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Sample records for ribosome inhibitors enhancement

  1. Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.

    PubMed

    Kawashima, Shigehiro A; Chen, Zhen; Aoi, Yuki; Patgiri, Anupam; Kobayashi, Yuki; Nurse, Paul; Kapoor, Tarun M

    2016-10-01

    All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.

  2. Increased ribosome density associated to positively charged residues is evident in ribosome profiling experiments performed in the absence of translation inhibitors.

    PubMed

    Requião, Rodrigo D; de Souza, Henrique José Araujo; Rossetto, Silvana; Domitrovic, Tatiana; Palhano, Fernando L

    2016-06-01

    It has been proposed that polybasic peptides cause slower movement of ribosomes through an electrostatic interaction with the highly negative ribosome exit tunnel. Ribosome profiling data-the sequencing of short ribosome-bound fragments of mRNA-is a powerful tool for the analysis of mRNA translation. Using the yeast Saccharomyces cerevisiae as a model, we showed that reduced translation efficiency associated with polybasic protein sequences could be inferred from ribosome profiling. However, an increase in ribosome density at polybasic sequences was evident only when the commonly used translational inhibitors cycloheximide and anisomycin were omitted during mRNA isolation. Since ribosome profiling performed without inhibitors agrees with experimental evidence obtained by other methods, we conclude that cycloheximide and anisomycin must be avoided in ribosome profiling experiments.

  3. Enhancement of internal ribosome entry site-mediated translation and replication of hepatitis C virus by PD98059

    SciTech Connect

    Murata, Takayuki; Hijikata, Makoto; Shimotohno, Kunitada . E-mail: kshimoto@virus.kyoto-u.ac.jp

    2005-09-15

    Translation initiation of hepatitis C virus (HCV) occurs in an internal ribosome entry site (IRES)-dependent manner. We found that HCV IRES-dependent protein synthesis is enhanced by PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) signaling pathway, while cellular cap-dependent translation was relatively unaffected by the compound. Treatment of cells with PD98059 allowed for robust HCV replication following cellular incubation with HCV-positive serum. Though the molecular mechanism underlying IRES enhancement remains elusive, PD98059 is a potent accelerator of HCV RNA replication.

  4. Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

    PubMed

    Cetin, R; Krab, I M; Anborgh, P H; Cool, R H; Watanabe, T; Sugiyama, T; Izaki, K; Parmeggiani, A

    1996-05-15

    This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF-Tu-GTP is strongly retarded, causing the Kd of EF- Tu-GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP- and GDP-bound EF-Tu on native PAGE is increased. The stimulation of EF-Tu-GDP dissociation by EF-Ts is inhibited. EF- Tu-GTP can still form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no longer protects aa-tRNA against spontaneous deacylation, showing that the EF-Tu-GTP orientation with respect to the 3' end of aa-tRNA is modified. However, the EF-Tu-dependent binding of aa-tRNA to the ribosomal A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-site directly, inducing an anomalous positioning of aa-tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF-Tu and the ribosome.

  5. Biocatalysts with enhanced inhibitor tolerance

    DOEpatents

    Yang, Shihui; Linger, Jeffrey; Franden, Mary Ann; Pienkos, Philip T.; Zhang, Min

    2015-12-08

    Disclosed herein are biocatalysts for the production of biofuels, including microorganisms that contain genetic modifications conferring tolerance to growth and fermentation inhibitors found in many cellulosic feedstocks. Methods of converting cellulose-containing materials to fuels and chemicals, as well as methods of fermenting sugars to fuels and chemicals, using these biocatalysts are also disclosed.

  6. New features of the ribosome and ribosomal inhibitors: non-enzymatic recycling, misreading and back-translocation.

    PubMed

    Szaflarski, Witold; Vesper, Oliver; Teraoka, Yoshika; Plitta, Beata; Wilson, Daniel N; Nierhaus, Knud H

    2008-06-27

    We describe the optimization of a poly(Phe) synthesis system, the conditions of which have been applied for efficient translation of heteropolymeric mRNAs. Here we identify two parameters that are essential to obtain translation at efficiency and accuracy levels equivalent to those in vivo, viz., the fine-tuning of the energy-rich components with an acetyl-phosphate substrate for energy regeneration, as well as the ionic conditions. Applying this system revealed a number of new features: (i) 70S ribosomes are able to recycle within 300 s in a non-enzymatic fashion in the absence of tmRNA. This observation might explain the fact that a knockout of the tmRNA gene ssrA is not lethal for Escherichia coli cells in contrast to other bacterial strains, such as Bacillus subtilis. (ii) The high efficiency of the system was exploited to analyze the misincorporation of various amino acids (resolution limit=1:15,000). No misreading was observed at the middle codon position and only marginal effects were observed at the first one (even when misreading was artificially stimulated 20- to 30-fold), yielding an improved definition of the near-cognate and non-cognate aminoacyl-tRNAs. (iii) Aminoglycosides increase Phe and Lys incorporation about 2-fold in the presence of poly(U) or poly(UUC) and poly(A), respectively, and induce a back-translocation (except hygromycin B) exclusively in the absence of EF-G*GTP, as do the non-related drugs viomycin and edeine.

  7. Single molecule fluorescence studies of ribosome dynamics: An application of metal enhanced fluorescence

    NASA Astrophysics Data System (ADS)

    Bharill, Shashank

    Metal enhanced fluorescence (MEF), in which a surface plasmon near a noble metal alters the spectral properties of an organic fluorophore, has been reported to increase fluorescence intensity without a concomitant increase in photobleaching rate. The fluorescence intensities of Cy3- and Cy5-labeled ribosomal initiation complexes (ICs) near 50 nm silver particles were increased 4 - 7-fold compared to ICs in the absence of silver colloids. Photobleaching lifetime was not significantly decreased, resulting in 4 - 5.5-fold enhancement in total photon emission prior to photobleaching. Fluorophores showing enhanced fluorescence were located within ˜280 nm of the colloidal particles, as detected by light scattering and scanning probe microscopy. Aggregates of silver particles or larger colloids themselves produced wavelength-shifted luminescence similar to fluorescence, presumably due to resonant extinction between nearby metal particles. Intensity fluctuations above shot noise, at 0.1 - 5 Hz, were greater from slides containing colloidal particles than from plain glass. Overall signal to noise ratio was similar or slightly better near the silver particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA to the A site of fluorescent labeled ribosomes, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosomal A and P sites, and elongation factor G catalyzed translocation.

  8. Common Pharmacophore of Structurally Distinct Small-Molecule Inhibitors of Intracellular Retrograde Trafficking of Ribosome Inactivating Proteins

    NASA Astrophysics Data System (ADS)

    Yu, Shichao; Park, Jewn Giew; Kahn, Jennifer Nielsen; Tumer, Nilgun E.; Pang, Yuan-Ping

    2013-12-01

    We reported previously (+/-)-2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one [(+/-)-Retro-2cycl] as the chemical structure of Retro-2 that showed mouse protection against ricin, a notorious ribosome inactivating protein (RIP). Herein we report our chemical resolution of (+/-)-Retro-2cycl, analog synthesis, and cell-based evaluation showing that the two optically pure enantiomers and their achiral analog have nearly the same degree of cell protection against ricin as (+/-)-Retro-2cycl. We also report our computational studies explaining the lack of stereo preference and revealing a common pharmacophore of structurally distinct inhibitors of intracellular retrograde trafficking of RIPs. This pharmacophore comprises a central aromatic ring o-substituted by an aromatic ring and a moiety bearing an O or S atom attached to sp2 C atom(s). These results offer new insights into lead identification and optimization for RIP antidote development to minimize the global health threat caused by ribosome-inactivating proteins.

  9. Fluorescence enhancement on silver nanostructures: studies of components of ribosomal translation in vitro

    NASA Astrophysics Data System (ADS)

    Mandecki, Wlodek; Bharill, Shashank; Borejdo, Julian; Cabral, Diana; Cooperman, Barry S.; Farrell, Ian; Fetter, Linus; Goldman, Emanuel; Gryczynski, Zygmunt; Jakubowski, Hieronim; Liu, Hanqing; Luchowski, Rafal; Matveeva, Evgenia; Pan, Dongli; Qin, Haiou; Tennant, Donald; Gryczynski, Ignacy

    2008-02-01

    Metallic particles, silver in particular, can significantly enhance the fluorescence of dye molecules in the immediate vicinity (5-20 nm) of the particle. This magnifying effect can be theoretically explained/predicted by considering the change of photonic mode density near the fluorophore due to coupling to the conducting surface. We are using this method to observe fluorescence from a single ribosomal particle in a project aimed at acquiring sequence information from the translating ribosome (NIH's $1000 Genome Initiative). Several quartz slides with silver nanostructures were made using electron beam lithography techniques. The structures were approximately 50 nm high silver tiles measuring 400-700 nm on the side, and were spaced differently over a total area of 1 mm x 1 mm on any given quartz slide. In a preliminary experiment, we coated this surface with the Alexa 647-labeled antibodies and collected single molecule images using the MicroTime 200 (PicoQuant) confocal system. We showed that the fluorescence intensity measured over the silver islands film was more than 100-fold higher than fluorescence from a comparable site on uncoated section of the quartz slide. No noticeable photobleaching was seen. The fluorescence lifetime was very short, about 200 ps or less (this is the resolution limit of the system). The method has great promise for investigations of biologically relevant single molecules.

  10. Enhancement of procollagen biosynthesis by p180 through augmented ribosome association on the endoplasmic reticulum in response to stimulated secretion.

    PubMed

    Ueno, Tomonori; Tanaka, Keisuke; Kaneko, Keiko; Taga, Yuki; Sata, Tetsutaro; Irie, Shinkichi; Hattori, Shunji; Ogawa-Goto, Kiyoko

    2010-09-24

    A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER. PMID:20647306

  11. The kissing-loop T-shaped structure translational enhancer of Pea enation mosaic virus can bind simultaneously to ribosomes and a 5' proximal hairpin.

    PubMed

    Gao, Feng; Gulay, Suna P; Kasprzak, Wojciech; Dinman, Jonathan D; Shapiro, Bruce A; Simon, Anne E

    2013-11-01

    The Pea enation mosaic virus (PEMV) 3' translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5'-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5' and 3' PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3' translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5' hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5' end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation.

  12. Ribosome Assembly as Antimicrobial Target.

    PubMed

    Nikolay, Rainer; Schmidt, Sabine; Schlömer, Renate; Deuerling, Elke; Nierhaus, Knud H

    2016-01-01

    Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors. PMID:27240412

  13. Ribosome Assembly as Antimicrobial Target

    PubMed Central

    Nikolay, Rainer; Schmidt, Sabine; Schlömer, Renate; Deuerling, Elke; Nierhaus, Knud H.

    2016-01-01

    Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors. PMID:27240412

  14. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA.

    PubMed

    Wang, Minshi; Parshin, Andrey V; Shcherbik, Natalia; Pestov, Dimitri G

    2015-07-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

  15. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

    PubMed Central

    Wang, Minshi; Parshin, Andrey V.; Shcherbik, Natalia; Pestov, Dimitri G.

    2015-01-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5′-truncated 5.8S rRNA, which we named 5.8SC. The 5′ exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. PMID:25995445

  16. High intake of dietary sugar enhances bisphenol A (BPA) disruption and reveals ribosome-mediated pathways of toxicity.

    PubMed

    Branco, Alan T; Lemos, Bernardo

    2014-05-01

    Bisphenol A (BPA) is an organic compound to which human populations are ubiquitously exposed. Epidemiological data suggest BPA exposure might be associated with higher rates of diabetes and reproductive anomalies. Health concerns also include transgenerational consequences, but these mechanisms are crudely defined. Similarly, little is known about synergistic interactions between BPA and other substances. Here we show that acute and chronic exposure to BPA causes genome-wide modulation of several functionally coherent genetic pathways in the fruit fly Drosophila melanogaster. In particular, BPA exposure causes massive downregulation of testis-specific genes and upregulation of ribosome-associated genes widely expressed across tissues. In addition, it causes the modulation of transposable elements that are specific to the ribosomal DNA loci, suggesting that nucleolar stress might contribute to BPA toxicity. The upregulation of ribosome-associated genes and the impairment of testis-specific gene expression are significantly enhanced upon BPA exposure with a high-sugar diet. Our results suggest that BPA and dietary sugar might functionally interact, with consequences to regulatory programs in both reproductive and somatic tissues.

