätskriterien werden durch Monte Carlo Verfahren hergeleitet, wobei zunächst eine große Anzahl struktureller kinetische Modelle (SK-Modelle) mit zufällig gezogenen Parametermengen generiert, und anschließend die resultierenden Jacobimatrizen evaluiert werden. Im Anschluss kann der Parameterraum statistisch analysiert werden, um Enzyme und Metabolite mit signifikantem Einfluss auf die Stabilität zu detektieren. In der vorliegenden Arbeit wurde das bisherige SKM-Verfahren durch neue methodische Verbesserungen erweitert. Diese Verbesserungen wurden anhand einer Simulationsstudie evaluiert, welche auf kleinen Beispielsystemen mit einfachen Michaelis Menten Kinetiken basierte. Im Anschluss wurden sie für eine detaillierte Analyse der dynamischen Eigenschaften des Zitratzyklus verwendet. Die erste Erweiterung der bestehenden Methodik wurde durch Untersuchung der biologischen Machbarkeit der zufällig erzeugten Elastizitäten erreicht. Es konnte gezeigt werden, dass die Mehrheit der zufällig erzeugten SK-Modelle zu negativen Michaeliskonstanten führt. Um dieses Problem anzugehen, wurde ein einfaches Kriterium formuliert, welches das Auftreten solcher biologisch unrealistischer SK-Modelle verhindert. Es konnte gezeigt werden, dass die Anwendung des Kriteriums die Ergebnisse von SKM Experimenten stark beeinflussen kann. Der zweite Beitrag bezog sich auf die Analyse von SKM-Experimenten mit Hilfe überwachter maschineller Lernverfahren. Bisherige SKM-Studien konzentrierten sich meist auf die Detektion individueller Elastizitäten, um einzelne Reaktionen mit Einfluss auf das Stabilitäts- oder oszillatorische Verhalten zu identifizieren. In dieser Arbeit wurde demonstriert, wie SKM Experimente im Hinblick auf multivariate Muster analysiert werden können, um Elastizitäten zu entdecken, die gemeinsam auf orchestrierte und koordinierte Weise die Eigenschaften des Systems bestimmen. Sowohl Entscheidungsbäume als auch Relevanzvektormaschinen (RVMs) wurden als Klassifikatoren eingesetzt. Während Entscheidungsbäume im allgemeinen gute Klassifikationsergebnisse lieferten, scheiterten RVMs an ihren großen Laufzeitbedürfnissen bei Anwendung auf ein komplexes System wie den Zitratzyklus. Hergeleitete Entscheidungsbaumregeln wurden sowohl statistisch als auch individuell analysiert, um die Koordination von Enzymen und Metaboliten in der Kontrolle von Trajektorien des Systems zu untersuchen. Der dritte Beitrag, welcher in dieser Arbeit vorgestellt wurde, war die Etablierung der Beziehung zwischen SKM und der metabolischer Kontrollanalyse. Insbesondere wurde gezeigt, wie die zufällig generierten Elastizitäten in Flusskontrollkoeffizienten umgewandelt werden. Diese wurden im Anschluss bezüglich ihres Informationsgehaltes zum Klassifikationstraining untersucht. Nach der Evaluierung anhand einiger kleiner Beispielsysteme wurde die neue Methodik auf die Studie zweier Fließgleichgewichte des neuronalen Zitratzyklus angewandt, um intrinsische Mechanismen für Stabilität oder Instabilität zu finden. Die Ergebnisse identifizierten Mutationen im Enzym alpha-ketoglutarate dehydrogenase als wahrscheinlichste Quelle füur Instabilitäten. KW - Systembiologie KW - mathematische Modellierung KW - systems biology KW - mathematical modeling Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-72017 ER - TY - JOUR A1 - Kuekenshoener, Tim A1 - Hagemann, Urs B. A1 - Wohlwend, Daniel A1 - Raeuber, Christina A1 - Baumann, Tobias A1 - Keller, Sandro A1 - Einsle, Oliver A1 - Mueller, Kristian M. A1 - Arndt, Katja Maren T1 - Analysis of Selected and Designed Chimeric D- and L-alpha-Helix Assemblies JF - Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences N2 - D-Peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base. Y1 - 2014 U6 - https://doi.org/10.1021/bm5006883 SN - 1525-7797 SN - 1526-4602 VL - 15 IS - 9 SP - 3296 EP - 3305 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Brust, Henrike A1 - Lehmann, Tanja A1 - Fettke, Jörg T1 - Analysis of the functional interaction of arabidopsis starch synthase and branching enzyme isoforms reveals that the cooperative action of SSI and BEs results in glucans with polymodal chain length distribution similar to amylopectin JF - PLoS one N2 - Starch synthase (SS) and branching enzyme (BE) establish the two glycosidic linkages existing in starch. Both enzymes exist as several isoforms. Enzymes derived from several species were studied extensively both in vivo and in vitro over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from in vitro studies including both interaction of leaf derived and heterologously expressed SS and BE isoforms. We found that SSI activity in native PAGE without addition of glucans was dependent on at least one of the two BE isoforms active in Arabidopsis leaves. This interaction is most likely not based on a physical association of the enzymes, as demonstrated by immunodetection and native PAGE mobility analysis of SSI, BE2, and BE3. The glucans formed by the action of SSI/BEs were analysed using leaf protein extracts from wild type and be single mutants (Atbe2 and Atbe3 mutant lines) and by different combinations of recombinant proteins. Chain length distribution (CLD) patterns of the formed glucans were irrespective of SSI and BE isoforms origin and still independent of assay conditions. Furthermore, we show that all SS isoforms (SSI-SSIV) were able to interact with BEs and form branched glucans. However, only SSI/BEs generated a polymodal distribution of glucans which was similar to CLD pattern detected in amylopectin of Arabidopsis leaf starch. We discuss the impact of the SSI/BEs interplay for the CLD pattern of amylopectin. Y1 - 2014 U6 - https://doi.org/10.1371/journal.pone.0102364 SN - 1932-6203 VL - 9 IS - 7 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Horn, Susanne A1 - Prost, Stefan A1 - Stiller, Mathias A1 - Makowiecki, Daniel A1 - Kuznetsova, Tatiana A1 - Benecke, Norbert A1 - Pucher, Erich A1 - Hufthammer, Anne K. A1 - Schouwenburg, Charles A1 - Shapiro, Beth A1 - Hofreiter, Michael T1 - Ancient mitochondrial DNA and the genetic history of Eurasian beaver (Castor fiber) in Europe JF - Molecular ecology N2 - After centuries of human hunting, the Eurasian beaver Castor fiber had disappeared from most of its original range by the end of the 19th century. The surviving relict populations are characterized by both low genetic diversity and strong phylogeographical structure. However, it remains unclear whether these attributes are the result of a human-induced, late Holocene bottleneck or already existed prior to this reduction in range. To investigate genetic diversity in Eurasian beaver populations during the Holocene, we obtained mitochondrial control region DNA sequences from 48 ancient beaver samples and added 152 modern sequences from GenBank. Phylogeographical analyses of the data indicate a differentiation of European beaver populations into three mitochondrial clades. The two main clades occur in western and eastern Europe, respectively, with an early Holocene contact zone in eastern Europe near a present-day contact zone. A divergent and previously unknown clade of beavers from the Danube Basin survived until at least 6000years ago, but went extinct during the transition to modern times. Finally, we identify a recent decline in effective population size of Eurasian beavers, with a stronger bottleneck signal in the western than in the eastern clade. Our results suggest that the low genetic diversity and the strong phylogeographical structure in recent beavers are artefacts of human hunting-associated population reductions. While beaver populations have been growing rapidly since the late 19th century, genetic diversity within modern beaver populations remains considerably reduced compared to what was present prior to the period of human hunting and habitat reduction. KW - Conservation Biology KW - Phylogeography KW - Conservation Genetics KW - Population Genetics - Empirical Y1 - 2014 U6 - https://doi.org/10.1111/mec.12691 SN - 0962-1083 SN - 1365-294X VL - 23 IS - 7 SP - 1717 EP - 1729 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Roder, Phillip A1 - Hille, Carsten T1 - ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy JF - Photochemical & photobiological sciences N2 - Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+](i)), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+](i) recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+](i) rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl-cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems. Y1 - 2014 U6 - https://doi.org/10.1039/c4pp00061g SN - 1474-905X SN - 1474-9092 VL - 13 IS - 12 SP - 1699 EP - 1710 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Radchuk, Viktoriia A1 - Johst, Karin A1 - Groeneveld, Jürgen A1 - Turlure, Camille A1 - Grimm, Volker A1 - Schtickzelle, Nicolas T1 - Appropriate resolution in time and model structure for population viability analysis: Insights from a butterfly metapopulation JF - : an international journal N2 - The importance of a careful choice of the appropriate scale for studying ecological phenomena has been stressed repeatedly. However, issues of spatial scale in metapopulation dynamics received much more attention compared to temporal scale. Moreover, multiple calls were made to carefully choose the appropriate model structure for Population Viability Analysis (PVA). We assessed the effect of using coarser resolution in time and model structure on population dynamics. For this purpose, we compared outcomes of two PVA models differing in their time step: daily individual-based model (dIBM) and yearly stage-based model (ySBM), loaded with empirical data on a well-known metapopulation of the butterfly Boloria eunomia. Both models included the same environmental drivers of population dynamics that were previously identified as being the most important for this species. Under temperature change scenarios, both models yielded the same qualitative scenario ranking, but they quite substantially differed quantitatively with dIBM being more pessimistic in absolute viability measures. We showed that these differences stemmed from inter-individual heterogeneity in dIBM allowing for phenological shifts of individual appearance. We conclude that a finer temporal resolution and an individual-based model structure allow capturing the essential mechanisms necessary to go beyond mere PVA scenario ranking. We encourage researchers to carefully chose the temporal resolution and structure of their model aiming at (1) depicting the processes important for (meta)population dynamics of the species and (2) implementing the environmental change scenarios expected for their study system in the future, using the temporal resolution at which such changes are predicted to operate. KW - Temporal grain KW - Model complexity KW - Model comparison KW - Population dynamics KW - Individual-based model KW - Stage-based model Y1 - 2014 U6 - https://doi.org/10.1016/j.biocon.2013.12.004 SN - 0006-3207 SN - 1873-2917 VL - 169 SP - 345 EP - 354 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Stief, Anna A1 - Altmann, Simone A1 - Hoffmann, Karen A1 - Pant, Bikram Datt A1 - Scheible, Wolf-Rüdiger A1 - Bäurle, Isabel T1 - Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors JF - The plant cell N2 - Plants are sessile organisms that gauge stressful conditions to ensure survival and reproductive success. While plants in nature often encounter chronic or recurring stressful conditions, the strategies to cope with those are poorly understood. Here, we demonstrate the involvement of ARGONAUTE1 and the microRNA pathway in the adaptation to recurring heat stress (HS memory) at the physiological and molecular level. We show that miR156 isoforms are highly induced after HS and are functionally important for HS memory. miR156 promotes sustained expression of HS-responsive genes and is critical only after HS, demonstrating that the effects of modulating miR156 on HS memory do not reflect preexisting developmental alterations. miR156 targets SPL transcription factor genes that are master regulators of developmental transitions. SPL genes are posttranscriptionally downregulated by miR156 after HS, and this is critical for HS memory. Altogether, the miR156-SPL module mediates the response to recurring HS in Arabidopsis thaliana and thus may serve to integrate stress responses with development. Y1 - 2014 U6 - https://doi.org/10.1105/tpc.114.123851 SN - 1040-4651 SN - 1532-298X VL - 26 IS - 4 SP - 1792 EP - 1807 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Trost, Gerda A1 - Vi, Son Lang A1 - Czesnick, Hjördis A1 - Lange, Peggy A1 - Holton, Nick A1 - Giavalisco, Patrick A1 - Zipfel, Cyril A1 - Kappel, Christian A1 - Lenhard, Michael T1 - Arabidopsis poly(A) polymerase PAPS1 limits founder-cell recruitment to organ primordia and suppresses the salicylic acid-independent immune response downstream of EDS1/PAD4 JF - The plant journal N2 - Polyadenylation of pre-mRNAs by poly(A) polymerase (PAPS) is a critical process in eukaryotic gene expression. As found in vertebrates, plant genomes encode several isoforms of canonical nuclear PAPS enzymes. In Arabidopsis thaliana these isoforms are functionally specialized, with PAPS1 affecting both organ growth and immune response, at least in part by the preferential polyadenylation of subsets of pre-mRNAs. Here, we demonstrate that the opposite effects of PAPS1 on leaf and flower growth reflect the different identities of these organs, and identify a role for PAPS1 in the elusive connection between organ identity and growth patterns. The overgrowth of paps1 mutant petals is due to increased recruitment of founder cells into early organ primordia, and suggests that PAPS1 activity plays unique roles in influencing organ growth. By contrast, the leaf phenotype of paps1 mutants is dominated by a constitutive immune response that leads to increased resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis and reflects activation of the salicylic acid-independent signalling pathway downstream of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)/PHYTOALEXIN DEFICIENT4 (PAD4). These findings provide an insight into the developmental and physiological basis of the functional specialization amongst plant PAPS isoforms. KW - poly(A) polymerase KW - founder-cell recruitment KW - organ growth KW - polyadenylation Y1 - 2014 U6 - https://doi.org/10.1111/tpj.12421 SN - 0960-7412 SN - 1365-313X VL - 77 IS - 5 SP - 688 EP - 699 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Roethlein, Christoph A1 - Miettinen, Markus S. A1 - Borwankar, Tejas A1 - Buerger, Joerg A1 - Mielke, Thorsten A1 - Kumke, Michael Uwe A1 - Ignatova, Zoya T1 - Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay JF - The journal of biological chemistry N2 - The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered beta-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure. Y1 - 2014 U6 - https://doi.org/10.1074/jbc.M114.581991 SN - 0021-9258 SN - 1083-351X VL - 289 IS - 39 SP - 26817 EP - 26828 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Meyer, Sören A1 - Schulz, J. A1 - Jeibmann, A. A1 - Taleshi, M. S. A1 - Ebert, Franziska A1 - Francesconi, Kevin A. A1 - Schwerdtle, Tanja T1 - Arsenic-containing hydrocarbons are toxic in the in vivo model Drosophila melanogaster JF - Metallomics : integrated biometal science N2 - Arsenic-containing hydrocarbons (AsHC) constitute one group of arsenolipids that have been identified in seafood. In this first in vivo toxicity study for AsHCs, we show that AsHCs exert toxic effects in Drosophila melanogaster in a concentration range similar to that of arsenite. In contrast to arsenite, however, AsHCs cause developmental toxicity in the late developmental stages of Drosophila melanogaster. This work illustrates the need for a full characterisation of the