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Oxidative stress attenuates the NO-cGMP pathway, e.g. in the vascular system, through scavenging of free NO radicals by superoxide O2•-, by inactivation of soluble guanylyl cyclase (sGC) via oxidation of its central Fe2+ ion, and by down-regulation of sGC protein levels. While the former pathways are well established, the molecular mechanisms underlying the latter are still obscure. Using oxidative sGC inhibitor ODQ we demonstrate rapid down-regulation of sGC protein in mammalian cells. Co-incubation with proteasomal inhibitor MG132 results in accumulation of ubiquitinated sGC whereas sGC activator BAY 58–2667 prevents ubiquitination. ODQ-induced down-regulation of sGC is mediated through selective ubiquitination of its b subunit, and BAY 58–2667 abrogates this effect. Ubiquitination of sGC-b is dramatically enhanced by E3 ligase CHIP. Our data indicate that oxidative stress promotes ubiquitination of sGC b subunit through E3 ligase CHIP, and that sGC activator 58–2667 reverts this effect, most likely through stabilization of the heme-free b subunit. Thus the deleterious effects of oxidative stress can be counter-balanced by an activator of a key enzyme of vascular homeostasis.
Much has been written on the success of the Indian software industry, enumerating systemic factors like first-class higher education and research institutions, both public and private; low labour costs, stimulating (state) policies etc. However, although most studies analyzing the 'Indian' software industry cover essentially the South (and West) Indian clusters, this issue has not been tackled explicitly. This paper supplements the economic geography explanations mentioned above with the additional factor social capital, which is not only important within the region, but also in transnational (ethnic) networks linking Indian software clusters with the Silicon Valley. In other words, spatial proximity is complemented with cultural proximity, thereby, extending the system of innovation. The main hypothesis is that some Indian regions are more apt to economic development and innovation due to their higher affinity to education and learning, as well as, their more general openness, which has been a main finding of my interviews. In addition, the transnational networks of Silicon Valley Indians seem to be dominated by South Indians, thus, corroborating the regional clustering of the Indian software industry. JEL Classifications: O30, R12, Z13, L86
Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase {eta} (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase {eta} itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain.
To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed crosscoupling on column. For this purpose 5-iodouridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE (R) chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed.
Riboswitches are highly structured elements in the 50-untranslated regions (50-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/ hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by longrange base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using highresolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.
Background Cryptic species are two or more distinct but morphologically similar species that were classified as a single species. During the past two decades we observed an exponential growth of publications on cryptic species. Recently published reviews have demonstrated cryptic species have profound consequences on many biological disciplines. It has been proposed that their distribution is non-random across taxa and biomes. Results We analysed a literature database for the taxonomic and biogeographical distribution of cryptic animal species reports. Results from regression analysis indicate that cryptic species are almost evenly distributed among major metazoan taxa and biogeographical regions when corrected for species richness and study intensity. Conclusion This indicates that morphological stasis represents an evolutionary constant and that cryptic metazoan diversity does predictably affect estimates of earth´s animal diversity. Our findings have direct theoretical and practical consequences for a number of prevailing biological questions with regard to global biodiversity estimates, conservation efforts and global taxonomic initiatives.