  17. Microorganisms having enhanced tolerance to inhibitors and stress

    SciTech Connect

    Brown, Steven D.; Yang, Shihui

    2014-07-29

    The present invention provides genetically modified strains of microorganisms that display enhanced tolerance to stress and/or inhibitors such as sodium acetate and vanillin. The enhanced tolerance can be achieved by increasing the expression of a protein of the Sm-like superfamily such as a bacterial Hfq protein and a fungal Sm or Lsm protein. Further, the present invention provides methods of producing alcohol from biomass materials by using the genetically modified microorganisms of the present invention.

  18. Enhancers and Inhibitors of Teacher Change among Secondary Physical Educators

    ERIC Educational Resources Information Center

    Bechtel, Pamela A.; O'Sullivan, Mary

    2007-01-01

    The purpose of this study was to explore enhancers and inhibitors that impacted 4 secondary physical education teachers to make changes in their programs. An interpretivist approach was used to understand the physical educators' change process. Data were collected from document analyses, participant information sheets, interviews, discussion…

  19. Ribosomal Biogenesis and Translational Flux Inhibition by the Selective Inhibitor of Nuclear Export (SINE) XPO1 Antagonist KPT-185

    PubMed Central

    Yamamoto, Shinichi; Sekihara, Kazumasa; Matsushita, Hiromichi; Davis, Richard Eric; Wang, Zhiqiang; Ma, Wencai; Ishizawa, Jo; Kazuno, Saiko; Kauffman, Michael; Shacham, Sharon; Fujimura, Tsutomu; Ueno, Takashi; Miida, Takashi; Andreeff, Michael

    2015-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the aberrant expression of several growth-regulating, oncogenic effectors. Exportin 1 (XPO1) mediates the nucleocytoplasmic transport of numerous molecules including oncogenic growth-regulating factors, RNAs, and ribosomal subunits. In MCL cells, the small molecule KPT-185 blocks XPO1 function and exerts anti-proliferative effects. In this study, we investigated the molecular mechanisms of this putative anti-tumor effect on MCL cells using cell growth/viability assays, immunoblotting, gene expression analysis, and absolute quantification proteomics. KPT-185 exhibited a p53-independent anti-lymphoma effect on MCL cells, by suppression of oncogenic mediators (e.g., XPO1, cyclin D1, c-Myc, PIM1, and Bcl-2 family members), repression of ribosomal biogenesis, and downregulation of translation/chaperone proteins (e.g., PIM2, EEF1A1, EEF2, and HSP70) that are part of the translational/transcriptional network regulated by heat shock factor 1. These results elucidate a novel mechanism in which ribosomal biogenesis appears to be a key component through which XPO1 contributes to tumor cell survival. Thus, we propose that the blockade of XPO1 could be a promising, novel strategy for the treatment of MCL and other malignancies overexpressing XPO1. PMID:26340096

  20. Ribosomal Biogenesis and Translational Flux Inhibition by the Selective Inhibitor of Nuclear Export (SINE) XPO1 Antagonist KPT-185.

    PubMed

    Tabe, Yoko; Kojima, Kensuke; Yamamoto, Shinichi; Sekihara, Kazumasa; Matsushita, Hiromichi; Davis, Richard Eric; Wang, Zhiqiang; Ma, Wencai; Ishizawa, Jo; Kazuno, Saiko; Kauffman, Michael; Shacham, Sharon; Fujimura, Tsutomu; Ueno, Takashi; Miida, Takashi; Andreeff, Michael

    2015-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the aberrant expression of several growth-regulating, oncogenic effectors. Exportin 1 (XPO1) mediates the nucleocytoplasmic transport of numerous molecules including oncogenic growth-regulating factors, RNAs, and ribosomal subunits. In MCL cells, the small molecule KPT-185 blocks XPO1 function and exerts anti-proliferative effects. In this study, we investigated the molecular mechanisms of this putative anti-tumor effect on MCL cells using cell growth/viability assays, immunoblotting, gene expression analysis, and absolute quantification proteomics. KPT-185 exhibited a p53-independent anti-lymphoma effect on MCL cells, by suppression of oncogenic mediators (e.g., XPO1, cyclin D1, c-Myc, PIM1, and Bcl-2 family members), repression of ribosomal biogenesis, and downregulation of translation/chaperone proteins (e.g., PIM2, EEF1A1, EEF2, and HSP70) that are part of the translational/transcriptional network regulated by heat shock factor 1. These results elucidate a novel mechanism in which ribosomal biogenesis appears to be a key component through which XPO1 contributes to tumor cell survival. Thus, we propose that the blockade of XPO1 could be a promising, novel strategy for the treatment of MCL and other malignancies overexpressing XPO1. PMID:26340096

  1. Comparative genomics of bacterial zinc regulons: enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins.

    PubMed

    Panina, Ekaterina M; Mironov, Andrey A; Gelfand, Mikhail S

    2003-08-19

    Zinc is an important component of many proteins, but in large concentrations it is poisonous to the cell. Thus its transport is regulated by zinc repressors ZUR of proteobacteria and Gram-positive bacteria from the Bacillus group and AdcR of bacteria from the Streptococcus group. Comparative computational analysis allowed us to identify binding signals of ZUR repressors GAAATGTTATANTATAACATTTC for gamma-proteobacteria, GTAATGTAATAACATTAC for the Agrobacterium group, GATATGTTATAACATATC for the Rhododoccus group, TAAATCGTAATNATTACGATTTA for Gram-positive bacteria, and TTAACYRGTTAA of the streptococcal AdcR repressor. In addition to known transporters and their paralogs, zinc regulons were predicted to contain a candidate component of the ATP binding cassette, zinT (b1995 in Escherichia coli and yrpE in Bacillus subtilis). Candidate AdcR-binding sites were identified upstream of genes encoding pneumococcal histidine triad (PHT) proteins from a number of pathogenic streptococci. Protein functional analysis of this family suggests that PHT proteins are involved in the invasion process. Finally, repression by zinc was predicted for genes encoding a variety of paralogs of ribosomal proteins. The original copies of all these proteins contain zinc-ribbon motifs and thus likely bind zinc, whereas these motifs are destroyed in zinc-regulated paralogs. We suggest that the induction of these paralogs in conditions of zinc starvation leads to their incorporation in a fraction of ribosomes instead of the original ribosomal proteins; the latter are then degraded with subsequent release of some zinc for the utilization by other proteins. Thus we predict a mechanism for maintaining zinc availability for essential enzymes. PMID:12904577

  2. Functional Phylogeny: the Use of the Sensitivity of Ribosomes to Protein Synthesis Inhibitors as a Tool to Study the Evolution of Organisms

    NASA Astrophysics Data System (ADS)

    Briones, Carlos; Koroutchev, Kostadin; Amils, Ricardo

    1998-10-01

    In order to study the functional phylogeny of organisms, forty different protein synthesis inhibitors with diverse domain and funcional specificities have been used to analyze forty archaeal, bacterial and eukaryotic translational systems. The inhibition curves generated with the different ribosome-antibiotic pairs have shown very interesting similarities among organisms belonging to the same phylogenetic group, confirming the feasibility of using such information in the development of evolutionary studies. A new method to extract most of the information contained in the inhibition curves is presented. Using a statistical treatment based on the principal components analysis of the data, we have defined coordinates for the organisms which have allowed us to perform a functional clustering of them. The phenograms obtained are very similar to those generated by 16/18S rRNA sequence comparison. These results prove the phylogenetic value of our functional analysis and suggest an interesting intersection between genotypic and phenotypic (functional) information.

  3. The Kissing-Loop T-Shaped Structure Translational Enhancer of Pea Enation Mosaic Virus Can Bind Simultaneously to Ribosomes and a 5′ Proximal Hairpin

    PubMed Central

    Gao, Feng; Gulay, Suna P.; Kasprzak, Wojciech; Dinman, Jonathan D.

    2013-01-01

    The Pea Enation Mosaic Virus (PEMV) 3′ translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5′-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5′ and 3′ PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3′ translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5′ hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5′ end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation. PMID:23986599

  4. PDE5 inhibitors enhance celecoxib killing in multiple tumor types.

    PubMed

    Booth, Laurence; Roberts, Jane L; Cruickshanks, Nichola; Tavallai, Seyedmehrad; Webb, Timothy; Samuel, Peter; Conley, Adam; Binion, Brittany; Young, Harold F; Poklepovic, Andrew; Spiegel, Sarah; Dent, Paul

    2015-05-01

    The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. PMID:25303541

  5. PDE5 Inhibitors Enhance Celecoxib Killing in Multiple Tumor Types

    PubMed Central

    BOOTH, LAURENCE; ROBERTS, JANE L.; CRUICKSHANKS, NICHOLA; TAVALLAI, SEYEDMEHRAD; WEBB, TIMOTHY; SAMUEL, PETER; CONLEY, ADAM; BINION, BRITTANY; YOUNG, HAROLD F.; POKLEPOVIC, ANDREW; SPIEGEL, SARAH; DENT, PAUL

    2015-01-01

    The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. PMID:25303541

  6. Isolation of ribosomes and polysomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-01

    Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

  7. Regulation of ribosomal DNA amplification by the TOR pathway

    PubMed Central

    Jack, Carmen V.; Cruz, Cristina; Hull, Ryan M.; Keller, Markus A.; Ralser, Markus; Houseley, Jonathan

    2015-01-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions. PMID:26195783

  8. Regulation of ribosomal DNA amplification by the TOR pathway.

    PubMed

    Jack, Carmen V; Cruz, Cristina; Hull, Ryan M; Keller, Markus A; Ralser, Markus; Houseley, Jonathan

    2015-08-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions.

  9. The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway.

    PubMed

    Bailly, A; Perrin, A; Bou Malhab, L J; Pion, E; Larance, M; Nagala, M; Smith, P; O'Donohue, M-F; Gleizes, P-E; Zomerdijk, J; Lamond, A I; Xirodimas, D P

    2016-01-28

    The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

  10. Sodium borohydride removes aldehyde inhibitors for enhancing biohydrogen fermentation.

    PubMed

    Lin, Richen; Cheng, Jun; Ding, Lingkan; Song, Wenlu; Zhou, Junhu; Cen, Kefa

    2015-12-01

    To enhance biohydrogen production from glucose and xylose in the presence of aldehyde inhibitors, reducing agent (i.e., sodium borohydride) was in situ added for effective detoxification. The detoxification efficiencies of furfural (96.7%) and 5-hydroxymethylfurfural (5-HMF, 91.7%) with 30mM NaBH4 were much higher than those of vanillin (77.3%) and syringaldehyde (69.3%). Biohydrogen fermentation was completely inhibited without detoxification, probably because of the consumption of nicotinamide adenine dinucleotide (NADH) by inhibitors reduction (R-CHO+2NADH→R-CH2OH+2NAD(+)). Addition of 30mM NaBH4 provided the reducing power necessary for inhibitors reduction (4R-CHO+NaBH4+2H2O→4R-CH2OH+NaBO2). The recovered reducing power in fermentation resulted in 99.3% recovery of the hydrogen yield and 64.6% recovery of peak production rate. Metabolite production and carbon conversion after detoxification significantly increased to 63.7mM and 81.9%, respectively.