toxicity of AsHCs in experimental animals to finally assess the risk to human health related to the presence of arsenolipids in seafood. Y1 - 2014 U6 - https://doi.org/10.1039/c4mt00249k SN - 1756-5901 SN - 1756-591X VL - 6 IS - 11 SP - 2010 EP - 2014 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Fujikura, Ushio A1 - Elsaesser, Lore A1 - Breuninger, Holger A1 - Sanchez-Rodriguez, Clara A1 - Ivakov, Alexander A1 - Laux, Thomas A1 - Findlay, Kim A1 - Persson, Staffan A1 - Lenhard, Michael T1 - Atkinesin-13A modulates cell-wall synthesis and cell expansion in arabidopsis thaliana via the THESEUS1 pathway JF - PLoS Genetics : a peer-reviewed, open-access journal N2 - Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, ion atkinl3a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of he two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent his function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtIGN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling he THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkinl3a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion. Y1 - 2014 U6 - https://doi.org/10.1371/journal.pgen.1004627 SN - 1553-7390 SN - 1553-7404 VL - 10 IS - 9 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Müller-Röber, Bernd A1 - Balazadeh, Salma T1 - Auxin and its role in plant senescence JF - Journal of plant growth regulation N2 - Leaf senescence represents a key developmental process through which resources trapped in the photosynthetic organ are degraded in an organized manner and transported away to sustain the growth of other organs including newly forming leaves, roots, seeds, and fruits. The optimal timing of the initiation and progression of senescence are thus prerequisites for controlled plant growth, biomass accumulation, and evolutionary success through seed dispersal. Recent research has uncovered a multitude of regulatory factors including transcription factors, micro-RNAs, protein kinases, and others that constitute the molecular networks that regulate senescence in plants. The timing of senescence is affected by environmental conditions and abiotic or biotic stresses typically trigger a faster senescence. Various phytohormones, including for example ethylene, abscisic acid, and salicylic acid, promote senescence, whereas cytokinins delay it. Recently, several reports have indicated an involvement of auxin in the control of senescence, however, its mode of action and point of interference with senescence control mechanisms remain vaguely defined at present and contrasting observations regarding the effect of auxin on senescence have so far hindered the establishment of a coherent model. Here, we summarize recent studies on auxin-related genes that affect senescence in plants and highlight how these findings might be integrated into current molecular-regulatory models of senescence. KW - ARF KW - Auxin KW - Chloroplast KW - Development KW - Leaf KW - SAUR KW - Senescence KW - Signaling KW - Transcription factor KW - YUCCA Y1 - 2014 U6 - https://doi.org/10.1007/s00344-013-9398-5 SN - 0721-7595 SN - 1435-8107 VL - 33 IS - 1 SP - 21 EP - 33 PB - Springer CY - New York ER - TY - JOUR A1 - Seul, Anait A1 - Müller, Jürgen J. A1 - Andres, Dorothee A1 - Stettner, Eva A1 - Heinemann, Udo A1 - Seckler, Robert T1 - Bacteriophage P22 tailspike: structure of the complete protein and function of the interdomain linker JF - Acta crystallographica : Section D, Biological crystallography N2 - Attachment of phages to host cells, followed by phage DNA ejection, represents the first stage of viral infection of bacteria. Salmonella phage P22 has been extensively studied, serving as an experimental model for bacterial infection by phages. P22 engages bacteria by binding to the sugar moiety of lipopolysaccharides using the viral tailspike protein for attachment. While the structures of the N-terminal particle-binding domain and the major receptor-binding domain of the tailspike have been analyzed individually, the three-dimensional organization of the intact protein, including the highly conserved linker region between the two domains, remained unknown. A single amino-acid exchange in the linker sequence made it possible to crystallize the full-length protein. Two crystal structures of the linker region are presented: one attached to the N-terminal domain and the other present within the complete tailspike protein. Both retain their biological function, but the mutated full-length tailspike displays a retarded folding pathway. Fitting of the full-length tailspike into a published cryo-electron microscopy map of the P22 virion requires an elastic distortion of the crystal structure. The conservation of the linker suggests a role in signal transmission from the distal tip of the molecule to the phage head, eventually leading to DNA ejection. Y1 - 2014 U6 - https://doi.org/10.1107/S1399004714002685 SN - 1399-0047 VL - 70 SP - 1336 EP - 1345 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Becher, Matthias A. A1 - Grimm, Volker A1 - Thorbek, Pernille A1 - Horn, Juliane A1 - Kennedy, Peter J. A1 - Osborne, Juliet L. T1 - BEEHAVE: a systems model of honeybee colony dynamics and foraging to explore multifactorial causes of colony failure JF - Journal of applied ecology : an official journal of the British Ecological Society N2 - BEEHAVE offers a valuable tool for researchers to design and focus field experiments, for regulators to explore the relative importance of stressors to devise management and policy advice and for beekeepers to understand and predict varroa dynamics and effects of management interventions. We expect that scientists and stakeholders will find a variety of applications for BEEHAVE, stimulating further model development and the possible inclusion of other stressors of potential importance to honeybee colony dynamics. KW - Apis mellifera KW - colony decline KW - cross-level interactions KW - feedbacks KW - foraging KW - modelling KW - multiple stressors KW - multi-agent simulation KW - predictive systems ecology KW - Varroa destructor Y1 - 2014 U6 - https://doi.org/10.1111/1365-2664.12222 SN - 0021-8901 SN - 1365-2664 VL - 51 IS - 2 SP - 470 EP - 482 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Boit, Alice A1 - Gaedke, Ursula T1 - Benchmarking successional progress in a quantitative food web JF - PLoS one N2 - Central to ecology and ecosystem management, succession theory aims to mechanistically explain and predict the assembly and development of ecological communities. Yet processes at lower hierarchical levels, e. g. at the species and functional group level, are rarely mechanistically linked to the under-investigated system-level processes which drive changes in ecosystem properties and functioning and are comparable across ecosystems. As a model system for secondary succession, seasonal plankton succession during the growing season is readily observable and largely driven autogenically. We used a long-term dataset from large, deep Lake Constance comprising biomasses, auto-and heterotrophic production, food quality, functional diversity, and mass-balanced food webs of the energy and nutrient flows between functional guilds of plankton and partly fish. Extracting population-and system-level indices from this dataset, we tested current hypotheses about the directionality of successional progress which are rooted in ecosystem theory, the metabolic theory of ecology, quantitative food web theory, thermodynamics, and information theory. Our results indicate that successional progress in Lake Constance is quantifiable, passing through predictable stages. Mean body mass, functional diversity, predator-prey weight ratios, trophic positions, system residence times of carbon and nutrients, and the complexity of the energy flow patterns increased during succession. In contrast, both the mass-specific metabolic activity and the system export decreased, while the