Sample records for g418-mediated ribosomal read-through

  1. Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

    PubMed Central

    Salvatori, Francesca; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Brognara, Eleonora; Lampronti, Ilaria; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2013-01-01

    In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations. PMID:19810011

  2. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    PubMed

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

  3. Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

    PubMed Central

    Au, Hilda H.; Cornilescu, Gabriel; Mouzakis, Kathryn D.; Ren, Qian; Burke, Jordan E.; Lee, Seonghoon; Butcher, Samuel E.; Jan, Eric

    2015-01-01

    The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame. PMID:26554019

  4. Translational read-through of a nonsense mutation causing Bartter syndrome.

    PubMed

    Cho, Hee Yeon; Lee, Beom Hee; Cheong, Hae Il

    2013-06-01

    Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.

  5. Structural determinants of an internal ribosome entry site that direct translational reading frame selection

    PubMed Central

    Ren, Qian; Au, Hilda H.T.; Wang, Qing S.; Lee, Seonghoon; Jan, Eric

    2014-01-01

    The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome. PMID:25038250

  6. Increased Selectivity towards Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations

    PubMed Central

    Kandasamy, Jeyakumar; Atia-Glikin, Dana; Shulman, Eli; Shapira, Katya; Shavit, Michal; Belakhov, Valery; Baasov, Timor

    2012-01-01

    Compelling evidence is now available that gentamicin and geneticin (G418) can induce mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity towards cytoplasmic ribosome correlates with the increased activity, and that the decreased specificity towards mitochondrial ribosome confers to the lowered cytotoxicity. PMID:23148581

  7. Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

    NASA Astrophysics Data System (ADS)

    Cho, Samuel

    DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K + or Na + ions. We optimized the CHARMM36 force field K + parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K + ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics.

  8. Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

    NASA Astrophysics Data System (ADS)

    Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2015-06-01

    The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

  9. Exploring Reading Comprehension Skill Relationships through the G-DINA Model

    ERIC Educational Resources Information Center

    Chen, Huilin; Chen, Jinsong

    2016-01-01

    By analysing the test data of 1029 British secondary school students' performance on 20 Programme for International Student Assessment English reading items through the generalised deterministic input, noisy "and" gate (G-DINA) model, the study conducted two investigations on exploring the relationships among the five reading…

  10. What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?

    PubMed Central

    Pöyry, Tuija A.A.; Kaminski, Ann; Jackson, Richard J.

    2004-01-01

    If the 5′-proximal AUG triplet in a mammalian mRNA is followed by a short open reading frame (sORF), a significant fraction of ribosomes resume scanning after termination of sORF translation, and reinitiate at a downstream AUG. To examine the underlying mechanism, we examined reinitiation in vitro using a series of mRNAs that differed only in the 5′-untranslated region (UTR). Efficient reinitiation was found to occur only if the eIF4F complex, or at a minimum the central one-third fragment of eIF4G, participated in the primary initiation event at the sORF initiation codon. It did not occur, however, when sORF translation was driven by the classical swine fever virus or cricket paralysis virus internal ribosome entry sites (IRESs), which do not use eIF4A, 4B, 4E, or 4G. A critical test was provided by an mRNA with an unstructured 5′-UTR, which is translated by scanning but does not absolutely need eIF4G and eIF4A: There was efficient reinitiation in a standard reticulocyte lysate, when initiation would be largely driven by eIF4F, but no reinitiation in an eIF4G-depleted lysate. These results suggest that resumption of scanning may depend on the interaction between eIF4F (or the eIF4G central domain) and the ribosome being maintained while the ribosome translates the sORF. PMID:14701882

  11. Automated synthesis of 4-[(18)F]fluoroanisole, [(18)F]DAA1106 and 4-[(18)F]FPhe using Cu-mediated radiofluorination under "minimalist" conditions.

    PubMed

    Zischler, Johannes; Krapf, Philipp; Richarz, Raphael; Zlatopolskiy, Boris D; Neumaier, Bernd

    2016-09-01

    The application of the "minimalist" approach to Cu-mediated radiofluorination allows the efficient preparation of (18)F-labeled arenes regardless of their electronic properties. The implementation of this methodology on a commercially available synthesis module (hotbox(three), Scintomics, Germany) enabled the automated production of 4-[(18)F]fluoroanisole as well as the clinically relevant PET-tracers, 4-[(18)F]FPhe and [(18)F]DAA1106, in radiochemical yields of 41-61% and radiochemical purities of >95% within 30-60min. These results demonstrated the high efficacy and versatility of the developed method that will open up opportunities for a broad application of Cu-mediated radiofluorination in PET-chemistry. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Translational profiling of B cells infected with the Epstein-Barr virus reveals 5' leader ribosome recruitment through upstream open reading frames.

    PubMed

    Bencun, Maja; Klinke, Olaf; Hotz-Wagenblatt, Agnes; Klaus, Severina; Tsai, Ming-Han; Poirey, Remy; Delecluse, Henri-Jacques

    2018-04-06

    The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. We have used ribosome profiling to characterize viral translation in infected cells and map new translation initiation sites. We show here that EBV transcripts are translated with highly variable efficiency, owing to variable transcription and translation rates, variable ribosome recruitment to the leader region and coverage by monosomes versus polysomes. Some transcripts were hardly translated, others mainly carried monosomes, showed ribosome accumulation in leader regions and most likely represent non-coding RNAs. A similar process was visible for a subset of lytic genes including the key transactivators BZLF1 and BRLF1 in cells infected with weakly replicating EBV strains. This suggests that ribosome trapping, particularly in the leader region, represents a new checkpoint for the repression of lytic replication. We could identify 25 upstream open reading frames (uORFs) located upstream of coding transcripts that displayed 5' leader ribosome trapping, six of which were located in the leader region shared by many latent transcripts. These uORFs repressed viral translation and are likely to play an important role in the regulation of EBV translation.

  13. 43 CFR 418.21 - Diversion of Truckee River water to Lahontan Reservoir, July through December.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Lahontan Reservoir, July through December. 418.21 Section 418.21 Public Lands: Interior Regulations... PROCEDURES FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Operations and Management § 418.21 Diversion of Truckee River water to Lahontan Reservoir, July through December. Truckee River diversions through the...

  14. 43 CFR 418.21 - Diversion of Truckee River water to Lahontan Reservoir, July through December.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Lahontan Reservoir, July through December. 418.21 Section 418.21 Public Lands: Interior Regulations... PROCEDURES FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Operations and Management § 418.21 Diversion of Truckee River water to Lahontan Reservoir, July through December. Truckee River diversions through the...

  15. 43 CFR 418.20 - Diversions from the Truckee River to Lahontan Reservoir, January through June.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Lahontan Reservoir, January through June. 418.20 Section 418.20 Public Lands: Interior Regulations Relating... FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Operations and Management § 418.20 Diversions from the Truckee River to Lahontan Reservoir, January through June. (a) Truckee River diversions through the...

  16. 43 CFR 418.20 - Diversions from the Truckee River to Lahontan Reservoir, January through June.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Lahontan Reservoir, January through June. 418.20 Section 418.20 Public Lands: Interior Regulations Relating... FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Operations and Management § 418.20 Diversions from the Truckee River to Lahontan Reservoir, January through June. (a) Truckee River diversions through the...

  17. UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation.

    PubMed

    Salvatori, Francesca; Pappadà, Mariangela; Breveglieri, Giulia; D'Aversa, Elisabetta; Finotti, Alessia; Lampronti, Ilaria; Gambari, Roberto; Borgatti, Monica

    2018-05-15

    Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β 0 39 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β 0 39 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. We developed a human cellular model of the β 0 39 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.

  18. Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation.

    PubMed

    Wasserman, Michael R; Alejo, Jose L; Altman, Roger B; Blanchard, Scott C

    2016-04-01

    Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

  19. The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form

    PubMed Central

    Hauryliuk, Vasili; Mitkevich, Vladimir A.; Eliseeva, Natalia A.; Petrushanko, Irina Yu.; Ehrenberg, Måns; Makarov, Alexander A.

    2008-01-01

    Translocation of the tRNA·mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4°C, 20°C, and 37°C. The binding affinity of EF-G is higher to GDP than to GTP at 4°C, but lower at 37°C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the γ-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an “activity chimera,” with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its “ratcheted state,” with hybrid tRNA binding sites. PMID:18836081

  20. Ribosome rearrangements at the onset of translational bypassing

    PubMed Central

    Agirrezabala, Xabier; Samatova, Ekaterina; Klimova, Mariia; Zamora, Miguel; Gil-Carton, David; Rodnina, Marina V.; Valle, Mikel

    2017-01-01

    Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap. PMID:28630923

  1. Kinase Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer

    DTIC Science & Technology

    2017-02-01

    Ribosome assembly • Autophagy • CRISPR /Cas9 6 ACCOMPLISHMENTS Major goals The major goals in this reporting period were to use the CRISPR ...defects on ribosome assembly, drug sensitivity etc. Accomplishments under these goals To set up the CRISPR /Cas9 experiment, the Karbstein...recombinant Cas9, and then assayed CRISPR /Cas9 mediated cleavage of a PCR-generated DNA. This demonstrated that the guide RNAs we had designed based on

  2. Nom1 Mediates Pancreas Development by Regulating Ribosome Biogenesis in Zebrafish

    PubMed Central

    Qin, Wei; Chen, Zelin; Zhang, Yihan; Yan, Ruibin; Yan, Guanrong; Li, Song; Zhong, Hanbing; Lin, Shuo

    2014-01-01

    Ribosome biogenesis is an important biological process for proper cellular function and development. Defects leading to improper ribosome biogenesis can cause diseases such as Diamond-Blackfan anemia and Shwachman-Bodian-Diamond syndrome. Nucleolar proteins are a large family of proteins and are involved in many cellular processes, including the regulation of ribosome biogenesis. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish line carrying mutation in nucleolar protein with MIF4G domain 1 (nom1), which encodes a conserved nulceolar protein with a role in pre-rRNA processing. Zebrafish nom1 mutants exhibit major defects in endoderm development, especially in exocrine pancreas. Further studies revealed that impaired proliferation of ptf1a-expressing pancreatic progenitor cells mainly contributed to the phenotype. RNA-seq and molecular analysis showed that ribosome biogenesis and pre-mRNA splicing were both affected in the mutant embryos. Several defects of ribosome assembly have been shown to have a p53-dependent mechanism. In the nom1 mutant, loss of p53 did not rescue the pancreatic defect, suggesting a p53-independent role. Further studies indicate that protein phosphatase 1 alpha, an interacting protein to Nom1, could partially rescue the pancreatic defect in nom1 morphants if a human nucleolar localization signal sequence was artificially added. This suggests that targeting Pp1α into the nucleolus by Nom1 is important for pancreatic proliferation. Altogether, our studies revealed a new mechanism involving Nom1 in controlling vertebrate exocrine pancreas formation. PMID:24967912

  3. The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning.

    PubMed

    Cuchalová, Lucie; Kouba, Tomás; Herrmannová, Anna; Dányi, István; Chiu, Wen-Ling; Valásek, Leos

    2010-10-01

    Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

  4. BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription

    PubMed Central

    Grierson, Patrick M.; Lillard, Kate; Behbehani, Gregory K.; Combs, Kelly A.; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

    2012-01-01

    Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. 3H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS. PMID:22106380

  5. BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription.

    PubMed

    Grierson, Patrick M; Lillard, Kate; Behbehani, Gregory K; Combs, Kelly A; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

    2012-03-01

    Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

  6. Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

    PubMed

    Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

    2007-04-01

    During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

  7. Slip of grip of a molecular motor on a crowded track: Modeling shift of reading frame of ribosome on RNA template

    NASA Astrophysics Data System (ADS)

    Mishra, Bhavya; Schütz, Gunter M.; Chowdhury, Debashish

    2016-06-01

    We develop a stochastic model for the programmed frameshift of ribosomes synthesizing a protein while moving along a mRNA template. Normally the reading frame of a ribosome decodes successive triplets of nucleotides on the mRNA in a step-by-step manner. We focus on the programmed shift of the ribosomal reading frame, forward or backward, by only one nucleotide which results in a fusion protein; it occurs when a ribosome temporarily loses its grip to its mRNA track. Special “slippery” sequences of nucleotides and also downstream secondary structures of the mRNA strand are believed to play key roles in programmed frameshift. Here we explore the role of an hitherto neglected parameter in regulating -1 programmed frameshift. Specifically, we demonstrate that the frameshift frequency can be strongly regulated also by the density of the ribosomes, all of which are engaged in simultaneous translation of the same mRNA, at and around the slippery sequence. Monte Carlo simulations support the analytical predictions obtained from a mean-field analysis of the stochastic dynamics.

  8. Morphine drives internal ribosome entry site-mediated hnRNP K translation in neurons through opioid receptor-dependent signaling

    PubMed Central

    Lee, Pin-Tse; Chao, Po-Kuan; Ou, Li-Chin; Chuang, Jian-Ying; Lin, Yen-Chang; Chen, Shu-Chun; Chang, Hsiao-Fu; Law, Ping-Yee; Loh, Horace H.; Chao, Yu-Sheng; Su, Tsung-Ping; Yeh, Shiu-Hwa

    2014-01-01

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the promoter region of mu-opioid receptor (MOR) to regulate its transcriptional activity. How hnRNP K contributes to the analgesic effects of morphine, however, is largely unknown. We provide evidence that morphine increases hnRNP K protein expression via MOR activation in rat primary cortical neurons and HEK-293 cells expressing MORs, without increasing mRNA levels. Using the bicistronic reporter assay, we examined whether morphine-mediated accumulation of hnRNP K resulted from translational control. We identified potential internal ribosome entry site elements located in the 5′ untranslated regions of hnRNP K transcripts that were regulated by morphine. This finding suggests that internal translation contributes to the morphine-induced accumulation of hnRNP K protein in regions of the central nervous system correlated with nociceptive and antinociceptive modulatory systems in mice. Finally, we found that down-regulation of hnRNP K mediated by siRNA attenuated morphine-induced hyperpolarization of membrane potential in AtT20 cells. Silencing hnRNP K expression in the spinal cord increased nociceptive sensitivity in wild-type mice, but not in MOR-knockout mice. Thus, our findings identify the role of translational control of hnRNP K in morphine-induced analgesia through activation of MOR. PMID:25361975

  9. Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing

    PubMed Central

    Sloan, Katherine E.; Mattijssen, Sandy; Lebaron, Simon; Tollervey, David; Pruijn, Ger J.M.

    2013-01-01

    Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3′ to 5′ exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference–mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells. PMID:23439679

  10. Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation

    PubMed Central

    Ramrath, David J. F.; Lancaster, Laura; Sprink, Thiemo; Mielke, Thorsten; Loerke, Justus; Noller, Harry F.; Spahn, Christian M. T.

    2013-01-01

    During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA. PMID:24324168

  11. A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes.

    PubMed

    Crossland, Hannah; Timmons, James A; Atherton, Philip J

    2017-12-01

    Increased ribosomal DNA transcription has been proposed to limit muscle protein synthesis, making ribosome biogenesis central to skeletal muscle hypertrophy. We examined the relationship between ribosomal RNA (rRNA) production and IGF-1-mediated myotube hypertrophy in vitro Primary skeletal myotubes were treated with IGF-1 (50 ng/ml) with or without 0.5 µM CX-5461 (CX), an inhibitor of RNA polymerase I. Myotube diameter, total protein, and RNA and DNA levels were measured along with markers of RNA polymerase I regulatory factors and regulators of protein synthesis. CX treatment reduced 45S pre-rRNA expression (-64 ± 5% vs. IGF-1; P < 0.001) and total RNA content (-16 ± 2% vs. IGF-1; P < 0.001) in IGF-1-treated myotubes. IGF-1-mediated increases in myotube diameter (1.27 ± 0.09-fold, P < 0.05 vs. control) and total protein (+20 ± 2%; P < 0.001 vs. control) were not prevented by CX treatment. Suppression of rRNA synthesis during IGF-1 treatment did not prevent early increases in AKT (+203 ± 39% vs. CX; P < 0.001) and p70 S6K1 (269 ± 41% vs. CX; P < 0.001) phosphorylation. Despite robust inhibition of the dynamic ribosomal biogenesis response to IGF-1, myotube diameter and protein accretion were sustained. Thus, while ribosome biogenesis represents a potential site for the regulation of skeletal muscle protein synthesis and muscle mass, it does not appear to be a prerequisite for IGF-1-induced myotube hypertrophy in vitro. -Crossland, H., Timmons, J. A., Atherton, P. J. A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes. © The Author(s).

  12. The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation.

    PubMed

    Rodnina, Marina V; Wintermeyer, Wolfgang

    2011-04-01

    Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.

  13. Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation†

    PubMed Central

    Svitkin, Yuri V.; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

    2005-01-01

    Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection. PMID:16287867

  14. Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.

    PubMed

    Svitkin, Yuri V; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

    2005-12-01

    Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

  15. The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis

    PubMed Central

    Koch, Miriam; Clementi, Nina; Rusca, Nicola; Vögele, Paul; Erlacher, Matthias; Polacek, Norbert

    2015-01-01

    During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis. PMID:25826414

  16. Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms

    PubMed Central

    Ye, Xu; Pan, Ting; Wang, Dang; Fang, Liurong; Ma, Jun; Zhu, Xinyu; Shi, Yanling; Zhang, Keshan; Zheng, Haixue; Chen, Huanchun; Li, Kui; Xiao, Shaobo

    2018-01-01

    Foot-and-mouth disease (FMD) is a highly contagious, severe viral illness notifiable to the World Organization for Animal Health. The causative agent, FMD virus (FMDV), replicates rapidly and efficiently inhibits host translation and the innate immune response for it has developed multiple tactics to evade host defenses and takes over gene expression machinery in the host cell. Here, we report a systemic analysis of the proteome and phosphoproteome of FMDV-infected cells. Bioinformatics analysis suggested that FMDV infection shuts off host cap-dependent translation, but leaves intact internal ribosome entry site (IRES)-mediated translation for viral proteins. Interestingly, several FMDV IRES-transacting factors, including G3BP stress granule assembly factor 1 (G3BP1), were dephosphorylated during FMDV infection. Ectopic expression of G3BP1 inhibited FMDV IRES activity, promoted assembly of stress granules, and activated innate immune responses, collectively suppressing FMDV replication. To counteract these host protective responses, FMDV-induced dephosphorylation of G3BP1, compromising its inhibitory effect on viral IRES. In addition, FMDV also proteolytically cleaved G3BP1 by its 3C protease (3Cpro). G3BP1 was cleaved at glutamic acid-284 (E284) by FMDV 3Cpro, and this cleavage completely lost the abilities of G3BP1 to activate innate immunity and to inhibit FMDV replication. Together, these data provide new insights into the post-translational mechanisms by which FMDV limits host stress and antiviral responses and indicate that G3BP1 dephosphorylation and its proteolysis by viral protease are important factors in the failure of host defense against FMDV infection.

  17. Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 – new vectors for in vitro and in vivo delivery

    PubMed Central

    Magin-Lachmann, Christine; Kotzamanis, George; D'Aiuto, Leonardo; Wagner, Ernst; Huxley, Clare

    2003-01-01

    Background Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells. Results We describe a series of retrofitting plasmids and a protocol for in vivo loxP/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE carries G418 resistance and oriP/EBNA-1. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice in vivo. We also show that a BAC carrying GFP, oriP and EBNA-1 can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks. Conclusion The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or oriP/EBNA-1 using in vivo expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle in vivo. OriP/EBNA-1 allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks. PMID:12609052

  18. Ribosomal Translocation: One Step Closer to the Molecular Mechanism

    PubMed Central

    Shoji, Shinichiro; Walker, Sarah E.; Fredrick, Kurt

    2010-01-01

    Protein synthesis occurs in ribosomes, the targets of numerous antibiotics. How these large and complex machines read and move along mRNA have proven to be challenging questions. In this Review, we focus on translocation, the last step of the elongation cycle in which movement of tRNA and mRNA is catalyzed by elongation factor G. Translocation entails large-scale movements of the tRNAs and conformational changes in the ribosome that require numerous tertiary contacts to be disrupted and reformed. We highlight recent progress toward elucidating the molecular basis of translocation and how various antibiotics influence tRNA–mRNA movement. PMID:19173642

  19. Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain

    PubMed Central

    Lintner, Nathanael G.; McClure, Kim F.; Petersen, Donna; Londregan, Allyn T.; Piotrowski, David W.; Wei, Liuqing; Xiao, Jun; Bolt, Michael; Loria, Paula M.; Maguire, Bruce; Geoghegan, Kieran F.; Huang, Austin; Rolph, Tim; Liras, Spiros; Doudna, Jennifer A.; Dullea, Robert G.

    2017-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts. PMID:28323820

  20. The Unexplored Mechanisms and Regulatory Functions of Ribosomal Translocation

    NASA Astrophysics Data System (ADS)

    Alejo, Jose Luis

    In every cell, protein synthesis is carried out by the ribosome, a complex macromolecular RNA-protein assembly. Decades of structural and kinetic studies have increased our understanding of ribosome initiation, decoding, translocation and termination. Yet, the underlying mechanism of these fundamental processes has yet to be fully delineated. Hence, the molecular basis of regulation remains obscure. Here, single-molecule fluorescence methods are applied to decipher the mechanism and regulatory roles of the multi-step process of directional substrate translocation on the ribosome that accompanies every round of protein synthesis. In Chapter 1, single-molecule fluorescence resonance energy transfer (smFRET) is introduced as a tool for studying bacterial ribosome translocation. Chapter 2 details the experimental methods. In Chapter 3, the elongation factor G(EF-G)-catalyzed movement of substrates through the ribosome is examined from several perspectives or signals reporting on various degrees of freedom of ribosome dynamics. Two ribosomal states interconvert in the presence of EF-G(GDP), displaying novel head domain motions, until relocking takes place. In Chapter 4, in order to test if the mentioned fluctuations leading to relocking are correlated to the engagement of the P-site by the peptidyl-tRNA, the translocation of miscoded tRNAs is studied. Severe defects in the relocking stages of translocation reveal the correlation between this new stage of translocation and P-site tRNA engagement.

  1. Structure of IgG and IgY molecules in ribosome-antibody complexes as studied by electron microscopy.

    PubMed

    Noll, F; Lutsch, G; Bielka, H

    1982-03-01

    The overall shape and dimensions of IgG (rabbit) and IgY (chicken) antibodies against ribosomal proteins have been studied in electron micrographs of ribosome-antibody complexes. The antibodies appear as Y-shaped molecules with an angle of about 90 degrees between their Fab arms. The length of one Fab arm amounts to about 10 nm. No differences between the IgG and IgY molecules could be detected electron microscopically. The data obtained on the shape of IgG and IgY correlate with those of earlier electron microscopic studies while the determined size of the Fab arms is in the range found by scattering methods.

  2. Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

    PubMed

    Nishimura, Kazuho; Kumazawa, Takuya; Kuroda, Takao; Katagiri, Naohiro; Tsuchiya, Mai; Goto, Natsuka; Furumai, Ryohei; Murayama, Akiko; Yanagisawa, Junn; Kimura, Keiji

    2015-03-03

    The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus

    PubMed Central

    Finch, Leanne K.; Ling, Roger; Napthine, Sawsan; Olspert, Allan; Michiels, Thomas; Lardinois, Cécile; Bell, Susanne; Loughran, Gary; Brierley, Ian

    2015-01-01

    ABSTRACT Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses. IMPORTANCE Many viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of

  4. 20 CFR 418.2105 - What is the threshold?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false What is the threshold? 418.2105 Section 418... Adjustment Amount § 418.2105 What is the threshold? (a) The threshold is a level of modified adjusted gross... years 2011 through and including 2019, the modified adjusted gross income threshold is $85,000 for...

  5. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    NASA Astrophysics Data System (ADS)

    Poirot, Olivier; Timsit, Youri

    2016-05-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing.

  6. Reading Amount as a Mediator of the Effects of Intrinsic and Extrinsic Reading Motivation on Reading Comprehension

    ERIC Educational Resources Information Center

    Schaffner, Ellen; Schiefele, Ulrich; Ulferts, Hannah

    2013-01-01

    This study examined the role of reading amount as a mediator of the effects of intrinsic and extrinsic reading motivation on higher order reading comprehension (comprised of paragraph-and passage-level comprehension) in a sample of 159 fifth-grade elementary students. A positive association between intrinsic reading motivation and reading amount…

  7. Multi-perspective smFRET reveals rate-determining late intermediates of ribosomal translocation

    PubMed Central

    Wasserman, Michael R.; Alejo, Jose L.; Altman, Roger B.; Blanchard, Scott C.

    2016-01-01

    Directional translocation of the ribosome through the messenger RNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of transfer and messenger RNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we have tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations reveal direct evidence of structurally and kinetically distinct, late intermediates during substrate movement, whose resolution is rate-determining to the translocation mechanism. These steps involve intra-molecular events within the EFG(GDP)-bound ribosome, including exaggerated, reversible fluctuations of the small subunit head domain, which ultimately facilitate peptidyl-tRNA’s movement into its final post-translocation position. PMID:26926435

  8. Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex.

    PubMed

    Xie, Ping

    2015-10-09

    Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by "hungry" codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.

  9. Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex

    PubMed Central

    Xie, Ping

    2015-01-01

    Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by “hungry” codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel. PMID:26473825

  10. Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bae, Hee Kyong; Shinozuka, Junko; Islam, Zahidul

    2009-06-01

    Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultantmore » fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.« less

  11. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

    PubMed Central

    Harada, Yoichiro; Li, Hua; Li, Huilin; Lennarz, William J.

    2009-01-01

    Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of ≈1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes. PMID:19365066

  12. Complete kinetic mechanism for recycling of the bacterial ribosome

    PubMed Central

    Borg, Anneli; Pavlov, Michael

    2016-01-01

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec−1, is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec−1, are discussed in the perspective of rapidly growing bacterial cells. PMID:26527791

  13. One Teacher's Journey through the Mediated Intersections

    ERIC Educational Resources Information Center

    Beach, Crystal L.

    2015-01-01

    Today's classrooms often have a plethora of new ways of reading and writing entering the room, but too often these new ways of "doing" are disregarded and checked at the door. For this reason, one educator shares her journey through the mediated intersections of media, culture, and education. In this piece, she explores how literacy…

  14. The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation

    PubMed Central

    Koloteva-Levine, Nadejda; Pinchasi, Dalia; Pereman, Idan; Zur, Amit; Brandeis, Michael; Elroy-Stein, Orna

    2004-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G1. We used a yeast three-hybrid screen to identify proteins that interact with the internal ribosome entry site (IRES) of platelet-derived growth factor 2 mRNA. Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain. We found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element. PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect. In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction. In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation. Expression of Apc5 in differentiated cells abolished IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex. PMID:15082755

  15. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Harada, Y.; Li, H.; Li, Hua

    2009-04-28

    Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of {approx}1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings,more » we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.« less

  16. 20 CFR 418.3220 - When is your application considered filed?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...? 418.3220 Section 418.3220 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES... the day it is submitted electronically through our Internet Web site. If a State Medicaid agency... subsidy application from our Internet Web site where the requirements set forth in § 418.3230 are met. ...