  11. Resveratrol and STAT inhibitor enhance autophagy in ovarian cancer cells

    PubMed Central

    Zhong, L-X; Zhang, Y; Wu, M-L; Liu, Y-N; Zhang, P; Chen, X-Y; Kong, Q-Y; Liu, J; Li, H

    2016-01-01

    Autophagic activity reflects cellular response to drug treatment and can be regulated by STAT3 signaling. Resveratrol inhibits STAT3 activation and causes remarkable growth arrest and cell death of ovarian cancer (OC) cells. However, the autophagic status and its relevance with resveratrol’s anti-OC effects remain unclear. We analyzed the states of autophagic activities, the nature of autophagosomes and the levels of autophagy-related proteins (LC-3, Beclin 1 and STAT3) in resveratrol-treated CAOV-3 and OVCAR-3 OC cells using multiple approaches. We elucidated the correlation of STAT3 inhibition with autophagic activity by treating OC cells with an upstream inhibitor of STAT proteins, AG490. Resveratrol efficiently suppressed growth, induced apoptosis and inactivated STAT3 signaling of the two OC cell lines. We found enhanced autophagic activity accompanied with Beclin-1 upregulation and LC3 enzymatic cleavage in resveratrol-treated OC cells. Immunofluorescent (IF) microscopic and IF-based confocal examinations demonstrated the accumulation of cytoplasmic granules co-labeled with LC3 and cytochrome C in resveratrol- or AG490-treated OC cells. Using electron microscopy, we confirmed an increase in autophagosomes and mitochondrial spheroids in either resveratrol- or AG490-treated OC cells. This study demonstrates the abilities of resveratrol to enhance apoptotic and autophagic activities in OC cells, presumably via inactivating STAT3 signaling. Resveratrol or the selective JAK2 inhibitor also leads to mitochondrial turnover, which would be unfavorable for OC cell survival and sensitize OC cells to resveratrol. PMID:27551495

  12. Resveratrol and STAT inhibitor enhance autophagy in ovarian cancer cells.

    PubMed

    Zhong, L-X; Zhang, Y; Wu, M-L; Liu, Y-N; Zhang, P; Chen, X-Y; Kong, Q-Y; Liu, J; Li, H

    2016-01-01

    Autophagic activity reflects cellular response to drug treatment and can be regulated by STAT3 signaling. Resveratrol inhibits STAT3 activation and causes remarkable growth arrest and cell death of ovarian cancer (OC) cells. However, the autophagic status and its relevance with resveratrol's anti-OC effects remain unclear. We analyzed the states of autophagic activities, the nature of autophagosomes and the levels of autophagy-related proteins (LC-3, Beclin 1 and STAT3) in resveratrol-treated CAOV-3 and OVCAR-3 OC cells using multiple approaches. We elucidated the correlation of STAT3 inhibition with autophagic activity by treating OC cells with an upstream inhibitor of STAT proteins, AG490. Resveratrol efficiently suppressed growth, induced apoptosis and inactivated STAT3 signaling of the two OC cell lines. We found enhanced autophagic activity accompanied with Beclin-1 upregulation and LC3 enzymatic cleavage in resveratrol-treated OC cells. Immunofluorescent (IF) microscopic and IF-based confocal examinations demonstrated the accumulation of cytoplasmic granules co-labeled with LC3 and cytochrome C in resveratrol- or AG490-treated OC cells. Using electron microscopy, we confirmed an increase in autophagosomes and mitochondrial spheroids in either resveratrol- or AG490-treated OC cells. This study demonstrates the abilities of resveratrol to enhance apoptotic and autophagic activities in OC cells, presumably via inactivating STAT3 signaling. Resveratrol or the selective JAK2 inhibitor also leads to mitochondrial turnover, which would be unfavorable for OC cell survival and sensitize OC cells to resveratrol. PMID:27551495

  13. Inhibitors of the interferon response enhance virus replication in vitro.

    PubMed

    Stewart, Claire E; Randall, Richard E; Adamson, Catherine S

    2014-01-01

    Virus replication efficiency is influenced by two conflicting factors, kinetics of the cellular interferon (IFN) response and induction of an antiviral state versus speed of virus replication and virus-induced inhibition of the IFN response. Disablement of a virus's capacity to circumvent the IFN response enables both basic research and various practical applications. However, such IFN-sensitive viruses can be difficult to grow to high-titer in cells that produce and respond to IFN. The current default option for growing IFN-sensitive viruses is restricted to a limited selection of cell-lines (e.g. Vero cells) that have lost their ability to produce IFN. This study demonstrates that supplementing tissue-culture medium with an IFN inhibitor provides a simple, effective and flexible approach to increase the growth of IFN-sensitive viruses in a cell-line of choice. We report that IFN inhibitors targeting components of the IFN response (TBK1, IKK2, JAK1) significantly increased virus replication. More specifically, the JAK1/2 inhibitor Ruxolitinib enhances the growth of viruses that are sensitive to IFN due to (i) loss of function of the viral IFN antagonist (due to mutation or species-specific constraints) or (ii) mutations/host cell constraints that slow virus spread such that it can be controlled by the IFN response. This was demonstrated for a variety of viruses, including, viruses with disabled IFN antagonists that represent live-attenuated vaccine candidates (Respiratory Syncytial Virus (RSV), Influenza Virus), traditionally attenuated vaccine strains (Measles, Mumps) and a slow-growing wild-type virus (RSV). In conclusion, supplementing tissue culture-medium with an IFN inhibitor to increase the growth of IFN-sensitive viruses in a cell-line of choice represents an approach, which is broadly applicable to research investigating the importance of the IFN response in controlling virus infections and has utility in a number of practical applications including

  14. The 3′ Untranslated Region of Pea Enation Mosaic Virus Contains Two T-Shaped, Ribosome-Binding, Cap-Independent Translation Enhancers

    PubMed Central

    Gao, Feng; Kasprzak, Wojciech K.; Szarko, Christine; Shapiro, Bruce A.

    2014-01-01

    ABSTRACT Many plant viruses without 5′caps or 3′ poly(A) tails contain 3′ proximal, cap-independent translation enhancers (3′CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3′CITEs participate in a long-distance kissing-loop interaction with a 5′ proximal hairpin to deliver ribosomal subunits to the 5′ end for translation initiation. Pea Enation Mosaic Virus (PEMV) contains two adjacent 3′CITEs in the center of its 703-nucleotide 3′ untranslated region (3′UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3′CITE located near the 3′ terminus. This 3′CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3′CITE of the carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3′TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3′TSS contains an apical loop capable of forming a kissing-loop interaction with a 5′ proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3′TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3′UTR in vitro, deleting the normally required kl-TSS and PTE 3′CITEs and placing the kl-H and 3′TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3′CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3′CITE identification. IMPORTANCE The rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3′ cap-independent translation

  15. Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor.

    PubMed

    Dumble, Melissa; Crouthamel, Ming-Chih; Zhang, Shu-Yun; Schaber, Michael; Levy, Dana; Robell, Kimberly; Liu, Qi; Figueroa, David J; Minthorn, Elisabeth A; Seefeld, Mark A; Rouse, Meagan B; Rabindran, Sridhar K; Heerding, Dirk A; Kumar, Rakesh

    2014-01-01

    Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.

  16. Ribosomal proteins: functions beyond the ribosome

    PubMed Central

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

    2015-01-01

    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. PMID:25735597

  17. A Ribosome-Binding, 3′ Translational Enhancer Has a T-Shaped Structure and Engages in a Long-Distance RNA-RNA Interaction

    PubMed Central

    Gao, Feng; Kasprzak, Wojciech; Stupina, Vera A.

    2012-01-01

    Many plant RNA viruses contain elements in their 3′ untranslated regions (3′ UTRs) that enhance translation. The PTE (Panicum mosaic virus-like translational enhancer) of Pea enation mosaic virus (PEMV) binds to eukaryotic initiation factor 4E (eIF4E), but how this affects translation from the 5′ end is unknown. We have discovered a three-way branched element just upstream of the PEMV PTE that engages in a long-distance kissing-loop interaction with a coding sequence hairpin that is critical for the translation of a reporter construct and the accumulation of the viral genome in vivo. Loss of the long-distance interaction was more detrimental than elimination of the adjacent PTE, indicating that the RNA-RNA interaction supports additional translation functions besides relocating the PTE to the 5′ end. The branched element is predicted by molecular modeling and molecular dynamics to form a T-shaped structure (TSS) similar to the ribosome-binding TSS of Turnip crinkle virus (TCV). The PEMV element binds to plant 80S ribosomes with a Kd (dissociation constant) of 0.52 μM and to 60S subunits with a Kd of 0.30 μM. Unlike the TCV TSS, the PEMV element also binds 40S subunits (Kd, 0.36 μM). Mutations in the element that suppressed translation reduced either ribosome binding or the RNA-RNA interaction, suggesting that ribosome binding is important for function. This novel, multifunctional element is designated a kl-TSS (kissing-loop T-shaped structure) to distinguish it from the TCV TSS. The kl-TSS has sequence and structural features conserved with the upper portion of most PTE-type elements, which, with the exception of the PEMV PTE, can engage in similar long-distance RNA-RNA interactions. PMID:22761367

  18. BRAF inhibitor resistance enhances vulnerability to arginine deprivation in melanoma

    PubMed Central

    Li, Ying-Ying; Wu, Chunjing; Chen, Shu-Mei; Shah, Sumedh S.; Wangpaichitr, Medhi; Feun, Lynn G.; Kuo, Macus T.; Suarez, Miguel; Prince, Jeffrey; Savaraj, Niramol

    2016-01-01

    BRAF inhibitor (BRAFi) has been used for treatment of melanomas harboring V600E mutation. Despite a high initial response rate, resistance to BRAFi is inevitable. Here, we demonstrate that BRAFi-resistant (BR) melanomas are susceptible to arginine deprivation due to inability to initiate re-expression of argininosuccinate synthetase (ASS1, a key enzyme for arginine synthesis) as well as ineffective autophagy. Autophagy and ASS1 re-expression are known to protect melanoma cells from cell death upon arginine deprivation. When melanoma cells become BR cells by long-term in vitro incubation with BRAFi, c-Myc-mediated ASS1 re-expression and the levels of autophagy-associated proteins (AMPK-α1 and Atg5) are attenuated. Furthermore, our study uncovers that downregulation of deubiquitinase USP28 which results in more active c-Myc degradation via ubiquitin-proteasome machinery is the primary mechanism for inability to re-express ASS1 upon arginine deprivation in BR cells. Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival. On the other hand, overexpression of Atg5 or AMPK-α1 in BR cells can redirect arginine deprivation-induced apoptosis toward autophagy. The xenograft models also confirm that BR tumors possess lower expression of ASS1 and are hypersensitive to arginine deprivation. These biochemical changes in BRAFi resistance which make them vulnerable to arginine deprivation can be exploited for the future treatment of BR melanoma patients. PMID:26771234

  19. Characterizing inactive ribosomes in translational profiling.

    PubMed

    Liu, Botao; Qian, Shu-Bing

    2016-01-01

    The broad impact of translational regulation has emerged explosively in the last few years in part due to the technological advance in genome-wide interrogation of gene expression. During mRNA translation, the majority of actively translating ribosomes exist as polysomes in cells with multiple ribosomes loaded on a single transcript. The importance of the monosome, however, has been less appreciated in translational profiling analysis. Here we report that the monosome fraction isolated by sucrose sedimentation contains a large quantity of inactive ribosomes that do not engage on mRNAs to direct translation. We found that the elongation factor eEF2, but not eEF1A, stably resides in these non-translating ribosomes. This unique feature permits direct evaluation of ribosome status under various stress conditions and in the presence of translation inhibitors. Ribosome profiling reveals that the monosome has a similar but not identical pattern of ribosome footprints compared to the polysome. We show that the association of free ribosomal subunits minimally contributes to ribosome occupancy outside of the coding region. Our results not only offer a quantitative method to monitor ribosome availability, but also uncover additional layers of ribosome status needed to be considered in translational profiling analysis. PMID:27335722

  20. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

    PubMed

    Dresios, John; Aschrafi, Armaz; Owens, Geoffrey C; Vanderklish, Peter W; Edelman, Gerald M; Mauro, Vincent P

    2005-02-01

    The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

  1. 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. PMID:26801560

  2. 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.