Membranes are essential for life, because a cell must separate itself from the environment to keep its molecules from dissipating away and also must keep out foreign molecules that disturb them or their cell components. However, the cell must communicate with the environment and adapt to the external conditions, needs to pump in nutrients and release toxic products of its metabolism. Membrane proteins present in the membranes of the cell and cell organelles, help the cell to gather information about the environment and perform various biological processes. Membrane proteins perform a wide range of biological functions including respiration, signal transduction and transport. Despite their high importance in biological function, only few structures have been determined because of the difficulties in producing high amounts of membrane proteins and obtaining good quality crystals. This Ph. D. thesis involves the study of different kinds of cytochrome oxidases and a membrane anchored cytochrome oxidase electron donor. Though structures of many cytochrome oxidases are known to date, there exist many different types of oxidases in different organisms, which help the organism to survive under unfavorable environmental conditions. The structural differences between these terminal oxidases which make the organism to survive in extreme environments are unclear. To investigate these, structures of different types of oxidases are necessary. Therefore, we are interested in revealing the structural details of different types of oxidases. The different types of oxidase I worked with were the caa3 HiPIP:oxygen oxidoreductase from Rhodothermus marinus, the aa3-type quinol oxidase from Acidianus ambivalens and bd-type quinol oxidase from three different organisms (Escherichia coli, Bacillus thermodenitrificans and Aquifex aeolicus). Besides the protein from E. coli all other proteins are from thermophilic organisms from which the proteins obtained are generally believed to be highly stable. The presence of a high content of charged amino acids that enhances the occurrence of salt bridges contributes to the stability of thermophilic proteins. ....
In this thesis the three dimensional solution strucutre of the RbfA protein from Thermotoga maritima was solved using multidimensional heteronuclear NMR spectroscopy. The RbfA protein binds to the helix I region of the 16S rRNA. To gain insights into the binding mode of RbfA to its target, a second RbfA construct from Helicobacter pylori was used. Comparison of the RbfA proteins with the published structure of RbfA from Escherichia coli, led to studies concerning the differences between proteins from thermophile and mesophile systems. In the second part of this thesis the native binding motive of the RbfA protein was identified. The RbfA protein binds to an alternate helix fold within the pre-sequence of the immature 16S rRNA.
Membrane proteins play vital role in a variety of cellular processes, such as signal transduction, transport and recognition. In turn they are involved in numerous human diseases and currently represent one of the most prevalent drug targets. A comprehensive understanding of the mechanisms mediated by membrane proteins requires information about their structures at near-atomic resolution, although structural studies of membrane proteins remain behind those of soluble proteins. A bottleneck in the study of membrane proteins resides in the difficulties that are encountered during their high-level production in cell based systems. However, many toxic effects attributed to the over production of membrane proteins are eliminated by cell-free expression, as viable host cells are no longer required. Therefore, the objective of this study was to obtain adequate amounts of selected membrane transport proteins for their structural studies using a cell-free expression system. For the establishment of the cell-free system for membrane proteins, the transporters YbgR and YiiP from Salmonella typhimurium LT2, PF0558 and PF1373 from Pyrococcus furiosus, from the cation diffusion family (CDF), BetP from Corynebacterium glutamicum from the betaine/carnitine/choline transporter (BCCT) family and Aq-2030 from Aquifex aeolicus VF5 from the monovalent cation/proton antiporter-2 (CPA2) family were selected. An Escherichia coli S-30 extract based cellfree system was established by generating the best expression constructs of the target proteins, preparing T7 RNA polymerase and an S-30 extract with high translation efficiency. The functionality of the S-30 extract was shown by the cell-free expression of correctly folded Green Fluorescent Protein (GFP). Essential factors of the cell-free system such as the Mg2+ concentration, the bacterial S-30 extract proportion in the reaction mixture and the time-course of cell-free reactions have been optimized. For the cell-free production of membrane proteins in soluble form, the possibility to supplement cell-free reactions with detergents was explored. A wide range of non-ionic or zwitterionic detergents, were found to be compatible with cell-free synthesis, while ionic detergents and non-ionic detergents at high concentrations had an inhibitory effect. Moreover, high concentrations of polyoxyethylene-alkyl-ethers (Brij) detergents were found to have enhancing effect on the production levels as well as on the solubility of cell-free produced proteins. As membrane proteins tend to misfold and aggregate in a membrane-free translation system, the possibility to supplement the cell-free reactions with inner membrane vesicles (IMVs) to obtain correctly folded target transport