  17. 20 CFR 418.3220 - When is your application considered filed?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...? 418.3220 Section 418.3220 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES... the day it is submitted electronically through our Internet Web site. If a State Medicaid agency... subsidy application from our Internet Web site where the requirements set forth in § 418.3230 are met. ...

  18. Preservice Teacher Sense-Making as They Learn to Teach Reading as Seen through Computer-Mediated Discourse

    ERIC Educational Resources Information Center

    Stefanski, Angela J.; Leitze, Amy; Fife-Demski, Veronica M.

    2018-01-01

    This collective case study used methods of discourse analysis to consider what computer-mediated collaboration might reveal about preservice teachers' sense-making in a field-based practicum as they learn to teach reading to children identified as struggling readers. Researchers agree that field-based experiences coupled with time for reflection…

  19. Elucidation of Motifs in Ribosomal Protein S9 That Mediate Its Nucleolar Localization and Binding to NPM1/Nucleophosmin

    PubMed Central

    Lindström, Mikael S.

    2012-01-01

    Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site. PMID:23285058

  20. G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase

    PubMed Central

    Wallgren, Marcus; Mohammad, Jani B.; Yan, Kok-Phen; Pourbozorgi-Langroudi, Parham; Ebrahimi, Mahsa; Sabouri, Nasim

    2016-01-01

    Certain guanine-rich sequences have an inherent propensity to form G-quadruplex (G4) structures. G4 structures are e.g. involved in telomere protection and gene regulation. However, they also constitute obstacles during replication if they remain unresolved. To overcome these threats to genome integrity, organisms harbor specialized G4 unwinding helicases. In Schizosaccharomyces pombe, one such candidate helicase is Pfh1, an evolutionarily conserved Pif1 homolog. Here, we addressed whether putative G4 sequences in S. pombe can adopt G4 structures and, if so, whether Pfh1 can resolve them. We tested two G4 sequences, derived from S. pombe ribosomal and telomeric DNA regions, and demonstrated that they form inter- and intramolecular G4 structures, respectively. Also, Pfh1 was enriched in vivo at the ribosomal G4 DNA and telomeric sites. The nuclear isoform of Pfh1 (nPfh1) unwound both types of structure, and although the G4-stabilizing compound Phen-DC3 significantly enhanced their stability, nPfh1 still resolved them efficiently. However, stable G4 structures significantly inhibited adenosine triphosphate hydrolysis by nPfh1. Because ribosomal and telomeric DNA contain putative G4 regions conserved from yeasts to humans, our studies support the important role of G4 structure formation in these regions and provide further evidence for a conserved role for Pif1 helicases in resolving G4 structures. PMID:27185885

  1. Dual effect of chloramphenicol peptides on ribosome inhibition.

    PubMed

    Bougas, Anthony; Vlachogiannis, Ioannis A; Gatos, Dimitrios; Arenz, Stefan; Dinos, George P

    2017-05-01

    Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

  2. Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.

    PubMed

    Weir, Dawn L; Laing, Eric D; Smith, Ina L; Wang, Lin-Fa; Broder, Christopher C

    2014-02-27

    Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

  3. Text (Oral) Reading Fluency as a Construct in Reading Development: An Investigation of its Mediating Role for Children from Grades 1 to 4

    PubMed Central

    Kim, Young-Suk Grace; Wagner, Richard K.

    2015-01-01

    In the present study we investigated a developmentally changing role of text reading fluency in mediating the relations of word reading fluency and listening comprehension to reading comprehension. We addressed this question by using longitudinal data from Grades 1 to 4, and employing structural equation models. Results showed that the role of text reading fluency changes over time as children’s reading proficiency develops. In the beginning phase of reading development (Grade 1), text reading fluency was not independently related to reading comprehension over and above word reading fluency and listening comprehension. In Grades 2 to 4, however, text reading fluency completely mediated the relation between word reading fluency and reading comprehension whereas it partially mediated the relation between listening comprehension and reading comprehension. These results suggest that text reading fluency is a dissociable construct that plays a developmentally changing role in reading acquisition. PMID:25848201

  4. Ribosome flow model with positive feedback

    PubMed Central

    Margaliot, Michael; Tuller, Tamir

    2013-01-01

    Eukaryotic mRNAs usually form a circular structure; thus, ribosomes that terminatae translation at the 3′ end can diffuse with increased probability to the 5′ end of the transcript, initiating another cycle of translation. This phenomenon describes ribosomal flow with positive feedback—an increase in the flow of ribosomes terminating translating the open reading frame increases the ribosomal initiation rate. The aim of this paper is to model and rigorously analyse translation with feedback. We suggest a modified version of the ribosome flow model, called the ribosome flow model with input and output. In this model, the input is the initiation rate and the output is the translation rate. We analyse this model after closing the loop with a positive linear feedback. We show that the closed-loop system admits a unique globally asymptotically stable equilibrium point. From a biophysical point of view, this means that there exists a unique steady state of ribosome distributions along the mRNA, and thus a unique steady-state translation rate. The solution from any initial distribution will converge to this steady state. The steady-state distribution demonstrates a decrease in ribosome density along the coding sequence. For the case of constant elongation rates, we obtain expressions relating the model parameters to the equilibrium point. These results may perhaps be used to re-engineer the biological system in order to obtain a desired translation rate. PMID:23720534

  5. Multiple Mediation Analysis of the Relationship between Rapid Naming and Reading

    ERIC Educational Resources Information Center

    Poulsen, Mads; Juul, Holger; Elbro, Carsten

    2015-01-01

    It is well established that rapid automatised naming (RAN) correlates with reading ability. Despite several attempts, no single component process (mediator) has been identified that fully accounts for the correlation. The present paper estimated the explanatory value of several mediators for the RAN--reading correlation. One hundred and sixty-nine…

  6. Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

    PubMed Central

    Abeyrathne, Priyanka D; Koh, Cha San; Grant, Timothy; Grigorieff, Nikolaus; Korostelev, Andrei A

    2016-01-01

    Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation. DOI: http://dx.doi.org/10.7554/eLife.14874.001 PMID:27159452

  7. Effects of metalinguistic awareness on reading comprehension and the mediator role of reading fluency from grades 2 to 4.

    PubMed

    Li, Liping; Wu, Xinchun

    2015-01-01

    This study examined the contribution of metalinguistic awareness including morphological awareness, phonological awareness and orthographical awareness to reading comprehension, and the role of reading fluency as a mediator of the effects of metalinguistic awareness on reading comprehension from grades 2 to 4. Four hundred and fifteen elementary students in China mainland were administered a test battery that included measures of morphological awareness, phonological awareness, orthographical awareness, reading fluency, reading comprehension and IQ. Hierarchical regression and structural equation models (SEM) were used to analyze the data. Morphological awareness uniquely explained 9%, 10% and 13% variance of reading comprehension respectively from grade 2 to grade 4, however, phonological awareness and orthographical awareness did not contribute to reading comprehension; Reading fluency partially mediated the effect of morphological awareness on reading comprehension in grades 2-4. These findings indicated that reading fluency and morphological awareness should be facilitated in the Chinese instruction. Morphological awareness played an important role in Chinese reading and affected reading comprehension in grades 2 to 4; Reading fluency was a significant link between morphological awareness and reading comprehension in grades 2-4.

  8. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

    PubMed

    Nguyen, Le Xuan Truong; Mitchell, Beverly S

    2013-12-17

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

  9. Ribosomal proteins: functions beyond the ribosome.

    PubMed

    Zhou, Xiang; Liao, Wen-Juan; Liao, Jun-Ming; Liao, Peng; Lu, Hua

    2015-04-01

    Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation, their ribosome-independent functions have also been greatly appreciated. Over the past decade, more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress. In addition, these ribosomal proteins are involved in various physiological and pathological processes. This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins, as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis, immune signaling, and development. We also propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  10. Integrating Art and Reading--Learning to Read through the Arts.

    ERIC Educational Resources Information Center

    O'Brien, Bernadette C.

    1982-01-01

    The New York City Board of Education's Title I program, "Learning to Read through the Arts," teaches skills in reading through involvement in the arts, builds self-confidence, improves self-image, and adds to the experiences of the participating children. If children are able to read material and apply the information thus acquired to…

  11. Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

    PubMed

    Alejo, Jose L; Blanchard, Scott C

    2017-10-10

    Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

  12. Impact of a Technology-Mediated Reading Intervention on Adolescents' Reading Comprehension

    ERIC Educational Resources Information Center

    Fogarty, Melissa; Clemens, Nathan; Simmons, Deborah; Anderson, Leah; Davis, John; Smith, Ashley; Wang, Huan; Kwok, Oi-man; Simmons, Leslie E.; Oslund, Eric

    2017-01-01

    In this experimental study we examined the effects of a technology-mediated, multicomponent reading comprehension intervention, Comprehension Circuit Training (CCT), for middle school students, the majority of whom were struggling readers. The study was conducted in three schools, involving three teachers and 228 students. Using a within-teacher…

  13. Longitudinal Mediators of Achievement in Mathematics and Reading in Typical and Atypical Development

    PubMed Central

    Barnes, Marcia A.; Raghubar, Kimberly P.; English, Lianne; Williams, Jeffrey M.; Taylor, Heather; Landry, Susan

    2014-01-01

    Longitudinal studies of neurodevelopmental disorders that are diagnosed at or before birth and which are associated with specific learning difficulties at school-age provide one method for investigating developmental precursors of later-emerging academic disabilities. Spina bifida myelomeningocele (SBM) is a neurodevelopmental disorder associated with particular problems in mathematics, in contrast to well-developed word reading. Children with SBM (n = 30) and typically developing children (n = 35) were used to determine whether cognitive abilities measured at 36 and 60 months of age mediated the effect of group on mathematical and reading achievement outcomes at 8.5 and 9.5 years of age. A series of multiple mediator models showed that: visual-spatial working memory at 36 months and phonological awareness at 60 months partially mediated the effect of group on math calculations; phonological awareness partially mediated the effect of group on small addition and subtraction problems on a test of math fluency; and visual-spatial working memory mediated the effect of group on a test of math problem solving. Groups did not differ on word reading, and phonological awareness was the only mediator for reading fluency and reading comprehension. The findings are discussed with reference to theories of mathematical development and disability and with respect to both common and differing cognitive correlates of math and reading. PMID:24269579

  14. Longitudinal mediators of achievement in mathematics and reading in typical and atypical development.

    PubMed

    Barnes, Marcia A; Raghubar, Kimberly P; English, Lianne; Williams, Jeffrey M; Taylor, Heather; Landry, Susan

    2014-03-01

    Longitudinal studies of neurodevelopmental disorders that are diagnosed at or before birth and are associated with specific learning difficulties at school-age provide one method for investigating developmental precursors of later-emerging academic disabilities. Spina bifida myelomeningocele (SBM) is a neurodevelopmental disorder associated with particular problems in mathematics, in contrast to well-developed word reading. Children with SBM (n=30) and typically developing children (n=35) were used to determine whether cognitive abilities measured at 36 and 60 months of age mediated the effect of group on mathematical and reading achievement outcomes at 8.5 and 9.5 years of age. A series of multiple mediator models showed that: visual-spatial working memory at 36 months and phonological awareness at 60 months partially mediated the effect of group on math calculations, phonological awareness partially mediated the effect of group on small addition and subtraction problems on a test of math fluency, and visual-spatial working memory mediated the effect of group on a test of math problem solving. Groups did not differ on word reading, and phonological awareness was the only mediator for reading fluency and reading comprehension. The findings are discussed with reference to theories of mathematical development and disability and with respect to both common and differing cognitive correlates of math and reading. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Pseudo-Synesthesia through Reading Books with Colored Letters

    PubMed Central

    Colizoli, Olympia; Murre, Jaap M. J.; Rouw, Romke

    2012-01-01

    Background Synesthesia is a phenomenon where a stimulus produces consistent extraordinary subjective experiences. A relatively common type of synesthesia involves perception of color when viewing letters (e.g. the letter ‘a’ always appears as light blue). In this study, we examine whether traits typically regarded as markers of synesthesia can be acquired by simply reading in color. Methodology/Principal Findings Non-synesthetes were given specially prepared colored books to read. A modified Stroop task was administered before and after reading. A perceptual crowding task was administered after reading. Reading one book (>49,000 words) was sufficient to induce effects regarded as behavioral markers for synesthesia. The results of the Stroop tasks indicate that it is possible to learn letter-color associations through reading in color (F(1, 14) = 5.85, p = .030). Furthermore, Stroop effects correlated with subjective reports about experiencing letters in color (r(13) = 0.51, p = .05). The frequency of viewing letters is related to the level of association as seen by the difference in the Stroop effect size between upper- and lower-case letters (t(14) = 2.79, p = .014) and in a subgroup of participants whose Stroop effects increased as they continued to read in color. Readers did not show significant performance advantages on the crowding task compared to controls. Acknowledging the many differences between trainees and synesthetes, results suggest that it may be possible to acquire a subset of synesthetic behavioral traits in adulthood through training. Conclusion/Significance To our knowledge, this is the first evidence of acquiring letter-color associations through reading in color. Reading in color appears to be a promising avenue in which we may explore the differences and similarities between synesthetes and non-synesthetes. Additionally, reading in color is a plausible method for a long-term ‘synesthetic’ training program. PMID

  16. 20 CFR 418.1305 - What is not an initial determination regarding your income-related monthly adjustment amount?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Amount Determinations and the Administrative Review Process § 418.1305 What is not an initial... process as provided by §§ 418.1320 through 418.1325 and §§ 418.1340 through 418.1355, and they are not subject to judicial review. These actions include, but are not limited to, our dismissal of a request for...

  17. Effects of Metalinguistic Awareness on Reading Comprehension and the Mediator Role of Reading Fluency from Grades 2 to 4

    PubMed Central

    Li, Liping; Wu, Xinchun

    2015-01-01

    Purpose This study examined the contribution of metalinguistic awareness including morphological awareness, phonological awareness and orthographical awareness to reading comprehension, and the role of reading fluency as a mediator of the effects of metalinguistic awareness on reading comprehension from grades 2 to 4. Methods Four hundred and fifteen elementary students in China mainland were administered a test battery that included measures of morphological awareness, phonological awareness, orthographical awareness, reading fluency, reading comprehension and IQ. Hierarchical regression and structural equation models (SEM) were used to analyze the data. Results Morphological awareness uniquely explained 9%, 10% and 13% variance of reading comprehension respectively from grade 2 to grade 4, however, phonological awareness and orthographical awareness did not contribute to reading comprehension; Reading fluency partially mediated the effect of morphological awareness on reading comprehension in grades 2-4. Conclusions These findings indicated that reading fluency and morphological awareness should be facilitated in the Chinese instruction. Morphological awareness played an important role in Chinese reading and affected reading comprehension in grades 2 to 4; Reading fluency was a significant link between morphological awareness and reading comprehension in grades 2-4. PMID:25799530

  18. Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

    PubMed Central

    Dunn, Joshua G; Foo, Catherine K; Belletier, Nicolette G; Gavis, Elizabeth R; Weissman, Jonathan S

    2013-01-01

    Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. DOI: http://dx.doi.org/10.7554/eLife.01179.001 PMID:24302569

  19. Reading Comprehension in Boys with ADHD: The Mediating Roles of Working Memory and Orthographic Conversion.

    PubMed

    Friedman, Lauren M; Rapport, Mark D; Raiker, Joseph S; Orban, Sarah A; Eckrich, Samuel J

    2017-02-01

    Reading comprehension difficulties in children with ADHD are well established; however, limited information exists concerning the cognitive mechanisms that contribute to these difficulties and the extent to which they interact with one another. The current study examines two broad cognitive processes known to be involved in children's reading comprehension abilities-(a) working memory (i.e., central executive processes [CE], phonological short-term memory [PH STM], and visuospatial short-term memory [VS STM]) and (b) orthographic conversion (i.e., conversion of visually presented text to a phonological code)-to elucidate their unique and interactive contribution to ADHD-related reading comprehension differences. Thirty-one boys with ADHD-combined type and 30 typically developing (TD) boys aged 8 to 12 years (M = 9.64, SD = 1.22) were administered multiple counterbalanced tasks assessing WM and orthographic conversion processes. Relative to TD boys, boys with ADHD exhibited significant deficits in PH STM (d = -0.70), VS STM (d = -0.92), CE (d = -1.58), and orthographic conversion (d = -0.93). Bias-corrected, bootstrapped mediation analyses revealed that CE and orthographic conversion processes modeled separately mediated ADHD-related reading comprehension differences partially, whereas PH STM and VS STM did not. CE and orthographic conversion modeled jointly mediated ADHD-related reading comprehension differences fully wherein orthographic conversion's large magnitude influence on reading comprehension occurred indirectly through CE's impact on the orthographic system. The findings suggest that adaptive cognitive interventions designed to improve reading-related outcomes in children with ADHD may benefit by including modules that train CE and orthographic conversion processes independently and interactively.

  20. Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

    PubMed Central

    Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M.; Kemmler, Stefan; Panse, Vikram G.

    2013-01-01

    Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel. PMID:23907389

  1. Investigating an Intelligent System for Vocabulary Learning through Reading

    ERIC Educational Resources Information Center

    Stockwell, Glenn

    2013-01-01

    While learners can acquire vocabulary through extensive reading (Pigada & Schmitt, 2006), research suggests that acquisition can be more effective when supplemented with targeted vocabulary activities (e.g., Paribakht & Wesche, 1997). Problems arise, however, in determining what vocabulary learners have acquired, and what items should be…

  2. 20 CFR 418.3220 - When is your application considered filed?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...? 418.3220 Section 418.3220 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES... the day it is submitted electronically through our Internet Web site. If a State Medicaid agency... inquiry about your subsidy eligibility is made, or the date we receive a partially completed Internet...

  3. 20 CFR 418.3220 - When is your application considered filed?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...? 418.3220 Section 418.3220 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES... the day it is submitted electronically through our Internet Web site. If a State Medicaid agency... inquiry about your subsidy eligibility is made, or the date we receive a partially completed Internet...

  4. 20 CFR 418.3220 - When is your application considered filed?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...? 418.3220 Section 418.3220 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES... the day it is submitted electronically through our Internet Web site. If a State Medicaid agency... inquiry about your subsidy eligibility is made, or the date we receive a partially completed Internet...

  5. Collaborative Strategic Reading as a Means To Enhance Peer-Mediated Instruction for Reading Comprehension and Content-Area Learning.

    ERIC Educational Resources Information Center

    Vaughn, Sharon; Klingner, Janette K.; Bryant, Diane P.

    2001-01-01

    This article summarizes studies conducted with Collaborative Strategic Reading (CSR), a program designed to enhance reading comprehension and content-area reading for diverse learners. It describes the stages of CSR development and discusses the role of peer-mediated learning in improving the social and academic outcomes of participating students.…

  6. The Mediating Role of Mind Wandering in the Relationship between Working Memory Capacity and Reading Comprehension

    ERIC Educational Resources Information Center

    McVay, Jennifer C.

    2010-01-01

    The primary goal of this study was to investigate the mediating role of mind wandering in the relationship between working memory capacity (WMC) and reading comprehension as predicted by the executive-attention theory of WMC (e.g., Kane & Engle, 2003). I used a latent-variable, structural-equation-model approach with three WMC span tasks, seven…

  7. Neural networks mediating sentence reading in the deaf

    PubMed Central

    Hirshorn, Elizabeth A.; Dye, Matthew W. G.; Hauser, Peter C.; Supalla, Ted R.; Bavelier, Daphne

    2014-01-01

    The present work addresses the neural bases of sentence reading in deaf populations. To better understand the relative role of deafness and spoken language knowledge in shaping the neural networks that mediate sentence reading, three populations with different degrees of English knowledge and depth of hearing loss were included—deaf signers, oral deaf and hearing individuals. The three groups were matched for reading comprehension and scanned while reading sentences. A similar neural network of left perisylvian areas was observed, supporting the view of a shared network of areas for reading despite differences in hearing and English knowledge. However, differences were observed, in particular in the auditory cortex, with deaf signers and oral deaf showing greatest bilateral superior temporal gyrus (STG) recruitment as compared to hearing individuals. Importantly, within deaf individuals, the same STG area in the left hemisphere showed greater recruitment as hearing loss increased. To further understand the functional role of such auditory cortex re-organization after deafness, connectivity analyses were performed from the STG regions identified above. Connectivity from the left STG toward areas typically associated with semantic processing (BA45 and thalami) was greater in deaf signers and in oral deaf as compared to hearing. In contrast, connectivity from left STG toward areas identified with speech-based processing was greater in hearing and in oral deaf as compared to deaf signers. These results support the growing literature indicating recruitment of auditory areas after congenital deafness for visually-mediated language functions, and establish that both auditory deprivation and language experience shape its functional reorganization. Implications for differential reliance on semantic vs. phonological pathways during reading in the three groups is discussed. PMID:24959127

  8. Positively Charged Residues Are the Major Determinants of Ribosomal Velocity

    PubMed Central

    Charneski, Catherine A.; Hurst, Laurence D.

    2013-01-01

    Both for understanding mechanisms of disease and for the design of transgenes, it is important to understand the determinants of ribosome velocity, as changes in the rate of translation are important for protein folding, error attenuation, and localization. While there is great variation in ribosomal occupancy along even a single transcript, what determines a ribosome's occupancy is unclear. We examine this issue using data from a ribosomal footprinting assay in yeast. While codon usage is classically considered a major determinant, we find no evidence for this. By contrast, we find that positively charged amino acids greatly retard ribosomes downstream from where they are encoded, consistent with the suggestion that positively charged residues interact with the negatively charged ribosomal exit tunnel. Such slowing is independent of and greater than the average effect owing to mRNA folding. The effect of charged amino acids is additive, with ribosomal occupancy well-predicted by a linear fit to the density of positively charged residues. We thus expect that a translated poly-A tail, encoding for positively charged lysines regardless of the reading frame, would act as a sandtrap for the ribosome, consistent with experimental data. PMID:23554576

  9. Ribosome profiling reveals the rhythmic liver translatome and circadian clock regulation by upstream open reading frames

    PubMed Central

    Janich, Peggy; Arpat, Alaaddin Bulak; Castelo-Szekely, Violeta; Lopes, Maykel; Gatfield, David

    2015-01-01

    Mammalian gene expression displays widespread circadian oscillations. Rhythmic transcription underlies the core clock mechanism, but it cannot explain numerous observations made at the level of protein rhythmicity. We have used ribosome profiling in mouse liver to measure the translation of mRNAs into protein around the clock and at high temporal and nucleotide resolution. We discovered, transcriptome-wide, extensive rhythms in ribosome occupancy and identified a core set of approximately 150 mRNAs subject to particularly robust daily changes in translation efficiency. Cycling proteins produced from nonoscillating transcripts revealed thus-far-unknown rhythmic regulation associated with specific pathways (notably in iron metabolism, through the rhythmic translation of transcripts containing iron responsive elements), and indicated feedback to the rhythmic transcriptome through novel rhythmic transcription factors. Moreover, estimates of relative levels of core clock protein biosynthesis that we deduced from the data explained known features of the circadian clock better than did mRNA expression alone. Finally, we identified uORF translation as a novel regulatory mechanism within the clock circuitry. Consistent with the occurrence of translated uORFs in several core clock transcripts, loss-of-function of Denr, a known regulator of reinitiation after uORF usage and of ribosome recycling, led to circadian period shortening in cells. In summary, our data offer a framework for understanding the dynamics of translational regulation, circadian gene expression, and metabolic control in a solid mammalian organ. PMID:26486724

  10. The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

    PubMed

    Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

    2015-10-01

    One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. A Numbers Game: Ribosome Densities, Bacterial Growth, and Antibiotic-Mediated Stasis and Death

    PubMed Central

    McCall, Ingrid C.; Perrot, Véronique; Weiss, Howard; Ovesepian, Armen; Baquero, Fernando

    2017-01-01

    ABSTRACT We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn) operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding “bacteriostatic” antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE), is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures. PMID:28174311

  12. 40 CFR 418.24 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 418.24 Section 418.24 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.24 [Reserved] ...

  13. 40 CFR 418.24 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 418.24 Section 418.24 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.24 [Reserved] ...

  14. 40 CFR 418.24 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 418.24 Section 418.24 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.24 [Reserved] ...

  15. 40 CFR 418.24 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 418.24 Section 418.24 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.24 [Reserved] ...

  16. 40 CFR 418.24 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 418.24 Section 418.24 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.24 [Reserved] ...

  17. 40 CFR 418.14 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 418.14 Section 418.14 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.14 [Reserved] ...

  18. 40 CFR 418.14 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 418.14 Section 418.14 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.14 [Reserved] ...

  19. 40 CFR 418.14 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 418.14 Section 418.14 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.14 [Reserved] ...

  20. 40 CFR 418.34 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 418.34 Section 418.34 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.34 [Reserved] ...

  1. 40 CFR 418.54 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 418.54 Section 418.54 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Nitric Acid Subcategory § 418.54 [Reserved] ...

  2. 40 CFR 418.34 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 418.34 Section 418.34 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.34 [Reserved] ...

  3. 40 CFR 418.34 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 418.34 Section 418.34 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.34 [Reserved] ...

  4. 40 CFR 418.54 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 418.54 Section 418.54 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Nitric Acid Subcategory § 418.54 [Reserved] ...

  5. 40 CFR 418.54 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 418.54 Section 418.54 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Nitric Acid Subcategory § 418.54 [Reserved] ...

  6. 40 CFR 418.34 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 418.34 Section 418.34 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.34 [Reserved] ...

  7. 40 CFR 418.54 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 418.54 Section 418.54 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Nitric Acid Subcategory § 418.54 [Reserved] ...

  8. 40 CFR 418.34 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 418.34 Section 418.34 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.34 [Reserved] ...

  9. 40 CFR 418.54 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 418.54 Section 418.54 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Nitric Acid Subcategory § 418.54 [Reserved] ...

  10. 40 CFR 418.14 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 418.14 Section 418.14 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.14 [Reserved] ...

  11. 40 CFR 418.14 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 418.14 Section 418.14 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.14 [Reserved] ...

  12. 40 CFR 418.44 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 418.44 Section 418.44 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Nitrate Subcategory § 418.44 [Reserved] ...

  13. 40 CFR 418.44 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 418.44 Section 418.44 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Nitrate Subcategory § 418.44 [Reserved] ...

  14. 40 CFR 418.44 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 418.44 Section 418.44 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Nitrate Subcategory § 418.44 [Reserved] ...

  15. 40 CFR 418.44 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 418.44 Section 418.44 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Nitrate Subcategory § 418.44 [Reserved] ...

  16. 40 CFR 418.44 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 418.44 Section 418.44 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Nitrate Subcategory § 418.44 [Reserved] ...

  17. How Does Parental Reading Influence Children's Reading? A Study of Cognitive Mediation

    ERIC Educational Resources Information Center

    van Bergen, Elsje; Bishop, Dorothy; van Zuijen, Titia; de Jong, Peter F.

