Structural basis for the antagonistic roles of RNP-8 and GLD-3 in GLD-2 poly(A)-polymerase activity

Cytoplasmic polyadenylation drives the translational activation of specific mRNAs in early metazoan development and is performed by distinct complexes that share the same catalytic poly(A)-polymerase subunit, GLD-2. The activity and specificity of GLD-2 depend on its binding partners. In Caenorhabditis elegans, GLD-2 promotes spermatogenesis when bound to GLD-3 and oogenesis when bound to RNP-8. GLD-3 and RNP-8 antagonize each other and compete for GLD-2 binding. Following up on our previous mechanistic studies of GLD-2-GLD-3, we report here the 2.5 resolution structure and biochemical characterization of a GLD-2-RNP-8core complex. In the structure, RNP-8 embraces the poly(A)-polymerase, docking onto several conserved hydrophobic hotspots present on the GLD-2 surface. RNP-8 stabilizes GLD-2 and indirectly stimulates polyadenylation. RNP-8 has a different amino-acid sequence and structure as compared to GLD-3. Yet, it binds the same surfaces of GLD-2 by forming alternative interactions, rationalizing the remarkable versatility of GLD-2 complexes

The Genomics of Isolated Populations of Gampsocleis glabra (Orthoptera: Tettigoniidae) in Central and Western Europe

Habitat destruction and fragmentation are among the major current threats to global biodiversity. Fragmentation may also affect species with good dispersal abilities. We study the heath bushcricket Gampsocleis glabra, a specialist of steppe-like habitats across Europe that are highly fragmented, investigating if these isolated populations can be distinguished using population genomics and if there are any traces of admixture or dispersal among them. We try to answer these questions using genome-wide SNP data generated with ddRAD sequencing. We calculated F-statistics and visualized differentiation using STRUCTURE plots. While limited by the difficulty of sampling this threatened species, our results show that all populations except one that was represented by a singleton were clearly distinct, with pairwise FST values between 0.010 and 0.181. STRUCTURE indicated limited but visible admixture across most populations and probably also an exchange of individuals between populations of Germany and The Netherlands. We conclude that in G. glabra, a certain amount of gene flow has persisted, at least in the past, also among populations that are isolated today. We also detect a possibly more recent dispersal event between a population in The Netherlands and one in Germany, which may be human aided. We suggest that the conservation of larger populations should be maintained, that efforts should be taken to restore abandoned habitat, that the preservation even of small habitat fragments may be beneficial for the conservation of this species, and that these habitats should be regularly monitored for possible (re-)colonization

Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma

Background: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. Methods: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. Results: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. Conclusions: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation

PEBSI - A Monte Carlo simulator for bremsstrahlung arising from electrons colliding with thin solid-state targets

We present a Monte Carlo code dedicated to the simulation of bremsstrahlung arising in collisions of polarized electrons with thin target foils. The program consists of an electron transport algorithm taking into account elastic electron-nucleus scattering and inelastic collisions with target electrons as well as a treatment of polarized-electron bremsstrahlung emission. Good agreement is found between the predictions of the electron transport code and data stemming from other simulation programs and experiments. In addition, we present first results from the bremsstrahlung simulation which indicate a significant decrease in the degree of linear polarization of bremsstrahlung even for the thinnest gold targets considered

Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling

Specificity factors in cytoplasmic polyadenylation

Poly(A) tail elongation after export of an messenger RNA (mRNA) to the cytoplasm is called cytoplasmic polyadenylation. It was first discovered in oocytes and embryos, where it has roles in meiosis and development. In recent years, however, has been implicated in many other processes, including synaptic plasticity and mitosis. This review aims to introduce cytoplasmic polyadenylation with an emphasis on the factors and elements mediating this process for different mRNAs and in different animal species. We will discuss the RNA sequence elements mediating cytoplasmic polyadenylation in the 3′ untranslated regions of mRNAs, including the CPE, MBE, TCS, eCPE, and C-CPE. In addition to describing the role of general polyadenylation factors, we discuss the specific RNA binding protein families associated with cytoplasmic polyadenylation elements, including CPEB (CPEB1, CPEB2, CPEB3, and CPEB4), Pumilio (PUM2), Musashi (MSI1, MSI2), zygote arrest (ZAR2), ELAV like proteins (ELAVL1, HuR), poly(C) binding proteins (PCBP2, αCP2, hnRNP-E2), and Bicaudal C (BICC1). Some emerging themes in cytoplasmic polyadenylation will be highlighted. To facilitate understanding for those working in different organisms and fields, particularly those who are analyzing high throughput data, HUGO gene nomenclature for the human orthologs is used throughout. Where human orthologs have not been clearly identified, reference is made to protein families identified in man