proteins was explored. All the target proteins were successfully produced in the batch cell-free reactions and were found to be incorporated in the IMVs. A continuous exchange cell-free (CECF) system was established, where consumable substrates (amino acids, nucleotides and energy regenerating compounds) were supplied to the cell-free reaction mixture through a dialysis membrane, which in consequence resulted in high-level production of target proteins compared to the batch system. The osmosensing and osmoregulated sodium-coupled symporter BetP from C. glutamicum was chosen for the large scale production in CECF set-up. The protein is easily produced in E. coli and is functional as assayed by its transport activity, after purification and reconstitution in liposomes. It is therefore possible to compare in-vivo and cell-free production. High-level cell-free production of BetP was achieved in CECF mode in different forms: (i) as precipitate, (ii) as soluble form in detergent, and (iii) incorporated in IMVs. Cell-free production of BetP resulted in the yield of about 0.5 mg of purified BetP from 1 ml of CECF reaction. The yield of purified BetP was increased to 1.6 fold by addition of 1% polyoxyethylene-(20)-cetyl-ether (Brij58) detergent in the reaction mixture. Moreover, the high level cell-free production of BetP (0.5 mg purified BetP/ml reaction mixture) incorporated in IMVs was shown for the first time in this work.However, it was observed that oligomerization of BetP was not efficient in the cell-free system. Factors that can promote the folding of membrane proteins such as lipids and chaperones were investigated. Addition of lipids and molecular chaperone GroE facilitated correct folding of BetP resulting in increased yield and stability of cell-free produced BetP. The results obtained indicate that most of the cell-free produced BetP exists in functional oligomeric form. The possibility of obtaining milligram amounts of BetP, a 12 trans-membrane protein from the cell-free reactions holds promise for structural and functional studies of other membrane proteins. In any case, the strategies adapted in this study should prove extremely valuable for the production of membrane proteins in the E. coli cell-free expression system.
First milestone of this Ph.D. thesis was the successful extension of conventional NTA/His-tag technique to self-assembling, multivalent chelator thiols for high-affinity recognition as well as stable and uniform immobilization of His-tagged proteins on chip surfaces. Bis-NTA was linked via an oligoethylene glycol to alkyl thiols by an efficient modular synthesis strategy yielding a novel, multivalent compound for formation of mixed SAMs with anti-adsorptive matrix thiols on gold. Multivalent chelator chips allow a specific, high-affinity, reversible, long-term immobilization of His-tagged proteins. In AFM studies reversibility of the specific protein immobilization process was visualized at single molecule level. The entire control over the orientation of the immobilized protein promotes this chip surface to an optimal platform for studies focusing on research targets at single molecule level and nanobiotechnology. Based on the constructed protein chip platform above and a novel AFM mode (contact oscillation mode, COM) – developed during the current Ph.D. work – protein nanolithography under physiological conditions enabling fabrication of active biomolecular patterns in countless variety has been established. Reversible COM-mediated nanostructuring is exceptionally suitable for multiplexed patterning of protein assemblies in situ. The first selfassembled protein layer acts as a biocompatible and ductile patterning material. Immobilized proteins can be replaced by the AFM tip applying COM, and the generated structures can be erased and refilled with different proteins, which are immobilized in a uniform and functional manner. Multi-protein arrays can be systematically fabricated by iterative erase-and-write processes, and employed for protein-protein interaction analysis. Fabrication of two-dimensionally arranged nanocatalytic centres with biological activity will establish a versatile tool for nanobiotechnology. As an alternative chip fabrication approach, the combined application of methodologies from surface chemistry, semiconductor technology, and chemical biology demonstrated successfully how pre-patterned templates for micro- and nanoarrays for protein chips are fabricated. The surface physical, as well the biophysical experiments, proved the functionality of this technology. The promises of such process technology are fast and economic fabrication of ready-to-use nanostructured biochips at industrial scale. Membrane proteins are complicated in handling and hence require sophisticated solutions for chip technological application. A silicon-on-insulator (SOI) chip substrate with microcavities and nanopores was employed for first technological investigation to construct a protein chip suitable for membrane proteins. The formation of an artificial lipid bilayer using vesicle fusion on oxidized SOI cavity substrates was verified by CLSM. Future AFM experiments will give further insights into the chip architecture and topography. This will provide last evidence of the sealing of the cavity by the lipid bilayer. Transmembrane proteins will be employed for reconstitution experiments on this membrane protein chip platform. Highly integrated microdevices will find application in basic biomedical and pharmaceutical research, whereas robust and portable point-of-care devices will be used in clinical settings.