    2015-01-01

    Cognitive processes underlying a behavioural outcome (like reading ability) and the impact of familial risk (e.g., for dyslexia) have been studied in isolation. We present a novel design, linking the two avenues. How do familial influences impact on children's cognitive skills, which subsequently underlie reading development? Participants from the…

  18. Activation of Elongation Factor G by Phosphate Analogues

    PubMed Central

    Salsi, Enea; Farah, Elie

    2016-01-01

    EF-G is a universally conserved translational GTPase that promotes the translocation of tRNA and mRNA through the ribosome. EF-G binds to the ribosome in a GTP-bound form and subsequently catalyzes GTP hydrolysis. The contribution of the ribosome-stimulated GTP hydrolysis by EF-G to tRNA/mRNA translocation remains debated. Here, we show that while EF-G•GDP does not stably bind to the ribosome and induce translocation, EFG• GDP in complex with phosphate group analogues BeF3− and AlF4− promotes the translocation of tRNA and mRNA. Furthermore, the rates of mRNA translocation induced by EF-G in the presence of GTP and a non-hydrolysable analogue of GTP, GDP•BeF3−are similar. Our results are consistent with the model suggesting that GTP hydrolysis is not directly coupled to mRNA/tRNA translocation. Hence, GTP binding is required to induce the activated, translocation-competent conformation of EF-G while GTP hydrolysis triggers EF-G release from the ribosome. PMID:27063503

  19. Trypanosome RNA Editing Mediator Complex proteins have distinct functions in gRNA utilization.

    PubMed

    Simpson, Rachel M; Bruno, Andrew E; Chen, Runpu; Lott, Kaylen; Tylec, Brianna L; Bard, Jonathan E; Sun, Yijun; Buck, Michael J; Read, Laurie K

    2017-07-27

    Uridine insertion/deletion RNA editing is an essential process in kinetoplastid parasites whereby mitochondrial mRNAs are modified through the specific insertion and deletion of uridines to generate functional open reading frames, many of which encode components of the mitochondrial respiratory chain. The roles of numerous non-enzymatic editing factors have remained opaque given the limitations of conventional methods to interrogate the order and mechanism by which editing progresses and thus roles of individual proteins. Here, we examined whole populations of partially edited sequences using high throughput sequencing and a novel bioinformatic platform, the Trypanosome RNA Editing Alignment Tool (TREAT), to elucidate the roles of three proteins in the RNA Editing Mediator Complex (REMC). We determined that the factors examined function in the progression of editing through a gRNA; however, they have distinct roles and REMC is likely heterogeneous in composition. We provide the first evidence that editing can proceed through numerous paths within a single gRNA and that non-linear modifications are essential, generating commonly observed junction regions. Our data support a model in which RNA editing is executed via multiple paths that necessitate successive re-modification of junction regions facilitated, in part, by the REMC variant containing TbRGG2 and MRB8180. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Active Reading Documents (ARDs): A Tool to Facilitate Meaningful Learning through Reading

    ERIC Educational Resources Information Center

    Dubas, Justin M.; Toledo, Santiago A.

    2015-01-01

    Presented here is a practical tool called the Active Reading Document (ARD) that can give students the necessary incentive to engage with the text/readings. By designing the tool to incrementally develop student understanding of the material through reading using Marzano's Taxonomy as a framework, the ARD offers support through scaffolding as they…

  1. 40 CFR 418.64 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 418.64 Section 418.64 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.64 [Reserved] ...

  2. 7 CFR 1944.418 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REGULATIONS (CONTINUED) HOUSING Self-Help Technical Assistance Grants § 1944.418 [Reserved] ... 7 Agriculture 13 2010-01-01 2009-01-01 true [Reserved] 1944.418 Section 1944.418 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS-COOPERATIVE...

  3. 40 CFR 418.64 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 418.64 Section 418.64 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.64 [Reserved] ...

  4. 40 CFR 418.64 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 418.64 Section 418.64 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.64 [Reserved] ...

  5. 40 CFR 418.64 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 418.64 Section 418.64 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.64 [Reserved] ...

  6. 40 CFR 418.64 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 418.64 Section 418.64 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.64 [Reserved] ...

  7. 40 CFR 418.74 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 418.74 Section 418.74 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer Production Subcategory § 418.74...

  8. 40 CFR 418.74 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 418.74 Section 418.74 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer Production Subcategory § 418.74...

  9. 40 CFR 418.74 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 418.74 Section 418.74 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer Production Subcategory § 418.74...

  10. 40 CFR 418.74 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 418.74 Section 418.74 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer Production Subcategory § 418.74...

  11. 40 CFR 418.74 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 418.74 Section 418.74 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer Production Subcategory § 418.74...

  12. Using Peer-Mediated Repeated Readings as a Fluency-Building Activity for Urban Learners

    ERIC Educational Resources Information Center

    Yurick, Amanda L.; Robinson, Porsha D.; Cartledge, Gwendolyn; Lo, Ya-yu; Evans, Trisha L.

    2006-01-01

    We conducted three experiments examining the effects of peer-mediated repeated readings on students' oral reading fluency and comprehension. Each repeated reading session consisted of students reading in pairs, alternating paragraphs, for 10 minutes. Students used a scripted correction procedure when errors occurred. Students then participated in…

  13. 40 CFR 98.418 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Definitions. 98.418 Section 98.418 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Suppliers of Industrial Greenhouse Gases § 98.418 Definitions. All terms used in...

  14. 40 CFR 98.418 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 21 2011-07-01 2011-07-01 false Definitions. 98.418 Section 98.418 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Suppliers of Industrial Greenhouse Gases § 98.418 Definitions. Except as provided...

  15. 40 CFR 98.418 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 22 2013-07-01 2013-07-01 false Definitions. 98.418 Section 98.418 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Suppliers of Industrial Greenhouse Gases § 98.418 Definitions. Except as provided...

  16. 40 CFR 98.418 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 21 2014-07-01 2014-07-01 false Definitions. 98.418 Section 98.418 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Suppliers of Industrial Greenhouse Gases § 98.418 Definitions. Except as provided...

  17. 40 CFR 98.418 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 22 2012-07-01 2012-07-01 false Definitions. 98.418 Section 98.418 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Suppliers of Industrial Greenhouse Gases § 98.418 Definitions. Except as provided...

  18. 43 CFR 418.17 - Truckee and Carson River water use.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Truckee and Carson River water use. 418.17... Operations and Management § 418.17 Truckee and Carson River water use. Project water must be managed to make maximum use of Carson River water and to minimize diversions of Truckee River water through the Truckee...

  19. 43 CFR 418.17 - Truckee and Carson River water use.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 1 2011-10-01 2011-10-01 false Truckee and Carson River water use. 418.17... Operations and Management § 418.17 Truckee and Carson River water use. Project water must be managed to make maximum use of Carson River water and to minimize diversions of Truckee River water through the Truckee...

  20. 43 CFR 418.29 - Project management.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Project management. 418.29 Section 418.29... INTERIOR OPERATING CRITERIA AND PROCEDURES FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Enforcement § 418.29 Project management. In addition to the provisions of § 418.28, if the District is found to be...

  1. 43 CFR 418.29 - Project management.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 1 2012-10-01 2011-10-01 true Project management. 418.29 Section 418.29... INTERIOR OPERATING CRITERIA AND PROCEDURES FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Enforcement § 418.29 Project management. In addition to the provisions of § 418.28, if the District is found to be...

  2. 43 CFR 418.29 - Project management.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 1 2014-10-01 2014-10-01 false Project management. 418.29 Section 418.29... INTERIOR OPERATING CRITERIA AND PROCEDURES FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Enforcement § 418.29 Project management. In addition to the provisions of § 418.28, if the District is found to be...

  3. 43 CFR 418.29 - Project management.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 1 2013-10-01 2013-10-01 false Project management. 418.29 Section 418.29... INTERIOR OPERATING CRITERIA AND PROCEDURES FOR THE NEWLANDS RECLAMATION PROJECT, NEVADA Enforcement § 418.29 Project management. In addition to the provisions of § 418.28, if the District is found to be...

  4. Structure of ratcheted ribosomes with tRNAs in hybrid states

    PubMed Central

    Julián, Patricia; Konevega, Andrey L.; Scheres, Sjors H. W.; Lázaro, Melisa; Gil, David; Wintermeyer, Wolfgang; Rodnina, Marina V.; Valle, Mikel

    2008-01-01

    During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G. PMID:18971332

  5. Ribosome-inactivating proteins

    PubMed Central

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms. PMID:24071927

  6. The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity.

    PubMed

    Andrade, José M; Dos Santos, Ricardo F; Chelysheva, Irina; Ignatova, Zoya; Arraiano, Cecília M

    2018-06-01

    Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA-mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA Hfq assists ribosome assembly and associates with pre-30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq-mediated regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm-like protein Hfq beyond its function in small RNA-mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation. © 2018 The Authors.

  7. Preschoolers' Emergent Literacy Skills: The Mediating Role of Maternal Reading Beliefs

    ERIC Educational Resources Information Center

    Cottone, Elizabeth Ann

    2012-01-01

    Research Findings: The purpose of this paper is to explore the association between maternal reading beliefs and children's emergent literacy outcomes in light of maternal education. Furthermore, I consider whether maternal reading beliefs may mediate the association between maternal education level and children's print knowledge and phonological…

  8. Reading Through Paint

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Two-dimensional data matrix symbols, which contain encoded letters and numbers, are permanently etched on items for identification. They can store up to 100 times more information than traditional bar codes. While the symbols provide several advantages over bar codes, once they are covered by paint they can no longer be read by optical scanners. Since most products are painted eventually, this presents a problem for industries relying on the symbols for identification and tracking. In 1987, NASA s Marshall Space Flight Center began studying direct parts marking with matrix symbols in order to track millions of Space Shuttle parts. Advances in the technology proved that by incorporating magnetic properties into the paints, inks, and pastes used to apply the matrix symbols, the codes could be read by a magnetic scanner even after being covered with paint or other coatings. NASA received a patent for such a scanner in 1998, but the system it used for development was not portable and was too costly. A prototype was needed as a lead-in to a production model. In the summer of 2000, NASA began seeking companies to build a hand-held scanner that would detect the Read Through Paint data matrix identification marks containing magnetic materials through coatings.

  9. Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex.

    PubMed

    Cukras, Anthony R; Southworth, Daniel R; Brunelle, Julie L; Culver, Gloria M; Green, Rachel

    2003-08-01

    Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.

  10. Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway.

    PubMed

    Llanos, Susana; Serrano, Manuel

    2010-10-01

    Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.

  11. Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

    PubMed Central

    Llanos, Susana; Serrano, Manuel

    2013-01-01

    Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage. PMID:20935493

  12. 43 CFR 418.21 - Diversion of Truckee River water to Lahontan Reservoir, July through December.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Adjustments to Lahontan Reservoir Storage Targets table and § 418.22. Diversions shall be started to achieve the end-of-month storage targets listed in the table in § 418.22 and will be discontinued when storage is forecast to meet or exceed the end-of-month storage targets at the end of the month. Diversions...

  13. 43 CFR 418.21 - Diversion of Truckee River water to Lahontan Reservoir, July through December.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Adjustments to Lahontan Reservoir Storage Targets table and § 418.22. Diversions shall be started to achieve the end-of-month storage targets listed in the table in § 418.22 and will be discontinued when storage is forecast to meet or exceed the end-of-month storage targets at the end of the month. Diversions...

  14. 43 CFR 418.21 - Diversion of Truckee River water to Lahontan Reservoir, July through December.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Adjustments to Lahontan Reservoir Storage Targets table and § 418.22. Diversions shall be started to achieve the end-of-month storage targets listed in the table in § 418.22 and will be discontinued when storage is forecast to meet or exceed the end-of-month storage targets at the end of the month. Diversions...

  15. Specific Function of the Met-Tyr-Trp Adduct Radical and Residues Arg-418 and Asp-137 in the Atypical Catalase Reaction of Catalase-Peroxidase KatG*

    PubMed Central

    Zhao, Xiangbo; Khajo, Abdelahad; Jarrett, Sanchez; Suarez, Javier; Levitsky, Yan; Burger, Richard M.; Jarzecki, Andrzej A.; Magliozzo, Richard S.

    2012-01-01

    Catalase activity of the dual-function heme enzyme catalase-peroxidase (KatG) depends on several structural elements, including a unique adduct formed from covalently linked side chains of three conserved amino acids (Met-255, Tyr-229, and Trp-107, Mycobacterium tuberculosis KatG numbering) (MYW). Mutagenesis, electron paramagnetic resonance, and optical stopped-flow experiments, along with calculations using density functional theory (DFT) methods revealed the basis of the requirement for a radical on the MYW-adduct, for oxyferrous heme, and for conserved residues Arg-418 and Asp-137 in the rapid catalase reaction. The participation of an oxyferrous heme intermediate (dioxyheme) throughout the pH range of catalase activity is suggested from our finding that carbon monoxide inhibits the activity at both acidic and alkaline pH. In the presence of H2O2, the MYW-adduct radical is formed normally in KatG[D137S] but this mutant is defective in forming dioxyheme and lacks catalase activity. KatG[R418L] is also catalase deficient but exhibits normal formation of the adduct radical and dioxyheme. Both mutants exhibit a coincidence between MYW-adduct radical persistence and H2O2 consumption as a function of time, and enhanced subunit oligomerization during turnover, suggesting that the two mutations disrupting catalase turnover allow increased migration of the MYW-adduct radical to protein surface residues. DFT calculations showed that an interaction between the side chain of residue Arg-418 and Tyr-229 in the MYW-adduct radical favors reaction of the radical with the adjacent dioxyheme intermediate present throughout turnover in WT KatG. Release of molecular oxygen and regeneration of resting enzyme are thereby catalyzed in the last step of a proposed catalase reaction. PMID:22918833

  16. Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui.

    PubMed

    Hatakeyama, T; Kimura, M

    1988-03-15

    Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.

  17. Mediating Effects of Working Memory in the Relation Between Rapid Automatized Naming and Chinese Reading Comprehension.

    PubMed

    Weng, Xiaoqian; Li, Guangze; Li, Rongbao

    2016-08-01

    This study examined the mediating role of working memory (WM) in the relation between rapid automatized naming (RAN) and Chinese reading comprehension. Three tasks assessing differentially visual and verbal components of WM were programmed by E-prime 2.0. Data collected from 55 Chinese college students were analyzed using correlations and hierarchical regression methods to determine the connection among RAN, reading comprehension, and WM components. Results showed that WM played a significant mediating role in the RAN-reading relation and that auditory WM made stronger contributions than visual WM. Taking into account of the multi-component nature of WM and the specificity of Chinese reading processing, this study discussed the mediating powers of the WM components, particularly auditory WM, further clarifying the possible components involved in the RAN-reading relation and thus providing some insight into the complicated Chinese reading process.

  18. Miscoding-induced stalling of substrate translocation on the bacterial ribosome

    PubMed Central

    Alejo, Jose L.; Blanchard, Scott C.

    2017-01-01

    Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

  19. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.

    PubMed

    Fischer, Niels; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V; Stark, Holger

    2010-07-15

    The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.

  20. Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

    PubMed

    Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

    2018-07-04

    The directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

  1. 40 CFR 418.31 - Specialized definitions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false Specialized definitions. 418.31 Section 418.31 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.31 Specialized...

  2. G-protein control of the ribosome-associated stress response protein SpoT.

    PubMed

    Jiang, Mengxi; Sullivan, Susan M; Wout, Patrice K; Maddock, Janine R

    2007-09-01

    The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.

  3. Early language mediates the relations between preschool inattention and school-age reading achievement.

    PubMed

    O'Neill, Sarah; Thornton, Veronica; Marks, David J; Rajendran, Khushmand; Halperin, Jeffrey M

    2016-05-01

    Early inattention is associated with later reading problems in children, but the mechanism by which this occurs is unclear. We investigated whether the negative relation between preschoolers' ADHD symptoms and 8-year-old reading achievement is directly related to the severity of inattention or is mediated by early language skills. Children (n = 150; 76% boys) were evaluated at 3 time points: preschool (T1), mean (SD) age = 4.24 (.49) years; 1 year later (T2), mean (SD) age = 5.28 (.50) years; and during school age (T3), mean (SD) age = 8.61 (.31) years. At T1, parents' Kiddie-SADS responses were dimensionalized to reflect ADHD severity. Children completed the Language domain of the NEPSY (i.e., A Developmental Neuropsychological Assessment) at T1 and again at T2. At T3, children completed the Wechsler Individual Achievement Test, Second Edition Word Reading, Pseudoword Decoding, Reading Comprehension, and Spelling subtests, and their teachers completed ratings of Reading and Written Expression performance in school. The mediating effect of T2 Language on the relation between preschool Inattention and age 8 Reading was examined using the nonparametric bootstrapping procedure, while controlling for T1 Language. Language ability at T2 mediated the path from preschool inattention (but not hyperactivity/impulsivity) to 8-year-old reading achievement (both test scores and ratings) after controlling for preschoolers' language ability. Early attentional deficits may negatively impact school-age reading outcomes by compromising the development of language skills, which in turn imperils later reading achievement. Screening children with attentional problems for language impairment, as well as implementing early intervention for both attentional and language problems may be critical to promote reading achievement during school years. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  4. 40 CFR 418.61 - Specialized definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true Specialized definitions. 418.61 Section 418.61 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.61...

  5. 40 CFR 418.61 - Specialized definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true Specialized definitions. 418.61 Section 418.61 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.61...

  6. 40 CFR 418.61 - Specialized definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true Specialized definitions. 418.61 Section 418.61 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.61...

  7. 40 CFR 418.61 - Specialized definitions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true Specialized definitions. 418.61 Section 418.61 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory § 418.61...

  8. 40 CFR 418.31 - Specialized definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true Specialized definitions. 418.31 Section 418.31 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.31 Specialized definitions...

  9. 40 CFR 418.31 - Specialized definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true Specialized definitions. 418.31 Section 418.31 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.31 Specialized definitions...

  10. Ribosomes slide on lysine-encoding homopolymeric A stretches

    PubMed Central

    Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

    2015-01-01

    Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

  11. Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

    PubMed Central

    Koch, Miriam; Flür, Sara; Kreutz, Christoph; Ennifar, Eric; Micura, Ronald; Polacek, Norbert

    2015-01-01

    Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases. PMID:25941362

  12. Cis-regulatory RNA elements that regulate specialized ribosome activity.

    PubMed

    Xue, Shifeng; Barna, Maria

    2015-01-01

    Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5'UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution.

  13. 15 CFR 971.418 - Diligence requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Diligence requirements. 971.418 Section 971.418 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued...: Terms, Conditions and Restrictions Terms, Conditions and Restrictions § 971.418 Diligence requirements...

  14. 48 CFR 9904.418-30 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... STANDARDS COST ACCOUNTING STANDARDS 9904.418-30 Definitions. (a) The following are definitions of terms... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Definitions. 9904.418-30 Section 9904.418-30 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD, OFFICE OF...

  15. Improving Reading Comprehension through Application and Transfer of Reading Strategies

    ERIC Educational Resources Information Center

    Pesa, Nicole; Somers, Sarah

    2007-01-01

    This study describes a program designed to improve reading comprehension through the selection, application, and transfer of appropriate reading strategies with both fictional and informational texts. The targeted population consisted of seventh and eighth grade middle school students in a middle-class community in the western suburbs of Chicago,…

  16. 37 CFR 41.8 - Mandatory notices.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2013-07-01 2013-07-01 false Mandatory notices. 41.8 Section 41.8 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE PRACTICE BEFORE THE PATENT TRIAL AND APPEAL BOARD General Provisions § 41.8 Mandatory notices. (a...

  17. The association of TIF-IA and polymerase I mediates promoter recruitment and regulation of ribosomal RNA transcription in Acanthamoeba castellanii.

    PubMed

    Gogain, Joseph C; Paule, Marvin R

    2005-01-01

    Large amounts of energy are expended for the construction of the ribosome during both transcription and processing, so it is of utmost importance for the cell to efficiently regulate ribosome production. Understanding how this regulation occurs will provide important insights into cellular growth control and into the coordination of gene expression mediated by all three transcription systems. Ribosomal RNA (rRNA) transcription rates closely parallel the need for protein synthesis; as a cell approaches stationary phase or encounters conditions that negatively affect either growth rate or protein synthesis, rRNA transcription is decreased. In eukaryotes, the interaction of RNA polymerase I (pol I) with the essential transcription initiation factor IA (TIF-IA) has been implicated in this downregulation of transcription. In agreement with the first observation that rRNA transcription is regulated by altering recruitment of pol I to the promoter in Acanthamoeba castellanii, we show here that pol I and an 80-kDa homologue of TIF-IA are found tightly associated in pol I fractions competent for specific transcription. Disruption of the pol I-TIF-IA complex is mediated by a specific dephosphorylation of either pol I or TIF-IA. Phosphatase treatment of TIF-IA-containing A. castellanii pol I fractions results in a downregulation of both transcriptional activity and promoter binding, reminiscent of the inactive pol I fractions purified from encysted cells. The fraction of pol I competent for promoter recruitment is enriched in TIF-IA relative to that not bound by immobilized promoter DNA. This downregulation coincides with an altered electrophoretic mobility of TIF-IA, suggesting at least it is phosphorylated.

  18. Ribosome profiling reveals the what, when, where and how of protein synthesis.

    PubMed

    Brar, Gloria A; Weissman, Jonathan S

    2015-11-01

    Ribosome profiling, which involves the deep sequencing of ribosome-protected mRNA fragments, is a powerful tool for globally monitoring translation in vivo. The method has facilitated discovery of the regulation of gene expression underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing a systematic approach for experimental annotation of coding regions. Here, we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including its prominent role in identifying thousands of novel translated short open reading frames and alternative translation products.

  19. Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

    PubMed Central

    Ll, Juan Modole; Cabrer, Bartolomé; Parmeggiani, Andrea; Azquez, David V

    1971-01-01

    Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G. PMID:4331558

  20. The phzA2-G2 Transcript Exhibits Direct RsmA-Mediated Activation in Pseudomonas aeruginosa M18

    PubMed Central

    Ren, Bin; Shen, Huifeng; Lu, Zhi John; Liu, Haiming; Xu, Yuquan

    2014-01-01

    In bacteria, RNA-binding proteins of the RsmA/CsrA family act as post-transcriptional regulators that modulate translation initiation at target transcripts. The Pseudomonas aeruginosa genome contains two phenazine biosynthetic (phz) gene clusters, phzA1-G1 (phz1) and phzA2-G2 (phz2), each of which is responsible for phenazine-1-carboxylic acid (PCA) biosynthesis. In the present study, we show that RsmA exhibits differential gene regulation on two phz clusters in P. aeruginosa M18 at the post-transcriptional level. Based on the sequence analysis, four GGA motifs, the potential RsmA binding sites, are found on the 5′-untranslated region (UTR) of the phz2 transcript. Studies with a series of lacZ reporter fusions, and gel mobility shift assays suggest that the third GGA motif (S3), located 21 nucleotides upstream of the Shine-Dalgarno (SD) sequence, is involved in direct RsmA-mediated activation of phz2 expression. We therefore propose a novel model in which the binding of RsmA to the target S3 results in the destabilization of the stem-loop structure and the enhancement of ribosome access. This model could be fully supported by RNA structure prediction, free energy calculations, and nucleotide replacement studies. In contrast, various RsmA-mediated translation repression mechanisms have been identified in which RsmA binds near the SD sequence of target transcripts, thereby blocking ribosome access. Similarly, RsmA is shown to negatively regulate phz1 expression. Our new findings suggest that the differential regulation exerted by RsmA on the two phz clusters may confer an advantage to P. aeruginosa over other pseudomonads containing only a single phz cluster in their genomes. PMID:24586939

  1. Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ahmad, Javed; Ahamed, Maqusood, E-mail: maqusood@gmail.com; Akhtar, Mohd Javed

    Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25–200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion ofmore » glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level. -- Highlights: ► We explored the mechanisms of toxicity caused by silica NPs in human liver HepG2 cells. ► Silica NPs induced a dose-dependent cytotoxicity in HepG2 cells. ► Silica NPs induced ROS generation and oxidative stress in a dose-dependent manner. ► Silica NPs were also modulated apoptosis markers both at mRNA and protein levels. ► ROS mediated apoptosis induced by silica NPs was preserved by vitamin C.« less

  2. An analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators.

    PubMed

    Ling, Roger; Firth, Andrew E

    2017-08-01

    Programmed -1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3'-adjacent to a 'slippery' heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of -1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3'-adjacent stem-loop structure, but is not appreciably affected by the 'StopGo' sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46-76 % efficient.

  3. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

    2016-01-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  4. Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

    PubMed Central

    Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

    2016-01-01

    Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

  5. Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

    PubMed

    Nikolaitchik, Olga A; Hu, Wei-Shau

    2014-04-01

    A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1

  6. Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging

    PubMed Central

    Nikolaitchik, Olga A.

    2014-01-01

    ABSTRACT A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5′ untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. IMPORTANCE To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5′ end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important

  7. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism

    PubMed Central

    Reilman, Ewoud; Mars, Ruben A. T.; van Dijl, Jan Maarten; Denham, Emma L.

    2014-01-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. PMID:25217586

  8. The Effect of Summary Writing on Reading Comprehension: The Role of Mediation in EFL Classroom

    ERIC Educational Resources Information Center

    Gao, Yang

    2013-01-01

    Reading teachers focus more on the instruction of reading content or strategies, but pay relatively less attention to the impact of writing on reading comprehension. Based on mediation theory, the author examined the effect of summary writing about reading texts on readers' comprehension. By reviewing relevant literatures on the topic of…

  9. Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus *

    PubMed Central

    Koripella, Ravi Kiran; Chen, Yang; Peisker, Kristin; Koh, Cha San; Selmer, Maria; Sanyal, Suparna

    2012-01-01

    Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. PMID:22767604

  10. 29 CFR 1926.418-1926.430 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 8 2010-07-01 2010-07-01 false [Reserved] 1926.418-1926.430 Section 1926.418-1926.430 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION... Practices §§ 1926.418-1926.430 [Reserved] Safety-Related Maintenance and Environmental Considerations ...

  11. Text (Oral) Reading Fluency as a Construct in Reading Development: An Investigation of Its Mediating Role for Children from Grades 1 to 4

    ERIC Educational Resources Information Center

    Kim, Young-Suk Grace; Wagner, Richard K.