  3. Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

    PubMed

    Parenteau, Julie; Lavoie, Mathieu; Catala, Mathieu; Malik-Ghulam, Mustafa; Gagnon, Jules; Abou Elela, Sherif

    2015-12-22

    In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress. PMID:26686636

  4. The Class I HDAC inhibitor RGFP963 enhances consolidation of cued fear extinction

    PubMed Central

    Bowers, Mallory E.; Xia, Bing; Carreiro, Samantha

    2015-01-01

    Evidence indicates that broad, nonspecific histone deacetylase (HDAC) inhibition enhances learning and memory, however, the contribution of the various HDACs to specific forms of learning is incompletely understood. Here, we show that the Class I HDAC inhibitor, RGFP963, enhances consolidation of cued fear extinction. However, RGFP966, a strong inhibitor of HDAC3, does not significantly enhance consolidation of cued fear extinction. These data extend previous evidence that demonstrate the Class I HDACs play a role in the consolidation of long-term memory, suggesting that HDAC1 and/or HDAC2, but less likely HDAC3, may function as negative regulators of extinction retention. The development of specific HDAC inhibitors, such as RGFP963, will further illuminate the role of specific HDACs in various types of learning and memory. Moreover, HDAC inhibitors that enhance cued fear extinction may show translational promise for the treatment of fear-related disorders, including post-traumatic stress disorder (PTSD). PMID:25776040

  5. Chemical modulators of ribosome biogenesis as biological probes.

    PubMed

    Stokes, Jonathan M; Brown, Eric D

    2015-12-01

    Small-molecule inhibitors of protein biosynthesis have been instrumental in the dissection of the complexities of ribosome structure and function. Ribosome biogenesis, on the other hand, is a complex and largely enigmatic process for which there is a paucity of chemical probes. Indeed, ribosome biogenesis has been studied almost exclusively using genetic and biochemical approaches without the benefit of small-molecule inhibitors of this process. Here, we provide a perspective on the promise of chemical inhibitors of ribosome assembly for future research. We explore key obstacles that complicate the interpretation of studies aimed at perturbing ribosome biogenesis in vivo using genetic methods, and we argue that chemical inhibitors are especially powerful because they can be used to induce perturbations in a manner that obviates these difficulties. Thus, in combination with leading-edge biochemical and structural methods, chemical probes offer unique advantages toward elucidating the molecular events that define the assembly of ribosomes. PMID:26575239

  6. Ribosome Binding to a 5′ Translational Enhancer Is Altered in the Presence of the 3′ Untranslated Region in Cap-Independent Translation of Turnip Crinkle Virus ▿

    PubMed Central

    Stupina, Vera A.; Yuan, Xuefeng; Meskauskas, Arturas; Dinman, Jonathan D.; Simon, Anne E.

    2011-01-01

    Plus-strand RNA viruses without 5′ caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3′ untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5′ UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5′ UTR sequences. Addition of 80S ribosomes to the 5′ UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5′ end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5′-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5′ and 3′ sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5′ and 3′ UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5′ UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5′ and 3′ UTRs of TCV. PMID:21389125

  7. PDE5 inhibitors enhance the lethality of standard of care chemotherapy in pediatric CNS tumor cells.

    PubMed

    Roberts, Jane L; Booth, Laurence; Conley, Adam; Cruickshanks, Nichola; Malkin, Mark; Kukreja, Rakesh C; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2014-06-01

    We determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill medulloblastoma cells. In medulloblastoma cells PDE5 inhibitors interacted in a greater than additive fashion with vincristine/etoposide/cisplatin to cause cell death. Knockdown of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of dominant negative caspase 9 did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with the PDE5 inhibitor sildenafil. Overexpression of BCL-XL and c-FLIP-s suppressed individual and combination drug toxicities. Knockdown of CD95 or FADD suppressed drug combination toxicity. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy which was maximal at ~12 h post-treatment, and in a cell type-dependent manner knockdown of Beclin1 or ATG5 either suppressed or enhanced drug combination lethality. PDE5 inhibitors enhanced the induction of chemotherapy-induced DNA damage in a nitric oxide synthase-dependent fashion. In conclusion, our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for medulloblastoma represents a possible novel modality for future treatment of this disease. PMID:24651037

  8. Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

    PubMed Central

    Gilabert-Oriol, Roger; Weng, Alexander; von Mallinckrodt, Benedicta; Melzig, Matthias F; Fuchs, Hendrik; Thakur, Mayank

    2014-01-01

    The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success. PMID:25341935

  9. The Class I HDAC Inhibitor RGFP963 Enhances Consolidation of Cued Fear Extinction

    ERIC Educational Resources Information Center

    Bowers, Mallory E.; Xia, Bing; Carreiro, Samantha; Ressler, Kerry J.

    2015-01-01

    Evidence indicates that broad, nonspecific histone deacetylase (HDAC) inhibition enhances learning and memory, however, the contribution of the various HDACs to specific forms of learning is incompletely understood. Here, we show that the Class I HDAC inhibitor, RGFP963, enhances consolidation of cued fear extinction. However, RGFP966, a strong…

  10. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  11. Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

    SciTech Connect

    Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S.

    2012-09-11

    The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-a-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 {angstrom} resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the aD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

  12. Novel JAK1-selective benzimidazole inhibitors with enhanced membrane permeability.

    PubMed

    Kim, Hyungmi; Kim, Mi Kyoung; Choo, Hyunah; Chong, Youhoon

    2016-07-15

    The previously identified Janus kinase 1 (JAK1)-selective inhibitor, 1-(2-aminoethyl)-2-(piperidin-4-yl)-1H-benzo[d]imidazole-5-carboxamide (2), suffered from low cell permeability, which resulted in poor pharmacokinetic properties. In this study, by introducing less polar hydrogen bond donors at N(1) (a hydroxyalkyl or a methylaminoalkyl group) and C2 (a cyclohexanol group) positions, a series of novel benzimidazole derivatives were prepared, which exhibited selective JAK1 inhibitory activity (IC50 against JAK1=0.08-0.15μM; JAK1-selectivity=26-40 fold vs JAK2, 12-23 fold vs JAK3, and 38-54 fold vs Tyk2) along with significantly increased lipophilicity (3.3-15.8 times) as well as membrane permeability (6.3-12 times). PMID:27261178

  13. p53 Small Molecule Inhibitor Enhances Temozolomide Cytotoxic Activity against Intracranial Glioblastoma Xenografts

    PubMed Central

    Dinca, Eduard B.; Lu, Kan V.; Sarkaria, Jann N.; Pieper, Russell O.; Prados, Michael D.; Haas-Kogan, Daphne A.; VandenBerg, Scott R.; Berger, Mitchel S.; James, C. David

    2010-01-01

    In this study we investigated corresponding precursor and active forms of a p53 small molecule inhibitor for effect on temozolomide (TMZ) anti-tumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when GBMs with wild-type p53 were co-treated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased PARP cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, that is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: i.e., TMZ + p53 inhibitor precursor co-treatment, of three distinct wild-type p53 GBM xenografts, resulted in significant enhancement of TMZ anti-tumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (p < 0.001 for co-treatment survival benefit in each case). Mice receiving intracranial injection with p53 null GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhance TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner. PMID:19074867

  14. p53 Small-molecule inhibitor enhances temozolomide cytotoxic activity against intracranial glioblastoma xenografts.

    PubMed

    Dinca, Eduard B; Lu, Kan V; Sarkaria, Jann N; Pieper, Russell O; Prados, Michael D; Haas-Kogan, Daphne A; Vandenberg, Scott R; Berger, Mitchel S; James, C David

    2008-12-15

    In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53(wt)) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53(wt) GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53(null) GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner.

  15. Enhanced resistance to blast fungus in rice (Oryza sativa L.) by expressing the ribosome-inactivating protein α-momorcharin.

    PubMed

    Qian, Qian; Huang, Lin; Yi, Rong; Wang, Shuzhen; Ding, Yi

    2014-03-01

    Rice blast caused by Magnaporthe grisea is one of the three major diseases that seriously affect the rice production. Alpha-momorcharin (α-MC), a ribosome-inactivating protein (RIP) isolated from Momordica charantia seeds, has antifungal effects in vitro. In this study, the α-MC gene was constitutively expressed under the control of the 2×35S promoter in transgenic rice (Oryza sativa L.) using an Agrobacterium tumefaciens-mediated method. The nine transgenic plants were obtained and confirmed by PCR and RT-PCR, and the four (B2, B4, B7 and B9) of them whose copy numbers were 1, 2, 3 and 3, respectively, were shown to express the α-MC protein by Western blot. The molecular weight of α-MC in transgenic plants was approximately 38 kDa larger than the purified α-MC protein (28 kDa) in vitro. When the confirmed T1 generations were inoculated with a suspension of M. grisea spores for ten days, the lesions on leaves of transgenic plants were much lesser than those found on wild type (WT). According to the criteria of International Rice Research Institute standard, the mean values for morbidity and disease index numbers were 29.8% and 14.9%, respectively, which were lower than for WT. It is unclear whether RIPs could impact plant fitness and however our results suggest that the α-MC protein is an effective antifungal protein preventing rice blast in transgenic rice.

  16. Enhanced responsiveness to selective serotonin reuptake inhibitors during lactation.

    PubMed

    Jury, Nicholas J; McCormick, Betsy A; Horseman, Nelson D; Benoit, Stephen C; Gregerson, Karen A

    2015-01-01

    The physiology of mood regulation in the postpartum is poorly understood despite the fact that postpartum depression (PPD) is a common pathology. Serotonergic mechanisms and their dysfunction are widely presumed to be involved, which has led us to investigate whether lactation induces changes in central or peripheral serotonin (5-HT) systems and related affective behaviors. Brain sections from lactating (day 10 postpartum) and age-matched nulliparous (non-pregnant) C57BL/6J mice were processed for 5-HT immunohistochemistry. The total number of 5-HT immunostained cells and optical density were measured. Lactating mice exhibited lower immunoreactive 5-HT and intensity in the dorsal raphe nucleus when compared with nulliparous controls. Serum 5-HT was quantified from lactating and nulliparous mice using radioimmunoassay. Serum 5-HT concentrations were higher in lactating mice than in nulliparous controls. Affective behavior was assessed in lactating and non-lactating females ten days postpartum, as well as in nulliparous controls using the forced swim test (FST) and marble burying task (MBT). Animals were treated for the preceding five days with a selective serotonin reuptake inhibitor (SSRI, citalopram, 5mg/kg/day) or vehicle. Lactating mice exhibited a lower baseline immobility time during the FST and buried fewer marbles during the MBT as compared to nulliparous controls. Citalopram treatment changed these behaviors in lactating mice with further reductions in immobility during the FST and decreased marble burying. In contrast, the same regimen of citalopram treatment had no effect on these behaviors in either non-lactating postpartum or nulliparous females. Our findings demonstrate changes in both central and peripheral 5-HT systems associated with lactation, independent of pregnancy. They also demonstrate a significant interaction of lactation and responsiveness to SSRI treatment, which has important implications in the treatment of PPD. Although recent evidence

  17. Structural basis for the inhibition of the eukaryotic ribosome.

    PubMed

    Garreau de Loubresse, Nicolas; Prokhorova, Irina; Holtkamp, Wolf; Rodnina, Marina V; Yusupova, Gulnara; Yusupov, Marat

    2014-09-25

    The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development.

  18. A computational perspective of molecular interactions through virtual screening, pharmacokinetic and dynamic prediction on ribosome toxin A chain and inhibitors of Ricinus communis

    PubMed Central

    Kumar, R. Barani; Suresh, M. Xavier

    2012-01-01

    Background: Ricin is considered to be one of the most deadly toxins and gained its favor as a bioweapon that has a serious social and biological impact, due to its widespread nature and abundant availability. The hazardous effects of this toxin in human being are seen in almost all parts of the organ system. The severe consequences of the toxin necessitate the need for developing potential inhibitors that can effectively block its interaction with the host system. Materials and Methods: In order to identify potential inhibitors that can effectively block ricin, we employed various computational approaches. In this work, we computationally screened and analyzed 66 analogs and further tested their ADME/T profiles. From the kinetic and toxicity studies we selected six analogs that possessed appropriate pharmacokinetic and dynamic property. We have also performed a computational docking of these analogs with the target. Results: On the basis of the dock scores and hydrogen bond interactions we have identified analog 64 to be the best interacting molecule. Molecule 64 seems to have stable interaction with the residues Tyr80, Arg180, and Val81. The pharmacophore feature that describes the key functional features of a molecule was also studied and presented. Conclusion: The pharmacophore features of the drugs provided suggests the key functional groups that can aid in the design and synthesis of more potential inhibitors. PMID:22224054

  19. Dopamine β-Hydroxylase Inhibitors Enhance the Discriminative Stimulus Effects of Cocaine in Rats

    PubMed Central

    Manvich, Daniel F.; DePoy, Lauren M.