Background Synchronous neuronal firing has been discussed as a potential neuronal code. For testing first, if synchronous firing exists, second if it is modulated by the behaviour, and third if it is not by chance, a large set of tools has been developed. However, to test whether synchronous neuronal firing is really involved in information processing one needs a direct comparison of the amount of synchronous firing for different factors like experimental or behavioural conditions. To this end we present an extended version of a previously published method NeuroXidence [1], which tests, based on a bi- and multivariate test design, whether the amount of synchronous firing above the chance level is different for different factors.
Background The synchrony hypothesis postulates that precise temporal synchronization of different pools of neurons conveys information that is not contained in their firing rates. The synchrony hypothesis had been supported by experimental findings demonstrating that millisecond precise synchrony of neuronal oscillations across well separated brain regions plays an essential role in visual perception and other higher cognitive tasks [1]. Albeit, more evidence is being accumulated in favour of its role as a binding mechanism of distributed neural responses, the physical and anatomical substrate for such a dynamic and precise synchrony, especially zero-lag even in the presence of non-negligible delays, remains unclear. Here we propose a simple network motif that naturally accounts for zero-lag synchronization for a wide range of temporal delays [3]. We demonstrate that zero-lag synchronization between two distant neurons or neural populations can be achieved by relaying the dynamics via a third mediating single neuron or population. Methods We simulated the dynamics of two Hodgkin-Huxley neurons that interact with each other via an intermediate third neuron. The synaptic coupling was mediated through alpha-functions. Individual temporal delays of the arrival of pre-synaptic potentials were modelled by a gamma distribution. The strength of the synchronization and the phase-difference between each individual pairs were derived by cross-correlation of the membrane potentials. Results In the regular spiking regime the two outer neurons consistently synchronize with zero phase lag irrespective of the initial conditions. This robust zero-lag synchronization naturally arises as a consequence of the relay and redistribution of the dynamics performed by the central neuron. This result is independent on whether the coupling is excitatory or inhibitory and can be maintained for arbitrarily long time delays (see Fig. 1). Conclusion We have presented a simple and extremely robust network motif able to account for the isochronous synchronization of distant neural elements in a natural way. As opposed to other possible mechanisms of neural synchronization, neither inhibitory coupling, gap junctions nor precise tuning of morphological parameters are required to obtain zero-lag synchronized neuronal oscillation.