Influence of medicinal plants on vascular smooth muscle cell proliferation

Arteriosklerose ist die zugrundeliegende Ursache für Erkrankungen, die weltweit die meisten Todesfälle verursachen. Zu diesen zählen koronare Herzkrankheiten und Schlaganfälle. Die Akkumulation und Einwanderung von vaskulären glatten Muskelzellen (VSMC) in die Intima, sowie deren starke Produktion an extrazellulärer Matrix trägt zum Prozess der Angiostenose (Gefäßverengung) bei. Eine Strategie zur Entwicklung neuer Medikamente und Therapien ist die Zellzyklusregulation von glatten Muskelzellen. Das primäre Ziel dieser Arbeit war, die Identifizierung und Charakterisierung von Naturstoffen, die die Proliferation von VSMC inhibieren. Diese Arbeit wurde als Teil des nationalen Forschungsnetzwerkes „Drugs from Nature Targeting Inflammation“ (DNTI), welches vom FWF (Wissenschaftsfond) finanziert wird, durchgeführt. Dieses interdisziplinäre Netzwerk verbindet das Wissen und Können verschiedener Bereiche aus Pharmazie, Chemie, Veterinär- und Humanmedizin und verknüpft Universitäten in ganz Österreich. Im Zuge des Projektes wurden Pflanzen einerseits durch „Pharmacophore modelling“, also durch einen computergestützten Ansatz ausgewählt. Andererseits basierte die Auswahl auf einem ethnopharmakologischen Ansatz, welcher auf der Auswertung traditionellen Wissens beruht. Das Screening von 150 verschiedenen Pflanzenextrakten mittels ‚Resazurin Conversion assay‘ ergab einige interessante Ergebnisse. Folgende Pflanzenextrakte konnten das VSMC Wachstum hemmen; Sideritis hyssopifolia, Aristolochia debilis, Stephania tetrandra, Melia toosendan und Peucedanum ostruthium. In einer parallelen Studie wurde von unseren Partnern eine quantitative und qualitative Analyse der Cumarine von Peucedanum ostruthium durchgeführt und die 7 bedeutendsten Cumarine identifiziert. Diese Cumarine wurden uns zur Verfügung gestellt und via Resazurin Assay überprüft. Dabei wurde Ostruthin als die aktive Substanz identifiziert. Die Untersuchung von strutkurell ähnlichen Furanocumarinen führte zum Schluss dass die Geranyl–Gruppe an der C-6 Position für den inhibierenden Effekt notwendig ist. Somit konnten wir die antiproliferative Wirkung des Peucedanum ostruthium Extraktes identifizieren und eine neue Bioaktivität von Ostruthin enthüllen.Atherosclerosis is the underlying cause of the diseases responsible for the most deaths worldwide, including ischemic heart disease and cerebrovascular disease. The accumulation of vascular smooth muscle cells (VSMC) within the intima of the arterial walls and the increase in extracellular matrix they produce, contributes to vessel narrowing in the pathogenesis of atherosclerosis and restenosis. One strategy for the treatment of vascular disease is to target cell cycle regulation of smooth muscle cells. The primary aim of this work is the identification and characterization of compounds which derive from natural sources and are capable to inhibit VSMC proliferation. This work was done as part of the national research network ‘drugs from nature targeting inflammation (DNTI)’, project an interdisciplinary network, which interlinks the knowledge and the skills of members of pharmacy, chemistry, veterinarian and human medicine from different universities in Austria. The plants were selected either by a computational approach using pharmacophore models of relevant target proteins or an ethnopharmacological approach using traditional knowledge. The screening of about 150 different plant extracts via the resazurin conversion assay yielded several interesting candidate plant extracts, which inhibited the growth of VSMC, including Sideritis hyssopifolia, Aristolochia debilis, Stephania tetrandra, Melia toosendan and Peucedanum ostruthium. Furthermore, in a parallel study, a comprehensive qualitative and quantitative analysis of the main coumarins in extracts from P. ostruthium was performed by our collaboration partners at the University of Vienna and led to the identification of 7 major coumarins in those extracts. These coumarins were further forwarded to our group and testing via the resazurin conversion method revealed that the active principle inhibiting the growth of VSMC is ostruthin. The investigation of structurally similar linear furanocoumarins led to the conclusion that the geranyl group at the C-6 position is necessary for the inhibitory effect. Thus, we identified the anti-proliferative principle of the Peucedanum ostruthium extract and uncovered a novel bioactivity for ostruthin