    2013-01-01

    Inhibitors of dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic cells, have shown promise for the treatment of cocaine abuse disorders. However, the mechanisms underlying the beneficial effects of these compounds have not been fully elucidated. We used the drug discrimination paradigm to determine the impact of DBH inhibitors on the interoceptive stimulus properties of cocaine. Sprague-Dawley rats were trained to discriminate cocaine (5.6 mg/kg) from saline using a multicomponent, food-reinforced discrimination procedure. On test days, subjects were pretreated with the nonselective DBH inhibitor disulfiram (0–100.0 mg/kg i.p.) or the selective DBH inhibitor nepicastat (0–56.0 mg/kg i.p.) 2 hours prior to a test session either alone or in combination with cumulatively administered cocaine (0–5.6 mg/kg i.p.). Neither disulfiram nor nepicastat substituted for the cocaine stimulus when tested up to doses that nonspecifically reduced responding. However, in combination studies, pretreatment with either disulfiram or nepicastat produced leftward shifts in the cocaine dose-response function and also conferred cocaine-like stimulus effects to the selective NE transporter inhibitor, reboxetine (0.3–5.6 mg/kg i.p.). These results indicate that pharmacological inhibition of DBH does not produce cocaine-like interoceptive stimulus effects alone, but functionally enhances the interoceptive stimulus effects of cocaine, possibly due to facilitated increases in DA released from noradrenergic terminals. These findings suggest that DBH inhibitors have low abuse liability and provide support to clinical reports that some subjective effects produced by cocaine, particularly aversive effects, are enhanced after DBH inhibition. PMID:24068832

  20. Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

    SciTech Connect

    Plechanovová, Anna; Byun, Youngjoo; Alquicer, Glenda; Škultétyová, L; ubica; Ml; #269; ochová, Petra; N; #283; mcová, Adriana; Kim, Hyung-Joon; Navrátil, Michal; Mease, Ronnie; Lubkowski, Jacek; Pomper, Martin; Konvalinka, Jan; Rulíšek, Lubomír; Ba; #345; inka, Cyril

    2012-10-09

    Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.

  1. Histone deacetylase inhibitors enhance phosphorylation of histone H2AX after ionizing radiation

    SciTech Connect

    Zhang Yubin; Adachi, Masaaki . E-mail: adachi@sapmed.ac.jp; Zou Huichao; Hareyama, Masato; Imai, Kohzoh; Shinomura, Yasuhisa

    2006-07-01

    Purpose Histone deacetylase (HDAC) inhibitors are believed to be promising radiosensitizers. To explore their effects on ionizing radiation (IR), we examined whether the HDAC inhibitors m-carboxycinnamic acid bis-hydroxamide (CBHA) and depsipeptide FK228 affect H2AX phosphorylation ({gamma}-H2AX), a landmark of DNA double-strand breaks after IR exposure. Methods and Materials We evaluated the effects of the HDAC inhibitors on clonogenic assay in human lung carcinoma A549 cells and progression of A549 xenograft tumors. IR-induced DNA damage was evaluated by histone {gamma}-H2AX. Histone hyperacetylation was induced by overexpression of histone acetyltransferase p300 and evaluated by Western blots. Results M-carboxycinnamic acid bishydroxyamide pretreatment radiosensitized A549 cells and strongly inhibited A549 xenograft tumor progression. CBHA and FK228, but not 5-fluorouracil, enhanced IR-induced {gamma}-H2AX in A549 and other cancer cell lines. Overexpression of p300 similarly augmented IR-induced {gamma}-H2AX. Conclusion The results of this study suggest that HDAC inhibitors enhance IR-induced {gamma}-H2AX, most likely through histone hyperacetylation, and radiosensitize various cancers.

  2. IGF1R tyrosine kinase inhibitor enhances the cytotoxic effect of methyl jasmonate in endometrial cancer.

    PubMed

    Bruchim, Ilan; Sarfstein, Rive; Reiss, Ari; Flescher, Eliezer; Werner, Haim

    2014-10-01

    The present study evaluated the cytotoxic activity of methyl jasmonate (MJ) in endometrial cancer cells and examined the hypothesis that the apoptotic and anti-proliferative actions of MJ in these cell lines can be enhanced by co-targeting the insulin-like growth factor-1 receptor (IGF1R) signaling pathway. MJ had a potent pro-apoptotic effect and exhibited significant toxicity in all cell lines tested. MJ in combination with NVP-AEW541, a selective IGF1R tyrosine kinase inhibitor, had significantly increased cytotoxicity. MJ decreased IGF1R phosphorylation, however, it enhanced AKT phosphorylation and abolished the anti-apoptotic effect of IGF1. These findings suggest that combined IGF1R inhibitor and MJ administration may constitute an attractive modality for treating endometrial cancer.

  3. Ribosome regulation by the nascent peptide.

    PubMed Central

    Lovett, P S; Rogers, E J

    1996-01-01

    Studies of bacterial and eukaryotic systems have identified two-gene operons in which the translation product of the upstream gene influences translation of the downstream gene. The upstream gene, referred to as a leader (gene) in bacterial systems or an upstream open reading frame (uORF) in eukaryotes, encodes a peptide that interferes with a function(s) of its translating ribosome. The peptides are therefore cis-acting negative regulators of translation. The inhibitory peptides typically consist of fewer than 25 residues and function prior to emergence from the ribosome. A biological role for this class of translation inhibitor is demonstrated in translation attenuation, a form or regulation that controls the inducible translation of the chloramphenicol resistance genes cat and cmlA in bacteria. Induction of cat or cmlA requires ribosome stalling at a particular codon in the leader region of the mRNA. Stalling destabilizes an adjacent, downstream mRNA secondary structure that normally sequesters the ribosome-binding site for the cat or cmlA coding regions. Genetic studies indicate that the nascent, leader-encoded peptide is the selector of the site of ribosome stalling in leader mRNA by cis interference with translation. Synthetic leader peptides inhibit ribosomal peptidyltransferase in vitro, leading to the prediction that this activity is the basis for stall site selection. Recent studies have shown that the leader peptides are rRNA-binding peptides with targets at the peptidyl transferase center of 23S rRNA. uORFs associated with several eukaryotic genes inhibit downstream translation. When inhibition depends on the specific codon sequence of the uORF, it has been proposed that the uORF-encoded nascent peptide prevents ribosome release from the mRNA at the uORF stop codon. This sets up a blockade to ribosome scanning which minimizes downstream translation. Segments within large proteins also appear to regulate ribosome activity in cis, although in most of the

  4. Pharmacology of novel small-molecule tubulin inhibitors in glioblastoma cells with enhanced EGFR signalling.

    PubMed

    Phoa, Athena F; Browne, Stephen; Gurgis, Fadi M S; Åkerfeldt, Mia C; Döbber, Alexander; Renn, Christian; Peifer, Christian; Stringer, Brett W; Day, Bryan W; Wong, Chin; Chircop, Megan; Johns, Terrance G; Kassiou, Michael; Munoz, Lenka

    2015-12-15

    We recently reported that CMPD1, originally developed as an inhibitor of MK2 activation, primarily inhibits tubulin polymerisation and induces apoptosis in glioblastoma cells. In the present study we provide detailed pharmacological investigation of CMPD1 analogues with improved molecular properties. We determined their anti-cancer efficacy in glioblastoma cells with enhanced EGFR signalling, as deregulated EGFR often leads to chemoresistance. Eight analogues of CMPD1 with varying lipophilicity and basicity were synthesised and tested for efficacy in the cell viability assay using established glioblastoma cell lines and patient-derived primary glioblastoma cells. The mechanism of action for the most potent analogue 15 was determined using MK2 activation and tubulin polymerisation assays, together with the immunofluorescence analysis of the mitotic spindle formation. Apoptosis was analysed by Annexin V staining, immunoblotting analysis of bcl-2 proteins and PARP cleavage. The apoptotic activity of CMPD1 and analogue 15 was comparable across glioblastoma cell lines regardless of the EGFR status. Primary glioblastoma cells of the classical subtype that are characterized by enhanced EGFR activity were most sensitive to the treatment with CMPD1 and 15. In summary, we present mechanism of action for a novel small molecule tubulin inhibitor, compound 15 that inhibits tubulin polymerisation and mitotic spindle formation, induces degradation of anti-apoptotic bcl-2 proteins and leads to apoptosis of glioblastoma cells. We also demonstrate that the enhanced EGFR activity does not decrease the efficacy of tubulin inhibitors developed in this study.

  5. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

    PubMed Central

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers. PMID:24955955

  6. A calpain-2 selective inhibitor enhances learning & memory by prolonging ERK activation.

    PubMed

    Liu, Yan; Wang, Yubin; Zhu, Guoqi; Sun, Jiandong; Bi, Xiaoning; Baudry, Michel

    2016-06-01

    While calpain-1 activation is required for LTP induction by theta burst stimulation (TBS), calpain-2 activation limits its magnitude during the consolidation period. A selective calpain-2 inhibitor applied either before or shortly after TBS enhanced the degree of potentiation. In the present study, we tested whether the selective calpain-2 inhibitor, Z-Leu-Abu-CONH-CH2-C6H3 (3, 5-(OMe)2 (C2I), could enhance learning and memory in wild-type (WT) and calpain-1 knock-out (C1KO) mice. We first showed that C2I could reestablish TBS-LTP in hippocampal slices from C1KO mice, and this effect was blocked by PD98059, an inhibitor of ERK. TBS resulted in PTEN degradation in hippocampal slices from both WT and C1KO mice, and C2I treatment blocked this effect in both mouse genotypes. Systemic injection of C2I 30 min before training in the fear-conditioning paradigm resulted in a biphasic dose-response curve, with low doses enhancing and high doses inhibiting freezing behavior. The difference between the doses needed to enhance and inhibit learning matches the difference in concentrations producing inhibition of calpain-2 and calpain-1. A low dose of C2I also restored normal learning in a novel object recognition task in C1KO mice. Levels of SCOP, a ERK phosphatase known to be cleaved by calpain-1, were decreased in dorsal hippocampus early but not late following training in WT mice; C2I treatment did not affect the early decrease in SCOP levels but prevented its recovery at the later time-point and prolonged ERK activation. The results indicate that calpain-2 activation limits the extent of learning, an effect possibly due to temporal limitation of ERK activation, as a result of SCOP synthesis induced by calpain-2-mediated PTEN degradation. PMID:26907807

  7. The Ribosomal Database Project.

    PubMed Central

    Maidak, B L; Larsen, N; McCaughey, M J; Overbeek, R; Olsen, G J; Fogel, K; Blandy, J; Woese, C R

    1994-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services, and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server/rdp.life.uiuc.edu) and gopher (rdpgopher.life.uiuc.edu). The electronic mail server also provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for chimeric nature of newly sequenced rRNAs, and automated alignment. PMID:7524021

  8. Enhancement of active corrosion protection via combination of inhibitor-loaded nanocontainers.

    PubMed

    Tedim, J; Poznyak, S K; Kuznetsova, A; Raps, D; Hack, T; Zheludkevich, M L; Ferreira, M G S

    2010-05-01

    The present work reports the synthesis of layered double hydroxides (LDHs) nanocontainers loaded with different corrosion inhibitors (vanadate, phosphate, and 2-mercaptobenzothiazolate) and the characterization of the resulting pigments by X-ray diffraction (XRD) and transmission electron microscopy (TEM). The anticorrosion activity of these nanocontainers with respect to aluminum alloy AA2024 was investigated by electrochemical impedance spectroscopy (EIS). The bare metallic substrates were immersed in dispersions of nanocontainers in sodium chloride solution and tested to understand the inhibition mechanisms and efficiency. The nanocontainers were also incorporated into commercial coatings used for aeronautical applications to study the active corrosion protection properties in systems of industrial relevance. The results show that an enhancement of the active protection effect can be reached when nanocontainers loaded with different inhibitors are combined in the same protective coating system.

  9. Exploiting translational stalling peptides in an effort to extend azithromycin interaction within the prokaryotic ribosome nascent peptide exit tunnel.

    PubMed

    Washington, Arren Z; Tapadar, Subhasish; George, Alex; Oyelere, Adegboyega K

    2015-08-15

    The ribosome is the primary protein synthesis machine in the cell and is a target for treatment of a variety of diseases including bacterial infection and cancer. The ribosomal peptide exit tunnel, the route of egress for the nascent peptide, is an inviting site for drug design. Toward a rational engagement of the nascent peptide components for the design of small molecule inhibitors of ribosome function, we designed and disclosed herein a set of N-10 indole functionalized azithromycin analogs. The indole moiety of these compounds is designed to mimic the translation stalling interaction of SecM W155 side-chain with the prokaryotic (Escherichia coli) ribosome A751 residue. Many of these N-10 functionalized compounds have enhanced translation inhibition activities against E. coli ribosome relative to azithromycin while a subset inhibited the growth of representative susceptible bacteria strains to about the same extent as azithromycin. Moreover, the inclusion of bovine serum in the bacterial growth media enhanced the anti-bacterial potency of the N-10 functionalized azithromycin analogs by as high as 10-fold. PMID:26037612

  10. Exploiting translational stalling peptides in an effort to extend azithromycin interaction within the prokaryotic ribosome nascent peptide exit tunnel.