succession rate exhibited a bimodal pattern. The weighted connectance introduced here represents a suitable index for assessing the evenness and interconnectedness of energy flows during succession. Diverging from earlier predictions, ascendency and eco-exergy did not increase during succession. Linking aspects of functional diversity to metabolic theory and food web complexity, we reconcile previously disjoint bodies of ecological theory to form a complete picture of successional progress within a pelagic food web. This comprehensive synthesis may be used as a benchmark for quantifying successional progress in other ecosystems. Y1 - 2014 U6 - https://doi.org/10.1371/journal.pone.0090404 SN - 1932-6203 VL - 9 IS - 2 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Otrelo-Cardoso, Ana Rita A1 - Schwuchow, Viola A1 - Rodrigues, David A1 - Cabrita, Eurico J. A1 - Leimkühler, Silke A1 - Romao, Maria Joao A1 - Santos-Silva, Teresa T1 - Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes JF - PLoS one N2 - Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C(4)mim]Cl and [C(2)OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD. Y1 - 2014 U6 - https://doi.org/10.1371/journal.pone.0087295 SN - 1932-6203 VL - 9 IS - 1 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Klie, Sebastian A1 - Nikoloski, Zoran A1 - Selbig, Joachim T1 - Biological cluster evaluation for gene function prediction JF - Journal of computational biology N2 - Recent advances in high-throughput omics techniques render it possible to decode the function of genes by using the "guilt-by-association" principle on biologically meaningful clusters of gene expression data. However, the existing frameworks for biological evaluation of gene clusters are hindered by two bottleneck issues: (1) the choice for the number of clusters, and (2) the external measures which do not take in consideration the structure of the analyzed data and the ontology of the existing biological knowledge. Here, we address the identified bottlenecks by developing a novel framework that allows not only for biological evaluation of gene expression clusters based on existing structured knowledge, but also for prediction of putative gene functions. The proposed framework facilitates propagation of statistical significance at each of the following steps: (1) estimating the number of clusters, (2) evaluating the clusters in terms of novel external structural measures, (3) selecting an optimal clustering algorithm, and (4) predicting gene functions. The framework also includes a method for evaluation of gene clusters based on the structure of the employed ontology. Moreover, our method for obtaining a probabilistic range for the number of clusters is demonstrated valid on synthetic data and available gene expression profiles from Saccharomyces cerevisiae. Finally, we propose a network-based approach for gene function prediction which relies on the clustering of optimal score and the employed ontology. Our approach effectively predicts gene function on the Saccharomyces cerevisiae data set and is also employed to obtain putative gene functions for an Arabidopsis thaliana data set. KW - algorithms KW - biochemical networks KW - combinatorics KW - computational molecular biology KW - databases KW - functional genomics KW - gene expression KW - NP-completeness Y1 - 2014 U6 - https://doi.org/10.1089/cmb.2009.0129 SN - 1066-5277 SN - 1557-8666 VL - 21 IS - 6 SP - 428 EP - 445 PB - Liebert CY - New Rochelle ER - TY - THES A1 - Sachse, Rita T1 - Biological membranes in cell-free systems BT - characterisation and functionalisation of spodoptera frugiperda derived microsomes Y1 - 2014 ER - TY - JOUR A1 - Ni, Jian A1 - Cao, Xianyong A1 - Jeltsch, Florian A1 - Herzschuh, Ulrike T1 - Biome distribution over the last 22,000 yr in China JF - Palaeogeography, palaeoclimatology, palaeoecology : an international journal for the geo-sciences N2 - Patterns of past vegetation changes over time and space can help facilitate better understanding of the interactions among climate, ecosystem, and human impact. Biome changes in China over the last 22,000 yr (calibrated radiocarbon date, a BP) were numerically reconstructed by using a standard approach of pollen-plant functional type-biome assignment (biomization). The biomization procedure involves pollen data from 2434 surface sites and 228 fossil sites with a high quality of pollen count and C-14 dating, 51 natural and three anthropogenic plant functional types (PFTs), as well as 19 natural and one anthropogenic biome. Surface pollen-based reconstruction of modern natural biome patterns is in good agreement (74.4%) with actual vegetation distribution in China. However, modem large-scale anthropogenic biome reconstruction has not been successful based on the current setup of three anthropogenic PFTs (plantation, secondary, and disturbed PFT) because of the limitation of non-species level pollen identification and the difficulty in the clear assignment of disturbed PFTs. The non-anthropogenic biome distributions of 44 time slices at 500-year intervals show large-scale discrepant and changed vegetation patterns from the last glacial maximum (LGM) to the Holocene throughout China. From 22 ka BP to 19 ka BP, temperate grassland, xerophytic shrubland, and desert dominated northern China, whereas cold or cool forests flourished in central China. Warm-temperate evergreen forests were restricted to far southern China, and tropical forests were absent During 18.5 ka BP to 12 ka BP, cold, cool, and dry biomes extended to some parts of northern, westem, and eastern China. Warm-temperate evergreen and mixed forests gradually expanded to occupy the whole of southern China. A slight northward shift of forest biomes occurred from 15 ka BP to 12 lea BP. During 11.5 ka BP to 9 ka BP, temperate grassland and shrubland gradually stretched to northern and western China. Cold and cool forests widely expanded into northern and central China, as well as in the northern margin of South China along with temperate deciduous forest. Since the early mid-Holocene (approximately 8.5 ka BP to 5.5 ka BP), all forest biomes shifted northward at the expense of herbaceous and shrubby biomes. Simultaneously, cold and cool forest biomes occupied the marginal areas of the Tibetan Plateau and the high mountains in western China. During the middle to late Holocene, from 5 ka to the present, temperate grassland and xerophytic shrubland expanded to the south and east, whereas temperate deciduous forests slightly shifted southward. After 3 lea BP, forest biomes were absent in western China and on the Tibetan plateau surface. Dramatic biome shifts from the LGM to the Holocene were observed in the forest-grassland ecotone and transitional zones between temperate and subtropical climates, between subtropical and tropical regions, and in the mountainous margins of the eastern Tibetan Plateau. Evidence showed more human disturbances during the late Holocene. More pollen records and historical documents are therefore further needed to understand fully the human disturbance-induced large-scale forest changes. In addition, more classifications of anthropogenic biome or land cover, more distinct assignment of pollen taxa to anthropogenic PFTs, and more effective numerical and/or mechanistic techniques in building large-scale human disturbances are required. (C) 2014 Elsevier B.V. All rights reserved. KW - Anthropogenic biome KW - Biomization KW - Holocene KW - Last glacial maximum KW - Plant functional types KW - Pollen dataset Y1 - 2014 U6 - https://doi.org/10.1016/j.palaeo.2014.04.023 SN - 0031-0182 SN - 1872-616X VL - 409 SP - 33 EP - 47 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Cortes-Avizanda, Ainara A1 - Jovani, Roger A1 - Antonio Donazar, Jose A1 - Grimm, Volker T1 - Bird sky networks: How do avian scavengers use social information to find carrion? JF - Ecology : a publication of the Ecological