    2015-01-01

    In the present study we investigated a developmentally changing role of text reading fluency in mediating the relations of word reading fluency and listening comprehension to reading comprehension. We addressed this question by using longitudinal data from Grades 1 to 4 and employing structural equation models. Results showed that the role of text…

  12. Structure of Ribosomal Silencing Factor Bound to Mycobacterium tuberculosis Ribosome.

    PubMed

    Li, Xiaojun; Sun, Qingan; Jiang, Cai; Yang, Kailu; Hung, Li-Wei; Zhang, Junjie; Sacchettini, James C

    2015-10-06

    The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during periods of diminished nutrient availability. The crystal structure of Mycobacterium tuberculosis (Mtb) RsfS, together with the cryo-electron microscopy (EM) structure of the large subunit 50S of Mtb ribosome, reveals how inhibition of protein synthesis by RsfS occurs. RsfS binds to the 50S at L14, which, when occupied, blocks the association of the small subunit 30S. Although Mtb RsfS is a dimer in solution, only a single subunit binds to 50S. The overlap between the dimer interface and the L14 binding interface confirms that the RsfS dimer must first dissociate to a monomer in order to bind to L14. RsfS interacts primarily through electrostatic and hydrogen bonding to L14. The EM structure shows extended rRNA density that it is not found in the Escherichia coli ribosome, the most striking of these being the extended RNA helix of H54a. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. 48 CFR 9904.418-63 - Effective date.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Effective date. 9904.418-63 Section 9904.418-63 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD, OFFICE... ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.418-63 Effective date. This Standard is effective as of...

  14. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility

    PubMed Central

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-01-01

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmAII enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmAII, rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmAII in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmAII activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmAII, thereby facilitating TEL binding to the ribosome. PMID:26365244

  15. Teaching Students to Read through Their Individual Learning Styles.

    ERIC Educational Resources Information Center

    Carbo, Marie; And Others

    Designed to assist parents, classroom teachers, reading specialists, and special educators, this book describes effective reading programs that promote reading success and achievement for children at all reading levels. The 10 chapters of the book are as follows: (1) "Preventing Reading Failure and Increasing Reading Achievement through Learning…

  16. Hypermethylation of 28S ribosomal RNA in β-thalassemia trait carriers.

    PubMed

    Sornjai, Wannapa; Lithanatudom, Pathrapol; Erales, Jenny; Joly, Philippe; Francina, Alain; Hacot, Sabine; Fucharoen, Suthat; Svasti, Saovaros; Diaz, Jean Jacques; Mertani, Hichem C; Smith, Duncan R

    2017-01-01

    Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2'-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of β-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in β-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in β-thalassemia trait carriers. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. The Circadian Clock Coordinates Ribosome Biogenesis

    PubMed Central

    Symul, Laura; Martin, Eva; Atger, Florian; Naef, Felix; Gachon, Frédéric

    2013-01-01

    Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. PMID:23300384

  18. Morphological awareness and reading comprehension: Examining mediating factors.

    PubMed

    Levesque, Kyle C; Kieffer, Michael J; Deacon, S Hélène

    2017-08-01

    The relation between morphological awareness-defined as the awareness of and ability to manipulate the smallest units of meaning in language-and reading comprehension remains in need of specification. In this study, we evaluated four potential intervening variables through which morphological awareness may contribute indirectly to reading comprehension. We assessed word reading and vocabulary as well as children's ability to read and analyze the meaning of morphologically complex words (morphological decoding and morphological analysis, respectively). Controls of phonological awareness and nonverbal ability were included in the model. Participants were 221 English-speaking children in Grade 3. Multivariate path analyses revealed evidence of two indirect relations and one direct relation between morphological awareness and reading comprehension. In the first indirect path, morphological awareness contributed to morphological decoding, which then influenced word reading and finally reading comprehension. In a second indirect path, morphological awareness contributed to morphological analysis, which contributed to reading comprehension. Finally, in a direct path, morphological awareness contributed to reading comprehension beyond all other variables. These findings inform as to the potential mechanisms underlying the relation between morphological awareness and reading comprehension in children. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

    PubMed Central

    Lill, R; Robertson, J M; Wintermeyer, W

    1989-01-01

    A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA. PMID:2583120

  20. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  1. LPS-treated bone marrow-derived dendritic cells induce immune tolerance through modulating differentiation of CD4+ regulatory T cell subpopulations mediated by 3G11 and CD127.

    PubMed

    Zhou, Fang; Zhang, Guang-Xian; Rostami, Abdolmohamad

    2017-06-01

    Intravenous transfer of LPS-treated bone marrow-derived dendritic cells blocks development of autoimmunity induced by CD4 + T cells in vivo. However, cellular mechanisms of dendritic cell-mediated immune tolerance have not yet been fully elucidated. Here, we report that there are two new subpopulations of CD4 + CD25 + FoxP3 + GITR + regulatory T cells (CD127 + 3G11 + and CD127 + 3G11 - cells). LPS-treated dendritic cells facilitate development of CD4 + CD127 + 3G11 - regulatory T cells but inhibit that of CD4 + CD127 + 3G11 + regulatory T cells. LPS-induced tolerogenic dendritic cells may cause immune tolerance through modulating balance of different subsets of CD4 + regulatory T cells mediated by CD127 and 3G11. Our results imply a new potential cellular mechanism of dendritic cell-mediated immune tolerance.

  2. Influence of the stringent control system on the transcription of ribosomal ribonucleic acid and ribosomal protein genes in Escherichia coli.

    PubMed Central

    Dennis, P P

    1977-01-01

    The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relA1 relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.5 degrees C to the semipermissive temperature of 35.5 degrees C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorpoarted 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages gammaspc1, which carries about 15 r-protein genes and lambdailv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is r protein mRNA and rRNA; in the rel+ strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate. PMID:320185

  3. Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale

    PubMed Central

    Michel, Audrey M; Baranov, Pavel V

    2013-01-01

    Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. © 2013 John Wiley & Sons, Ltd. PMID:23696005

  4. Eukaryotic ribosome display with in situ DNA recovery.

    PubMed

    He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

    2012-01-01

    Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

  5. Increasing Academic Growth through Motivating Students To Read.

    ERIC Educational Resources Information Center

    Duignan, Sandra; Klioris, Ann; Porter, Jennifer; Rockett, Nicole; Vogwill, Kathy

    This report describes a program for increasing academic growth through motivating students to read. The targeted population includes kindergarten, first, third, and high school special education students. The lack of motivation in reading was documented through data revealed by pre-surveys and post-surveys of students' interest in books. Analysis…

  6. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

    PubMed Central

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

  7. Promoting toddlers' vegetable consumption through interactive reading and puppetry.

    PubMed

    de Droog, Simone M; van Nee, Roselinde; Govers, Mieke; Buijzen, Moniek

    2017-09-01

    Picture books with characters that promote healthy eating are increasingly being used to make this behavior more attractive. The first aim of this study was to investigate whether the effect of vegetable-promoting picture books on toddlers' vegetable consumption differed according to the reading style and the use of a hand puppet during reading. The second aim was to investigate whether these effects were mediated by toddlers' narrative involvement and character imitation. In a 2 (reading style: interactive vs. passive) x 2 (puppet use: with vs. without puppet) between-subjects design, 163 toddlers (2-3 years) were randomly assigned to one of the four reading conditions. The story was about a rabbit that loves to eat carrots. After the fourth reading day, the eating task was conducted in which children could eat freely from four different snacks, including carrots. The main finding was that interactive reading produced the greatest carrot consumption. The explanation for this effect was that interactive reading stimulated toddlers to imitate poses of the book characters, even more when interactive reading was supported by the use of a hand puppet. The findings underline that young children should be actively involved with health interventions in order for them to be effective. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Home environmental influences on children's language and reading skills in a genetically sensitive design: Are socioeconomic status and home literacy environment environmental mediators and moderators?

    PubMed

    Chow, Bonnie Wing-Yin; Ho, Connie Suk-Han; Wong, Simpson W L; Waye, Mary M Y; Zheng, Mo

    2017-12-01

    This twin study examined how family socioeconomic status (SES) and home literacy environment (HLE) contributes to Chinese language and reading skills. It included 312 Chinese twin pairs aged 3 to 11. Children were individually administered tasks of Chinese word reading, receptive vocabulary and reading-related cognitive skills, and nonverbal reasoning ability. Information on home environment was collected through parent-reported questionnaires. Results showed that SES and HLE mediated shared environmental influences but did not moderate genetic influences on general language and reading abilities. Also, SES and HLE mediated shared environmental contributions to receptive vocabulary and syllable and rhyme awareness, but not orthographic skills. The findings of this study add to past twin studies that focused on alphabetic languages, suggesting that these links could be universal across languages. They also extend existing findings on SES and HLE's contributions to reading-related cognitive skills. © 2017 Scandinavian Psychological Associations and John Wiley & Sons Ltd.

  9. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

    PubMed

    Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

    2014-10-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. 40 CFR 418.61 - Specialized definitions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false Specialized definitions. 418.61 Section 418.61 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate Production Subcategory...

  11. Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana

    PubMed Central

    Merret, Rémy; Nagarajan, Vinay K.; Carpentier, Marie-Christine; Park, Sunhee; Favory, Jean-Jacques; Descombin, Julie; Picart, Claire; Charng, Yee-yung; Green, Pamela J.; Deragon, Jean-Marc; Bousquet-Antonelli, Cécile

    2015-01-01

    The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5′-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5′-ribosome pausing leading to the XRN4-mediated 5′-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘non-aberrant’ mRNAs. PMID:25845591

  12. Ribosomal RNA and ribosomal proteins in corynebacteria.

    PubMed

    Martín, Juan F; Barreiro, Carlos; González-Lavado, Eva; Barriuso, Mónica

    2003-09-04

    Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.

  13. 48 CFR 9904.418-60 - Illustrations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Section 9904.418-60 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD, OFFICE OF FEDERAL PROCUREMENT POLICY, OFFICE OF MANAGEMENT AND BUDGET PROCUREMENT PRACTICES AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.418-60 Illustrations. (a) Business Unit A has various...

  14. 7 CFR 58.418 - Automatic cheese making equipment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 3 2011-01-01 2011-01-01 false Automatic cheese making equipment. 58.418 Section 58.418 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING... Service 1 Equipment and Utensils § 58.418 Automatic cheese making equipment. (a) Automatic Curd Maker. The...

  15. 7 CFR 58.418 - Automatic cheese making equipment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Automatic cheese making equipment. 58.418 Section 58.418 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING... Service 1 Equipment and Utensils § 58.418 Automatic cheese making equipment. (a) Automatic Curd Maker. The...

  16. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility.

    PubMed

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-10-15

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA(II) enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA(II), rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA(II) in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA(II) activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA(II), thereby facilitating TEL binding to the ribosome. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Modular Assembly of the Bacterial Large Ribosomal Subunit

    PubMed Central

    Davis, Joseph H.; Tan, Yong Zi; Carragher, Bridget; Potter, Clinton S.; Lyumkis, Dmitry; Williamson, James R.

    2016-01-01

    SUMMARY The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ~4–5Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be ‘re-routed’ through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines. PMID:27912064

  18. Parametric mediational g-formula approach to mediation analysis with time-varying exposures, mediators, and confounders

    PubMed Central

    Lin, Sheng-Hsuan; Young, Jessica; Logan, Roger; Tchetgen Tchetgen, Eric J.; VanderWeele, Tyler J.

    2016-01-01

    The assessment of direct and indirect effects with time-varying mediators and confounders is a common but challenging problem, and standard mediation analysis approaches are generally not applicable in this context. The mediational g-formula was recently proposed to address this problem, paired with a semi-parametric estimation approach to evaluate longitudinal mediation effects empirically. In this paper, we develop a parametric estimation approach to the mediational g-formula, including a feasible algorithm implemented in a freely available SAS macro. In the Framingham Heart Study data, we apply this method to estimate the interventional analogues of natural direct and indirect effects of smoking behaviors sustained over a 10-year period on blood pressure when considering weight change as a time-varying mediator. Compared with not smoking, smoking 20 cigarettes per day for 10 years was estimated to increase blood pressure by 1.2 (95 % CI: −0.7, 2.7) mm-Hg. The direct effect was estimated to increase blood pressure by 1.5 (95 % CI: −0.3, 2.9) mm-Hg, and the indirect effect was −0.3 (95% CI: −0.5, −0.1) mm-Hg, which is negative because smoking which is associated with lower weight is associated in turn with lower blood pressure. These results provide evidence that weight change in fact partially conceals the detrimental effects of cigarette smoking on blood pressure. Our work represents, to our knowledge, the first application of the parametric mediational g-formula in an epidemiologic cohort study. PMID:27984420

  19. Teachers' Theoretical Orientations toward Reading and Pupil Control Ideology: A National Survey.

    ERIC Educational Resources Information Center

    Morrison, Timothy G.; Wilcox, Brad; Madrigal, J. L.; Roberts, Susan; Hintze, Eric

    1999-01-01

    Examines relationships between 418 elementary school teachers' theoretical beliefs toward reading instruction and their attitudes about pupil control. Uses the Theoretical Orientation to Reading Profile (TORP) and the Pupil Control Ideology Form (PCI) for data collection. Finds as teachers' scores moved toward the whole language end of the TORP…

  20. 43 CFR 418.26 - Charges for water use.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 1 2011-10-01 2011-10-01 false Charges for water use. 418.26 Section 418... and Management § 418.26 Charges for water use. The District must maintain a financing and accounting... provides reasonable financial incentives for the economical and efficient use of water. ...

  1. 43 CFR 418.26 - Charges for water use.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Charges for water use. 418.26 Section 418... and Management § 418.26 Charges for water use. The District must maintain a financing and accounting... provides reasonable financial incentives for the economical and efficient use of water. ...

  2. 43 CFR 418.26 - Charges for water use.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 1 2012-10-01 2011-10-01 true Charges for water use. 418.26 Section 418... and Management § 418.26 Charges for water use. The District must maintain a financing and accounting... provides reasonable financial incentives for the economical and efficient use of water. ...

  3. 43 CFR 418.26 - Charges for water use.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 1 2013-10-01 2013-10-01 false Charges for water use. 418.26 Section 418... and Management § 418.26 Charges for water use. The District must maintain a financing and accounting... provides reasonable financial incentives for the economical and efficient use of water. ...

  4. 40 CFR 418.31 - Specialized definitions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true Specialized definitions. 418.31 Section... STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.31 Specialized definitions. For the purposes of this subpart: (a) Except as provided below, the general definitions, abbreviations...

  5. Reading and Grammar Learning through Mobile Phones

    ERIC Educational Resources Information Center

    Wang, Shudong; Smith, Simon

    2013-01-01

    This paper describes an ongoing language-learning project, three years into its development. We examine both the feasibility and the limitations of developing English reading and grammar skills through the interface of mobile phones. Throughout the project, reading and grammar materials were regularly sent to students' mobile phones. Students read…

  6. Adult Reading, Rated G.

    ERIC Educational Resources Information Center

    Lewis, Anne C.

    2003-01-01

    Suggests books for summer reading for Department of Education officials involved in closing all 16 ERIC Clearinghouses or touting the superiority of a Christian education, standardized test-makers, planners of the Gates Foundation, and the creator of the Algebra Project. For example, suggests that standardized test-makers read William Bennett's…

  7. Acquisition of Three Word Knowledge Aspects through Reading

    ERIC Educational Resources Information Center

    Daskalovska, Nina

    2016-01-01

    A number of studies have shown that second or foreign language learners can acquire vocabulary through reading. The aim of the study was to investigate (a) the effects of reading an authentic novel on the acquisition of 3 aspects of word knowledge: spelling, meaning, and collocation; (b) the influence of reading on the acquisition of partial and…

  8. 20 CFR 418.2105 - What is the threshold?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false What is the threshold? 418.2105 Section 418.2105 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income-Related Monthly Adjustments to Medicare Prescription Drug Coverage Premiums Determination of the Income-Related Monthly Adjustment Amount § 418.2105 What is the...

  9. 20 CFR 418.2105 - What is the threshold?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false What is the threshold? 418.2105 Section 418.2105 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income-Related Monthly Adjustments to Medicare Prescription Drug Coverage Premiums Determination of the Income-Related Monthly Adjustment Amount § 418.2105 What is the...

  10. 20 CFR 418.2105 - What is the threshold?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false What is the threshold? 418.2105 Section 418.2105 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income-Related Monthly Adjustments to Medicare Prescription Drug Coverage Premiums Determination of the Income-Related Monthly Adjustment Amount § 418.2105 What is the...

  11. Ribosomal vaccines. I. Immunogenicity of ribosomal fractions isolated from Salmonella typhimurium and Yersinia pestis.

    PubMed

    Johnson, W

    1972-06-01

    The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein.

  12. How logical reasoning mediates the relation between lexical quality and reading comprehension.

    PubMed

    Segers, Eliane; Verhoeven, Ludo

    The present study aimed to examine the role of logical reasoning in the relation between lexical quality and reading comprehension in 146 fourth grade Dutch children. We assessed their standardized reading comprehension measure, along with their decoding efficiency and vocabulary as measures of lexical quality, syllogistic reasoning as measure of (verbal) logical reasoning, and nonverbal reasoning as a control measure. Syllogistic reasoning was divided into a measure tapping basic, coherence inferencing skill using logical syllogisms, and a measure tapping elaborative inferencing skill using indeterminate syllogisms. Results showed that both types of syllogisms partly mediated the relation between lexical quality and reading comprehension, but also had a unique additional effect on reading comprehension. The indirect effect of lexical quality on reading comprehension via syllogisms was driven by vocabulary knowledge. It is concluded that measures of syllogistic reasoning account for higher-order thinking processes that are needed to make inferences in reading comprehension. The role of lexical quality appears to be pivotal in explaining the variation in reading comprehension both directly and indirectly via syllogistic reasoning.

  13. Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease.

    PubMed

    Büttner, Sabrina; Habernig, Lukas; Broeskamp, Filomena; Ruli, Doris; Vögtle, F Nora; Vlachos, Manolis; Macchi, Francesca; Küttner, Victoria; Carmona-Gutierrez, Didac; Eisenberg, Tobias; Ring, Julia; Markaki, Maria; Taskin, Asli Aras; Benke, Stefan; Ruckenstuhl, Christoph; Braun, Ralf; Van den Haute, Chris; Bammens, Tine; van der Perren, Anke; Fröhlich, Kai-Uwe; Winderickx, Joris; Kroemer, Guido; Baekelandt, Veerle; Tavernarakis, Nektarios; Kovacs, Gabor G; Dengjel, Jörn; Meisinger, Chris; Sigrist, Stephan J; Madeo, Frank

    2013-11-27

    Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

  14. Immunogenic Activity of a Ribosomal Fraction Obtained from Mycobacterium tuberculosis

    PubMed Central

    Youmans, Anne S.; Youmans, Guy P.

    1965-01-01

    Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Immunogenic activity of a ribosomal fraction obtained from Mycobacterium tuberculosis. J. Bacteriol. 89:1291–1298. 1965.—The highly immunogenic particulate fraction obtained from mechanically ruptured cells of the H37Ra strain of Mycobacterium tuberculosis was suspended and centrifuged at 20,360 × g. The supernatant liquid from this centrifugation was centrifuged at 56,550 × g to remove the larger particles, and the supernatant liquid from this was centrifuged at 144,000 × g to obtain a ribosomal fraction. The sediments from the first two centrifugations were highly immunogenic, but the ribosomal fraction showed only slight capacity to immunize mice. However, when the ribosomal fraction was mixed with Freund's incomplete adjuvant, the immunogenic activity was equivalent to the particulate fraction from which it was prepared. To test the hypothesis that some membranous substance in the particulate fraction was acting as an adjuvant for the smaller particles in the ribosomal fraction, portions of the particulate fraction were treated separately with each of the membrane-disrupting agents, sodium deoxycholate, sodium lauryl sulfate, and 1 m sodium chloride. The treated materials were then centrifuged at 144,000 × g, and the sediments were tested for immunogenicity both with and without the addition of Freund's incomplete adjuvant. Without the adjuvant, the immunizing activities were very weak or absent; with the adjuvant, they were equivalent to that of the particulate fraction from which they were prepared. Other factors which have been found to damage or destroy membranes, such as freezing and thawing, and heat, also significantly decreased the immunogenic activity of the particulate fraction unless it was incorporated into Freund's incomplete adjuvant. The larger particles which sedimented at 56,550 × g were also treated with sodium lauryl sulfate and sodium

  15. 48 CFR 9904.418-20 - Purpose.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Purpose. 9904.418-20... FEDERAL PROCUREMENT POLICY, OFFICE OF MANAGEMENT AND BUDGET PROCUREMENT PRACTICES AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.418-20 Purpose. The purpose of this Cost Accounting Standard is to...

  16. Ribosomal Vaccines I. Immunogenicity of Ribosomal Fractions Isolated from Salmonella typhimurium and Yersinia pestis

    PubMed Central

    Johnson, William

    1972-01-01

    The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein. Images PMID:4564407

  17. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy

    PubMed Central

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2014-01-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knockin mice expressing non-phosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knockin mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knockin mice compared to their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knockin mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knockin and wild type mice, indicating that mTORC1 was still activated in the knockin mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knockin mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth. PMID:25229342

  18. 42 CFR 418.25 - Admission to hospice care.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Admission to hospice care. 418.25 Section 418.25... (CONTINUED) MEDICARE PROGRAM HOSPICE CARE Eligibility, Election and Duration of Benefits § 418.25 Admission to hospice care. (a) The hospice admits a patient only on the recommendation of the medical director...

  19. Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

    PubMed Central

    2016-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth. PMID:25706898

  20. Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism.

    PubMed

    Li, Ruixi; Sun, Ruobai; Hicks, Glenn R; Raikhel, Natasha V

    2015-01-06

    The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

  1. 20 CFR 418.1105 - What is the threshold?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false What is the threshold? 418.1105 Section 418.1105 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part B Income-Related Monthly Adjustment Amount Determination of the Income-Related Monthly Adjustment Amount § 418.1105 What is the threshold? (a) The threshold is a...

  2. 20 CFR 418.1105 - What is the threshold?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false What is the threshold? 418.1105 Section 418.1105 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part B Income-Related Monthly Adjustment Amount Determination of the Income-Related Monthly Adjustment Amount § 418.1105 What is the threshold? (a) The threshold is a...

  3. 20 CFR 418.1105 - What is the threshold?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false What is the threshold? 418.1105 Section 418.1105 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part B Income-Related Monthly Adjustment Amount Determination of the Income-Related Monthly Adjustment Amount § 418.1105 What is the threshold? (a) The threshold is a...

  4. 20 CFR 418.1105 - What is the threshold?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false What is the threshold? 418.1105 Section 418.1105 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part B Income-Related Monthly Adjustment Amount Determination of the Income-Related Monthly Adjustment Amount § 418.1105 What is the threshold? (a) The threshold is a...

  5. The ribosome as a missing link in the evolution of life.

    PubMed

    Root-Bernstein, Meredith; Root-Bernstein, Robert

    2015-02-21

    Many steps in the evolution of cellular life are still mysterious. We suggest that the ribosome may represent one important missing link between compositional (or metabolism-first), RNA-world (or genes-first) and cellular (last universal common ancestor) approaches to the evolution of cells. We present evidence that the entire set of transfer RNAs for all twenty amino acids are encoded in both the 16S and 23S rRNAs of Escherichia coli K12; that nucleotide sequences that could encode key fragments of ribosomal proteins, polymerases, ligases, synthetases, and phosphatases are to be found in each of the six possible reading frames of the 16S and 23S rRNAs; and that every sequence of bases in rRNA has information encoding more than one of these functions in addition to acting as a structural component of the ribosome. Ribosomal RNA, in short, is not just a structural scaffold for proteins, but the vestigial remnant of a primordial genome that may have encoded a self-organizing, self-replicating, auto-catalytic intermediary between macromolecules and cellular life. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Regulation of the protein-conducting channel by a bound ribosome

    PubMed Central

    Gumbart, James; Trabuco, Leonardo G.; Schreiner, Eduard; Villa, Elizabeth; Schulten, Klaus

    2009-01-01

    Summary During protein synthesis, it is often necessary for the ribosome to form a complex with a membrane-bound channel, the SecY/Sec61 complex, in order to translocate nascent proteins across a cellular membrane. Structural data on the ribosome-channel complex are currently limited to low-resolution cryo-electron microscopy maps, including one showing a bacterial ribosome bound to a monomeric SecY complex. Using that map along with available atomic-level models of the ribosome and SecY, we have determined, through molecular dynamics flexible fitting (MDFF), an atomic-resolution model of the ribosome-channel complex. We characterized computationally the sites of ribosome-SecY interaction within the complex and determined the effect of ribosome binding on the SecY channel. We also constructed a model of a ribosome in complex with a SecY dimer by adding a second copy of SecY to the MDFF-derived model. The study involved 2.7-million-atom simulations over altogether nearly 50 ns. PMID:19913480

  7. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

    PubMed

    Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

    2014-01-01

    Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

  8. Unique localization of the plastid-specific ribosomal proteins in the chloroplast ribosome small subunit provides mechanistic insights into the chloroplastic translation

    PubMed Central

    Ahmed, Tofayel; Shi, Jian

    2017-01-01

    Abstract Chloroplastic translation is mediated by a bacterial-type 70S chloroplast ribosome. During the evolution, chloroplast ribosomes have acquired five plastid-specific ribosomal proteins or PSRPs (cS22, cS23, bTHXc, cL37 and cL38) which have been suggested to play important regulatory roles in translation. However, their exact locations on the chloroplast ribosome remain elusive due to lack of a high-resolution structure, hindering our progress to understand their possible roles. Here we present a cryo-EM structure of the 70S chloroplast ribosome from spinach resolved to 3.4 Å and focus our discussion mainly on the architecture of the 30S small subunit (SSU) which is resolved to 3.7 Å. cS22 localizes at the SSU foot where it seems to compensate for the deletions in 16S rRNA. The mRNA exit site is highly remodeled due to the presence of cS23 suggesting an alternative mode of translation initiation. bTHXc is positioned at the SSU head and appears to stabilize the intersubunit bridge B1b during thermal fluctuations. The translation factor plastid pY binds to the SSU on the intersubunit side and interacts with the conserved nucleotide bases involved in decoding. Most of the intersubunit bridges are conserved compared to the bacteria, except for a new bridge involving uL2c and bS6c. PMID:28582576

  9. Ribosomal elongation factor 4 promotes cell death associated with lethal stress.

    PubMed

    Li, Liping; Hong, Yuzhi; Luan, Gan; Mosel, Michael; Malik, Muhammad; Drlica, Karl; Zhao, Xilin

    2014-12-09

    Ribosomal elongation factor 4 (EF4) is highly conserved among bacteria, mitochondria, and chloroplasts. However, the EF4-encoding gene, lepA, is nonessential and its deficiency shows no growth or fitness defect. In purified systems, EF4 back-translocates stalled, posttranslational ribosomes for efficient protein synthesis; consequently, EF4 has a protective role during moderate stress. We were surprised to find that EF4 also has a detrimental role during severe stress: deletion of lepA increased Escherichia coli survival following treatment with several antimicrobials. EF4 contributed to stress-mediated lethality through reactive oxygen species (ROS) because (i) the protective effect of a ΔlepA mutation against lethal antimicrobials was eliminated by anaerobic growth or by agents that block hydroxyl radical accumulation and (ii) the ΔlepA mutation decreased ROS levels stimulated by antimicrobial stress. Epistasis experiments showed that EF4 functions in the same genetic pathway as the MazF toxin, a stress response factor implicated in ROS-mediated cell death. The detrimental action of EF4 required transfer-messenger RNA (tmRNA, which tags truncated proteins for degradation and is known to be inhibited by EF4) and the ClpP protease. Inhibition of a protective, tmRNA/ClpP-mediated degradative activity would allow truncated proteins to indirectly perturb the respiratory chain and thereby provide a potential link between EF4 and ROS. The connection among EF4, MazF, tmRNA, and ROS expands a pathway leading from harsh stress to bacterial self-destruction. The destructive aspect of EF4 plus the protective properties described previously make EF4 a bifunctional factor in a stress response that promotes survival or death, depending on the severity of stress. Translation elongation factor 4 (EF4) is one of the most conserved proteins in nature, but it is dispensable. Lack of strong phenotypes for its genetic knockout has made EF4 an enigma. Recent biochemical work has

  10. Modular Assembly of the Bacterial Large Ribosomal Subunit.

    PubMed

    Davis, Joseph H; Tan, Yong Zi; Carragher, Bridget; Potter, Clinton S; Lyumkis, Dmitry; Williamson, James R

    2016-12-01

    The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes.