    PubMed

    Washington, Arren Z; Tapadar, Subhasish; George, Alex; Oyelere, Adegboyega K

    2015-08-15

    The ribosome is the primary protein synthesis machine in the cell and is a target for treatment of a variety of diseases including bacterial infection and cancer. The ribosomal peptide exit tunnel, the route of egress for the nascent peptide, is an inviting site for drug design. Toward a rational engagement of the nascent peptide components for the design of small molecule inhibitors of ribosome function, we designed and disclosed herein a set of N-10 indole functionalized azithromycin analogs. The indole moiety of these compounds is designed to mimic the translation stalling interaction of SecM W155 side-chain with the prokaryotic (Escherichia coli) ribosome A751 residue. Many of these N-10 functionalized compounds have enhanced translation inhibition activities against E. coli ribosome relative to azithromycin while a subset inhibited the growth of representative susceptible bacteria strains to about the same extent as azithromycin. Moreover, the inclusion of bovine serum in the bacterial growth media enhanced the anti-bacterial potency of the N-10 functionalized azithromycin analogs by as high as 10-fold.

  11. HDAC inhibitors as cognitive enhancers in fear, anxiety and trauma therapy: where do we stand?

    PubMed

    Whittle, Nigel; Singewald, Nicolas

    2014-04-01

    A novel strategy to treat anxiety and fear-related disorders such as phobias, panic and PTSD (post-traumatic stress disorder) is combining CBT (cognitive behavioural therapy), including extinction-based exposure therapy, with cognitive enhancers. By targeting and boosting mechanisms underlying learning, drug development in this field aims at designing CBT-augmenting compounds that help to overcome extinction learning deficits, promote long-term fear inhibition and thus support relapse prevention. Progress in revealing the role of epigenetic regulation of specific genes associated with extinction memory generation has opened new avenues in this direction. The present review examines recent evidence from pre-clinical studies showing that increasing histone acetylation, either via genetic or pharmacological inhibition of HDACs (histone deacetylases) by e.g. vorinostat/SAHA (suberoylanilide hydroxamic acid), entinostat/MS-275, sodium butyrate, TSA (trichostatin A) or VPA (valproic acid), or by targeting HATs (histone acetyltransferases), augments fear extinction and, importantly, generates a long-term extinction memory that can protect from return of fear phenomena. The molecular mechanisms and pathways involved including BDNF (brain-derived neurotrophic factor) and NMDA (N-methyl-D-aspartate) receptor signalling are just beginning to be revealed. First studies in healthy humans are in support of extinction-facilitating effects of HDAC inhibitors. Very recent evidence that HDAC inhibitors can rescue deficits in extinction-memory-impaired rodents indicates a potential clinical utility of this approach also for exposure therapy-resistant patients. Important future work includes investigation of the long-term safety aspects of HDAC inhibitor treatment, as well as design of isotype(s)-specific inhibitors. Taken together, HDAC inhibitors display promising potential as pharmacological adjuncts to augment the efficacy of exposure-based approaches in anxiety and trauma therapy.

  12. HDAC inhibitors as cognitive enhancers in fear, anxiety and trauma therapy: where do we stand?

    PubMed Central

    Whittle, Nigel; Singewald, Nicolas

    2014-01-01

    A novel strategy to treat anxiety and fear-related disorders such as phobias, panic and PTSD (post-traumatic stress disorder) is combining CBT (cognitive behavioural therapy), including extinction-based exposure therapy, with cognitive enhancers. By targeting and boosting mechanisms underlying learning, drug development in this field aims at designing CBT-augmenting compounds that help to overcome extinction learning deficits, promote long-term fear inhibition and thus support relapse prevention. Progress in revealing the role of epigenetic regulation of specific genes associated with extinction memory generation has opened new avenues in this direction. The present review examines recent evidence from pre-clinical studies showing that increasing histone acetylation, either via genetic or pharmacological inhibition of HDACs (histone deacetylases) by e.g. vorinostat/SAHA (suberoylanilide hydroxamic acid), entinostat/MS-275, sodium butyrate, TSA (trichostatin A) or VPA (valproic acid), or by targeting HATs (histone acetyltransferases), augments fear extinction and, importantly, generates a long-term extinction memory that can protect from return of fear phenomena. The molecular mechanisms and pathways involved including BDNF (brain-derived neurotrophic factor) and NMDA (N-methyl-D-aspartate) receptor signalling are just beginning to be revealed. First studies in healthy humans are in support of extinction-facilitating effects of HDAC inhibitors. Very recent evidence that HDAC inhibitors can rescue deficits in extinction-memory-impaired rodents indicates a potential clinical utility of this approach also for exposure therapy-resistant patients. Important future work includes investigation of the long-term safety aspects of HDAC inhibitor treatment, as well as design of isotype(s)-specific inhibitors. Taken together, HDAC inhibitors display promising potential as pharmacological adjuncts to augment the efficacy of exposure-based approaches in anxiety and trauma therapy

  13. Antitumor effects of immunotoxins are enhanced by lowering HCK or treatment with SRC kinase inhibitors.

    PubMed

    Liu, Xiu-Fen; Xiang, Laiman; FitzGerald, David J; Pastan, Ira

    2014-01-01

    Recombinant immunotoxins (RIT) are agents being developed for cancer treatment. They are composed of an Fv that binds to a cancer cell, fused to a 38-kDa fragment of Pseudomonas exotoxin A. SS1P is a RIT that targets mesothelin, a protein expressed on mesothelioma as well as pancreatic, ovarian, lung, and other cancers. Because the protein tyrosine kinase family regulates a variety of cellular processes and pathways, we hypothesized that tyrosine kinases might regulate susceptibility to immunotoxin killing. To investigate their role, we used siRNAs to lower the level of expression of the 88 known tyrosine kinases. We identified five tyrosine kinases, INSR, HCK, SRC, PDGFRβ, and BMX that enhance the activity of SS1P when their level of expression is lowered by siRNAs. We further investigated the Src family member HCK in this study. Knocking down of SRC slightly increased SS1P killing in A431/H9 cells, but knocking down HCK substantially enhanced killing by SS1P. We investigated the mechanism of enhancement and found that HCK knockdown enhanced SS1P cleavage by furin and lowered levels of Mcl-1 and raised Bax. We then found that Src inhibitors mimic the stimulatory effect of HCK knockdown; both SU6656 and SKI-606 (bosutinib) enhanced immunotoxin killing of mesothelin-expressing cells by SS1P and CD22-expressing cells by HA22 (moxetumomab pasudotox). SU6656 also enhanced the antitumor effects of SS1P and HA22 in mouse xenograft tumor models. Our data suggest that the combination of immunotoxin with tyrosine kinase inhibitors may be an effective way to treat some cancers. PMID:24145282

  14. Dyclonine and alverine citrate enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells

    PubMed Central

    JU, DONGHONG; WANG, XIAOGANG; XIE, YOUMING

    2014-01-01

    Proteasome is an important target in cancer therapy. To enhance the efficacy of proteasome inhibitors is a challenging task due to the paucity of understanding the functional interactions between proteasome and other cellular pathways in mammalian cells. Taking advantage of the knowledge gained from Saccharomyces cerevisiae, we show that dyclonine and alverine citrate, the major components of two over-the-counter medicines, can substantially enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells. This study also highlights an important yeast genetic approach to identification of potential therapeutics that can be used for combination therapy with proteasome inhibitors. PMID:19148544

  15. Dyclonine and alverine citrate enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells.

    PubMed

    Ju, Donghong; Wang, Xiaogang; Xie, Youming

    2009-02-01

    Proteasome is an important target in cancer therapy. To enhance the efficacy of proteasome inhibitors is a challenging task due to the paucity of understanding the functional interactions between proteasome and other cellular pathways in mammalian cells. Taking advantage of the knowledge gained from Saccharomyces cerevisiae, we show that dyclonine and alverine citrate, the major components of two over-the-counter medicines, can substantially enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells. This study also highlights an important yeast genetic approach to identification of potential therapeutics that can be used for combination therapy with proteasome inhibitors. PMID:19148544

  16. Immunogenicity of Ribosomal Preparations from Yeast Cells of Histoplasma capsulatum

    PubMed Central

    Feit, Carl; Tewari, Ram P.

    1974-01-01

    Protective immunity was elicited by immunization of mice with ribosomal preparations from yeast cells of Histoplasma capsulatum. Ribosomes from disrupted cells were isolated by differential centrifugation using sodium dodecyl sulfate. These preparations contained 55% protein and 45% ribonucleic acid and sedimented as a single peak with a sedimentation coefficient of 77S on sucrose density gradient analysis. Mice immunized subcutaneously with ribosomes, with or without adjuvant, were challenged intravenously with 8 × 106 yeast cells of H. capsulatum. Significant protection was induced by ribosomes and was greatly enhanced by adjuvants. Protection measured by 30-day survival compared favorably with the immunoprotection assessed by absence of lung lesions and negative spleen cultures. Treatment of ribosomes with ribonuclease before immunization reduced protection by 85%, whereas trypsin and Pronase reduced the protection by 50 to 55%. These findings indicate that both intact ribosomal ribonucleic acid and protein are necessary for maximal immunogenicity of Histoplasma ribosomes. PMID:16558095

  17. Resveratrol and P-glycoprotein Inhibitors Enhance the Anti-skin Cancer Effects of Ursolic Acid

    PubMed Central

    Junco, Jacob J.; Mancha, Anna; Malik, Gunjan; Wei, Sung-Jen; Kim, Dae Joon; Liang, Huiyun; Slaga, Thomas J.

    2013-01-01

    Ursolic acid (UA), present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to UA treatment. Herein, skin carcinoma cells (Ca3/7) as compared to skin papilloma cells (MT1/2) displayed more resistance to UA-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in pre-clinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate Rhodamine 123 (Rh123) indicating UA is transported by P-gp. To determine if P-gp inhibition can enhance UA-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil (VRP) or cyclosporin A (CsA). Alternatively, cells were pre-treated with the natural compound resveratrol (RES), a known chemotherapy sensitizer. VRP and RES enhanced the effects of UA in both cell lines, while CsA only did so in Ca3/7 cells. Similarly, VRP inhibited Rh123 efflux in both lines, while CsA only inhibited Rh123 efflux in Ca3/7 cells. RES did not inhibit Rh123 efflux in either line, indicating the synergistic effects of RES and UA are not manifest by inhibition of P-gp-mediated efflux of UA. These results indicate that the anti-skin cancer effects of UA are enhanced with P-gp inhibitors. In addition, RES and UA interact synergistically, but not through inhibition of P-gp. Implications Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen. PMID:24072817

  18. The Ribosomal Database Project

    NASA Technical Reports Server (NTRS)

    Olsen, G. J.; Overbeek, R.; Larsen, N.; Marsh, T. L.; McCaughey, M. J.; Maciukenas, M. A.; Kuan, W. M.; Macke, T. J.; Xing, Y.; Woese, C. R.

    1992-01-01

    The Ribosomal Database Project (RDP) complies ribosomal sequences and related data, and redistributes them in aligned and phylogenetically ordered form to its user community. It also offers various software packages for handling, analyzing and displaying sequences. In addition, the RDP offers (or will offer) certain analytic services. At present the project is in an intermediate stage of development.