Society of America N2 - The relative contribution of personal and social information to explain individual and collective behavior in different species and contexts is an open question in animal ecology. In particular, there is a major lack of studies combining theoretical and empirical approaches to test the relative relevance of different hypothesized individual behaviors to predict empirical collective patterns. We used an individual-based model to confront three hypotheses about the information transfer between social scavengers (Griffon Vultures, Gyps fulvus) when searching for carrion: (1) Vultures only use personal information during foraging ("nonsocial" hypothesis); (2) they create long chains of vultures by following both other vultures that are flying towards carcasses and vultures that are following other vultures that are flying towards carcasses ("chains of vultures" hypothesis); and (3) vultures are only attracted by other vultures that are sinking vertically to a carcass ("local enhancement" hypothesis). The chains of vultures hypothesis has been used in existing models, but never been confronted with field data. Testing is important, though, because these hypotheses could have different management implications. The model was parameterized to mimic the behavior and the densities of both Griffon Vultures and carcasses in a 10 000-km(2) study area in northeastern Spain. We compared the number of vultures attending simulated carcasses with those attending 25 continuously monitored experimental carcasses in the field. Social hypotheses outperformed the nonsocial hypothesis. The chains of vultures hypothesis overestimated the number of vultures feeding on carcasses; the local enhancement hypothesis fitted closely to the empirical data. Supported by our results, we discuss mechanistic and adaptive considerations that reveal that local enhancement may be the key social mechanism behind collective foraging in this and likely other avian scavengers and/or social birds. It also highlights the current need for more studies confronting alternative models of key behaviors with empirical patterns in order to understand how collective behavior emerges in animal societies. KW - carrion resources KW - foraging KW - group-living KW - pulsed resources KW - sociality KW - Spain KW - vultures Y1 - 2014 SN - 0012-9658 SN - 1939-9170 VL - 95 IS - 7 SP - 1799 EP - 1808 PB - Wiley CY - Washington ER - TY - JOUR A1 - Dietrich, Ann-Christin A1 - Lombardo, Veronica A. A1 - Abdelilah-Seyfried, Salim T1 - Blood flow and Bmp signaling control endocardial chamber morphogenesis JF - Developmental cell N2 - During heart development, the onset of heartbeat and blood flow coincides with a ballooning of the cardiac chambers. Here, we have used the zebrafish as a vertebrate model to characterize chamber ballooning morphogenesis of the endocardium, a specialized population of endothelial cells that line the interior of the heart. By combining functional manipulations, fate mapping studies, and high-resolution imaging, we show that endocardial growth occurs without an influx of external cells. Instead, endocardial cell proliferation is regulated, both by blood flow and by Bmp signaling, in a manner independent of vascular endothelial growth factor (VEGF) signaling. Similar to myocardial cells, endocardial cells obtain distinct chamber-specific and inner- versus outer-curvature-specific surface area sizes. We find that the hemodynamic-sensitive transcription factor Klf2a is involved in regulating endocardial cell morphology. These findings establish the endocardium as the flow-sensitive tissue in the heart with a key role in adapting chamber growth in response to the mechanical stimulus of blood flow. Y1 - 2014 U6 - https://doi.org/10.1016/j.devcel.2014.06.020 SN - 1534-5807 SN - 1878-1551 VL - 30 IS - 4 SP - 367 EP - 377 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Pieper, Christian A1 - Marek, Jasmin Jacqueline A1 - Unterberg, Marlies A1 - Schwerdtle, Tanja A1 - Galla, Hans-Joachim T1 - Brain capillary pericytes contribute to the immune defense in response to cytokines or LPS in vitro JF - Brain research : an international multidisciplinary journal devoted to fundamental research in the brain sciences N2 - The prevention of an inflammation in the brain is one of the most important goals the body has to achieve. As pericytes are located on the abluminal side of the capillaries in the brain, their role in fighting against invading pathogens has been investigated in some points, mostly in their ability to behave like macrophages. Here we studied the potential of pericytes to react as immune cells under inflammatory conditions, especially regarding the expression of the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), major histocompatibility complex II (MHC II) molecules, CD68, as well as the generation of reactive oxygen and nitrogen species (RONS), and their ability in phagocytosis. Quantitative real time PCR and western blot analysis showed that pericytes are able to increase the expression of typical inflammatory marker proteins after the stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1 beta), interferon-gamma (IFN-gamma), or lipopolysaccharides (LPS). Depending on the different specific pro-inflammatory factors pericytes changed the expression of alpha smooth muscle actin (alpha SMA), the most predominant pericyte marker. We conclude that the role of the pericytes within the immune system is regulated and fine-tuned by different cytokines strongly depending on the time when the cytokines are released and their concentration. The present results will help to understand the pericyte mediated defense mechanisms in the brain. KW - Pericytes KW - Cytokines KW - Inflammation KW - LPS KW - Macrophage-like phenotype Y1 - 2014 U6 - https://doi.org/10.1016/j.brainres.2014.01.004 SN - 0006-8993 SN - 1872-6240 VL - 1550 SP - 1 EP - 8 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Heindorff, Kristoffer A1 - Baumann, Otto T1 - Calcineurin is part of a negative feedback loop in the InsP(3)/Ca2+ signalling pathway in blowfly salivary glands JF - Cell calcium N2 - The ubiquitous InsP(3)/Ca2+ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca2+ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca2+] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP(3)-induced Ca2+ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP(3)/Ca2+ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca2+ signals; (2) cyclosporin A and FK506, inhibitors of Ca2+/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca2+ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca2+ responses does not involve Ca2+ entry into the cells; (4) cyclosporin A increases InsP(3)-dependent Ca2+ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca2+ responses, indicating that PKA and calcineurin act antagonistically on the InsP(3)/Ca2+ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP(3)/Ca2+ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations. (C) 2014 Elsevier Ltd. All rights reserved. KW - Calcineurin KW - Ca2+ KW - Ca2+ oscillations KW - cAMP KW - Protein kinase A KW - Intracellular signalling KW - Salivary gland KW - Blowfly KW - Insect Y1 - 2014 U6 - https://doi.org/10.1016/j.ceca.2014.07.009 SN - 0143-4160 SN - 1532-1991 VL - 56 IS - 3 SP - 215 EP - 224 PB - Churchill Livingstone CY - Edinburgh ER - TY - JOUR A1 - Bechi, Beatrice A1 - Herter, Susanne A1 - McKenna, Shane A1 - Riley, Christopher A1 - Leimkühler, Silke A1 - Turner, Nicholas J. A1 - Carnell, Andrew J. T1 - Catalytic bio-chemo and bio-bio tandem oxidation reactions for amide and carboxylic acid synthesis JF - Green chemistry : an international journal and green chemistry resource N2 - A catalytic toolbox for three different water-based one-pot cascades to convert aryl alcohols to amides and acids and cyclic amines to lactams, involving combination of oxidative enzymes (monoamine oxidase, xanthine dehydrogenase, galactose oxidase and laccase) and chemical oxidants (TBHP or Cul(cat)/H2O2) at mild temperatures, is presented. Mutually compatible conditions were found to afford products in good to excellent yields. Y1 - 2014 U6 - https://doi.org/10.1039/c4gc01321b SN - 1463-9262 SN - 1463-9270 VL - 16 IS - 10 SP - 4524 EP - 4529 PB - Royal Society of Chemistry CY - Cambridge ER - TY - GEN A1 - Bechi, Beatrice A1 - Herter, Susanne A1 - McKenna, Shane A1 - Riley, Christopher A1 - Leimkühler, Silke A1 - Turner, Nicholas J. A1 - Carnell, Andrew J. T1 - Catalytic bio–chemo and bio–bio tandem oxidation reactions for amide and carboxylic acid synthesis N2 - A catalytic toolbox for three different water-based one-pot cascades to convert aryl alcohols to amides and acids and cyclic amines to lactams, involving combination of oxidative enzymes (monoamine oxidase, xanthine dehydrogenase, galactose oxidase and laccase) and chemical oxidants (TBHP or CuI(cat)/H2O2) at mild temperatures, is presented. Mutually compatible conditions were found to afford products in good to excellent yields. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 282 Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-99414 ER - TY - JOUR A1 - Lehto, Taavi A1 - Alvarez, Alejandra Castillo A1 - Gauck, Sarah A1 - Gait, Michael J. A1 - Coursindel, Thibault A1 - Wood, Matthew J. A. A1 - Lebleu, Bernard A1 - Boisguerin, Prisca T1 - Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells JF - Nucleic acids research N2 - Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy-and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the predominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape. Y1 - 2014 U6 - https://doi.org/10.1093/nar/gkt1220 SN - 0305-1048 SN - 1362-4962 VL - 42 IS - 5 SP - 3207 EP - 3217 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Hagen, Sven A1 - Mattay, Dinah A1 - Raeuber, Christina A1 - Mueller, Kristian M. A1 - Arndt, Katja Maren T1 - Characterization and inhibition of AF10-mediated interaction JF - Journal of peptide science N2 - The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics. Copyright (c) 2014 European Peptide Society and John Wiley & Sons, Ltd. KW - protein-protein interaction KW - protein design and selection KW - protein engineering KW - coiled coil KW - leucine zipper KW - AF10 Y1 - 2014 U6 - https://doi.org/10.1002/psc.2626 SN - 1075-2617 SN - 1099-1387 VL - 20 IS - 6 SP - 385 EP - 397 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Fraesdorf, Benjamin A1 - Radon, Christin A1 - Leimkühler, Silke T1 - Characterization and interaction studies of two isoforms of the dual localized 3-mercaptopyruvate sulfurtransferase TUM1 from humans JF - The journal of biological chemistry N2 - Background: Localization and identification of interaction partners of two splice variants of the human 3-mercaptopyruvate sulfurtransferase TUM1. Results: We show that TUM1 interacts with proteins involved in Moco and FeS cluster biosynthesis. Conclusion: Human TUM1 is a dual localized protein in the cytosol and mitochondria with distinct roles in sulfur transfer and interaction partners. Significance: The study contributes to the sulfur transfer pathway for the biosynthesis of sulfur-containing biofactors. The human tRNA thiouridine modification protein (TUM1), also designated as 3-mercaptopyruvate sulfurtransferase (MPST), has been implicated in a wide range of physiological processes in the cell. The roles range from an involvement in thiolation of cytosolic tRNAs to the generation of H2S as signaling molecule both in mitochondria and the cytosol. TUM1 is a member of the sulfurtransferase family and catalyzes the conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide. Here, we purified and characterized two novel TUM1 splice variants, designated as TUM1-Iso1 and TUM1-Iso2. The purified proteins showed similar kinetic behavior and comparable pH and temperature dependence. Cellular localization studies, however, showed a different localization pattern between the isoforms. TUM1-Iso1 is exclusively localized in the cytosol, whereas TUM1-Iso2 showed a dual localization both in the cytosol and mitochondria. Interaction studies were performed with the isoforms both in vitro using the purified proteins and in vivo by fluorescence analysis in human cells, using the split-EGFP system. The studies showed that TUM1 interacts with the l-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, suggesting a dual function of TUM1 both in sulfur transfer for the biosynthesis of the molybdenum cofactor, and for the thiolation of tRNA. Our studies point to distinct roles of each TUM1 isoform in the sulfur transfer processes in the cell, with different compartmentalization of the two splice variants of TUM1. KW - Fluorescence KW - Mitochondria KW - Molybdenum KW - Sulfur KW - Transfer RNA (tRNA) Y1 - 2014 U6 - https://doi.org/10.1074/jbc.M114.605733 SN - 0021-9258 SN - 1083-351X VL - 289 IS - 50 SP - 34543 EP - 34556 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - THES A1 - Tenenboim, Yehezkel T1 - Characterization of a Chlamydomonas protein involved in cell division and autophagy T1 - Charakterisierung eines an Zellteilung und Autophagie beteiligten Chlamydomonas-Proteins N2 - The contractile vacuole (CV) is an osmoregulatory organelle found exclusively in algae and protists. In addition to expelling excessive water out of the cell, it also expels ions and other metabolites and thereby contributes to the cell's metabolic homeostasis. The interest in the CV reaches beyond its immediate cellular roles. The CV's function is tightly related to basic cellular processes such as membrane dynamics and vesicle budding and fusion; several physiological processes in animals, such as synaptic neurotransmission and blood filtration in the kidney, are related to the CV's function; and several pathogens, such as the causative agents of sleeping sickness, possess CVs, which may serve as pharmacological targets. The green alga Chlamydomonas reinhardtii has two CVs. They are the smallest known CVs in nature, and they remain relatively untouched in the CV-related literature. Many genes that have been shown to be related to the CV in other organisms have close homologues in C. reinhardtii. We attempted to silence some of these genes and observe the effect on the CV. One of our genes, VMP1, caused striking, severe phenotypes when silenced. Cells exhibited defective cytokinesis and aberrant morphologies. The CV, incidentally, remained unscathed. In addition, mutant cells showed some evidence of disrupted autophagy. Several important regulators of the cell cycle as well as autophagy were found to be underexpressed in the mutant. Lipidomic analysis revealed many meaningful changes between wild-type and mutant cells, reinforcing the compromised-autophagy observation. VMP1 is a singular protein, with homologues in numerous eukaryotic organisms (aside from fungi), but usually with no relatives in each particular genome. Since its first characterization in 2002 it has been associated with several cellular processes and functions, namely autophagy, programmed cell-death, secretion, cell adhesion, and organelle biogenesis. It has been implicated in several human diseases: pancreatitis, diabetes, and several types of cancer. Our results reiterate some of the observations in VMP1's six reported homologues, but, importantly, show for the first time an involvement of this protein in cell division. The mechanisms underlying this involvement in Chlamydomonas, as well as other key aspects, such as VMP1's subcellular localization and interaction partners, still await elucidation. N2 - Die kontraktile Vakuole ist ein osmoregulatorisches Organell, das ausschließlich in Algen und Protisten vorkommt. Zusätzlich zu ihrer Rolle als Ausstoßer überflüßigen Wassers aus der Zelle heraus, stößt sie auch Ionen und andere Metaboliten aus, und trägt