    PubMed

    Schäfer, Thorsten; Strauss, Daniela; Petfalski, Elisabeth; Tollervey, David; Hurt, Ed

    2003-03-17

    Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (approximately 30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.

  12. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

    2007-08-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as amore » Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.« less

  13. PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration

    PubMed Central

    Janjanam, Jagadeesh; Chandaka, Giri Kumar; Kotla, Sivareddy; Rao, Gadiparthi N.

    2015-01-01

    Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein–coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδ activation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin–WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration. PMID:26490115

  14. Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development

    PubMed Central

    Zhao, Chengtian; Andreeva, Viktoria; Gibert, Yann; LaBonty, Melissa; Lattanzi, Victoria; Prabhudesai, Shubhangi; Zhou, Yi; Zon, Leonard; McCann, Kathleen L.; Baserga, Susan; Yelick, Pamela C.

    2014-01-01

    During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome. PMID:24497835

  15. Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development.

    PubMed

    Zhao, Chengtian; Andreeva, Viktoria; Gibert, Yann; LaBonty, Melissa; Lattanzi, Victoria; Prabhudesai, Shubhangi; Zhou, Yi; Zon, Leonard; McCann, Kathleen L; Baserga, Susan; Yelick, Pamela C

    2014-01-01

    During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

  16. 40 CFR 418.36 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true Pretreatment standards for new sources. 418.36 Section 418.36 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.36...

  17. 40 CFR 418.36 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false Pretreatment standards for new sources. 418.36 Section 418.36 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.36...

  18. 40 CFR 418.36 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true Pretreatment standards for new sources. 418.36 Section 418.36 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.36...

  19. 40 CFR 418.16 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false Pretreatment standards for new sources. 418.16 Section 418.16 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.16...

  20. 40 CFR 418.16 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true Pretreatment standards for new sources. 418.16 Section 418.16 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.16...

  1. 40 CFR 418.16 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true Pretreatment standards for new sources. 418.16 Section 418.16 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.16...

  2. 40 CFR 418.36 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true Pretreatment standards for new sources. 418.36 Section 418.36 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Urea Subcategory § 418.36...

  3. 40 CFR 418.16 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true Pretreatment standards for new sources. 418.16 Section 418.16 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.16...

  4. 40 CFR 418.16 - Pretreatment standards for new sources.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true Pretreatment standards for new sources. 418.16 Section 418.16 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate Subcategory § 418.16...

  5. Ribosome protection by antibiotic resistance ATP-binding cassette protein.

    PubMed

    Su, Weixin; Kumar, Veerendra; Ding, Yichen; Ero, Rya; Serra, Aida; Lee, Benjamin Sian Teck; Wong, Andrew See Weng; Shi, Jian; Sze, Siu Kwan; Yang, Liang; Gao, Yong-Gui

    2018-05-15

    The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins. Copyright © 2018 the Author(s). Published by PNAS.

  6. sORFs.org: a repository of small ORFs identified by ribosome profiling.

    PubMed

    Olexiouk, Volodimir; Crappé, Jeroen; Verbruggen, Steven; Verhegen, Kenneth; Martens, Lennart; Menschaert, Gerben

    2016-01-04

    With the advent of ribosome profiling, a next generation sequencing technique providing a "snap-shot'' of translated mRNA in a cell, many short open reading frames (sORFs) with ribosomal activity were identified. Follow-up studies revealed the existence of functional peptides, so-called micropeptides, translated from these 'sORFs', indicating a new class of bio-active peptides. Over the last few years, several micropeptides exhibiting important cellular functions were discovered. However, ribosome occupancy does not necessarily imply an actual function of the translated peptide, leading to the development of various tools assessing the coding potential of sORFs. Here, we introduce sORFs.org (http://www.sorfs.org), a novel database for sORFs identified using ribosome profiling. Starting from ribosome profiling, sORFs.org identifies sORFs, incorporates state-of-the-art tools and metrics and stores results in a public database. Two query interfaces are provided, a default one enabling quick lookup of sORFs and a BioMart interface providing advanced query and export possibilities. At present, sORFs.org harbors 263 354 sORFs that demonstrate ribosome occupancy, originating from three different cell lines: HCT116 (human), E14_mESC (mouse) and S2 (fruit fly). sORFs.org aims to provide an extensive sORFs database accessible to researchers with limited bioinformatics knowledge, thus enabling easy integration into personal projects. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. sORFs.org: a repository of small ORFs identified by ribosome profiling

    PubMed Central

    Olexiouk, Volodimir; Crappé, Jeroen; Verbruggen, Steven; Verhegen, Kenneth; Martens, Lennart; Menschaert, Gerben

    2016-01-01

    With the advent of ribosome profiling, a next generation sequencing technique providing a “snap-shot’’ of translated mRNA in a cell, many short open reading frames (sORFs) with ribosomal activity were identified. Follow-up studies revealed the existence of functional peptides, so-called micropeptides, translated from these ‘sORFs’, indicating a new class of bio-active peptides. Over the last few years, several micropeptides exhibiting important cellular functions were discovered. However, ribosome occupancy does not necessarily imply an actual function of the translated peptide, leading to the development of various tools assessing the coding potential of sORFs. Here, we introduce sORFs.org (http://www.sorfs.org), a novel database for sORFs identified using ribosome profiling. Starting from ribosome profiling, sORFs.org identifies sORFs, incorporates state-of-the-art tools and metrics and stores results in a public database. Two query interfaces are provided, a default one enabling quick lookup of sORFs and a BioMart interface providing advanced query and export possibilities. At present, sORFs.org harbors 263 354 sORFs that demonstrate ribosome occupancy, originating from three different cell lines: HCT116 (human), E14_mESC (mouse) and S2 (fruit fly). sORFs.org aims to provide an extensive sORFs database accessible to researchers with limited bioinformatics knowledge, thus enabling easy integration into personal projects. PMID:26527729

  8. 42 CFR 418.104 - Condition of participation: Clinical records.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Condition of participation: Clinical records. 418.104 Section 418.104 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...: Organizational Environment § 418.104 Condition of participation: Clinical records. A clinical record containing...

  9. 42 CFR 418.102 - Condition of participation: Medical director.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Condition of participation: Medical director. 418.102 Section 418.102 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...: Organizational Environment § 418.102 Condition of participation: Medical director. The hospice must designate a...

  10. 42 CFR 418.102 - Condition of participation: Medical director.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Condition of participation: Medical director. 418.102 Section 418.102 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...: Organizational Environment § 418.102 Condition of participation: Medical director. The hospice must designate a...

  11. 42 CFR 418.104 - Condition of participation: Clinical records.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Condition of participation: Clinical records. 418.104 Section 418.104 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...: Organizational Environment § 418.104 Condition of participation: Clinical records. A clinical record containing...

  12. 42 CFR 418.104 - Condition of participation: Clinical records.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Condition of participation: Clinical records. 418.104 Section 418.104 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...: Organizational Environment § 418.104 Condition of participation: Clinical records. A clinical record containing...

  13. 42 CFR 418.102 - Condition of participation: Medical director.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Condition of participation: Medical director. 418.102 Section 418.102 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...: Organizational Environment § 418.102 Condition of participation: Medical director. The hospice must designate a...

  14. Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

    PubMed

    Finch, Leanne K; Ling, Roger; Napthine, Sawsan; Olspert, Allan; Michiels, Thomas; Lardinois, Cécile; Bell, Susanne; Loughran, Gary; Brierley, Ian; Firth, Andew E

    2015-08-01

    Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed-1 ribosomal frameshift (1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that-1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3= RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient-1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.

  15. 45 CFR 160.418 - Penalty not exclusive.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Penalty not exclusive. 160.418 Section 160.418 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES ADMINISTRATIVE DATA STANDARDS AND RELATED... addition to any other penalty prescribed by law. ...

  16. READING FOR THE GIFTED--GUIDED EXTENSION OF READING SKILLS THROUGH LITERATURE. PART 2, APPRECIATING THE CONTRIBUTIONS OF SCIENCE THROUGH BIOGRAPHY...

    ERIC Educational Resources Information Center

    PENROSE, ROBERT; AND OTHERS

    THIS TEACHING GUIDE IS DESIGNED FOR USE WITH GIFTED PUPILS AT GRADES FIVE AND SIX WHO ARE READING TWO OR MORE LEVELS ABOVE THEIR GRADE PLACEMENT. THE GUIDE ALSO PROVIDES GUIDANCE FOR THE STUDY OF BIOGRAPHY THROUGH SCIENCE LITERATURE. SUCH READING SKILLS AS ANALYZING THE AUTHOR'S PURPOSE, HIS ORGANIZATION, PERSONALITY AND STYLE, UNDERSTANDING…

  17. 43 CFR 418.6 - Fallon Paiute-Shoshone Indian Reservation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 1 2013-10-01 2013-10-01 false Fallon Paiute-Shoshone Indian Reservation. 418.6 Section 418.6 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION... General Provisions § 418.6 Fallon Paiute-Shoshone Indian Reservation. Nothing in this part affects: (a...

  18. 43 CFR 418.6 - Fallon Paiute-Shoshone Indian Reservation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 1 2011-10-01 2011-10-01 false Fallon Paiute-Shoshone Indian Reservation. 418.6 Section 418.6 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION... General Provisions § 418.6 Fallon Paiute-Shoshone Indian Reservation. Nothing in this part affects: (a...

  19. 43 CFR 418.6 - Fallon Paiute-Shoshone Indian Reservation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Fallon Paiute-Shoshone Indian Reservation. 418.6 Section 418.6 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION... General Provisions § 418.6 Fallon Paiute-Shoshone Indian Reservation. Nothing in this part affects: (a...

  20. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Before They Read: Teaching Language and Literacy Development through Conversations, Interactive Read-Alouds, and Listening Games

    ERIC Educational Resources Information Center

    Miller, Cathy Puett

    2010-01-01

    Preschool and kindergarten educators know that strong oral language skills must be in place before children can learn to read. In "Before They Read: Teaching Language and Literacy Development through Conversations, Interactive Read-Alouds, and Listening Games," Cathy Puett Miller helps educators teach those early literacy skills with engaging…

  2. Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.

    The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red stainingmore » suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.« less

  3. Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

    DOE PAGES

    Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.; ...

    2014-12-22

    The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red stainingmore » suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.« less

  4. Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.

    PubMed

    Kumar, Parimal; Hellen, Christopher U T; Pestova, Tatyana V

    2016-07-01

    Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5'-terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5' end of mRNA, implying that Met-tRNAi (Met) inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

  5. The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

    PubMed

    Yamaguchi, K; von Knoblauch, K; Subramanian, A R

    2000-09-15

    translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

  6. The essential nature of YqfG, a YbeY homologue required for 3' maturation of Bacillus subtilis 16S ribosomal RNA is suppressed by deletion of RNase R.

    PubMed

    Baumgardt, Kathrin; Gilet, Laetitia; Figaro, Sabine; Condon, Ciarán

    2018-06-05

    Ribosomal RNAs are processed from primary transcripts containing 16S, 23S and 5S rRNAs in most bacteria. Maturation generally occurs in a two-step process, consisting of a first crude separation of the major species by RNase III during transcription, followed by precise trimming of 5' and 3' extensions on each species upon accurate completion of subunit assembly. The various endo- and exoribonucleases involved in the final processing reactions are strikingly different in Escherichia coli and Bacillus subtilis, the two best studied representatives of Gram-negative and Gram-positive bacteria, respectively. Here, we show that the one exception to this rule is the protein involved in the maturation of the 3' end of 16S rRNA. Cells depleted for the essential B. subtilis YqfG protein, a homologue of E. coli YbeY, specifically accumulate 16S rRNA precursors bearing 3' extensions. Remarkably, the essential nature of YqfG can be suppressed by deleting the ribosomal RNA degrading enzyme RNase R, i.e. a ΔyqfG Δrnr mutant is viable. Our data suggest that 70S ribosomes containing 30S subunits with 3' extensions of 16S rRNA are functional to a degree, but become substrates for degradation by RNase R and are eliminated.

  7. Zinc-finger protein 418 overexpression protects against cardiac hypertrophy and fibrosis

    PubMed Central

    Huang, Zirui; Zhu, Zhilin; Xu, Chunli; Teng, Lin; He, Ling; Gu, Chen; Yi, Cai

    2017-01-01

    Background This study aimed to investigated the effect and mechanism of zinc-finger protein 418 (ZNF418) on cardiac hypertrophy caused by aortic banding (AB), phenylephrine (PE) or angiotensin II (Ang II) in vivo and in vitro. Methods The expression of ZNF418 in hearts of patients with dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) and AB-induced cardiac hypertrophy mice, as well as in Ang II- or PE-induced hypertrophic primary cardiomyocytes was detected by western blotting. Then, the expression of ZNF418 was up-regulated or down-regulated in AB-induced cardiac hypertrophy mice and Ang II -induced hypertrophic primary cardiomyocytes. The hypertrophic responses and fibrosis were evaluated by echocardiography and histological analysis. The mRNA levels of hypertrophy markers and fibrotic markers were detected by RT-qPCR. Furthermore, the phosphorylation and total levels of c-Jun were measured by western blotting. Results ZNF418 was markedly down-regulated in hearts of cardiac hypertrophy and hypertrophic primary cardiomyocytes. Down-regulated ZNF418 exacerbated the myocyte size and fibrosis, moreover increased the mRNA levels of ANP, BNP, β-MHC, MCIP1.4, collagen 1a, collagen III, MMP-2 and fibronection in hearts of AB-treated ZNF418 knockout mice or Ang II-treated cardiomyocytes with AdshZNF418. Conversely, these hypertrophic responses were reduced in the ZNF418 transgenic (TG) mice treated by AB and the AdZNF418-transfected primary cardiomyocytes treated by Ang II. Additionally, the deficiency of ZNF418 enhanced the phosphorylation level of c-jun, and overexpression of ZNF418 suppressed the phosphorylation level of c-jun in vivo and in vitro. Conclusion ZNF418 maybe attenuate hypertrophic responses by inhibiting the activity of c-jun/AP-1. PMID:29065170

  8. 42 CFR 414.418 - Opportunity for networks.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Opportunity for networks. 414.418 Section 414.418 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... not supersede any Federal law or regulation that regulates anticompetitive behavior. (5) A bid...

  9. 42 CFR 414.418 - Opportunity for networks.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Opportunity for networks. 414.418 Section 414.418 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... not supersede any Federal law or regulation that regulates anticompetitive behavior. (5) A bid...

  10. Sequential protein association with nascent 60S ribosomal particles.

    PubMed

    Saveanu, Cosmin; Namane, Abdelkader; Gleizes, Pierre-Emmanuel; Lebreton, Alice; Rousselle, Jean-Claude; Noaillac-Depeyre, Jacqueline; Gas, Nicole; Jacquier, Alain; Fromont-Racine, Micheline

    2003-07-01

    Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

  11. Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli.

    PubMed Central

    Oehler, R; Polacek, N; Steiner, G; Barta, A

    1997-01-01

    Photolysis of [3H]tetracycline in the presence of Escherichia coli ribosomes results in an approximately 1:1 ratio of labelling ribosomal proteins and RNAs. In this work we characterize crosslinks to both 16S and 23S RNAs. Previously, the main target of photoincorporation of [3H]tetracycline into ribosomal proteins was shown to be S7, which is also part of the one strong binding site of tetracycline on the 30S subunit. The crosslinks on 23S RNA map exclusively to the central loop of domain V (G2505, G2576 and G2608) which is part of the peptidyl transferase region. However, experiments performed with chimeric ribosomal subunits demonstrate that peptidyltransferase activity is not affected by tetracycline crosslinked solely to the 50S subunits. Three different positions are labelled on the 16S RNA, G693, G1300 and G1338. The positions of these crosslinked nucleotides correlate well with footprints on the 16S RNA produced either by tRNA or the protein S7. This suggests that the nucleotides are labelled by tetracycline bound to the strong binding site on the 30S subunit. In addition, our results demonstrate that the well known inhibition of tRNA binding to the A-site is solely due to tetracycline crosslinked to 30S subunits and furthermore suggest that interactions of the antibiotic with 16S RNA might be involved in its mode of action. PMID:9092632

  12. Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

    PubMed Central

    Ho, Jennifer Hei-Ngam; Kallstrom, George; Johnson, Arlen W.

    2000-01-01

    In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway. PMID:11086007

  13. Art Conquers All? Herbert Read's "Education through Art"

    ERIC Educational Resources Information Center

    Barchana-Lorand, Dorit

    2015-01-01

    Herbert Read's "Education through Art" (henceforth ETA) is a pioneering attempt to provide empirical evidence for the need for art in the public school system. Rooting for art education, Read applies the conclusions of the newly evolving psychological research to his thesis on education, which he holds to be a contemporary revival of…

  14. 42 CFR 418.309 - Hospice cap amount.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... change in the medical care expenditure category of the Consumer Price Index (CPI) for urban consumers... 42 Public Health 3 2010-10-01 2010-10-01 false Hospice cap amount. 418.309 Section 418.309 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

  15. 20 CFR 418.3401 - What are resources?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false What are resources? 418.3401 Section 418.3401 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part D Subsidies Resources... individual owns and could convert to cash to be used for his or her support and maintenance. ...

  16. 20 CFR 418.3401 - What are resources?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false What are resources? 418.3401 Section 418.3401 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part D Subsidies Resources... individual owns and could convert to cash to be used for his or her support and maintenance. ...

  17. Teaching Reading Comprehension through Collaborative Strategic Reading.

    ERIC Educational Resources Information Center

    Vaughn, Sharon; Klingner, Janette Kettman

    1999-01-01

    Provides an overview of collaborative strategic reading (CSR) as an approach to enhancing the reading-comprehension skills of students with learning disabilities. Procedures for implementing CSR with collaborative groups and techniques for teaching reading-comprehension skills are provided. The role of the teacher is described and sample teaching…

  18. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    NASA Astrophysics Data System (ADS)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

  19. 40 CFR 418.20 - Applicability; description of the ammonia subcategory.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ammonia subcategory. 418.20 Section 418.20 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.20 Applicability; description of the ammonia subcategory. The provisions of this subpart are...

  20. 40 CFR 418.20 - Applicability; description of the ammonia subcategory.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ammonia subcategory. 418.20 Section 418.20 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.20 Applicability; description of the ammonia subcategory. The provisions of this subpart are...

  1. 40 CFR 418.20 - Applicability; description of the ammonia subcategory.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ammonia subcategory. 418.20 Section 418.20 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.20 Applicability; description of the ammonia subcategory. The provisions of this subpart are...

  2. 40 CFR 418.20 - Applicability; description of the ammonia subcategory.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ammonia subcategory. 418.20 Section 418.20 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.20 Applicability; description of the ammonia subcategory. The provisions of this subpart are...

  3. 40 CFR 418.20 - Applicability; description of the ammonia subcategory.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ammonia subcategory. 418.20 Section 418.20 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.20 Applicability; description of the ammonia subcategory. The provisions of this subpart are...

  4. Ribosome A and P sites revealed by length analysis of ribosome profiling data

    PubMed Central

    Martens, Andrew T.; Taylor, James; Hilser, Vincent J.

    2015-01-01

    The high-throughput sequencing of nuclease-protected mRNA fragments bound to ribosomes, a technique known as ribosome profiling, quantifies the relative frequencies with which different regions of transcripts are translated. This technique has revealed novel translation initiation sites with unprecedented scope and has furthered investigations into the connections between codon biases and translation rates. Yet the location of the codon being decoded in ribosome footprints is still unknown, and has been complicated by the recent observation of footprints with non-canonical lengths. Here we show how taking into account the variations in ribosome footprint lengths can reveal the ribosome aminoacyl (A) and peptidyl (P) site locations. These location assignments are in agreement with the proposed mechanisms for various ribosome pauses and further enhance the resolution of the profiling data. We also show that GC-rich motifs at the 5′ ends of footprints are found in yeast, calling into question the anti-Shine-Dalgarno effect's role in ribosome pausing. PMID:25805170

  5. Analysis of immune responses in genital tracts of mice immunised with purified ribosomal fractions of Neisseria gonorrhoeae.

    PubMed Central

    Kita, E; Kashiba, S

    1984-01-01

    Immunisation of ddY mice with the purified ribosomal fraction of Neisseria gonorrhoeae was found to protect against intravaginal challenge with homologous organisms. This protection correlated with the presence of bactericidal antibody to purified ribosomal fraction in serum as well as in vaginal secretions. Analysis of the vaginal fluids from control mice and those immunised with purified ribosomal fraction showed that the enhanced elimination of gonococci in immune mice might be because of an early response of leucocytes generated by the reaction mediated by antibody and complement. Absorption studies showed that there was at least one major protective antigen in purified ribosomal fraction, other than cell surface substances such as lipopolysaccharide, outer membrane proteins, and pili. Bactericidal assays mediated by antibody and complement showed that matched samples of serum and vaginal fluid from immune mice had comparable gonococcidal activity, which was augmented by the effect of progesterone. Although delayed hypersensitivity was produced in immune mice that were resistant to N gonorrhoeae, the exact role of cellular immunity could not be clarified in this study. These results suggest that antibody to purified ribosomal fraction plays a major part in protection against gonococcal infection in the genital tract, and that such protection may entail both cellular immunity and hormonal changes. PMID:6430462

  6. 42 CFR 418.2 - Scope of part.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Scope of part. 418.2 Section 418.2 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE... policy. Subpart H specifies coinsurance amounts applicable to hospice care. [74 FR 39413, Aug. 6, 2009] ...

  7. Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

    PubMed

    Jha, Sujata; Rollins, Madeline G; Fuchs, Gabriele; Procter, Dean J; Hall, Elizabeth A; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N; Walsh, Derek

    2017-06-29

    Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

  8. Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    PubMed Central

    Sterk, Maaike; Romilly, Cédric; Wagner, E Gerhart H

    2018-01-01

    Abstract Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5′-tails—as standby sites—with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats—and to a lesser extent, AU-repeats—dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16–18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem–loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails. PMID:29420821

  9. Openness to Experience and Night-Sky Watching Interest as Predictors of Reading for Pleasure: Path Analysis of a Mediation Model

    ERIC Educational Resources Information Center

    Kelly, William E.

    2010-01-01

    The relation between reading for pleasure, night-sky watching interest, and openness to experience were examined in a sample of 129 college students. Results of a path analysis examining a mediation model indicated that the influence of night-sky interest on reading for pleasure was not mediated by the broad personality domain openness to…

  10. The Role of Trait Anxiety and Preoccupation with Reading Disabilities of Children and Their Mothers in Predicting Children's Reading Comprehension

    ERIC Educational Resources Information Center

    Blicher, Shira; Feingold, Liat; Shany, Michal

    2017-01-01

    This study investigated the relationship between reading comprehension (RC), trait anxiety, and preoccupation with reading disability (RD) in 88 school children in Grades 3 through 5 and in their mothers. Children's trait anxiety had a significant direct negative relationship with RC and also mediated the association between preoccupation with RD…

  11. Lupeol induces p53 and cyclin-B-mediated G2/M arrest and targets apoptosis through activation of caspase in mouse skin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nigam, Nidhi; Prasad, Sahdeo; George, Jasmine

    2009-04-03

    Lupeol, present in fruits and medicinal plants, is a biologically active compound that has been shown to have various pharmacological properties in experimental studies. In the present study, we demonstrated the modulatory effect of lupeol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced alterations on cell proliferation in the skin of Swiss albino mice. Lupeol treatment showed significant (p < 0.05) preventive effects with marked inhibition at 48, 72, and 96 h against DMBA-mediated neoplastic events. Cell-cycle analysis showed that lupeol-induced G2/M-phase arrest (16-37%) until 72 h, and these inhibitory effects were mediated through inhibition of the cyclin-B-regulated signaling pathway involving p53, p21/WAF1, cdc25C, cdc2,more » and cyclin-B gene expression. Further lupeol-induced apoptosis was observed, as shown by an increased sub-G1 peak (28%) at 96 h, with upregulation of bax and caspase-3 genes and downregulation of anti-apoptotic bcl-2 and survivin genes. Thus, our results indicate that lupeol has novel anti-proliferative and apoptotic potential that may be helpful in designing strategies to fight skin cancer.« less

  12. Ribosomal protein S14 transcripts are edited in Oenothera mitochondria.

    PubMed Central

    Schuster, W; Unseld, M; Wissinger, B; Brennicke, A

    1990-01-01

    The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci. Images PMID:2326162

  13. Improving EFL Learners' Reading Levels through Extensive Reading

    ERIC Educational Resources Information Center

    Mermelstein, Aaron David

    2014-01-01

    Today there is an increasing amount of research promoting the effectiveness of extensive reading (ER) towards increasing learners' vocabulary, comprehension, reading speed, and motivation towards reading. However, little has been done to measure the effects of ER on learners' reading levels. This quantitative study examined the effects of ER on…

  14. Free-Energy Landscape of Reverse tRNA Translocation through the Ribosome Analyzed by Electron Microscopy Density Maps and Molecular Dynamics Simulations

    PubMed Central

    Ishida, Hisashi; Matsumoto, Atsushi

    2014-01-01

    To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA. PMID:24999999

  15. Free-energy landscape of reverse tRNA translocation through the ribosome analyzed by electron microscopy density maps and molecular dynamics simulations.

    PubMed

    Ishida, Hisashi; Matsumoto, Atsushi

    2014-01-01

    To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA.