Die Verarbeitung von Informationen im zentralen Nervensystem beruht auf dem Zusammenspiel von erregender und hemmender Neurotransmission. Die Übertragung von Signalen zwischen Neuronen erfolgt chemisch über die Ausschüttung von Neurotransmittern an spezialisierten Kontaktstellen, den Synapsen. Glyzin und gamma-Aminobuttersäure (GABA) sind die bedeutendsten inhibitorischen Neurotransmitter im zentralen Nervensystem von Säugern, welche Rezeptoren vom Glyzin- (GlyR) und GABAA-Typ (GABAAR) aktivieren. Diese ligandengesteuerten Ionenkanäle sind in postsynaptischen Membranen angereichert und mit intrazellulären Proteinen assoziiert. Die Rekrutierung der Rezeptoren in postsynaptischen Domänen ist ein an das zytoplasmatisch lokalisierte Protein Gephyrin gekoppelter Prozess. So bindet Gephyrin spezifisch an die intrazelluläre Domäne der beta-Untereinheit des GlyR (GlyR beta) und bildet für die Verankerung des Rezeptors ein gerüstartiges Netzwerk unterhalb der synaptischen Membran. Die gezielte Inaktivierung des Gephyrin-Gens führt in Mäusen zu einem postnatal letalen Phänotyp und zu dem Verlust der synaptischen Anreicherung des GlyR und bestimmter GABAA-Rezeptoren auf zellulärer Ebene. Gephyrin ist ein 93 kDa großes Protein, das nicht nur im zentralen Nervensystem (ZNS), sondern auch in anderen Organen wie Leber und Niere exprimiert wird, in denen es an der Synthese des Molybdän-Kofaktors von Oxido-Reduktasen beteiligt ist. Das Gephyrin-Protein wird durch 30 Exons codiert, von denen zehn als sogenannte Kassetten alternativ gespleißt werden können. Die bestuntersuchte Spleißvariante besitzt 736 Aminosäuren und ist in eine N- und eine C-terminale Domäne (Aminosäuren 1-181 bzw. 318-736) sowie eine zentrale Linker-Domäne unterteilt. Die N- und die C-terminalen Bereiche von Gephyrin sind den Proteinen MogA und MoeA aus E. coli homolog und werden daher auch als G-Domäne (N-terminal) bzw. E-Domäne (C-terminal) bezeichnet. In kristallographischen Untersuchungen wurde gezeigt, dass die G- und E-Domänen zur Tri- bzw. Dimerisierung befähigt sind. Diese speziellen Oligomerisierungseigenschaften der beiden Gephyrindomänen bilden wahrscheinlich die Grundlage für die Entstehung von Gephyrin-Clustern sowie eines hexagonalen Gephyrin-Gerüstes. Dieses Gerüst stellt den Verknüpfungspunkt zwischen Rezeptoren und dem Zytoskelett dar und ermöglicht somit die effiziente Clusterbildung und die zielgerichtete Anordnung einer großen Anzahl inhibitorischer Rezeptoren. In der vorliegenden Arbeit sollten die Rolle dieser beiden Domänen bei der Bildung membranassoziierter Gephyrinaggregate und die molekularen Mechanismen der Clusterbildung des Gephyrinmoleküls untersucht werden. Zu diesem Zweck wurden durch zielgerichtete Mutagenese unterschiedliche Gephyrin-Mutanten hergestellt, um die Fähigkeit der Oligomerisierung der G- und E-Domäne gezielt zu modifizieren. Dadurch sollte die Bedeutung der Oligomerisierung hinsichtlich der Aggregat- bzw. Clusterbildung untersucht werden. Außerdem sollten die Wechselwirkungen zwischen Gephyrin und anderen Proteinen und deren Einfluss auf die synaptische Lokalisation analysiert werden. Für diese Untersuchungen wurden auf der Basis von Röntgenstruktur-Daten spezifische Aminosäurereste an den bei der Oligomerisierung beteiligten Kontaktstellen ausgetauscht. In der G-Domäne wurden zu diesem Zweck vier separate Aminosäuren des Trimer-Interface durch Arginin ersetzt (GephRRRR). Analog hierzu wurden in der EDomäne einzelne Aminosäuren durch Arginin bzw. Glutamat substituiert (GephRER), um dadurch eine Dimersierung zu verhindern. Für die Kassette C5’ wird angenommen, dass deren Vorhandensein die Interaktion zwischen Gephyrin und GlyR beeinträchtigt, wodurch GlyR aus GABAergenen Synapsen ausgeschlossen wird. Daher wurde der Einfluss dieser Gephyrin-Spleißvariante (GephC5’), die zu einer Peptidinsertion innerhalb der G-Domäne führt, und einer Gephyrin-Mutante (Gephmut), die den Verlust der Wechselwirkung mit dem GlyR bedingt, auf die Aggregatbildung von Gephyrinoligomeren untersucht. Bei dem Konstrukt Gephmut