dabei zur metabolischen Homöostase der Zelle bei. Das Interesse an der kontraktilen Vakuole erstreckt sich über seine unmittelbare zelluläre Rolle hinaus. Die Funktion der kontraktilen Vakuole ist mit einigen grundsätzlichen zellulären Verfahren, wie Membrandynamik und Vesikelknospung und -fusion, verwandt; einige physiologische Verfahren in Tieren, zum Beispiel synaptische Neurotransmission und das Filtrieren des Blutes in den Nieren, sind mit der Funktion der Vakuole eng verwandt; und einige Pathogene—der Ursacher der Schlafkrankheit als Beispiel—besitzen kontraktile Vakuolen, die als Ziele von Medikamenten dienen könnten. Die grüne Alge Chlamydomonas reinhardtii verfügt über zwei Vakuolen. Sie sind die kleinsten bekannten in der Natur, und bleiben bisher verhältnismäßig unerforscht. Viele Gene, die in anderen Organismen als kontraktile-Vakuole-bezogen erwiesen wurden, haben Homologe in C. reinhardtii. Wir versuchten, diese Gene auszuschalten und den Einfluss auf die Vakuole zu beobachten. Die Ausschaltung eines unserer Gene, VMP1, verursachte starke, beachtliche Phänotype. Die Zellen zeigten gestörte Zytokinese und aberrante Zellformen. Die kontraktile Vakuole blieb jedoch verschont. Des Weiteren zeigten Mutantzellen einige Hinweise auf gestörte Autophagie. Einige wichtige Gene des Zellzyklus und der Autophagie waren unterexprimiert in Mutantzellen. Lipidomische Analyse zeigte mehrere bedeutsame Unterschiede zwischen Wildtyp und Mutant, die die Beobachtungen der gestörten Autophagie verstärkten. VMP1 ist ein singularisches Protein, mit Homologen in zähligen eukaryotischen Organismen (jedoch nicht in Pilzen), aber üblicherweise ohne Verwandte in den jeweiligen Genomen. Seit seiner Erstcharakterisierung 2002 wurde es mit etlichen zellulären Verfahren, wie Autophagie, programmiertem Zelltod, Sekretion, Zelladhäsion, und Biogenese der Organellen, assoziiert. Es wurde auch mit einigen menschlichen Krankheiten wie Diabetes, Pankreatitis, und einigen Arten von Krebs in Verbindung gebracht. Unsere Ergebnisse wiederholen einige Beobachtungen in anderen Organismen, zeigen dennoch zum ersten Mal eine Beteiligung von VMP1 an der Zellteilung. Die unterliegenden Mechanismen dieser Beteiligung in Chlamydomonas, sowie andere wichtige Aspekte, etwa die subzelluläre Lokalisierung von VMP1 und dessen Interaktionspartner, warten noch auf Aufklärung. KW - VMP1 KW - autophagy KW - cytokinesis KW - chlamydomonas Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-70650 ER - TY - JOUR A1 - Hovestaedt, Marc A1 - Memczak, Henry A1 - Pleiner, Dennis A1 - Zhang, Xin A1 - Rappich, Joerg A1 - Bier, Frank Fabian A1 - Stöcklein, Walter F. M. T1 - Characterization of a new maleimido functionalization of gold for surface plasmon resonance spectroscopy JF - Journal of molecular recognition : an international journal devoted to research on specific molecular recognition in chemistry, biology, biotechnology and medicine N2 - Para-maleimidophenyl (p-MP) modified gold surfaces have been prepared by one-step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N-terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p-MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read-out for a broad variety of biomolecular interactions on the same chip. Copyright (c) 2014 John Wiley & Sons, Ltd. KW - biosensor KW - surface plasmon resonance KW - diazonium coupling KW - maleimidophenyl KW - cys-peptide KW - aryl diazonium salts Y1 - 2014 U6 - https://doi.org/10.1002/jmr.2396 SN - 0952-3499 SN - 1099-1352 VL - 27 IS - 12 SP - 707 EP - 713 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Troppmann, Britta A1 - Balfanz, Sabine A1 - Krach, Christian A1 - Baumann, Arnd A1 - Blenau, Wolfgang T1 - Characterization of an Invertebrate-Type Dopamine Receptor of the American Cockroach, Periplaneta americana JF - International journal of molecular sciences N2 - We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology. KW - G-protein-coupled receptor KW - dopamine KW - insect KW - cellular signaling KW - salivary gland KW - biogenic amine Y1 - 2014 U6 - https://doi.org/10.3390/ijms15010629 SN - 1422-0067 VL - 15 IS - 1 SP - 629 EP - 653 PB - MDPI CY - Basel ER - TY - THES A1 - Sprenger, Heike T1 - Characterization of drought tolerance in potato cultivars for identification of molecular markers Y1 - 2014 ER - TY - THES A1 - Jüppner, Jessica T1 - Characterization of metabolomic dynamics in synchronized Chlamydomonas reinhardtii cell cultures and the impact of TOR inhibition on cell cycle, proliferation and growth T1 - Charakterisierung der metabolischen Dynamik in synchronisierten Chlamydomonas reinhardtii Zellkulturen und der Einfluss der TOR-Inhibition auf Zellzyklus, Proliferation und Wachstum N2 - The adaptation of cell growth and proliferation to environmental changes is essential for the surviving of biological systems. The evolutionary conserved Ser/Thr protein kinase “Target of Rapamycin” (TOR) has emerged as a major signaling node that integrates the sensing of numerous growth signals to the coordinated regulation of cellular metabolism and growth. Although the TOR signaling pathway has been widely studied in heterotrophic organisms, the research on TOR in photosynthetic eukaryotes has been hampered by the reported land plant resistance to rapamycin. Thus, the finding that Chlamydomonas reinhardtii is sensitive to rapamycin, establish this unicellular green alga as a useful model system to investigate TOR signaling in photosynthetic eukaryotes. The observation that rapamycin does not fully arrest Chlamydomonas growth, which is different from observations made in other organisms, prompted us to investigate the regulatory function of TOR in Chlamydomonas in context of the cell cycle. Therefore, a growth system that allowed synchronously growth under widely unperturbed cultivation in a fermenter system was set up and the synchronized cells were characterized in detail. In a highly resolved kinetic study, the synchronized cells were analyzed for their changes in cytological parameters as cell number and size distribution and their starch content. Furthermore, we applied mass spectrometric analysis for profiling of primary and lipid metabolism. This system was then used to analyze the response dynamics of the Chlamydomonas metabolome and lipidome to TOR-inhibition by rapamycin The results show that TOR inhibition reduces cell growth, delays cell division and daughter cell release and results in a 50% reduced cell number at the end of the cell cycle. Consistent with the growth phenotype we observed strong changes in carbon and nitrogen partitioning in the direction of rapid conversion into carbon and nitrogen storage through an accumulation of starch, triacylglycerol and arginine. Interestingly, it seems that the conversion of carbon into triacylglycerol occurred faster than into starch after TOR inhibition, which may indicate a more dominant role of TOR in the regulation of TAG biosynthesis than in the regulation of starch. This study clearly shows, for the first time, a complex picture of metabolic and lipidomic dynamically changes during the cell cycle of Chlamydomonas reinhardtii and furthermore reveals a complex regulation and adjustment of metabolite pools and lipid composition in response to TOR inhibition. N2 - Die Anpassung der Wachstumsrate an Umweltveränderungen ist essentiell für das Überleben biologischer Systeme. Mit der Identifikation der evolutionär konservierten Serin/Threonin Kinase “Target of Rapamycin” (TOR) war ein zentraler Regulator gefunden, der in Abhängigkeit einer Vielzahl von Wachstumsfaktoren den zellulären Metabolismus und das Wachstum reguliert. Während zum heutigen Zeitpunkt schon relativ gute Kenntnisse über die Funktionen und Signalwege dieser Kinase in heterotrophen Organismen gewonnen werden konnten, wurden die Untersuchungen des TOR-Signalweges in photoautotrophen Organismen durch deren Resistenz gegenüber dem TOR-spezifischen