  16. Reading books with young deaf children: strategies for mediating between American Sign Language and English.

    PubMed

    Berke, Michele

    2013-01-01

    Research on shared reading has shown positive results on children's literacy development in general and for deaf children specifically; however, reading techniques might differ between these two populations. Families with deaf children, especially those with deaf parents, often capitalize on their children's visual attributes rather than primarily auditory cues. These techniques are believed to provide a foundation for their deaf children's literacy skills. This study examined 10 deaf mother/deaf child dyads with children between 3 and 5 years of age. Dyads were videotaped in their homes on at least two occasions reading books that were provided by the researcher. Descriptive analysis showed specifically how deaf mothers mediate between the two languages, American Sign Language (ASL) and English, while reading. These techniques can be replicated and taught to all parents of deaf children so that they can engage in more effective shared reading activities. Research has shown that shared reading, or the interaction of a parent and child with a book, is an effective way to promote language and literacy, vocabulary, grammatical knowledge, and metalinguistic awareness (Snow, 1983), making it critical for educators to promote shared reading activities at home between parent and child. Not all parents read to their children in the same way. For example, parents of deaf children may present the information in the book differently due to the fact that signed languages are visual rather than spoken. In this vein, we can learn more about what specific connections deaf parents make to the English print. Exploring strategies deaf mothers may use to link the English print through the use of ASL will provide educators with additional tools when working with all parents of deaf children. This article will include a review of the literature on the benefits of shared reading activities for all children, the relationship between ASL and English skill development, and the techniques

  17. Contexts of Digital Reading: How Genres Affect Reading Practices

    ERIC Educational Resources Information Center

    Morris, Janine

    2016-01-01

    "Contexts of Digital Reading: How Genres Affect Reading Practices" is a study of the different ways university students use digital devices to read. Many mainstream representations of digital reading fail to distinguish between different contexts or genres of reading, making digital reading appear transparent rather than mediated by…

  18. Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

    PubMed Central

    Stage-Zimmermann, Tracy; Schmidt, Ute; Silver, Pamela A.

    2000-01-01

    In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. PMID:11071906

  19. 42 CFR 414.418 - Opportunity for networks.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Opportunity for networks. 414.418 Section 414.418... networks. (a) A network may be comprised of at least 2 but not more than 20 small suppliers. (b) The following rules apply to networks that seek contracts under this subpart: (1) Each network must form a...

  20. 42 CFR 414.418 - Opportunity for networks.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Opportunity for networks. 414.418 Section 414.418... networks. (a) A network may be comprised of at least 2 but not more than 20 small suppliers. (b) The following rules apply to networks that seek contracts under this subpart: (1) Each network must form a...

  1. Literacy Coaching to Improve Student Reading Achievement: A Multi-Level Mediation Model

    ERIC Educational Resources Information Center

    Matsumura, Lindsay Clare; Garnier, Helen E.; Spybrook, Jessaca

    2013-01-01

    In a longitudinal group-randomized trial, we explore the key role of the quality of classroom text discussions in mediating the effects of Content-Focused Coaching (CFC) on student reading achievement (2983 students, 167 teachers). Schools in the United States serving large numbers of minority and English language learning (ELL) students from…

  2. Peeling the onion: ribosomes are ancient molecular fossils.

    PubMed

    Hsiao, Chiaolong; Mohan, Srividya; Kalahar, Benson K; Williams, Loren Dean

    2009-11-01

    We describe a method to establish chronologies of ancient ribosomal evolution. The method uses structure-based and sequence-based comparison of the large subunits (LSUs) of Haloarcula marismortui and Thermus thermophilus. These are the highest resolution ribosome structures available and represent disparate regions of the evolutionary tree. We have sectioned the superimposed LSUs into concentric shells, like an onion, using the site of peptidyl transfer as the origin (the PT-origin). This spherical approximation combined with a shell-by-shell comparison captures significant information along the evolutionary time line revealing, for example, that sequence and conformational similarity of the 23S rRNAs are greatest near the PT-origin and diverge smoothly with distance from it. The results suggest that the conformation and interactions of both RNA and protein can be described as changing, in an observable manner, over evolutionary time. The tendency of macromolecules to assume regular secondary structural elements such as A-form helices with Watson-Crick base pairs (RNA) and alpha-helices and beta-sheets (protein) is low at early time points but increases as time progresses. The conformations of ribosomal protein components near the PT-origin suggest that they may be molecular fossils of the peptide ancestors of ribosomal proteins. Their abbreviated length may have proscribed formation of secondary structure, which is indeed nearly absent from the region of the LSU nearest the PT-origin. Formation and evolution of the early PT center may have involved Mg(2+)-mediated assembly of at least partially single-stranded RNA oligomers or polymers. As one moves from center to periphery, proteins appear to replace magnesium ions. The LSU is known to have undergone large-scale conformation changes upon assembly. The T. thermophilus LSU analyzed here is part of a fully assembled ribosome, whereas the H. marismortui LSU analyzed here is dissociated from other ribosomal components

  3. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

    PubMed Central

    Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

    2015-01-01

    In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

  4. Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

    PubMed

    Biedka, Stephanie; Micic, Jelena; Wilson, Daniel; Brown, Hailey; Diorio-Toth, Luke; Woolford, John L

    2018-04-24

    Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C 2 site of 27SB pre-rRNA. C 2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C 2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C 2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C 2 cleavage. Interestingly, when C 2 cleavage is directly blocked by depleting or inactivating the C 2 endonuclease, assembly progresses through all other subsequent steps. © 2018 Biedka et al.

  5. Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase

    PubMed Central

    Jha, Sujata; Rollins, Madeline G.; Fuchs, Gabriele; Procter, Dean J.; Hall, Elizabeth A.; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N.; Walsh, Derek

    2017-01-01

    Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements1. However, beyond differential subunit expression during development2,3, evidence for regulated ribosome specification within individual cells has remained elusive1. Here, we report that a poxvirus kinase phosphorylates serine/threonine residues in the small ribosomal subunit protein, Receptor for Activated C Kinase (RACK1) that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs whose 5’ untranslated regions (UTRs) contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analysis revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, where these leaders act as translational enhancers for poorly understood reasons. Phosphomimetics and inter-species chimeras demonstrated that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase4 confer a translational advantage. Our findings uncover ribosome customization through a novel trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie the enigmatic poxvirus polyA-leaders4. PMID:28636603

  6. Ribosome-inactivating proteins: potent poisons and molecular tools.

    PubMed

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-11-15

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms.

  7. The nuclear import of ribosomal proteins is regulated by mTOR

    PubMed Central

    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  8. Ribosomal proteins L5 and L11 co-operatively inactivate c-Myc via RNA-induced silencing complex.

    PubMed

    Liao, J-M; Zhou, X; Gatignol, A; Lu, H

    2014-10-09

    Oncogene MYC is highly expressed in many human cancers and functions as a global regulator of ribosome biogenesis. Previously, we reported that ribosomal protein (RP) L11 binds to c-Myc and inhibits its transcriptional activity in response to ribosomal stress. Here, we show that RPL5, co-operatively with RPL11, guides the RNA-induced silencing complex (RISC) to c-Myc mRNA and mediates the degradation of the mRNA, consequently leading to inhibition of c-Myc activity. Knocking down of RPL5 induced c-Myc expression at both mRNA and protein levels, whereas overexpression of RPL5 suppressed c-Myc expression and activity. Immunoprecipitation revealed that RPL5 binds to 3'UTR of c-Myc mRNA and two subunits of RISC, TRBP (HIV-1 TAR RNA-binding protein) and Ago2, mediating the targeting of c-Myc mRNA by miRNAs. Interestingly, RPL5 and RPL11 co-resided on c-Myc mRNA and suppressed c-Myc expression co-operatively. These findings uncover a mechanism by which these two RPs can co-operatively suppress c-Myc expression, allowing a tightly controlled ribosome biogenesis in cells.

  9. 42 CFR 418.78 - Conditions of participation-Volunteers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Conditions of participation-Volunteers. 418.78 Section 418.78 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HOSPICE CARE Conditions of Participation: Patient Care Non-Core...

  10. Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes

    PubMed Central

    Becker, Annemarie H.; Oh, Eugene; Weissman, Jonathan S.; Kramer, Günter; Bukau, Bernd

    2014-01-01

    A plethora of factors is involved in the maturation of newly synthesized proteins, including chaperones, membrane targeting factors, and enzymes. Many factors act cotranslationally through association with ribosome-nascent chain complexes (RNCs), but their target specificities and modes of action remain poorly understood. We developed selective ribosome profiling (SeRP) to identify substrate pools and points of RNC engagement of these factors. SeRP is based on sequencing mRNA fragments covered by translating ribosomes (general ribosome profiling, RP), combined with a procedure to selectively isolate RNCs whose nascent polypeptides are associated with the factor of interest. Factor–RNC interactions are stabilized by crosslinking, the resulting factor–RNC adducts are then nuclease-treated to generate monosomes, and affinity-purified. The ribosome-extracted mRNA footprints are converted to DNA libraries for deep sequencing. The protocol is specified for general RP and SeRP in bacteria. It was first applied to the chaperone trigger factor and is readily adaptable to other cotranslationally acting factors, including eukaryotic factors. Factor–RNC purification and sequencing library preparation takes 7–8 days, sequencing and data analysis can be completed in 5–6 days. PMID:24136347

  11. A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase

    PubMed Central

    2009-01-01

    Membrane protein integration occurs predominantly at the endoplasmic reticulum and is mediated by the translocon, which is formed by the Sec61p complex. The translocon binds to the ribosome at the polypeptide exit site such that integration occurs in a cotranslational manner. Ribosomal protein Rpl17 is positioned such that it contacts both the ribosome exit tunnel and the surface of the ribosome near the exit site, where it is intimately associated with the translocon. The presence of a trans-membrane (TM) segment inside the ribosomal exit tunnel leads to the recruitment of RAMP4 to the translocon at a site adjacent to Rpl17. This suggests a signaling function for Rpl17 such that it can recognize a TM segment inside the ribosome and triggers rearrangements of the translocon, priming it for subsequent TM segment integration. PMID:19468070

  12. Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sathiyamoorthy, Karthik; Jiang, Jiansen; Mohl, Britta S.

    Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studiedmore » the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. Furthermore, these data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.« less

  13. Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sathiyamoorthy, Karthik; Jiang, Jiansen; Möhl, Britta S.

    Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studiedmore » the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. These data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.« less

  14. Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

    DOE PAGES

    Sathiyamoorthy, Karthik; Jiang, Jiansen; Mohl, Britta S.; ...

    2017-09-22

    Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studiedmore » the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. Furthermore, these data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.« less

  15. Reading Attitude as a Mediator between Contextual Factors and Reading Behavior

    ERIC Educational Resources Information Center

    Lim, Hyo Jin; Bong, Mimi; Woo, Yeon-Kyung

    2015-01-01

    Background: Among the factors known to influence reading development and performance, attitude toward reading is shown to be particularly critical for developing learners. Reading attitude (McKenna, 1994; McKenna et al., 1995) enhances independent reading, levels of engagement in classroom reading activities, and the amount and variety of topics…

  16. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  17. Identification in a pseudoknot of a U.G motif essential for the regulation of the expression of ribosomal protein S15.

    PubMed

    Bénard, L; Mathy, N; Grunberg-Manago, M; Ehresmann, B; Ehresmann, C; Portier, C

    1998-03-03

    The ribosomal protein S15 from Escherichia coli binds to a pseudoknot in its own messenger. This interaction is an essential step in the mechanism of S15 translational autoregulation. In a previous study, a recognition determinant for S15 autoregulation, involving a U.G wobble pair, was located in the center of stem I of the pseudoknot. In this study, an extensive mutagenesis analysis has been conducted in and around this U.G pair by comparing the effects of these mutations on the expression level of S15. The results show that the U.G wobble pair cannot be substituted by A.G, C.A, A.C, G.U, or C.G without loss of the autocontrol. In addition, the base pair C.G, adjacent to the 5' side of U, cannot be flipped or changed to another complementary base pair without also inducing derepression of translation. A unique motif, made of only two adjacent base pairs, U.G/C.G, is essential for S15 autoregulation and is presumably involved in direct recognition by the S15 protein.

  18. From Words to Text: Inference Making Mediates the Role of Vocabulary in Children's Reading Comprehension

    ERIC Educational Resources Information Center

    Daugaard, Hanne Trebbien; Cain, Kate; Elbro, Carsten

    2017-01-01

    We examined the relationship between inference making, vocabulary knowledge, and verbal working memory on children's reading comprehension in 62 6th graders (aged 12). The effect of vocabulary knowledge on reading comprehension was predicted to be partly mediated by inference making for two reasons: Inference making often taps the semantic…

  19. RISSC: a novel database for ribosomal 16S–23S RNA genes spacer regions

    PubMed Central

    García-Martínez, Jesús; Bescós, Ignacio; Rodríguez-Sala, Jesús Javier; Rodríguez-Valera, Francisco

    2001-01-01

    A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S–23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documen­tation tool for studies on evolution, identification, typing and strain characterization, among others. PMID:11125084

  20. [Immunochemistry of eukaryotic ribosomes].

    PubMed

    Lopaczyński, W; Gałasiński, W

    1990-01-01

    Immunochemical investigations of ribosomes should correlate with basic knowledge of the function, structure and activity of organelles in the cell processes. Our paper presents data of immunochemical methods used to determine the structure, function and differences of ribosomes. We present the usefulness of immunochemical methods to test human ribosomes, diagnosis and therapy of many diseases.

  1. PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration.

    PubMed

    Janjanam, Jagadeesh; Chandaka, Giri Kumar; Kotla, Sivareddy; Rao, Gadiparthi N

    2015-12-15

    Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein-coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδ activation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin-WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration. © 2015 Janjanam et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  2. 40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

  3. 40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

  4. 40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

  5. 40 CFR 418.40 - Applicability; description of the ammonium nitrate subcategory.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ammonium nitrate subcategory. 418.40 Section 418.40 Protection of Environment ENVIRONMENTAL PROTECTION... Ammonium Nitrate Subcategory § 418.40 Applicability; description of the ammonium nitrate subcategory. The provisions of this subpart are applicable to discharges resulting from the manufacture of ammonium nitrate...

  6. Improving Reading Skills through Parental Involvement.

    ERIC Educational Resources Information Center

    Clark, Kathryn; Pillion, Jennifer

    This report describes a project for increasing parental involvement through the existing reading program. The targeted first and second grade students lived in growing rural, Midwestern, low to middle class communities located in north central Illinois. The problem was noted in literature by researchers who found that parents had a very limited…

  7. The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen

    2010-04-26

    Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, themore » streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.« less

  8. Ribosomal synthesis and folding of peptide-helical aromatic foldamer hybrids

    NASA Astrophysics Data System (ADS)

    Rogers, Joseph M.; Kwon, Sunbum; Dawson, Simon J.; Mandal, Pradeep K.; Suga, Hiroaki; Huc, Ivan

    2018-03-01

    Translation, the mRNA-templated synthesis of peptides by the ribosome, can be manipulated to incorporate variants of the 20 cognate amino acids. Such approaches for expanding the range of chemical entities that can be produced by the ribosome may accelerate the discovery of molecules that can perform functions for which poorly folded, short peptidic sequences are ill suited. Here, we show that the ribosome tolerates some artificial helical aromatic oligomers, so-called foldamers. Using a flexible tRNA-acylation ribozyme—flexizyme—foldamers were attached to tRNA, and the resulting acylated tRNAs were delivered to the ribosome to initiate the synthesis of non-cyclic and cyclic foldamer-peptide hybrid molecules. Passing through the ribosome exit tunnel requires the foldamers to unfold. Yet foldamers encode sufficient folding information to influence the peptide structure once translation is completed. We also show that in cyclic hybrids, the foldamer portion can fold into a helix and force the peptide segment to adopt a constrained and stretched conformation.

  9. Identification in a pseudoknot of a U⋅G motif essential for the regulation of the expression of ribosomal protein S15

    PubMed Central

    Bénard, Lionel; Mathy, Nathalie; Grunberg-Manago, Marianne; Ehresmann, Bernard; Ehresmann, Chantal; Portier, Claude

    1998-01-01

    The ribosomal protein S15 from Escherichia coli binds to a pseudoknot in its own messenger. This interaction is an essential step in the mechanism of S15 translational autoregulation. In a previous study, a recognition determinant for S15 autoregulation, involving a U⋅G wobble pair, was located in the center of stem I of the pseudoknot. In this study, an extensive mutagenesis analysis has been conducted in and around this U⋅G pair by comparing the effects of these mutations on the expression level of S15. The results show that the U⋅G wobble pair cannot be substituted by A⋅G, C⋅A, A⋅C, G⋅U, or C⋅G without loss of the autocontrol. In addition, the base pair C⋅G, adjacent to the 5′ side of U, cannot be flipped or changed to another complementary base pair without also inducing derepression of translation. A unique motif, made of only two adjacent base pairs, U⋅G/C⋅G, is essential for S15 autoregulation and is presumably involved in direct recognition by the S15 protein. PMID:9482926

  10. Gender Differences in How Family Income and Parental Education Relate to Reading Achievement in China: The Mediating Role of Parental Expectation and Parental Involvement

    PubMed Central

    Guo, Xiaolin; Lv, Bo; Zhou, Huan; Liu, Chunhui; Liu, Juan; Jiang, Kexin; Luo, Liang

    2018-01-01

    The impact of social economic status (SES) on children's academic outcomes has been well documented. However, the mechanisms underlying this relationship remain poorly understood. Furthermore, the process by which SES relates to academic achievement needs to be studied separately for boys and girls. Using a sample of 598 Chinese children (299 boys, 299 girls) in grades 4 to 6 and their parents, this study examined the process of how family SES, specifically family income and parental education, indirectly relates to children's reading achievement through parental expectation and parental involvement and whether this process differs between boys and girls. The results revealed that parental expectation and specific parental involvement behaviors played critical mediating roles between family SES and reading achievement. Moreover, the exact nature of these links differed by the gender of children. For boys, both the effect of parental education and the effect of family income were partially mediated by parental expectation and parent-child communication orderly. For girls, the effect of parental education was partially mediated by three separate pathways: (1) home monitoring; (2) parent-child communication; and (3) parental expectation followed by parent-child communication, while the effect of family income was fully mediated by parent-child communication. These findings suggest a process through which SES factors are related to children's academic development and identify a context under which these associations may differ. The practical implications of these findings are discussed, along with possible future research directions. PMID:29910752

  11. Gender Differences in How Family Income and Parental Education Relate to Reading Achievement in China: The Mediating Role of Parental Expectation and Parental Involvement.

    PubMed

    Guo, Xiaolin; Lv, Bo; Zhou, Huan; Liu, Chunhui; Liu, Juan; Jiang, Kexin; Luo, Liang

    2018-01-01

    The impact of social economic status (SES) on children's academic outcomes has been well documented. However, the mechanisms underlying this relationship remain poorly understood. Furthermore, the process by which SES relates to academic achievement needs to be studied separately for boys and girls. Using a sample of 598 Chinese children (299 boys, 299 girls) in grades 4 to 6 and their parents, this study examined the process of how family SES, specifically family income and parental education, indirectly relates to children's reading achievement through parental expectation and parental involvement and whether this process differs between boys and girls. The results revealed that parental expectation and specific parental involvement behaviors played critical mediating roles between family SES and reading achievement. Moreover, the exact nature of these links differed by the gender of children. For boys, both the effect of parental education and the effect of family income were partially mediated by parental expectation and parent-child communication orderly. For girls, the effect of parental education was partially mediated by three separate pathways: (1) home monitoring; (2) parent-child communication; and (3) parental expectation followed by parent-child communication, while the effect of family income was fully mediated by parent-child communication. These findings suggest a process through which SES factors are related to children's academic development and identify a context under which these associations may differ. The practical implications of these findings are discussed, along with possible future research directions.

  12. Engaging Sources through Reading-Writing Connections across the Disciplines

    ERIC Educational Resources Information Center

    Carillo, Ellen C.

    2016-01-01

    This essay argues that what might otherwise be considered "plagiarism" in student writing is a symptom of the difficulties students encounter in their reading and writing, moments in which students' inabilities to critically assess, read, and respond to sources through the act of writing come to the surface. Expanding the context within…

  13. MoYvh1 subverts rice defense through functions of ribosomal protein MoMrt4 in Magnaporthe oryzae.

    PubMed

    Liu, Xinyu; Yang, Jie; Qian, Bin; Cai, Yongchao; Zou, Xi; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2018-04-01

    The accumulation of the reactive oxygen species (ROS) in rice is important in its interaction with the rice blast fungus Magnaporthe oryzae during which the pathogen scavenges ROS through the production of extracellular enzymes that promote blast. We previously characterized the MoYvh1 protein phosphatase from M. oryzae that plays a role in scavenging of ROS. To understand the underlying mechanism, we found that MoYvh1 is translocated into the nucleus following oxidative stress and that this translocation is dependent on MoSsb1 and MoSsz1 that are homologous to heat-shock protein 70 (Hsp70) proteins. In addition, we established a link between MoYvh1 and MoMrt4, a ribosome maturation factor homolog whose function also involves shuttling between the cytoplasm and the nucleus. Moreover, we found that MoYvh1 regulates the production of extracellular proteins that modulate rice-immunity. Taking together, our evidence suggests that functions of MoYvh1 in regulating ROS scavenging require its nucleocytoplasmic shuttling and the partner proteins MoSsb1 and MoSsz1, as well as MoMrt4. Our findings provide novel insights into the mechanism by which M. oryzae responds to and subverts host immunity through the regulation of ribosome biogenesis and protein biosynthesis.

  14. 42 CFR 418.78 - Conditions of participation-Volunteers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Conditions of participation-Volunteers. 418.78... Non-Core Services § 418.78 Conditions of participation—Volunteers. The hospice must use volunteers to the extent specified in paragraph (e) of this section. These volunteers must be used in defined roles...

  15. 42 CFR 418.78 - Conditions of participation-Volunteers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Conditions of participation-Volunteers. 418.78... Non-Core Services § 418.78 Conditions of participation—Volunteers. The hospice must use volunteers to the extent specified in paragraph (e) of this section. These volunteers must be used in defined roles...

  16. 42 CFR 418.78 - Conditions of participation-Volunteers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Conditions of participation-Volunteers. 418.78... Non-Core Services § 418.78 Conditions of participation—Volunteers. The hospice must use volunteers to the extent specified in paragraph (e) of this section. These volunteers must be used in defined roles...

  17. 42 CFR 418.78 - Conditions of participation-Volunteers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Conditions of participation-Volunteers. 418.78... Services § 418.78 Conditions of participation—Volunteers. The hospice must use volunteers to the extent specified in paragraph (e) of this section. These volunteers must be used in defined roles and under the...

  18. Enhancing reading performance through action video games: the role of visual attention span.

    PubMed

    Antzaka, A; Lallier, M; Meyer, S; Diard, J; Carreiras, M; Valdois, S

    2017-11-06

    Recent studies reported that Action Video Game-AVG training improves not only certain attentional components, but also reading fluency in children with dyslexia. We aimed to investigate the shared attentional components of AVG playing and reading, by studying whether the Visual Attention (VA) span, a component of visual attention that has previously been linked to both reading development and dyslexia, is improved in frequent players of AVGs. Thirty-six French fluent adult readers, matched on chronological age and text reading proficiency, composed two groups: frequent AVG players and non-players. Participants performed behavioural tasks measuring the VA span, and a challenging reading task (reading of briefly presented pseudo-words). AVG players performed better on both tasks and performance on these tasks was correlated. These results further support the transfer of the attentional benefits of playing AVGs to reading, and indicate that the VA span could be a core component mediating this transfer. The correlation between VA span and pseudo-word reading also supports the involvement of VA span even in adult reading. Future studies could combine VA span training with defining features of AVGs, in order to build a new generation of remediation software.

  19. 43 CFR 418.24 - Precautionary drawdown and spills from Lahontan Reservoir.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Lahontan Reservoir. 418.24 Section 418.24 Public Lands: Interior Regulations Relating to Public Lands... RECLAMATION PROJECT, NEVADA Operations and Management § 418.24 Precautionary drawdown and spills from Lahontan Reservoir. (a) Even though flood control is not a specifically authorized purpose of the Project, at the...

  20. 43 CFR 418.24 - Precautionary drawdown and spills from Lahontan Reservoir.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Lahontan Reservoir. 418.24 Section 418.24 Public Lands: Interior Regulations Relating to Public Lands... RECLAMATION PROJECT, NEVADA Operations and Management § 418.24 Precautionary drawdown and spills from Lahontan Reservoir. (a) Even though flood control is not a specifically authorized purpose of the Project, at the...

  1. LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    PubMed Central

    Liu, Xiuju; Huang, Henry; Wilkinson, Scott C.; Zhong, Diansheng; Khuri, Fadlo R.; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

    2016-01-01

    We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells. PMID:26506235

  2. LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions.

    PubMed

    Liu, Fakeng; Jin, Rui; Liu, Xiuju; Huang, Henry; Wilkinson, Scott C; Zhong, Diansheng; Khuri, Fadlo R; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

    2016-01-19

    We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.

  3. 42 CFR 418.22 - Certification of terminal illness.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Certification of terminal illness. 418.22 Section... Certification of terminal illness. (a) Timing of certification—(1) General rule. The hospice must obtain written certification of terminal illness for each of the periods listed in § 418.21, even if a single election...

  4. 42 CFR 418.22 - Certification of terminal illness.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Certification of terminal illness. 418.22 Section... Certification of terminal illness. (a) Timing of certification—(1) General rule. The hospice must obtain written certification of terminal illness for each of the periods listed in § 418.21, even if a single election...

  5. 42 CFR 418.22 - Certification of terminal illness.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Certification of terminal illness. 418.22 Section... Certification of terminal illness. (a) Timing of certification—(1) General rule. The hospice must obtain written certification of terminal illness for each of the periods listed in § 418.21, even if a single election...

  6. 42 CFR 418.22 - Certification of terminal illness.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Certification of terminal illness. 418.22 Section... Certification of terminal illness. (a) Timing of certification—(1) General rule. The hospice must obtain written certification of terminal illness for each of the periods listed in § 418.21, even if a single election...

  7. Strong G-Protein-Mediated Inhibition of Sodium Channels.

    PubMed

    Mattheisen, Glynis B; Tsintsadze, Timur; Smith, Stephen M

    2018-05-29

    Voltage-gated sodium channels (VGSCs) are strategically positioned to mediate neuronal plasticity because of their influence on action potential waveform. VGSC function may be strongly inhibited by local anesthetic and antiepileptic drugs and modestly modulated via second messenger pathways. Here, we report that the allosteric modulators of the calcium-sensing receptor (CaSR) cinacalcet, calindol, calhex, and NPS 2143 completely inhibit VGSC current in the vast majority of cultured mouse neocortical neurons. This form of VGSC current block persisted in CaSR-deficient neurons, indicating a CaSR-independent mechanism. Cinacalcet-mediated blockade of VGSCs was prevented by the guanosine diphosphate (GDP) analog GDPβs, indicating that G-proteins mediated this effect. Cinacalcet inhibited VGSCs by increasing channel inactivation, and block was reversed by prolonged hyperpolarization. Strong cinacalcet inhibition of VGSC currents was also present in acutely isolated mouse cortical neurons. These data identify a dynamic signaling pathway by which G-proteins regulate VGSC current to indirectly modulate central neuronal excitability. Published by Elsevier Inc.

  8. 20 CFR 418.2205 - What is a major life-changing event?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false What is a major life-changing event? 418.2205 Section 418.2205 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income-Related... Year's Modified Adjusted Gross Income § 418.2205 What is a major life-changing event? We will follow...

  9. 20 CFR 418.2205 - What is a major life-changing event?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false What is a major life-changing event? 418.2205 Section 418.2205 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income-Related... Year's Modified Adjusted Gross Income § 418.2205 What is a major life-changing event? We will follow...