wurden, basierend auf Daten von Röntgenstrukturuntersuchungen, neun Aminosäuren (713-721) am Cterminalen Ende der E-Domäne durch den homologen Bereich des bakteriellen MoeA Proteins aus E. coli ersetzt. Zunächst wurden die einzelnen isolierten Domänen mittels Gelfiltration hinsichtlich ihres Oligomerisierungsverhaltens untersucht. Die Mutationen wurden hierzu in verkürzte Proteine eingeführt, bei denen nur die G- bzw. die E-Domäne exprimiert wurden. Diese Konstrukte wurden daher als GRRRR, GC5’ bzw. ERER und Emut bezeichnet. Bei diesen zeigte sich, dass die G-Domäne des Gephyrin-Wildtyps zu trimeren Proteinkomplexen oligomerisiert. Im Gegensatz hierzu war die Mutante GRRRR nicht in der Lage, Trimere zu bilden. Das Einfügen der C5’-Kassette führte ebenfalls zu einer Störung der Trimerisierung. Gelfiltrationsexperimente mit der E-Domäne ergaben, dass die mutierte Domäne ERER, im Gegensatz zum Wildtyp-Konstrukt, keine Dimere ausbildet. Bisherige Studien haben jedoch gezeigt, dass das Emut Polypeptid zur Dimerisierung befähigt ist. Das Oligomerisierungsverhalten des kompletten Gephyrin-Proteins wurde mittels blauer nativer Gelelektrophorese (BN-PAGE) analysiert. Für die hier beschriebenen Untersuchungen mit BN-PAGE wurde rekombinantes Gephyrin in Xenopus laevis Oozyten heterolog exprimiert. Die Analyse ergab, dass Wildtyp Gephyrin nativ als Hexamer vorliegt, welches durch ansteigende Konzentrationen des Detergenzes Natriumdodecylsulfat (SDS) in Trimere, Dimere und Monomere zerfällt. Sowohl GephRRRR und GephC5’ liegen nativ fast ausschließlich als Dimere vor, während GephRER nur trimere Aggregate formt. Die entsprechende Doppelmutante mit Mutationen in Gund E-Domäne war wie erwartet nur noch als Monomer existent. Die als Kontrolle eingesetzte Glyzinrezeptor-Bindungsmutante Gephmut bildete, ebenso wie der Wildtyp, Hexamere aus. Daraus folgt, dass die Oligomere der G- bzw E-Domäne Zwischenprodukte der Hexamerbildung darstellen. Die Analyse der Oligomerisierungseigenschaften der Mutanten wurde nachfolgend in humanen embryonalen Nierenzellen (HEK 293T) untersucht. Nach heterologer Expression von Wildtyp Gephyrin in HEK 293T-Zellen formen sich große, charakteristische Gephyrinaggregate. Die Oligomerisierungs-Mutanten GephRRRR, GephRER und GephC5’ aggregierten jedoch nicht, sondern waren diffus im Zytoplasma verteilt. Die wiederum als Kontrolle eingesetzte Bindungsmutante Gephmut hingegen wies eine normale Aggregation auf. Diese Ergebnisse bestätigen die grundlegende Rolle der Oligomerisierung von G- und E- Domänen für die Aggregatbildung von Gephyrin. Mittels GST-Pulldown und Kolokalisationsanalysen in HEK Zellen wurde die Wechselwirkung der Gephyrinmutanten mit der GlyR beta, dem Motorkomplexprotein Dynein light chain-1 (Dlc-1) und dem Guanin-Nukleotid-Austauschfaktor Collybistin (Cb) untersucht. Beide Ansätze weisen darauf hin, dass die Trimerisierung der G-Domäne an der Interaktion von Gephyrin mit Dlc-1 und die Dimerisierung der E-Domäne bei der Bindung an GlyR beta und Cb beteiligt ist. Die Mutante Gephmut zeigte in beiden Fällen einen totalen Verlust der Bindungsfähigkeit sowohl an das GlyR beta Bindungsmotiv als auch an Cb. Der Einbau der C5’ Kassette in Gephyrin scheint jedoch nicht dessen Bindung an den GlyR zu beeinflussen. Für die Analyse der Clusterbildung und des zielgerichteten Transports in Neuronen wurden Wildtyp und mutiertes Gephyrin in hippocampalen und spinalen Primärkulturen der Ratte exprimiert. Zur Überprüfung einer synaptischen Lokalisation wurde Gephyrin gemeinsam mit dem vesikulären inhibitorischen Aminosäure-Transporter (VIAAT), einem präsynaptischen Marker-Protein, detektiert. In beiden Kulturen wies Gephyrin eine punktartige Verteilung in den Neuriten auf und wurde gezielt an Synapsen angereichert. Im Kontrast dazu zeigten alle Oligomerisierungsmutanten, GephRRRR, GephC5’ und GephRER keine Ausbildung von Clustern sondern eine diffuse Verteilung im Zellkörper und in Dendriten. Das Konstrukt Gephmut wies jedoch Clusterbildung und eine punktförmige Verteilung auf. Diese Daten belegen, dass die Oligomerisierung der G- wie auch der E-Domänen für die Clusterbildung und synaptische Lokalisation von Gephyrin unerlässlich ist. Die Wechselwirkung mit dem GlyR und/oder Collybistin ist ebenfalls für die Anreicherung in der Synapse erforderlich, nicht jedoch für die Bildung der Gephyrin-Cluster. Die dargestellten Ergebnisse belegen die Rolle der spezifischen Oligomerisierungseigenschaften der G- und E-Domäne für die Ausbildung des hexagonalen Gephyringerüstes und dessen grundlegende Bedeutung für die spezifische Anreicherung von Gephyrin an inhibitorischen Synapsen in Neuronen.