Inhibitor Rapamycin für lange Zeit erschwert. Daher bietet die Entdeckung, dass die einzelligen Grünalge Chlamydomonas reinhardtii eine natürliche Sensitivität gegenüber Rapamycin aufweist, eine gute Grundlage zur Erforschung des TOR-Signalweges in photosynthetisch aktiven Eukaryoten. Aufgrund der Beobachtung, dass das Wachstum von Chlamydomonas nicht vollständig durch Rapamycin inhibiert werden konnte, was im Gegensatz zu Beobachtungen in anderen Organismen steht, entschieden wir uns für eine detailliertere Analyse des Einflusses von TOR auf den Zellzyklus. Dazu wurde ein System etabliert das eine Synchronisation der Zellen unter weitestgehend ungestörten Bedingungen in einem Fermenter-system erlaubte. Dieses System wurde dann für eine detaillierte Charakterisierung der synchronisierten Zellen genutzt. Mittels einer hochaufgelösten Zeitreihe wurden Veränderungen zytologischer Parameter (Zellzahl und Zellgrößenverteilung) und des Stärkegehalts analysiert. Zusätzlich wurden massenspektrometrische Verfahren zur Analyse des Primär- und Lipidmetabolismus verwendet. Dieses System wurde des Weiteren dazu genutzt dynamische Veränderungen im Metabolom und Lipidom von Chlamydomonas nach Inhibition der TOR Kinase durch Rapamycin zu untersuchen Die Ergebnisse der TOR-Inhibition zeigen ein vermindertes Wachstum, eine Verzögerung in der Zellteilung und der Entlassung der Tochterzellen und resultieren in einer um 50% verringerten Zellzahl am Ende des Zellzyklus. Des Weiteren konnte eine Akkumulation von Kohlenstoff– und Stickstoff-Reserven in Form von Stärke und Triacylglyceriden, sowie Arginin beobachtet werden. Dabei ist vor allem interessant, dass die der Einbau von Kohlenstoff in Triacylglyceride offenbar schneller erfolgt als der in Stärke, was auf eine dominantere Rolle von TOR in der Regulation der Triacylglycerid-Biosynthese gegenüber der Stärkesynthese hindeutet. Diese Studie zeigt zum ersten Mal eine komplexe Analyse der dynamischen Veränderungen im Primär- und Lipidmetabolismus im Verlauf des Zellzyklus von Chlamydomonas und zeigt weiterhin die komplexe Regulation und Adjustierung des Metabolit-Pools und der Lipidzusammensetzung als Antwort auf die Inhibition von TOR. KW - chlamydomonas reinhardtii KW - metabolites KW - Metaboliten KW - lipids KW - Lipide Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-76923 ER - TY - JOUR A1 - Neumann, Bettina A1 - Yarman, Aysu A1 - Wollenberger, Ursula A1 - Scheller, Frieder W. T1 - Characterization of the enhanced peroxidatic activity of amyloid beta peptide-hemin complexes towards neurotransmitters JF - Analytical & bioanalytical chemistry N2 - Binding of heme to the amyloid peptides A beta 40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer's disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the binding of hemin to A beta peptides might play an even more crucial role in the early stages of Alzheimer's disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide A beta 1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone. KW - Peroxidatic activity Y1 - 2014 U6 - https://doi.org/10.1007/s00216-014-7822-8 SN - 1618-2642 SN - 1618-2650 VL - 406 IS - 14 SP - 3359 EP - 3364 PB - Springer CY - Heidelberg ER - TY - JOUR A1 - Spijkerman, Elly A1 - Stojkovic, Slobodanka A1 - Beardall, John T1 - CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus, availability JF - Photosynthesis research N2 - The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3-3.4), where CO2 is the sole inorganic carbon source. Previous work has revealed that the species can accumulate inorganic carbon (Ci) and exhibits high affinity CO2 utilization under low-CO2 (air-equilibrium) conditions, similar to organisms with an active CO2 concentrating mechanism (CCM), whereas both processes are down-regulated under high CO2 (4.5 % CO2) conditions. Responses of this species to phosphorus (Pi)-limited conditions suggested a contrasting regulation of the CCM characteristics. Therefore, we measured external carbonic anhydrase (CA(ext)) activities and protein expression (CAH1), the internal pH, Ci accumulation, and CO2-utilization in cells adapted to high or low CO2 under Pi-replete and Pi-limited conditions. Results reveal that C. acidophila expressed CA(ext) activity and expressed a protein cross-reacting with CAH1 (the CA(ext) from Chlamydomonas reinhardtii). Although the function of this CA remains unclear, CA(ext) activity and high affinity CO2 utilization were the highest under low CO2 conditions. C. acidophila accumulated Ci and expressed the CAH1 protein under all conditions tested, and C. reinhardtii also contained substantial amounts of CAH1 protein under Pi-limitation. In conclusion, Ci utilization is optimized in C. acidophila under ecologically relevant conditions, which may enable optimal survival in its extreme Ci- and Pi-limited habitat. The exact physiological and biochemical acclimation remains to be further studied. KW - CO2 concentrating mechanism KW - Inorganic phosphorus limitation KW - Varying CO2 condition KW - Extremophilic green alga KW - Co-limitation KW - Internal pH KW - Inorganic carbon accumulation KW - Affinity for CO2 uptake Y1 - 2014 U6 - https://doi.org/10.1007/s11120-014-0016-6 SN - 0166-8595 SN - 1573-5079 VL - 121 IS - 2-3 SP - 213 EP - 221 PB - Springer CY - Dordrecht ER - TY - GEN A1 - Schedina, Ina Maria A1 - Hartmann, Stefanie A1 - Groth, Detlef A1 - Schlupp, Ingo A1 - Tiedemann, Ralph T1 - Comparative analysis of the gonadal transcriptomes of the all-female species Poecilia formosa and its maternal ancestor Poecilia mexicana N2 - Background The Amazon molly, Poecilia formosa (Teleostei: Poeciliinae) is an unisexual, all-female species. It evolved through the hybridisation of two closely related sexual species and exhibits clonal reproduction by sperm dependent parthenogenesis (or gynogenesis) where the sperm of a parental species is only used to activate embryogenesis of the apomictic, diploid eggs but does not contribute genetic material to the offspring. Here we provide and describe the first de novo assembled transcriptome of the Amazon molly in comparison with its maternal ancestor, the Atlantic molly Poecilia mexicana. The transcriptome data were produced through sequencing of single end libraries (100 bp) with the Illumina sequencing technique. Results 83,504,382 reads for the Amazon molly and 81,625,840 for the Atlantic molly were assembled into 127,283 and 78,961 contigs for the Amazon molly and the Atlantic molly, respectively. 63% resp. 57% of the contigs could be annotated with gene ontology terms after sequence similarity comparisons. Furthermore, we were able to identify genes normally involved in reproduction and especially in meiosis also in the transcriptome dataset of the apomictic reproducing Amazon molly. Conclusions We assembled and annotated the transcriptome of a non-model organism, the Amazon molly, without a reference genome (de novo). The obtained dataset is a fundamental resource for future research in functional and expression analysis. Also, the presence of 30 meiosis-specific genes within a species where no meiosis is known to take place is remarkable and raises new questions for future research. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 404 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-401420 ER - TY - JOUR A1 - Kuhne, Maren A1 - Dippong, Martin A1 - Flemig, Sabine A1 - Hoffmann, Katrin A1 - Petsch, Kristin A1 - Schenk, Jörg A. A1 - Kunte, Hans-Jörg A1 - Schneider, Rudolf J. T1 - Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA JF - Journal of immunological methods N2 - A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. (C) 2014 Elsevier B.V. All rights reserved. KW - Immunization KW - Hapten KW - Monoclonal antibodies KW - Hybridoma KW - Flow cytometry KW - ELISA Y1 - 2014 U6 - https://doi.org/10.1016/j.jim.2014.07.004 SN - 0022-1759 SN - 1872-7905 VL - 413 SP - 45 EP - 56 PB - Elsevier CY - Amsterdam ER -