  19. PDE5 inhibitors enhance tumor permeability and efficacy of chemotherapy in a rat brain tumor model.

    PubMed

    Black, Keith L; Yin, Dali; Ong, John M; Hu, Jinwei; Konda, Bindu M; Wang, Xiao; Ko, MinHee K; Bayan, Jennifer-Ann; Sacapano, Manuel R; Espinoza, Andreas; Irvin, Dwain K; Shu, Yan

    2008-09-16

    The blood-brain tumor barrier (BTB) significantly limits delivery of therapeutic concentrations of chemotherapy to brain tumors. A novel approach to selectively increase drug delivery is pharmacologic modulation of signaling molecules that regulate BTB permeability, such as those in cGMP signaling. Here we show that oral administration of sildenafil (Viagra) and vardenafil (Levitra), inhibitors of cGMP-specific PDE5, selectively increased tumor capillary permeability in 9L gliosarcoma-bearing rats with no significant increase in normal brain capillaries. Tumor-bearing rats treated with the chemotherapy agent, adriamycin, in combination with vardenafil survived significantly longer than rats treated with adriamycin alone. The selective increase in tumor capillary permeability appears to be mediated by a selective increase in tumor cGMP levels and increased vesicular transport through tumor capillaries, and could be attenuated by iberiotoxin, a selective inhibitor for calcium-dependent potassium (K(Ca)) channels, that are effectors in cGMP signaling. The effect by sildenafil could be further increased by simultaneously using another BTB "opener", bradykinin. Collectively, this data demonstrates that oral administration of PDE5 inhibitors selectively increases BTB permeability and enhances anti-tumor efficacy for a chemotherapeutic agent. These findings have significant implications for improving delivery of anti-tumor agents to brain tumors. PMID:18674521

  20. Luteolin is a novel p90 ribosomal S6 kinase (RSK) inhibitor that suppresses Notch4 signaling by blocking the activation of Y-box binding protein-1 (YB-1).

    PubMed

    Reipas, Kristen M; Law, Jennifer H; Couto, Nicole; Islam, Sumaiya; Li, Yvonne; Li, Huifang; Cherkasov, Artem; Jung, Karen; Cheema, Amarpal S; Jones, Steven J M; Hassell, John A; Dunn, Sandra E

    2013-02-01

    Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormone receptors and have limited targeted therapies. Recently, we demonstrated that p90 ribosomal S6 kinase (RSK) is essential for TNBC growth and survival indicating it as a target for therapeutic development. RSK phosphorylates Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, highly expressed in TNBC (~70% of cases) and associated with poor prognosis, drug resistance and tumor initiation. YB-1 regulates the tumor-initiating cell markers, CD44 and CD49f however its role in Notch signaling has not been explored. We sought to identify novel chemical entities with RSK inhibitory activity. The Prestwick Chemical Library of 1120 off-patent drugs was screened for RSK inhibitors using both in vitro kinase assays and molecular docking. The lead candidate, luteolin, inhibited RSK1 and RSK2 kinase activity and suppressed growth in TNBC, including TIC-enriched populations. Combining luteolin with paclitaxel increased cell death and unlike chemotherapy alone, did not enrich for CD44(+) cells. Luteolin's efficacy against drug-resistant cells was further indicated in the primary x43 cell line, where it suppressed monolayer growth and mammosphere formation. We next endeavored to understand how the inhibition of RSK/YB-1 signaling by luteolin elicited an effect on TIC-enriched populations. ChIP-on-ChIP experiments in SUM149 cells revealed a 12-fold enrichment of YB-1 binding to the Notch4 promoter. We chose to pursue this because there are several reports indicating that Notch4 maintains cells in an undifferentiated, TIC state. Herein we report that silencing YB-1 with siRNA decreased Notch4 mRNA. Conversely, transient expression of Flag:YB-1(WT) or the constitutively active mutant Flag:YB-1(D102) increased Notch4 mRNA. The levels of Notch4 transcript and the abundance of the Notch4 intracellular domain (N4ICD) correlated with activation of P-RSK(S221/7) and

  1. Object memory enhancement by combining sub-efficacious doses of specific phosphodiesterase inhibitors.

    PubMed

    Bollen, E; Akkerman, S; Puzzo, D; Gulisano, W; Palmeri, A; D'Hooge, R; Balschun, D; Steinbusch, H W M; Blokland, A; Prickaerts, J

    2015-08-01

    The second messengers cGMP and cAMP have a vital role in synaptic plasticity and memory processes. As such, phosphodiesterases inhibitors (PDE-Is), which prevent the breakdown of these cyclic nucleotides, represent a potential treatment strategy in memory decline. Recently it has been demonstrated that cGMP and cAMP signaling act in sequence during memory consolidation, with early cGMP signaling requiring subsequent cAMP signaling. Here, we sought to confirm this relationship, and to evaluate its therapeutic implications. Combining sub-efficacious doses of the cGMP-specific PDE type 5 inhibitor vardenafil (0.1 mg/kg) and cAMP-specific PDE type 4 inhibitor rolipram (0.01 mg/kg) during the early and late memory consolidation phase, respectively, led to improved memory performance in a 24 h interval object recognition task. Similarly, such a sub-efficacious combination treatment enhanced the transition of early-phase long-term potentiation (LTP) to late-phase LTP in hippocampal slices. In addition, both object memory and LTP were improved after administration of two sub-efficacious doses of the dual substrate PDE type 2 inhibitor BAY60 7550 (0.3 mg/kg) at the early and late consolidation phase, respectively. Taken together, combinations of sub-efficacious doses of cAMP- and cGMP-specific PDE-Is have an additive effect on long-term synaptic plasticity and memory formation and might prove a superior alternative to single PDE-I treatment. PMID:25896769

  2. Enhanced memory persistence is blocked by a DNA methyltransferase inhibitor in the snail Lymnaea stagnalis.

    PubMed

    Lukowiak, Ken; Heckler, Benjamin; Bennett, Thomas E; Schriner, Ellen K; Wyrick, Kathryn; Jewett, Cynthia; Todd, Ryan P; Sorg, Barbara A

    2014-08-15

    Lymnaea stagnalis provides an excellent model system for studying memory because these snails have a well-described set of neurons, a single one of which controls expression of long-term memory of operantly conditioned respiratory behavior. We have shown that several different manipulations, including pre-training exposure to serotonin (5-HT) or methamphetamine, submersion of snails after training to prevent memory interference, and exposure to effluent from predatory crayfish (CE), enhance memory persistence. Changes in DNA methylation underlie formation of strong memories in mammals and 5-HT-enhanced long-term facilitation in Aplysia. Here we determined the impact of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-AZA; 87 μmol l(-1)), on enhanced memory persistence by all four manipulations. We found that 5-HT (100 μmol l(-1)) enhanced memory persistence, which was blocked by 5-AZA pretreatment. Snails pre-exposed to 3.3 μmol l(-1) Meth 4 h prior to training demonstrated memory 72 h later, which was not present in controls. This memory-enhancing effect was blocked by pre-treatment with 87 μmol l(-1) 5-AZA. Similarly, submersion to prevent interference learning as well as training in CE produced memory that was not present in controls, and these effects were blocked by pre-treatment with 87 μmol l(-1) 5-AZA. In contrast, 5-AZA injection did not alter expression of normal (non-enhanced) memory, suggesting that these four stimuli enhance memory persistence by increasing DNA methyltransferase activity, which, in turn, increases expression of memory-enhancing genes and/or inhibits memory suppressor genes. These studies lay important groundwork for delineating gene methylation changes that are common to persistent memory produced by different stimuli.

  3. Bowman-Birk protease inhibitor from soybeans enhances cisplatin-induced cytotoxicity in human mesothelioma cells.

    PubMed

    Kashiwagi, Korehito; Virgona, Nantiga; Yamada, Jin; Sato, Ayami; Ota, Masako; Yazawa, Takuya; Yano, Tomohiro

    2011-07-01

    Malignant mesothelioma (MM) is an aggressive cancer with no effective treatment options. Enforced expression of the gap junction (GJ) component connexin 43 (Cx43) increases the sensitivity of MM cells to cisplatin. Bowman-Birk protease inhibitor (BBI) induces the restoration of Cx43 in several types of tumor cells. In this study, we examined the capability of BBI to enhance the cytotoxic effect of cisplatin in MM cells via the induction of Cx43. Human MM H28 cells were used. Cell viability was evaluated by a WST-1 assay and proteasomal activity was determined by fluorometric analysis. Protein and mRNA levels were determined by immunoblot analysis and real-time RT-PCR, respectively. GJ function mediated by Cx43 was evaluated using the scrape-loading method. BBI effectively inhibited H28 cell growth in a dose-dependent manner (200-400 μg/ml). In parallel with the growth inhibition, Cx43 levels (mRNA and protein) and GJ function were elevated by BBI treatment. Knockdown of BBI-induced Cx43 by an antisense nucleotide treatment almost cancelled the growth inhibition. BBI enhanced cisplatin-induced cytotoxicity in H28 cells, and down-regulation of Cx43 by the antisense nucleotide treatment abrogated the enhancing effect of BBI. The induction of Cx43 by BBI contributed to Src inactivation and subsequent induction of Bax. Furthermore, an Src inhibitor (SU6656) also enhanced cisplatin-induced cytotoxicity in H28 cells. These results suggest that BBI improves the cytotoxic efficacy of cisplatin in H28 cells via the inhibition of Src signaling.

  4. Bowman-Birk protease inhibitor from soybeans enhances cisplatin-induced cytotoxicity in human mesothelioma cells

    PubMed Central

    KASHIWAGI, KOREHITO; VIRGONA, NANTIGA; YAMADA, JIN; SATO, AYAMI; OTA, MASAKO; YAZAWA, TAKUYA; YANO, TOMOHIRO

    2011-01-01

    Malignant mesothelioma (MM) is an aggressive cancer with no effective treatment options. Enforced expression of the gap junction (GJ) component connexin 43 (Cx43) increases the sensitivity of MM cells to cisplatin. Bowman-Birk protease inhibitor (BBI) induces the restoration of Cx43 in several types of tumor cells. In this study, we examined the capability of BBI to enhance the cytotoxic effect of cisplatin in MM cells via the induction of Cx43. Human MM H28 cells were used. Cell viability was evaluated by a WST-1 assay and proteasomal activity was determined by fluorometric analysis. Protein and mRNA levels were determined by immunoblot analysis and real-time RT-PCR, respectively. GJ function mediated by Cx43 was evaluated using the scrape-loading method. BBI effectively inhibited H28 cell growth in a dose-dependent manner (200–400 μg/ml). In parallel with the growth inhibition, Cx43 levels (mRNA and protein) and GJ function were elevated by BBI treatment. Knockdown of BBI-induced Cx43 by an antisense nucleotide treatment almost cancelled the growth inhibition. BBI enhanced cisplatin-induced cytotoxicity in H28 cells, and down-regulation of Cx43 by the antisense nucleotide treatment abrogated the enhancing effect of BBI. The induction of Cx43 by BBI contributed to Src inactivation and subsequent induction of Bax. Furthermore, an Src inhibitor (SU6656) also enhanced cisplatin-induced cytotoxicity in H28 cells. These results suggest that BBI improves the cytotoxic efficacy of cisplatin in H28 cells via the inhibition of Src signaling. PMID:22977565

  5. Enhanced effects by 4-phenylbutyrate in combination with RTK inhibitors on proliferation in brain tumor cell models

    SciTech Connect

    Marino, Ana-Maria; Sofiadis, Anastasios; Baryawno, Ninib; Johnsen, John Inge; Larsson, Catharina; Vukojevic, Vladana; Ekstroem, Tomas J.

    2011-07-22

    Highlights: {yields} The histone deacetylase inhibitor 4-phenylbutyrate substantially enhance efficacy of the receptor tyrosine kinase inhibitors gefitinib or vandetanib in glioma and medulloblastoma cell lines. {yields} Cell death increases and clonogenic survival is reduced in the combination treatments, over mono-therapy. {yields} Combination treatments with these drugs may improve clinical outcome for cancer therapy. -- Abstract: We have investigated in vitro effects of anticancer therapy with the histone deacetylase inhibitor (HDACi) 4-phenylbutyrate (4-PB) combined with receptor tyrosine kinase inhibitors (RTKi) gefitinib or vandetanib on the survival of glioblastoma (U343MGa) and medulloblastoma (D324Med) cells. In comparison with individual effects of these drugs, combined treatment with gefitinib/4-PB or vandetanib/4-PB resulted in enhanced cell killing and reduced clonogenic survival in both cell lines. Our results suggest that combined treatment using HDACi and RTKi may beneficially affect the outcome of cancer therapy.

  6. Enzastaurin (LY317615), a Protein Kinase C Beta Selective Inhibitor, Enhances Antiangiogenic Effect of Radiation

    SciTech Connect

    Willey, Christopher D.; Xiao Dakai; Tu Tianxiang; Kim, Kwang Woon; Moretti, Luigi; Niermann, Kenneth J.; Tawtawy, Mohammed N.; Quarles, Chad C. Ph.D.; Lu Bo

    2010-08-01

    Purpose: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. Methods and Materials: The combination of the selective PKC{beta} inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. Results: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiation such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. Conclusion: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.