The Indian IT industry has received great attention. Although most studies cover South Indian locations, clustering has rarely been a topic. This study focuses on Bangalore addressing questions related to Bangalore’s successful development and lessons thereof for other regions in and outside India. The approach pertains to economic geography and international business; hypotheses have been developed from a multi-disciplinary literature survey and interview fieldwork in Bangalore. I emphasize human and social capital and networks. While the first chapter delineates cultural foundations of human capital formation, the second and third deal with bonding and bridging social capital (or dense and loose networks), respectively; the fourth is an outlook on future opportunities through intersectoral upgrading. The main hypothesis is that a combination of both forms of social networks - contingent upon sub-sectors – has helped Bangalore developing a successful IT industry. Positive attitudes towards education led to relatively more human capital spawning two positive feedbacks: 1) establishment of national research and educational institutes resulting in large inflows of a diversity of people providing the required setting for creativity and innovation; 2) transnational networks linking to Silicon Valley are dominated by people from South India, allowing for additional knowledge spillovers corroborating the regional clustering.
The goal of this thesis was to gain further insight into the binding behavior of ligands in the heptahelical domain (HD) of group I metabotropic glutamate receptors (mGluRs). This was realized by the establishment of strategies for the detection and optimization of molecules acting as non-competitive antagonists of group I mGluRs (mGluR1/5). These strategies should guarantee high diversity in the retrieved chemotypes of the detected compounds not resembling original reference molecules (“scaffold-hopping”). The detection of new scaffolds, in turn, was divided into two approaches: First the development of pharmacological assays to screen compounds at a certain target for bioactivity (here: affinity towards the allosteric recognition site of mGluR1 and mGluR5), and second the evaluation of computer assisted methods for the identification of virtual hits to be screened afterwards on the pharmacological assays established before. Promising molecules should be optimized with respect to activity/affinity and selectivity, their binding mode investigated and, finally, compared to existing lead compounds. Initially, membrane based binding assays for the HD of mGlu1 and mGlu5 receptors with enhanced throughput (shifting from 24-well plates to 96-well plates) were set up. For the mGluR1 assay the potent antagonist EMQMCM exhibited high affinity towards the binding site (Ki ~3nM), which is in accordance with published data from Mabire et al. (functional IC50 3nM). For mGluR5 the reference antagonist MPEP binds with high affinity to the receptor (binding IC50 13.8nM), which confirmed earlier findings from Anderson et al. (binding IC50 15nM). In another series of experiments the properties of rat cerebellar (mGluR1) and corticalmembranes (mGluR5) as well as of radiotracers were investigated by means of binding saturation studies and kinetic experiments. Furthermore, the influence of the solvent DMSO, necessary for compound screening of lipophilic substances, on positive and negative controls was evaluated. As the precise