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Poster presentation from Twentieth Annual Computational Neuroscience Meeting: CNS*2011 Stockholm, Sweden. 23-28 July 2011. Background: Oscillatory activity in high-beta and gamma bands (20-80Hz) is known to play an important role in cortical processing being linked to cognitive processes and behavior. Beta/gamma oscillations are thought to emerge in local cortical circuits via two mechanisms: the interaction between excitatory principal cells and inhibitory interneurons – the pyramidal-interneuron gamma (PING) [1], and in networks of coupled inhibitory interneurons under tonic excitation – the interneuronal gamma (ING) [2]. Experimental evidence underlines the important role of inhibitory interneurons and especially of the fast spiking (FS) interneurons [3,4]. We show in simulation that an important property of FS neurons, namely the membrane resonance (frequency preference), represents an additional mechanism – the resonance induced gamma (RING), i.e. modulation of oscillatory discharge by resonance. RING promotes frequency stability and enables oscillations in purely excitatory networks. Methods: Local circuits were modeled with small world networks of 80% excitatory and 20% inhibitory neuron populations interconnected in small-world topology by realistic conductance-based synapses. Neuron populations were leaky integrate and fire (LIF) or Izhikevich resonator (RES) neurons. We also tested networks of purely inhibitory and purely excitatory RES neurons. Networks were stimulated with miniature postsynaptic potentials (MINIs) [5] and with low frequency sinusoidal (0.5 Hz) input that mimics the effect of gratings passing trough the visual field. The activity was calibrated to match recordings from cat visual cortex (firing rate, oscillatory activity). Results: Sinusoidal input modulates network oscillation frequency. This effect is most prominent in IF excitatory and IF inhibitory (IF-IF) networks and less prominent (about 4 times) in IF-RES or RES-IF networks where frequency remains relatively stable. The most stable frequency was observed in networks of pure resonators (RES-RES, None-RES, RES-None). Interestingly, purely excitatory RES networks (RES-None) were also able to exhibit oscillations through RING. By contrast purely excitatory or inhibitory IF networks (IF-None, None-IF) were not able to express oscillations under these conditions, matching experimental parameters. Conclusions: In both PING and ING, adding membrane resonance to principal cells or inhibitory interneurons stabilizes network oscillation frequency via the RING mechanism. Notably, in networks of purely excitatory networks, where ING and PING are not defined, oscillations can emerge via the RING mechanism if membrane resonance is expressed. Thus, RING appears as a potentially important mechanism for promoting stable network oscillations.
This thesis is based on the following publications (in chronological order): 1. Biegel, E., S. Schmidt & V. Müller (2009) Genetic, immunological and biochemical evidence for a Rnf complex in the acetogen Acetobacterium woodii. Environ. Microbiol. 11: 1438-1443. My contribution: Amplification, sequence determination and analysis of Rnf homologues, enrichment of the Rnf complex 2. Biegel, E. & V. Müller (2010) Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase. Proc. Nat. Acad. Sci. U. S. A. 107: 18138-18142. My contribution: I designed and performed all experiments shown and interpreted the data. 3. Biegel, E., S. Schmidt, J. Gonzáles & V. Müller (2010) Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes. Cell. Mol. Life Sci., in press. DOI: 10.1007/s00018-010-0555-8. My contribution: I was involved in writing all chapters except chapters: „phylogenetic analyses of rnf genes“ and „distribution of rnf genes“. 4. Biegel, E. & V. Müller (2010) A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii. J. Biol. Chem., in press. DOI: 10.1074/jbc.M110.192823. My contribution: I designed and performed all experiments shown and interpreted the data.
In der vorliegenden Arbeit konzentrierte ich mich auf mediterrane wirbellose Tierarten, welche sich als Konsequenz ihrer Lebensweise nur schlecht ausbreiten können. Nichtsdestotrotz haben es Süßwasserkrabben der Gattung Potamon und Landschnecken der Gattung Tudorella geschafft, große Gebiete zu besiedeln, die heute durch das Mittelmeer getrennt sind. Für beide Gruppen wurde spekuliert, dass Menschen an ihrer Ausbreitung beteiligt waren. Es war mein Ziel die biogeographischen Muster dieser beiden Gattungen zu analysieren und abzuschätzen, ob Menschen tatsächlich Vektoren ihrer Ausbreitung waren. Meine Analysen fanden auf drei Ebenen statt: Taxonomie, Gattung und Art.
TIM23-mediated insertion of transmembrane alpha-helices into the mitochondrial inner membrane
(2011)
While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) alpha-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems. Keywords: CoxVa , membrane protein , Mgm1p , mitochondria , TIM23
G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence of molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well as Xenopus laevis egg extract by CD and NMR spectroscopy and native PAGE. Here, we show that the conformational behavior of the telomeric repeats in X. laevis egg extract or in Ficoll is notably different from that observed in the presence of PEG. While the behavior of the telomeric repeat in X. laevis egg extract or in Ficoll resembles results obtained under dilute conditions, PEG promotes the formation of high-order parallel topologies. Our data suggest that PEG should not be used as a MC mimetic.
Background: The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. The IRG protein, Irga6 (IIGP1), participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, Toxoplasma gondii, through which it communicates with its cellular hosts. Some aspects of the protein's behaviour have suggested a dynamin-like molecular mode of action, in that the energy released by GTP hydrolysis is transduced into mechanical work that results in deformation and ultimately rupture of the vacuolar membrane. Results: Irga6 forms GTP-dependent oligomers in vitro and thereby activates hydrolysis of the GTP substrate. In this study we define the catalytic G-domain interface by mutagenesis and present a structural model, of how GTP hydrolysis is activated in Irga6 complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle (SRP) and its receptor (SRalpha). In conformity with this model, we show that the bound nucleotide is part of the catalytic interface and that the 3'hydroxyl of the GTP ribose bound to each subunit is essential for trans-activation of hydrolysis of the GTP bound to the other subunit. We show that both positive and negative regulatory interactions between IRG proteins occur via the catalytic interface. Furthermore, mutations that disrupt the catalytic interface also prevent Irga6 from accumulating on the parasitophorous vacuole membrane of T. gondii, showing that GTP-dependent Irga6 activation is an essential component of the resistance mechanism. Conclusions: The catalytic interface of Irga6 defined in the present experiments can probably be used as a paradigm for the nucleotide-dependent interactions of all members of the large family of IRG GTPases, both activating and regulatory. Understanding the activation mechanism of Irga6 will help to explain the mechanism by which IRG proteins exercise their resistance function. We find no support from sequence or G-domain structure for the idea that IRG proteins and the SRP GTPases have a common phylogenetic origin. It therefore seems probable, if surprising, that the substrate-assisted catalytic mechanism has been independently evolved in the two protein families.
Background: The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level. Results: We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress. Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available. Conclusions: This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26bp tags by SuperSAGE.