  10. 20 CFR 418.2205 - What is a major life-changing event?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false What is a major life-changing event? 418.2205 Section 418.2205 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income-Related... Year's Modified Adjusted Gross Income § 418.2205 What is a major life-changing event? We will follow...

  11. 20 CFR 418.2205 - What is a major life-changing event?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false What is a major life-changing event? 418.2205 Section 418.2205 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income-Related... Year's Modified Adjusted Gross Income § 418.2205 What is a major life-changing event? We will follow...

  12. Niacin activates the G protein estrogen receptor (GPER)-mediated signalling.

    PubMed

    Santolla, Maria Francesca; De Francesco, Ernestina Marianna; Lappano, Rosamaria; Rosano, Camillo; Abonante, Sergio; Maggiolini, Marcello

    2014-07-01

    Nicotinic acid, also known as niacin, is the water soluble vitamin B3 used for decades for the treatment of dyslipidemic diseases. Its action is mainly mediated by the G protein-coupled receptor (GPR) 109A; however, certain regulatory effects on lipid levels occur in a GPR109A-independent manner. The amide form of nicotinic acid, named nicotinamide, acts as a vitamin although neither activates the GPR109A nor exhibits the pharmacological properties of nicotinic acid. In the present study, we demonstrate for the first time that nicotinic acid and nicotinamide bind to and activate the GPER-mediated signalling in breast cancer cells and cancer-associated fibroblasts (CAFs). In particular, we show that both molecules are able to promote the up-regulation of well established GPER target genes through the EGFR/ERK transduction pathway. As a biological counterpart, nicotinic acid and nicotinamide induce proliferative and migratory effects in breast cancer cells and CAFs in a GPER-dependent fashion. Moreover, nicotinic acid prevents the up-regulation of ICAM-1 triggered by the pro-inflammatory cytokine TNF-α and stimulates the formation of endothelial tubes through GPER in HUVECs. Together, our findings concerning the agonist activity for GPER displayed by both nicotinic acid and nicotinamide broaden the mechanisms involved in the biological action of these molecules and further support the potential of a ligand to induce different responses mediated in a promiscuous manner by distinct GPCRs. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. 20 CFR 418.1105 - What is the threshold?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false What is the threshold? 418.1105 Section 418... What is the threshold? (a) The threshold is a level of modified adjusted gross income above which the... gross income threshold is $80,000 for individuals with a Federal income tax filing status of single...

  14. Structural and functional organization of ribosomal genes within the mammalian cell nucleolus.

    PubMed

    Derenzini, Massimo; Pasquinelli, Gianandrea; O'Donohue, Marie-Françoise; Ploton, Dominique; Thiry, Marc

    2006-02-01

    Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA double-helix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell.

  15. ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

    PubMed Central

    Chauveau, J.; Moulé, Y.; Rouiller, C.; Schneebeli, J.

    1962-01-01

    Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry. PMID:13878497

  16. Improving Reading Rates and Comprehension through Timed Repeated Reading

    ERIC Educational Resources Information Center

    Chang, Anna C-S.; Millett, Sonia

    2013-01-01

    Thirteen English as a foreign language students read 26 passages during a 13-week period. Each passage was read five times, and students answered comprehension questions after the first and the fifth reading. Another 13 students read the same number of passages but without repetition and only answered the comprehension questions once. All students…

  17. Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome.

    PubMed

    Jetzt, Amanda E; Li, Xiao-Ping; Tumer, Nilgun E; Cohick, Wendie S

    2016-11-01

    Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. 20 CFR 418.1205 - What is a major life-changing event?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false What is a major life-changing event? 418.1205 Section 418.1205 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part B... Gross Income § 418.1205 What is a major life-changing event? For the purposes of this subpart, we will...

  19. Revealing Nature’s Synthetic Potential Through the Study of Ribosomal Natural Product Biosynthesis

    PubMed Central

    Dunbar, Kyle L.; Mitchell, Douglas A.

    2013-01-01

    Ribosomally synthesized posttranslationally modified peptides (RiPPs) are a rapidly growing class of natural products with diverse structures and activities. In recent years, a great deal of progress has been made in elucidating the biosynthesis of various RiPP family members. As with the study of nonribosomal peptide and polyketide biosynthetic enzymes, these investigations have led to the discovery of entirely new biological chemistry. With each unique enzyme investigated, a more complex picture of Nature’s synthetic potential is revealed. This review focuses on recent reports (since 2008) that have changed the way that we think about ribosomal natural product biosynthesis and the enzymology of complex bond-forming reactions. PMID:23286465

  20. Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

    PubMed Central

    Schawalder, James; Paric, Enesa; Neff, Norma F

    2003-01-01

    Background Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. Results Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. Conclusion These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres. PMID:14577841

  1. Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.

    PubMed

    Tsai, Chung-Che; Kuo, Ting-Yu; Hong, Zhi-Wei; Yeh, Ying-Chieh; Shih, Kuo-Shun; Du, Shin-Yi; Fu, Hua-Wen

    2015-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.

  2. Mechanisms of ribosome stalling by SecM at multiple elongation steps

    PubMed Central

    Zhang, Jun; Pan, Xijiang; Yan, Kaige; Sun, Shan; Gao, Ning; Sui, Sen-Fang

    2015-01-01

    Regulation of translating ribosomes is a major component of gene expression control network. In Escherichia coli, ribosome stalling by the C-terminal arrest sequence of SecM regulates the SecA-dependent secretion pathway. Previous studies reported many residues of SecM peptide and ribosome exit tunnel are critical for stalling. However, the underlying molecular mechanism is still not clear at the atomic level. Here, we present two cryo-EM structures of the SecM-stalled ribosomes at 3.3–3.7 Å resolution, which reveal two different stalling mechanisms at distinct elongation steps of the translation cycle: one is due to the inactivation of ribosomal peptidyl-transferase center which inhibits peptide bond formation with the incoming prolyl-tRNA; the other is the prolonged residence of the peptidyl-RNA at the hybrid A/P site which inhibits the full-scale tRNA translocation. These results demonstrate an elegant control of translation cycle by regulatory peptides through a continuous, dynamic reshaping of the functional center of the ribosome. DOI: http://dx.doi.org/10.7554/eLife.09684.001 PMID:26670735

  3. TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p.

    PubMed

    Bodem, J; Dobreva, G; Hoffmann-Rohrer, U; Iben, S; Zentgraf, H; Delius, H; Vingron, M; Grummt, I

    2000-08-01

    Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

  4. G-CNV: A GPU-Based Tool for Preparing Data to Detect CNVs with Read-Depth Methods.

    PubMed

    Manconi, Andrea; Manca, Emanuele; Moscatelli, Marco; Gnocchi, Matteo; Orro, Alessandro; Armano, Giuliano; Milanesi, Luciano

    2015-01-01

    Copy number variations (CNVs) are the most prevalent types of structural variations (SVs) in the human genome and are involved in a wide range of common human diseases. Different computational methods have been devised to detect this type of SVs and to study how they are implicated in human diseases. Recently, computational methods based on high-throughput sequencing (HTS) are increasingly used. The majority of these methods focus on mapping short-read sequences generated from a donor against a reference genome to detect signatures distinctive of CNVs. In particular, read-depth based methods detect CNVs by analyzing genomic regions with significantly different read-depth from the other ones. The pipeline analysis of these methods consists of four main stages: (i) data preparation, (ii) data normalization, (iii) CNV regions identification, and (iv) copy number estimation. However, available tools do not support most of the operations required at the first two stages of this pipeline. Typically, they start the analysis by building the read-depth signal from pre-processed alignments. Therefore, third-party tools must be used to perform most of the preliminary operations required to build the read-depth signal. These data-intensive operations can be efficiently parallelized on graphics processing units (GPUs). In this article, we present G-CNV, a GPU-based tool devised to perform the common operations required at the first two stages of the analysis pipeline. G-CNV is able to filter low-quality read sequences, to mask low-quality nucleotides, to remove adapter sequences, to remove duplicated read sequences, to map the short-reads, to resolve multiple mapping ambiguities, to build the read-depth signal, and to normalize it. G-CNV can be efficiently used as a third-party tool able to prepare data for the subsequent read-depth signal generation and analysis. Moreover, it can also be integrated in CNV detection tools to generate read-depth signals.

  5. Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jetzt, Amanda E.

    Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarlymore » to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. - Highlights: • Arginines 193 and 235 of RTA are critical for binding to the mammalian ribosome. • R193A/R235A has full catalytic activity on RNA but not on mammalian ribosomes. • R193A/R235A is less toxic than a mutant that targets the active site. • The toxin-ribosome interaction is a therapeutic target for ricin intoxication.« less

  6. 42 CFR 418.114 - Condition of participation: Personnel qualifications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Condition of participation: Personnel qualifications. 418.114 Section 418.114 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HOSPICE CARE Conditions of participation...

  7. 42 CFR 418.104 - Condition of participation: Clinical records.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Condition of participation: Clinical records. 418.104 Section 418.104 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HOSPICE CARE Conditions of participation: Organizational...

  8. 42 CFR 418.104 - Condition of participation: Clinical records.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Condition of participation: Clinical records. 418.104 Section 418.104 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HOSPICE CARE Conditions of participation: Organizational...

  9. 42 CFR 418.102 - Condition of participation: Medical director.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Condition of participation: Medical director. 418.102 Section 418.102 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HOSPICE CARE Conditions of participation: Organizational...

  10. 42 CFR 418.102 - Condition of participation: Medical director.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Condition of participation: Medical director. 418.102 Section 418.102 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HOSPICE CARE Conditions of participation: Organizational...

  11. 42 CFR 418.114 - Condition of participation: Personnel qualifications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Condition of participation: Personnel qualifications. 418.114 Section 418.114 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) HOSPICE CARE Conditions of...

  12. 42 CFR 418.114 - Condition of participation: Personnel qualifications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Condition of participation: Personnel qualifications. 418.114 Section 418.114 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) HOSPICE CARE Conditions of...

  13. 42 CFR 418.114 - Condition of participation: Personnel qualifications.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Condition of participation: Personnel qualifications. 418.114 Section 418.114 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) HOSPICE CARE Conditions of...

  14. Enhancing reading abilities of learners with intellectual impairments through computer technology

    PubMed Central

    Warnick, Albert M.

    2017-01-01

    Background Developments in the teaching of children with disabilities support pedagogy that emphasises learners’ strengths as opposed to their assumed deficiencies. Educators and mediators who advocate this view continually strive for tools and methodologies that enhance learner participation in academic environments. Computer technology is one of the tools recognised for its potential to enrich learning experiences of learners with an intellectual impairment. Objectives We sought to assess the influence of text-to-speech stories on the reading ability of intellectually challenged learners. Method A qualitative action research study that involves learners at a special school in Cape Town, South Africa. Pre- and post-test data of the reading performance of learners are analysed with a focus on how they demonstrate change. Results Although no claims can be made about the explicit influence on reading performance, computer-assisted learning has the potential in isolating reading processes that classroom-based interventions can address. In addition, computers enhance motivation and enthusiasm to learn. Conclusion A need for education based on inclusion and positive differentiation remains the key driver in any educational interventions. PMID:28951849

  15. LET-418/Mi2 and SPR-5/LSD1 cooperatively prevent somatic reprogramming of C. elegans germline stem cells.

    PubMed

    Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

    2014-04-08

    Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, "sensitization" of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

  16. The Effects of a Peer-Mediated Reading Intervention on Juvenile Offenders' Main Idea Statements about Informational Text

    ERIC Educational Resources Information Center

    Wexler, Jade; Reed, Deborah K.; Barton, Erin E.; Mitchell, Marisa; Clancy, Erin

    2018-01-01

    Many youth in the juvenile justice system with or at risk for emotional and behavioral disorders struggle with reading. A multiple-baseline-across-participants single-case research design was used to examine the relationship between a supplemental peer-mediated reading intervention and juvenile offenders' generation of main idea statements about…

  17. Mutation Detection with Next-Generation Resequencing through a Mediator Genome

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wurtzel, Omri; Dori-Bachash, Mally; Pietrokovski, Shmuel

    2010-12-31

    The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WTmore » and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.« less

  18. 47 CFR 54.418 - Digital Television Transition Notices by Eligible Telecommunications Carriers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 3 2014-10-01 2014-10-01 false Digital Television Transition Notices by Eligible Telecommunications Carriers. 54.418 Section 54.418 Telecommunication FEDERAL COMMUNICATIONS... Low-Income Consumers § 54.418 Digital Television Transition Notices by Eligible Telecommunications...

  19. 47 CFR 54.418 - Digital Television Transition Notices by Eligible Telecommunications Carriers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 3 2013-10-01 2013-10-01 false Digital Television Transition Notices by Eligible Telecommunications Carriers. 54.418 Section 54.418 Telecommunication FEDERAL COMMUNICATIONS... Low-Income Consumers § 54.418 Digital Television Transition Notices by Eligible Telecommunications...

  20. 47 CFR 54.418 - Digital Television Transition Notices by Eligible Telecommunications Carriers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 3 2012-10-01 2012-10-01 false Digital Television Transition Notices by Eligible Telecommunications Carriers. 54.418 Section 54.418 Telecommunication FEDERAL COMMUNICATIONS... Low-Income Consumers § 54.418 Digital Television Transition Notices by Eligible Telecommunications...

  1. 47 CFR 54.418 - Digital Television Transition Notices by Eligible Telecommunications Carriers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 3 2010-10-01 2010-10-01 false Digital Television Transition Notices by Eligible Telecommunications Carriers. 54.418 Section 54.418 Telecommunication FEDERAL COMMUNICATIONS... Low-Income Consumers § 54.418 Digital Television Transition Notices by Eligible Telecommunications...

  2. 47 CFR 54.418 - Digital Television Transition Notices by Eligible Telecommunications Carriers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 3 2011-10-01 2011-10-01 false Digital Television Transition Notices by Eligible Telecommunications Carriers. 54.418 Section 54.418 Telecommunication FEDERAL COMMUNICATIONS... Low-Income Consumers § 54.418 Digital Television Transition Notices by Eligible Telecommunications...

  3. Parent-mediated reading interventions with children up to four years old: a systematic review.

    PubMed

    Sloat, Elizabeth A; Letourneau, Nicole L; Joschko, Justin R; Schryer, Erin A; Colpitts, Jennifer E

    2015-03-01

    Research demonstrates that literacy and academic achievement are predicated on the emergent literacy knowledge and skills children acquire from birth up to 4 years of age. Parents are children's first and most important language and literacy teachers, yet not all parents have the capacity to establish an adequate early literacy foundation. Efforts to address this situation have resulted in numerous programs aimed at fostering emergent literacy development. This systematic review evaluates evidence on the effectiveness of parent-mediated interventions that increase the time parents spend reading with young children up to 4 years old. Four studies met inclusion criteria, reporting outcomes for 664 children. Three provided data for meta-analysis of effects on reading duration. The standardized mean difference in reading duration was 1.61 (95% CI, 1.03, 2.19 fixed-effect), favoring intervention over control. Results indicate that interventions aimed at increasing the amount of time parents spend reading interactively with their children yield positive results. Findings also demonstrate that pediatric primary care providers are well positioned to deliver reading promotion programs to parents and preschoolers.

  4. High-Efficiency "-1" and "-2" Ribosomal Frameshiftings Revealed by Force Spectroscopy.

    PubMed

    Tsai, Te-Wei; Yang, Haopeng; Yin, Heng; Xu, Shoujun; Wang, Yuhong

    2017-06-16

    Ribosomal frameshifting is a rare but ubiquitous process that is being studied extensively. Meanwhile, frameshifting motifs without any secondary mRNA structures were identified but rarely studied experimentally. We report unambiguous observation of highly efficient "-1" and "-2" frameshiftings on a GA 7 G slippery mRNA without the downstream secondary structure, using force-induced remnant magnetization spectroscopy combined with unique probing schemes. The result represents the first experimental evidence of multiple frameshifting steps. It is also one of the rare reports of the "-2" frameshifting. Our assay removed the ambiguity of transcriptional slippage involvement in other frameshifting assays. Two significant insights for the frameshifting mechanism were revealed. First, EF-G·GTP is indispensable to frameshifting. Although EFG·GDPCP has been shown to prompt translocation before, we found that it could not induce frameshifting. This implies that the GTP hydrolysis is responsible for the codon-anticodon re-pairing in frameshifting, which corroborates our previous mechanical force measurement of EF-G·GTP. Second, translation in all three reading frames of the slippery sequence can be induced by the corresponding in-frame aminoacyl tRNAs. Although A-site tRNA is known to affect the partition between "0" and "-1" frameshifting, it has not been reported that all three reading frames can be translated by their corresponding tRNAs. The in vitro results were confirmed by toe-printing assay and protein sequencing.

  5. 43 CFR Appendix A to Part 418 - Calculation of Efficiency Equation

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 1 2011-10-01 2011-10-01 false Calculation of Efficiency Equation A Appendix A to Part 418 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION.... 418, App. A Appendix A to Part 418—Calculation of Efficiency Equation ER18DE97.008 ER18DE97.009 ...

  6. 43 CFR Appendix A to Part 418 - Calculation of Efficiency Equation

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Calculation of Efficiency Equation A Appendix A to Part 418 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION.... 418, App. A Appendix A to Part 418—Calculation of Efficiency Equation ER18DE97.008 ER18DE97.009 ...

  7. 40 CFR 418.60 - Applicability; description of the ammonium sulfate production subcategory.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ammonium sulfate production subcategory. 418.60 Section 418.60 Protection of Environment ENVIRONMENTAL... CATEGORY Ammonium Sulfate Production Subcategory § 418.60 Applicability; description of the ammonium sulfate production subcategory. The provisions of this subpart apply to discharges resulting from the...

  8. 40 CFR 418.60 - Applicability; description of the ammonium sulfate production subcategory.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ammonium sulfate production subcategory. 418.60 Section 418.60 Protection of Environment ENVIRONMENTAL... CATEGORY Ammonium Sulfate Production Subcategory § 418.60 Applicability; description of the ammonium sulfate production subcategory. The provisions of this subpart apply to discharges resulting from the...

  9. 40 CFR 418.60 - Applicability; description of the ammonium sulfate production subcategory.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ammonium sulfate production subcategory. 418.60 Section 418.60 Protection of Environment ENVIRONMENTAL... CATEGORY Ammonium Sulfate Production Subcategory § 418.60 Applicability; description of the ammonium sulfate production subcategory. The provisions of this subpart apply to discharges resulting from the...

  10. 40 CFR 418.60 - Applicability; description of the ammonium sulfate production subcategory.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ammonium sulfate production subcategory. 418.60 Section 418.60 Protection of Environment ENVIRONMENTAL... CATEGORY Ammonium Sulfate Production Subcategory § 418.60 Applicability; description of the ammonium sulfate production subcategory. The provisions of this subpart apply to discharges resulting from the...

  11. Computer-Mediated Glosses in Second Language Reading Comprehension and Vocabulary Learning: A Meta-Analysis

    ERIC Educational Resources Information Center

    Abraham, Lee B.

    2008-01-01

    Language learners have unprecedented opportunities for developing second language literacy skills and intercultural understanding by reading authentic texts on the Internet and in multimedia computer-assisted language learning environments. This article presents findings from a meta-analysis of 11 studies of computer-mediated glosses in second…

  12. 42 CFR 418.52 - Condition of participation: Patient's rights.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Condition of participation: Patient's rights. 418.52 Section 418.52 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... alleged violations involving mistreatment, neglect, or verbal, mental, sexual, and physical abuse...

  13. 42 CFR 418.52 - Condition of participation: Patient's rights.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Condition of participation: Patient's rights. 418.52 Section 418.52 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... alleged violations involving mistreatment, neglect, or verbal, mental, sexual, and physical abuse...

  14. 42 CFR 418.52 - Condition of participation: Patient's rights.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Condition of participation: Patient's rights. 418.52 Section 418.52 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... alleged violations involving mistreatment, neglect, or verbal, mental, sexual, and physical abuse...

  15. Transcriptome-wide studies uncover the diversity of modes of mRNA recruitment to eukaryotic ribosomes.

    PubMed

    Shatsky, Ivan N; Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M

    2014-01-01

    The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5' untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5' ends without the involvement of either the 5' m(7)G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.

  16. TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

    PubMed

    Ghosh, Abhishek; Rideout, Elizabeth J; Grewal, Savraj S

    2014-10-01

    The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

  17. TIF-IA-Dependent Regulation of Ribosome Synthesis in Drosophila Muscle Is Required to Maintain Systemic Insulin Signaling and Larval Growth

    PubMed Central

    Ghosh, Abhishek; Rideout, Elizabeth J.; Grewal, Savraj S.

    2014-01-01

    The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis—a limiting step in ribosome biogenesis—via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2—a secreted factor that binds and inhibits dILP activity—from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis. PMID:25356674

  18. Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell.

    PubMed

    Kavitha, Nowroji; Ein Oon, Chern; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

    2017-04-06

    Phaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. The purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. MDA-MB-231 cells were incubated with PMEAF for 24h. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. The AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G 0 /G 1 and G 2 /M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆ Ψm ) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells. The results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and anti-apoptotic proteins, caspases, G 0 /G 1 and G 2 /M-phases cell cycle arrest by p53-mediated mechanism. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  19. Looking through an adolescent literacy lens at the narrow view of reading.

    PubMed

    Ehren, Barbara J

    2009-04-01

    This commentary is a personal reaction to A. G. Kamhi's (2007) article on the "narrow view" of reading and his suggestion that this view be adopted as a way to address the reading problems of children and adolescents. In this article, I consider the narrow view of reading from an adolescent literacy perspective and discuss the practical implications of adopting this view in the schools. Discussion revolves around the complexities of reading comprehension, comprehension as a teachable set of complex processes, and the speech-language pathologist's role in reading comprehension. Although I acknowledge that the narrow view of reading may have merit, I opine that it may create more problems than it solves.

  20. 5S rRNA and ribosome.

    PubMed

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  1. Ribosomal targets for antibiotic drug discovery

    DOEpatents

    Blanchard, Scott C.; Feldman, Michael Brian; Wang, Leyi; Doudna Cate, James H.; Pulk, Arto; Altman, Roger B.; Wasserman, Michael R

    2016-09-13

    The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.

  2. Viewing Eye Movements During Reading through the Lens of Chaos Theory: How Reading Is Like the Weather

    ERIC Educational Resources Information Center

    Paulson, Eric J.

    2005-01-01

    This theoretical article examines reading processes using chaos theory as an analogy. Three principles of chaos theory are identified and discussed, then related to reading processes as revealed through eye movement research. Used as an analogy, the chaos theory principle of sensitive dependence contributes to understanding the difficulty in…

  3. Big Dreams: A Family Book about Reading. Preschool through Grade Three

    ERIC Educational Resources Information Center

    Goldman, Elizabeth; Adler, C. Ralph

    2006-01-01

    This family booklet about reading is aimed at parents of children in Preschool through 3rd Grade. The simple text provides ideas for parents of all literacy skill levels to read with their children and find lessons for reading in everyday activities. Each page presents an exercise with the following page showing a black-and-white photograph of a…

  4. Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions

    PubMed Central

    Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří

    2017-01-01

    Abstract Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA. PMID:27986857

  5. In vitro degradation of ribosomes.

    PubMed

    Mora, G; Rivas, A

    1976-12-01

    The cytoplasmic ribosomes from Euglena gracilis var. bacillaris are found to be of two types taking into consideration their stability "in vitro". In the group of unstable ribosomes the large subunit is degraded. The other group apparently does not suffer any degradation under the conditions described. However the RNAs extracted from both types of ribosomes are degraded during sucrose density gradients. The degradation of the largest RNA species has been reported previously, but no comment has been made about the stability of the ribosome itself.

  6. Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

    PubMed

    Khajuria, Rajiv K; Munschauer, Mathias; Ulirsch, Jacob C; Fiorini, Claudia; Ludwig, Leif S; McFarland, Sean K; Abdulhay, Nour J; Specht, Harrison; Keshishian, Hasmik; Mani, D R; Jovanovic, Marko; Ellis, Steven R; Fulco, Charles P; Engreitz, Jesse M; Schütz, Sabina; Lian, John; Gripp, Karen W; Weinberg, Olga K; Pinkus, Geraldine S; Gehrke, Lee; Regev, Aviv; Lander, Eric S; Gazda, Hanna T; Lee, Winston Y; Panse, Vikram G; Carr, Steven A; Sankaran, Vijay G

    2018-03-22

    Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. 42 CFR 418.60 - Condition of participation: Infection control.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Condition of participation: Infection control. 418.60 Section 418.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND....60 Condition of participation: Infection control. The hospice must maintain and document an effective...

  8. 42 CFR 418.60 - Condition of participation: Infection control.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... communicable diseases, including the use of standard precautions. (b) Standard: Control. The hospice must... 42 Public Health 3 2012-10-01 2012-10-01 false Condition of participation: Infection control. 418.60 Section 418.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...

  9. 42 CFR 418.60 - Condition of participation: Infection control.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... communicable diseases, including the use of standard precautions. (b) Standard: Control. The hospice must... 42 Public Health 3 2014-10-01 2014-10-01 false Condition of participation: Infection control. 418.60 Section 418.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...

  10. 42 CFR 418.60 - Condition of participation: Infection control.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... communicable diseases, including the use of standard precautions. (b) Standard: Control. The hospice must... 42 Public Health 3 2013-10-01 2013-10-01 false Condition of participation: Infection control. 418.60 Section 418.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND...

  11. The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

    PubMed Central

    Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

    2017-01-01

    Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC. PMID:28685749

  12. The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs.

    PubMed

    Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

    2017-07-07

    Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNA Lys (UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

  13. Aldosterone mediates its rapid effects in vascular endothelial cells through GPER activation.

    PubMed

    Gros, Robert; Ding, Qingming; Liu, Bonan; Chorazyczewski, Jozef; Feldman, Ross D

    2013-03-01

    The importance of the rapid vascular effects of aldosterone is increasingly appreciated. Through these rapid pathways, aldosterone has been shown to regulate vascular contractility, cell growth, and apoptosis. In our most recent studies, we demonstrated the effects of aldosterone on cell growth and contractility in vascular smooth muscle cells. We showed that these effects could occur via activation of the classic mineralocorticoid receptor, as well the recently characterized G protein-coupled estrogen receptor (GPER), initially characterized as an estrogen-specific receptor. However, the mechanisms underlying aldosterone's endothelium-dependent actions are unknown. Furthermore, the ERK regulatory and proapoptotic effects of aldosterone mediated by GPER activation in cultured vascular smooth muscle cells were only apparent when GPER was reintroduced into these cells by gene transfer. Whether GPER activation via aldosterone might be an important regulator in native vascular cells has been questioned. Therefore, to determine the role of GPER in mediating aldosterone's effects on cell growth and vascular reactivity in native cells, we examined rat aortic vascular endothelial cells, a model characterized by persistent robust expression of GPER, but without detectable mineralocorticoid receptor expression. In these endothelial cells, the GPER agonist G1 mediates a rapid increase in ERK phosphorylation that is wholly GPER-dependent, paralleling the actions of aldosterone. The effects of G1 and aldosterone to stimulate ERK phosphorylation paralleled their proapoptotic and antiproliferative effects. In previous studies, we reported that aldosterone mediates a rapid endothelium-dependent vasodilatory effect, antagonistic to its direct vasoconstrictor effect in endothelium-denuded preparations. Using a rat aortic ring/organ bath preparation to determine the GPER dependence of aldosterone's endothelium-dependent vasodilator effects, we demonstrate that aldosterone inhibits

  14. Energetics of codon-anticodon recognition on the small ribosomal subunit.

    PubMed

    Almlöf, Martin; Andér, Martin; Aqvist, Johan

    2007-01-09

    Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

  15. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ostrup, Olga, E-mail: osvarcova@gmail.com; Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo; Norwegian Center for Stem Cell Research, Oslo

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression.more » This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.« less

  16. 42 CFR 418.52 - Condition of participation: Patient's rights.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Condition of participation: Patient's rights. 418.52 Section 418.52 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... her rights, and the hospice must protect and promote the exercise of these rights. (a) Standard...