architecture of the HD of mGluR1 is still not known our efforts in identifying new ligands for this receptor focused on the ligand-based approach. All computer assisted methods that were applied to virtually screen large compound collections and to retrieve potential hits (“activity-enriched subsets”) acting at the heptahelical domain of mGluR1 relied on the existence of a valid dataset of reference molecules. This was realized by an initial compilation of a mGluR reference data collection comprising in total 357 entries predominantly negative but also some positive allosteric modulators for mGluR1 and mGluR5. In the next step a pharmacophore model for non-competitive mGluR1 antagonists was constructed. It was based upon six selective, potent and structurally diverse ligands. Prospective virtual screening was performed using the CATS atom-pair descriptor. The Asinex Gold-Collection was screened for each seed compound and some of the most similar compounds (according to the CATS descriptor) were ordered and tested forbinding affinity and functional activity at mGluR1. A high hit rate of approximately 26% (IC50 < 15 micro M) was yielded confirming the applicability of this method. One compound exerted functional activity below one micro molar (IC50-value of C-07:362nM ± 0.03). Moreover, non-linear principal component analysis was employed. Again the Asinex vendor database served as test database and was filtered by the pharmacophore model for mGluR1 established before. Test molecules that were adjacently located with mGluR1 antagonist references were selected. 15 compounds were tested on mGluR1 in binding and functional assays and three of them exhibited functional activity (IC50) below 15 micro M. The most potent molecule P-06 revealed an IC50-value of 1.11 micro M (± 0.41). The COBRA database comprising 5,376 structurally diverse bioactive molecules affecting various targets was encoded with the CATS descriptor and used for training two selforganizing maps (SOM). The encoded mGluR reference data collection was projected onto this map according to the SOM algorithm. This projection allowed to clearly distinguish between antagonists of mGluR1 and mGluR5 subtype. 28 compounds were ordered and tested on activity and affinity for mGluR1. They exhibited functional activity down to the sub-micro molar range (IC50-value of S-08: 744nM ± 0.29) yielding a final hit rate of 46% (<15 micro M). Then, the Asinex collection was screened using the SOM approach. For a predicted target panel including the muscarinic mACh (M1) receptor, the histamine H1-receptor and the dopamine D2/D3 receptors, the tested mGluR ligands exhibited the calculated binding pattern. This virtual screening concept might provide a basis for early recognition of potential sideeffects in lead discovery. We superimposed a set of 39 quinoline derivatives as non-competitive mGluR1 antagonists that were recently published by Mabire and co-workers. A CoMFA model (QSAR) was established and the influence of several side chains on functional activity was investigated. The coumarine derivative C-07 was obtained as a result of similarity searching. Starting from this compound a series of chemical derivatives was synthesized. This led to the discovery of potent (B-28, IC50: 58nM ± 0.008; Ki: 293nM ± 0.022) and selective (rmGluR5 IC50: 28.6 micro M) mGluR1 antagonists. From a homology model of mGluR1 we derived a potential binding mode for coumarines within the allosteric transmembrane region. Potential interacting patterns with amino acids were proposed considering the difference of the binding pockets between rat and human receptors. The proposed binding modes for quinolines (here:EMQMCM) and coumarines (here:B-04) were compared and discussed considering in particular the influence on activity of several side chains of quinolines obtained from the QSAR studies. The present studies demonstrated the applicability of ligand-based virtual screening for non-competitive antagonists of a G-protein coupled receptor, resulting in novel, potent and selective agents.