  17. Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.

    PubMed

    Park, Sang-Wook; Stevens, Noah M; Vivanco, Jorge M

    2002-12-01

    Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.

  18. Unemployment and subsequent depression: A mediation analysis using the parametric G-formula.

    PubMed

    Bijlsma, Maarten J; Tarkiainen, Lasse; Myrskylä, Mikko; Martikainen, Pekka

    2017-12-01

    The effects of unemployment on depression are difficult to establish because of confounding and limited understanding of the mechanisms at the population level. In particular, due to longitudinal interdependencies between exposures, mediators and outcomes, intermediate confounding is an obstacle for mediation analyses. Using longitudinal Finnish register data on socio-economic characteristics and medication purchases, we extracted individuals who entered the labor market between ages 16 and 25 in the period 1996 to 2001 and followed them until the year 2007 (n = 42,172). With the parametric G-formula we estimated the population-averaged effect on first antidepressant purchase of a simulated intervention which set all unemployed person-years to employed. In the data, 74% of person-years were employed and 8% unemployed, the rest belonging to studying or other status. In the intervention scenario, employment rose to 85% and the hazard of first antidepressant purchase decreased by 7.6%. Of this reduction 61% was mediated, operating primarily through changes in income and household status, while mediation through other health conditions was negligible. These effects were negligible for women and particularly prominent among less educated men. By taking complex interdependencies into account in a framework of observed repeated measures data, we found that eradicating unemployment raises income levels, promotes family formation, and thereby reduces antidepressant consumption at the population-level. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. 20 CFR 418.3123 - When is a change in your subsidy effective?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false When is a change in your subsidy effective? 418.3123 Section 418.3123 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare Part D Subsidies Eligibility for A Medicare Prescription Drug Subsidy § 418.3123 When is a change...

  20. Targeting ricin to the ribosome.

    PubMed

    May, Kerrie L; Yan, Qing; Tumer, Nilgun E

    2013-07-01

    The plant toxin ricin is highly toxic for mammalian cells and is of concern for bioterrorism. Ricin belongs to a family of functionally related toxins, collectively referred to as ribosome inactivating proteins (RIPs), which disable ribosomes and halt protein synthesis. Currently there are no specific antidotes against ricin or related RIPs. The catalytic subunit of ricin is an N-glycosidase that depurinates a universally conserved adenine residue within the sarcin/ricin loop (SRL) of the 28S rRNA. This depurination activity inhibits translation and its biochemistry has been intensively studied. Yet, recent developments paint a more complex picture of toxicity, with ribosomal proteins and cellular signaling pathways contributing to the potency of ricin. In particular, several studies have now established the importance of the ribosomal stalk structure in facilitating the depurination activity and ribosome specificity of ricin and other RIPs. This review highlights recent developments defining toxin-ribosome interactions and examines the significance of these interactions for toxicity and therapeutic intervention. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. 42 CFR 418.52 - Condition of participation: Patient's rights.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Condition of participation: Patient's rights. 418...: Patient Care § 418.52 Condition of participation: Patient's rights. The patient has the right to be... written notice of the patient's rights and responsibilities in a language and manner that the patient...

  2. LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

    PubMed Central

    Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

    2014-01-01

    Summary Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, “sensitization” of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis. PMID:24749077

  3. 42 CFR 418.205 - Special requirements for hospice pre-election evaluation and counseling services.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... evaluation and counseling services. 418.205 Section 418.205 Public Health CENTERS FOR MEDICARE & MEDICAID... Covered Services § 418.205 Special requirements for hospice pre-election evaluation and counseling... pre-election evaluation and counseling services as specified in § 418.304(d) may be made to a hospice...

  4. 42 CFR 418.205 - Special requirements for hospice pre-election evaluation and counseling services.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... evaluation and counseling services. 418.205 Section 418.205 Public Health CENTERS FOR MEDICARE & MEDICAID... Covered Services § 418.205 Special requirements for hospice pre-election evaluation and counseling... pre-election evaluation and counseling services as specified in § 418.304(d) may be made to a hospice...

  5. 42 CFR 418.205 - Special requirements for hospice pre-election evaluation and counseling services.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... evaluation and counseling services. 418.205 Section 418.205 Public Health CENTERS FOR MEDICARE & MEDICAID... Covered Services § 418.205 Special requirements for hospice pre-election evaluation and counseling... pre-election evaluation and counseling services as specified in § 418.304(d) may be made to a hospice...

  6. Increasing Reading Comprehension of Elementary Students through Fluency-Based Interventions

    ERIC Educational Resources Information Center

    Neumann, Veda S.; Ross, Dorothy K.; Slaboch, Anita F.

    2008-01-01

    The authors of this action research project report implemented oral reading fluency-based interventions for the purpose of improving students' reading comprehension. Six students in grade three, six students in grade five and six students in grade six participated in the study from Monday, August 27 through Friday, December 7, 2007. Researchers…

  7. Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter.

    PubMed

    Huang, Shijie; Zhu, Xuechen; Melançon, Charles E

    2016-01-15

    The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome-controlled green fluorescent protein (GFP) reporter that produce fluorescent signal when the orthogonal ribosome is inhibited. As a proof of concept, we demonstrate that these strains, when coexpressing homogeneous populations of aminoglycoside resistant ribosomes, act as sensitive and quantitative detectors of ribosome inhibition by a set of 12 structurally diverse aminoglycoside antibiotics. We suggest that this strategy can be extended to quantifying ribosome inhibition by other drug classes.

  8. 42 CFR 418.70 - Condition of participation: Furnishing of non-core services.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Condition of participation: Furnishing of non-core services. 418.70 Section 418.70 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... Care Non-Core Services § 418.70 Condition of participation: Furnishing of non-core services. A hospice...

  9. 42 CFR 418.70 - Condition of participation: Furnishing of non-core services.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Condition of participation: Furnishing of non-core services. 418.70 Section 418.70 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... Care Non-Core Services § 418.70 Condition of participation: Furnishing of non-core services. A hospice...

  10. 20 CFR 418.2210 - What is not a major life-changing event?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false What is not a major life-changing event? 418.2210 Section 418.2210 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income... Recent Tax Year's Modified Adjusted Gross Income § 418.2210 What is not a major life-changing event? We...

  11. 20 CFR 418.2210 - What is not a major life-changing event?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false What is not a major life-changing event? 418.2210 Section 418.2210 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income... Recent Tax Year's Modified Adjusted Gross Income § 418.2210 What is not a major life-changing event? We...

  12. 20 CFR 418.2225 - Which more recent tax year will we use?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false Which more recent tax year will we use? 418.2225 Section 418.2225 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income... Recent Tax Year's Modified Adjusted Gross Income § 418.2225 Which more recent tax year will we use? We...

  13. 20 CFR 418.2225 - Which more recent tax year will we use?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false Which more recent tax year will we use? 418.2225 Section 418.2225 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income... Recent Tax Year's Modified Adjusted Gross Income § 418.2225 Which more recent tax year will we use? We...

  14. 20 CFR 418.2225 - Which more recent tax year will we use?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false Which more recent tax year will we use? 418.2225 Section 418.2225 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income... Recent Tax Year's Modified Adjusted Gross Income § 418.2225 Which more recent tax year will we use? We...

  15. 20 CFR 418.2225 - Which more recent tax year will we use?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false Which more recent tax year will we use? 418.2225 Section 418.2225 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income... Recent Tax Year's Modified Adjusted Gross Income § 418.2225 Which more recent tax year will we use? We...

  16. 20 CFR 418.2210 - What is not a major life-changing event?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false What is not a major life-changing event? 418.2210 Section 418.2210 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Income... Recent Tax Year's Modified Adjusted Gross Income § 418.2210 What is not a major life-changing event? We...

  17. TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p

    PubMed Central

    Bodem, Jochen; Dobreva, Gergana; Hoffmann-Rohrer, Urs; Iben, Sebastian; Zentgraf, Hanswalter; Delius, Hajo; Vingron, Martin; Grummt, Ingrid

    2000-01-01

    Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro. PMID:11265758

  18. RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor.

    PubMed

    Mikulík, K; Janda, I; Weiser, J; Stastná, J; Jiránová, A

    1984-12-03

    Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.

  19. Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain

    PubMed Central

    Koch, Miriam; Willi, Jessica; Pradère, Ugo; Hall, Jonathan

    2017-01-01

    Abstract The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation. PMID:28369621

  20. Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

    PubMed Central

    1996-01-01

    An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

  1. Differences in the ribosomes prepared from lactating and non-lactating bovine mammary gland

    PubMed Central

    Herrington, M. D.; Hawtrey, A. O.

    1971-01-01

    1. Ribosomes prepared from bovine lactating mammary gland are able to synthesize protein, whereas similar preparations from non-lactating glands are not. Washing the ribosome suspensions through a medium containing 0.5m-ammonium chloride enhanced their ability to incorporate phenylalanine into polyphenylalanine. 2. Ribosomes isolated from non-lactating bovine mammary gland, in contrast with those from rat liver and lactating mammary gland, contained significant amounts of extraneous nucleases. These enzymes could be removed by washing with a medium A buffer containing 0.5m-ammonium chloride. 3. Only those ribosomes from functionally active tissues were able to bind polyuridylic acid and phenylalanyl-tRNA. PMID:5165653

  2. Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

    PubMed Central

    Kuyumcu-Martinez, N. Muge; Joachims, Michelle; Lloyd, Richard E.

    2002-01-01

    Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases. PMID:11836384

  3. Proteomic analysis of rodent ribosomes revealed heterogeneity including ribosomal proteins L10-like, L22-like 1, and L39-like.

    PubMed

    Sugihara, Yoshihiko; Honda, Hiroki; Iida, Tomoharu; Morinaga, Takuma; Hino, Shingo; Okajima, Tetsuya; Matsuda, Tsukasa; Nadano, Daita

    2010-03-05

    Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome.

  4. 42 CFR 418.205 - Special requirements for hospice pre-election evaluation and counseling services.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... evaluation and counseling services. 418.205 Section 418.205 Public Health CENTERS FOR MEDICARE & MEDICAID... Services § 418.205 Special requirements for hospice pre-election evaluation and counseling services. (a... evaluation and counseling services as specified in § 418.304(d) may be made to a hospice on behalf of a...

  5. 42 CFR 418.205 - Special requirements for hospice pre-election evaluation and counseling services.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... evaluation and counseling services. 418.205 Section 418.205 Public Health CENTERS FOR MEDICARE & MEDICAID... Services § 418.205 Special requirements for hospice pre-election evaluation and counseling services. (a... evaluation and counseling services as specified in § 418.304(d) may be made to a hospice on behalf of a...

  6. Expression of Human Hemojuvelin (HJV) Is Tightly Regulated by Two Upstream Open Reading Frames in HJV mRNA That Respond to Iron Overload in Hepatic Cells

    PubMed Central

    Onofre, Cláudia; Tomé, Filipa; Barbosa, Cristina; Silva, Ana Luísa

    2015-01-01

    The gene encoding human hemojuvelin (HJV) is one of the genes that, when mutated, can cause juvenile hemochromatosis, an early-onset inherited disorder associated with iron overload. The 5′ untranslated region of the human HJV mRNA has two upstream open reading frames (uORFs), with 28 and 19 codons formed by two upstream AUGs (uAUGs) sharing the same in-frame stop codon. Here we show that these uORFs decrease the translational efficiency of the downstream main ORF in HeLa and HepG2 cells. Indeed, ribosomal access to the main AUG is conditioned by the strong uAUG context, which results in the first uORF being translated most frequently. The reach of the main ORF is then achieved by ribosomes that resume scanning after uORF translation. Furthermore, the amino acid sequences of the uORF-encoded peptides also reinforce the translational repression of the main ORF. Interestingly, when iron levels increase, translational repression is relieved specifically in hepatic cells. The upregulation of protein levels occurs along with phosphorylation of the eukaryotic initiation factor 2α. Nevertheless, our results support a model in which the increasing recognition of the main AUG is mediated by a tissue-specific factor that promotes uORF bypass. These results support a tight HJV translational regulation involved in iron homeostasis. PMID:25666510

  7. Association of the DAT1 Genotype with Inattentive Behavior Is Mediated by Reading Ability in a General Population Sample

    ERIC Educational Resources Information Center

    Cornish, Kim M.; Savage, Robert; Hocking, Darren R.; Hollis, Chris P.

    2011-01-01

    Attention deficit hyperactivity disorder (ADHD) and reading disability (RD) frequently co-occur in the child population and therefore raise the possibility of shared genetic etiology. We used a quantitative trait loci (QTL) approach to assess the involvement of the dopamine transporter (DAT1) gene polymorphism in mediating reading disability and…

  8. 20 CFR 418.1210 - What is not a major life-changing event?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false What is not a major life-changing event? 418.1210 Section 418.1210 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare... Adjusted Gross Income § 418.1210 What is not a major life-changing event? We will not consider events other...

  9. 20 CFR 418.1210 - What is not a major life-changing event?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false What is not a major life-changing event? 418.1210 Section 418.1210 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES Medicare... Adjusted Gross Income § 418.1210 What is not a major life-changing event? We will not consider events other...

  10. TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus.

    PubMed

    Wu, Yong; Gao, Tieli; Wang, Xiaolin; Hu, Youjin; Hu, Xuyun; Hu, Zhiqing; Pang, Jialun; Li, Zhuo; Xue, Jinfeng; Feng, Mai; Wu, Lingqian; Liang, Desheng

    2014-03-28

    Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Fully automated synthesis of 4-[18F]fluorobenzylamine based on borohydride/NiCl2 reduction.

    PubMed

    Way, Jenilee; Wuest, Frank

    2013-04-01

    4-[(18)F]Fluorobenzylamine ([(18)F]FBA) is an important building block for the synthesis of (18)F-labeled compounds. Synthesis of [(18)F]FBA usually involves application of strong reducing agents like LiAlH4 which is challenging to handle in automated synthesis units (ASUs). Therefore, alternative methods for the preparation of [(18)F]FBA compatible with remotely-controlled syntheses in ASUs are needed. (18)F]FBA was prepared in a remotely-controlled synthesis unit (GE TRACERlab™ FX) based on Ni(II)-mediated borohydride exchange resin (BER) reduction of 4-[(18)F]fluorobenzonitrile ([(18)F]FBN). [(18)F]FBA was used for the synthesis of novel thiol-reactive prosthetic group 4-[(18)F]fluorobenzyl)maleimide [(18)F]FBM and Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin [(18)F] GA. [(18)F]FBA could be prepared in high radiochemical yield greater than 80% (decay-corrected) within 60min. In a typical experiment, 7.4GBq of [(18)F]FBA could be obtained in high radiochemical purity of greater than 95% starting from 10GBq of cyclotron-produced n.c.a. [(18)F]fluoride. [(18)F]FBA was used for the preparation of 4-[(18)F]fluorobenzyl)maleimide as a novel prosthetic group for labeling of thiol groups as demonstrated with tripeptide glutathione. [(18)F]FBA was also used as building block for the syntheses of small molecules as exemplified by the preparation of Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin. The described remotely-controlled synthesis of [(18)F]FBA will significantly improve the availability of [(18)F]FBA as an important and versatile building block for the development of novel (18)F-labeled compounds containing a fluorobenzylamine moiety. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. The Ribosome Shape Directs mRNA Translocation through Entrance and Exit Dynamics

    USDA-ARS?s Scientific Manuscript database

    The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs (transfer ribonucleic acids) and mRNA (messenger ribonucleic acid); here the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal struc...

  13. Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans

    PubMed Central

    Holdt, Lesca M.; Stahringer, Anika; Sass, Kristina; Pichler, Garwin; Kulak, Nils A.; Wilfert, Wolfgang; Kohlmaier, Alexander; Herbst, Andreas; Northoff, Bernd H.; Nicolaou, Alexandros; Gäbel, Gabor; Beutner, Frank; Scholz, Markus; Thiery, Joachim; Musunuru, Kiran; Krohn, Knut; Mann, Matthias; Teupser, Daniel

    2016-01-01

    Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease. PMID:27539542

  14. Characterization of four species of Trichuris (Nematoda: Enoplida) by their second internal transcribed spacer ribosomal DNA sequence.

    PubMed

    Oliveros, R; Cutillas, C; De Rojas, M; Arias, P

    2000-12-01

    Adult worms of Trichuris ovis and T. globulosa were collected from Ovis aries (sheep) and Capra hircus (goats). T. suis was isolated from Sus scrofa domestica (swine) and T. leporis was isolated from Lepus europaeus (rabbits) in Spain. Genomic DNA was isolated and a ribosomal internal transcribed spacer (ITS2) was amplified and sequenced using polymerase-chain-reaction (PCR) techniques. The ITS2 of T. ovis and T. globulosa was 407 nucleotides in length and had a GC content of about 62%. Furthermore, the ITS2 of T. suis and T. leporis was 534 and 418 nucleotides in length and had a GC content of about 64.8% and 62.4%, respectively. There was evidence of slight variation in the sequence within individuals of all species analyzed, indicating intraindividual variation in the sequence of different copies of the ribosomal DNA. Furthermore, low-level intraspecific variation was detected. Sequence analyses of ITS2 products of T. ovis and T. globulosa demonstrated no sequence difference between them. Nevertheless, differences were detected between the ITS2 sequences of T. suis, T. leporis, and T. ovis, indicating that Trichuris species can reliably be differentiated by their ITS2 sequences and PCR-linked restriction-fragment-length polymorphism (RFLP).

  15. 43 CFR 418.9 - Reporting changes in eligible land.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Reporting changes in eligible land. 418.9... Conditions of Water Delivery § 418.9 Reporting changes in eligible land. (a) Eligible land anticipated to be irrigated. (1) Anticipated changes in irrigated eligible land from the prior year will be reported to the...

  16. A New Measure of Reading Habit: Going Beyond Behavioral Frequency

    PubMed Central

    Schmidt, Fabian T. C.; Retelsdorf, Jan

    2016-01-01

    Reading habit is considered an important construct in reading research as it serves as a significant predictor of reading achievement. However, there is still no consensus on how to best measure reading habit. In recent research, it has mostly been measured as behavioral frequency; this approach neglects the fact that repeated behavior does not cover the broad content of habitual behavior—such as automaticity and the expression of one’s identity. In this study, we aimed to adapt a 10-item scale on the basis of the Self-Report Habit Index by Verplanken and Orbell (2003) that is comprehensive but still economical for measuring reading habit. It was tested by drawing on a sample of N = 1,418 upper secondary school students. The scale showed good psychometric properties and the internal and external validity was supported. Moreover, the scale predicted reading achievement and decoding speed over and above reading frequency. The implications of an elaborated but still economical way of measuring reading habit are discussed giving new impetus on research on reading habit, challenging conventional approaches of traditional measures. PMID:27660619

  17. N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)-benzylamine (4-(18)F-ADAM): an improved PET radioligand for serotonin transporters.

    PubMed

    Shiue, Grace G; Choi, Seok-Rye; Fang, Ping; Hou, Catherine; Acton, Paul D; Cardi, Chris; Saffer, Janet R; Greenberg, Joel H; Karp, Joel S; Kung, Hank F; Shiue, Chyng-Yann

    2003-12-01

    There has been considerable interest in the development of PET radioligands that are useful for imaging serotonin transporter (SERT) in the living human brain. For the last decade, (11)C-(+)McN5652 has been the most promising PET agent for studying SERT in humans. However, this agent has some limitations. Recently, a new promising SERT PET radioligand, 3-(11)C-amino-4-(2-dimethylaminomethylphenylsulfanyl)benzonitrile, has been reported. We recently reported the synthesis of a new (18)F-labeled SERT PET radioligand, N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)benzylamine (4-(18)F-ADAM), which may have advantages over (11)C-labeled radioligands. The purpose of this study was to evaluate this newly developed (18)F-labeled PET radioligand as a promising agent for studying SERT in the living human brain. This agent was evaluated by studying its in vitro binding to different monoamine transporters, its in vivo biodistributions in rats, its integrity and pharmacologic profiles in rat brain, and its distribution in a female baboon brain. In vitro binding assays showed that 4-F-ADAM displayed high affinity to SERT sites (inhibition constant = 0.081 nmol/L, using membrane preparations of LLC-PK1 cells expressing the specific transporter) and showed more than 1,000- and 28,000-fold selectivity for SERT over norepinephrine transporter and dopamine transporter, respectively. Biodistribution of 4-(18)F-ADAM in rats showed a high initial uptake and slow clearance in the brain (2.13%, 1.90%, and 0.95% injected dose per organ at 2, 30, and 60 min after intravenous injection, respectively), with the specific binding peaking at 2 h after injection (hypothalamus/cerebellum = 12.49). The uptake in blood, muscle, lung, kidney, and liver was also initially high but cleared rapidly. The radioactivity in the femur increases with time for 4-(18)F-ADAM, indicating that in vivo defluorination may occur. In vivo metabolism studies in rats showed that 4-(18)F-ADAM was not metabolized in

  18. 40 CFR 418.65 - Standards of performance for new sources.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true Standards of performance for new sources. 418.65 Section 418.65 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate...

  19. 40 CFR 418.65 - Standards of performance for new sources.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true Standards of performance for new sources. 418.65 Section 418.65 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate...

  20. 40 CFR 418.65 - Standards of performance for new sources.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true Standards of performance for new sources. 418.65 Section 418.65 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate...

  1. 40 CFR 418.65 - Standards of performance for new sources.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true Standards of performance for new sources. 418.65 Section 418.65 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate...

  2. 40 CFR 418.65 - Standards of performance for new sources.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false Standards of performance for new sources. 418.65 Section 418.65 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonium Sulfate...

  3. 40 CFR 418.10 - Applicability; description of the phosphate subcategory.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true Applicability; description of the phosphate subcategory. 418.10 Section 418.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate...

  4. 40 CFR 418.10 - Applicability; description of the phosphate subcategory.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false Applicability; description of the phosphate subcategory. 418.10 Section 418.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate...

  5. 40 CFR 418.10 - Applicability; description of the phosphate subcategory.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true Applicability; description of the phosphate subcategory. 418.10 Section 418.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Phosphate...

  6. Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

    PubMed

    Running, William E; Reilly, James P

    2010-10-01

    Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

  7. Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Woolford, John L.; Baserga, Susan J.

    2013-01-01

    Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. PMID:24190922

  8. 20 CFR 418.1210 - What is not a major life-changing event?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false What is not a major life-changing event? 418... Adjusted Gross Income § 418.1210 What is not a major life-changing event? We will not consider events other than those described in § 418.1205 to be major life-changing events. Certain types of events are not...

  9. 20 CFR 418.1210 - What is not a major life-changing event?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false What is not a major life-changing event? 418... Adjusted Gross Income § 418.1210 What is not a major life-changing event? We will not consider events other than those described in § 418.1205 to be major life-changing events. Certain types of events are not...

  10. Single Molecule Force Measurement for Protein Synthesis on the Ribosome

    NASA Astrophysics Data System (ADS)

    Uemura, Sotaro

    2008-04-01

    The ribosome is a molecular machine that translates the genetic code described on the messenger RNA (mRNA) into an amino acid sequence through repetitive cycles of transfer RNA (tRNA) selection, peptide bond formation and translocation. Although the detailed interactions between the translation components have been revealed by extensive structural and biochemical studies, it is not known how the precise regulation of macromolecular movements required at each stage of translation is achieved. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence. The removal of the SD sequence significantly reduced the rupture force, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA in a pre-peptidyl transfer state. In contrast, the post-peptidyl transfer state weakened the rupture force as compared to the complex in a pre-peptidyl transfer state and it was the same for both the SD-containing and SD-deficient mRNAs. The results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step. In this article, we discuss about the above new results including the introduction of the ribosome translation mechanism and the optical tweezer method.

  11. 40 CFR 418.75 - Standards of performance for new sources.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true Standards of performance for new sources. 418.75 Section 418.75 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer...

  12. 40 CFR 418.75 - Standards of performance for new sources.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 30 2012-07-01 2012-07-01 false Standards of performance for new sources. 418.75 Section 418.75 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer...

  13. 40 CFR 418.75 - Standards of performance for new sources.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 29 2011-07-01 2009-07-01 true Standards of performance for new sources. 418.75 Section 418.75 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer...

  14. 40 CFR 418.75 - Standards of performance for new sources.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 30 2013-07-01 2012-07-01 true Standards of performance for new sources. 418.75 Section 418.75 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer...

  15. 40 CFR 418.75 - Standards of performance for new sources.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 29 2014-07-01 2012-07-01 true Standards of performance for new sources. 418.75 Section 418.75 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Mixed and Blend Fertilizer...

  16. 47 CFR 68.418 - Procedure; designation of agents for service.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 3 2010-10-01 2010-10-01 false Procedure; designation of agents for service. 68.418 Section 68.418 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) CONNECTION OF TERMINAL EQUIPMENT TO THE TELEPHONE NETWORK Complaint Procedures § 68...

  17. 20 CFR 418.3005 - Purpose and administration of the program.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Purpose and administration of the program. 418.3005 Section 418.3005 Employees' Benefits SOCIAL SECURITY ADMINISTRATION MEDICARE SUBSIDIES... and Human Services has responsibility for administration of the Medicare program, including the new...

  18. Book Reading Mediation, SES, Home Literacy Environment, and Children's Literacy: Evidence from Arabic-Speaking Families

    ERIC Educational Resources Information Center

    Korat, Ofra; Arafat, Safieh H.; Aram, Dorit; Klein, Pnina

    2013-01-01

    This article investigates the contribution of maternal mediation in storybook reading, socioeconomic status (SES), and home literacy environment (HLE) to children's literacy level in kindergarten and first grade in Israeli Arabic-speaking families. A total of 109 kindergarten children and their mothers participated. Children's literacy level was…

  19. 40 CFR 418.21 - Specialized definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.21 Specialized... product shall mean the anhydrous ammonia content of the compound manufactured. (c) The term shipping...

  20. 40 CFR 418.21 - Specialized definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... STANDARDS FERTILIZER MANUFACTURING POINT SOURCE CATEGORY Ammonia Subcategory § 418.21 Specialized... product shall mean the anhydrous ammonia content of the compound manufactured. (c) The term shipping...