  7. Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors

    SciTech Connect

    Hagiwara, Hiroki; Saito, Fumiaki; Masaki, Toshihiro; Ikeda, Miki; Nakamura-Ohkuma, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of TSA, one of most potent HDACIs, on myogenesis using the C2C12 skeletal muscle cell line. Black-Right-Pointing-Pointer TSA enhances the expression of myosin heavy chain without affecting DAPC expression. Black-Right-Pointing-Pointer TSA enhances the expression of the early MRFs, Myf5 and MEF2, and suppresses the late MRF, myogenin, after 24 h treatment. Black-Right-Pointing-Pointer TSA enhances the expression of the myogenic repressors, Ids, which inhibit myogenic differentiation. Black-Right-Pointing-Pointer TSA promotes myogenesis by coordinating the expression of MRFs and myogenic repressors. -- Abstract: Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.

  8. The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome.

    PubMed

    Kisly, Ivan; Gulay, Suna P; Mäeorg, Uno; Dinman, Jonathan D; Remme, Jaanus; Tamm, Tiina

    2016-05-22

    During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality. PMID:27038511

  9. 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

  10. TAK1 inhibitor NG25 enhances doxorubicin-mediated apoptosis in breast cancer cells.

    PubMed

    Wang, Zhenyu; Zhang, Huiyuan; Shi, Minghao; Yu, Yang; Wang, Hao; Cao, Wen-Ming; Zhao, Yanling; Zhang, Hong

    2016-01-01

    Doxorubicin (Dox, Adriamycin) has been widely used in breast cancer treatment. But its severe cardio-toxic side effects limited the clinical use. Dox treatment can induce DNA damage and other accompanying effects in cancer cells, and subsequently activates nuclear factor κB (NF-κB) pathway which has a strong pro-survival role in different types of malignancy. We hypothesize that blocking NF-κB pathway may sensitize breast cancer cells to Dox chemotherapy. TGFβ-activated kinase-1 (TAK1) is a key intracellular molecule participating in genotoxic stresses-induced NF-κB activation. Targeting TAK1 as a strategy to enhance cancer treatment efficacy has been studied in several malignancies. We showed that NG25, a synthesized TAK1 inhibitor, greatly enhanced Dox treatment efficacy in a panel of breast cancer cell lines. In this pre-clinical study, we found that NG25 partially blocked Dox-induced p38 phosphorylation and IκBα degradation and enhanced Dox-induced cytotoxic effects and apoptosis in all breast cancer cell lines tested. Taken together, we provided clear evidence that NG25 sensitizes the breast cancer cells to Dox treatment in vitro. This combination may be an effective and feasible therapeutic option maximizing Dox efficacy and meanwhile minimizing Dox side effects in treating breast cancer. PMID:27599572

  11. TAK1 inhibitor NG25 enhances doxorubicin-mediated apoptosis in breast cancer cells

    PubMed Central

    Wang, Zhenyu; Zhang, Huiyuan; Shi, Minghao; Yu, Yang; Wang, Hao; Cao, Wen-Ming; Zhao, Yanling; Zhang, Hong

    2016-01-01

    Doxorubicin (Dox, Adriamycin) has been widely used in breast cancer treatment. But its severe cardio-toxic side effects limited the clinical use. Dox treatment can induce DNA damage and other accompanying effects in cancer cells, and subsequently activates nuclear factor κB (NF-κB) pathway which has a strong pro-survival role in different types of malignancy. We hypothesize that blocking NF-κB pathway may sensitize breast cancer cells to Dox chemotherapy. TGFβ-activated kinase-1 (TAK1) is a key intracellular molecule participating in genotoxic stresses-induced NF-κB activation. Targeting TAK1 as a strategy to enhance cancer treatment efficacy has been studied in several malignancies. We showed that NG25, a synthesized TAK1 inhibitor, greatly enhanced Dox treatment efficacy in a panel of breast cancer cell lines. In this pre-clinical study, we found that NG25 partially blocked Dox-induced p38 phosphorylation and IκBα degradation and enhanced Dox-induced cytotoxic effects and apoptosis in all breast cancer cell lines tested. Taken together, we provided clear evidence that NG25 sensitizes the breast cancer cells to Dox treatment in vitro. This combination may be an effective and feasible therapeutic option maximizing Dox efficacy and meanwhile minimizing Dox side effects in treating breast cancer. PMID:27599572

  12. Proteasome Inhibitor YSY01A Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant Human Ovarian Cancer Cells

    PubMed Central

    Huang, Wei; Zhou, Quan; Yuan, Xia; Ge, Ze-mei; Ran, Fu-xiang; Yang, Hua-yu; Qiang, Guang-liang; Li, Run-tao; Cui, Jing-rong

    2016-01-01

    Cisplatin is one of the most common drugs used for treatment of solid tumors such as ovarian cancer. Unfortunately, the development of resistance against this cytotoxic agent limits its clinical use. Here we report that YSY01A, a novel proteasome inhibitor, is capable of suppressing survival of cisplatin-resistant ovarian cancer cells by inducing apoptosis. And YSY01A treatment enhances the cytotoxicity of cisplatin in drug-resistant ovarian cancer cells. Specifically, YSY01A abrogates regulatory proteins important for cell proliferation and anti-apoptosis including NF-κB p65 and STAT3, resulting in down-regulation of Bcl-2. A dramatic increase in cisplatin uptake was also observed by inductively coupled plasma-mass spectrometry following exposure to YSY01A. Taken together, YSY01A serves as a potential candidate for further development as anticancer therapeutics targeting the proteasome. PMID:27326257

  13. Enhancement of a pentacyclic tyrosine kinase inhibitor production in Cladosporium cf. cladosporioides by Cladosporol.

    PubMed

    Assante, Gemma; Bava, Adriana; Nasini, Gianluca

    2006-02-01

    The binaphthyl derivative cladosporol 3 was supplied from 60 to 200 mg l(-1) to shaken cultures of Cladosporium cf. cladosporioides. Compared to blank, fungal biomass was not affected by adding cladosporol till 100 mg l(-1): it rather increased at higher ratios between 150 and 200 mg l(-1). The production of the major pentacyclic metabolite 1, a cytokine production and tyrosine kinase inhibitor, was enhanced tenfold when cladosporol was supplied at the highest ratio (200 mg l(-1)) to shaken growing cultures of the fungus. The bioconversion of cladosporol to cladosporol D through reductive cleavage of the epoxide group was also observed. Interest in this kind of metabolites lies in their potential activity vs DNA topoisomerase I.

  14. Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to Hepatocyte Growth Factor.

    PubMed

    Frasca, F; Vigneri, P; Vella, V; Vigneri, R; Wang, J Y

    2001-06-28

    The Hepatocyte Growth Factor (HGF) and its receptor Met are physiological regulators of cell migration. HGF and Met have also been implicated in tumor progression and metastasis. We show here that the tyrosine kinase inhibitor STI571 has a stimulatory effect on HGF-induced migration and branching morphogenesis in thyroid cancer but not in primary or immortalized thyroid epithelial cells. These stimulatory effects of STI571 are observed at a concentration that is clinically relevant. The STI571-enhanced motile response can be correlated with an increase in the Met receptor tyrosine phosphorylation as well as ERK and Akt activation by HGF. Interestingly, one of the targets of STI571, namely the c-Abl tyrosine kinase, is activated by HGF and is recruited at the migrating edge of thyroid cancer cells. These data suggests that c-Abl and/or STI571-inhibited tyrosine kinases can negatively regulate the Met receptor to restrain the motile response in thyroid cancer cells.

  15. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins

    PubMed Central

    Gamalinda, Michael; Woolford, John L

    2015-01-01

    The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders. PMID:26779413

  16. Phospholipase Cϵ Activates Nuclear Factor-κB Signaling by Causing Cytoplasmic Localization of Ribosomal S6 Kinase and Facilitating Its Phosphorylation of Inhibitor κB in Colon Epithelial Cells.

    PubMed

    Wakita, Masahiro; Edamatsu, Hironori; Li, Mingzhen; Emi, Aki; Kitazawa, Sohei; Kataoka, Tohru

    2016-06-10

    Phospholipase Cϵ (PLCϵ), an effector of Ras and Rap small GTPases, plays a crucial role in inflammation by augmenting proinflammatory cytokine expression. This proinflammatory function of PLCϵ is implicated in its facilitative role in tumor promotion and progression during skin and colorectal carcinogenesis, although their direct link remains to be established. Moreover, the molecular mechanism underlying these functions of PLCϵ remains unknown except that PKD works downstream of PLCϵ. Here we show by employing the colitis-induced colorectal carcinogenesis model, where Apc(Min) (/+) mice are administered with dextran sulfate sodium, that PLCϵ knock-out alleviates the colitis and suppresses the following tumorigenesis concomitant with marked attenuation of proinflammatory cytokine expression. In human colon epithelial Caco2 cells, TNF-α induces sustained expression of proinflammatory molecules and sustained activation of nuclear factor-κB (NF-κB) and PKD, the late phases of which are suppressed by not only siRNA-mediated PLCϵ knockdown but also treatment with a lysophosphatidic acid (LPA) receptor antagonist. Also, LPA stimulation induces these events in an early time course, suggesting that LPA mediates TNF-α signaling in an autocrine manner. Moreover, PLCϵ knockdown results in inhibition of phosphorylation of IκB by ribosomal S6 kinase (RSK) but not by IκB kinases. Subcellular fractionation suggests that enhanced phosphorylation of a scaffolding protein, PEA15 (phosphoprotein enriched in astrocytes 15), downstream of the PLCϵ-PKD axis causes sustained cytoplasmic localization of phosphorylated RSK, thereby facilitating IκB phosphorylation in the cytoplasm. These results suggest the crucial role of the TNF-α-LPA-LPA receptor-PLCϵ-PKD-PEA15-RSK-IκB-NF-κB pathway in facilitating inflammation and inflammation-associated carcinogenesis in the colon. PMID:27053111

  17. The non-Geldanamycin Hsp90 inhibitors enhanced the antifungal activity of fluconazole

    PubMed Central

    Li, Liping; An, Maomao; Shen, Hui; Huang, Xin; Yao, Xueya; Liu, Jian; Zhu, Fang; Zhang, Shiqun; Chen, Simin; He, Lijuan; Zhang, Jundong; Zou, Zui; Jiang, Yuanying

    2015-01-01

    The molecular chaperone heat shock protein 90 (Hsp90) is highly conserved in eukaryotes and facilitates the correct folding, productive assembly and maturation of a diverse cellular proteins. In fungi, especially the most prevalent human fungal pathogen Candida albicans, Hsp90 influences development and modulates drug resistance. Here, we mainly explore the effect of non-Geldanamycin Hsp90 inhibitor HSP990 on the activity of fluconazole (FLC) against Candida albicans and investigate the underlying mechanism. We demonstrate that HSP990 has potent synergistic antifungal activity with FLC against FLC-resistant C. albicans through the checkerboard microdilution assay,agar diffusion tests and time-kill curves, and shows low cytotoxicity to human umbilical vein endothelial cells. Further study shows that the activity of FLC against C. albicans biofilm formation in vitro is significantly enhanced when used in combination with HSP990. In a murine model of disseminated candidiasis, the therapeutic efficacy of FLC is also enhanced by the pharmacological inhibition of C. albicans Hsp90 function with HSP990. Thus, the combined use of small molecule compound and existing antifungal drugs may provide a potential therapeutic strategy for fungal infectious disease. PMID:26885259

  18. Heat shock protein inhibitors, 17-DMAG and KNK437, enhance arsenic trioxide-induced mitotic apoptosis

    SciTech Connect

    Wu Yichen; Yen Wenyen; Lee, T.-C. Yih, L.-H.

    2009-04-15

    Arsenic trioxide (ATO) has recently emerged as a promising therapeutic agent in leukemia because of its ability to induce apoptosis. However, there is no sufficient evidence to support its therapeutic use for other types of cancers. In this study, we investigated if, and how, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), an antagonist of heat shock protein 90 (HSP90), and KNK437, a HSP synthesis inhibitor, potentiated the cytotoxic effect of ATO. Our results showed that cotreatment with ATO and either 17-DMAG or KNK437 significantly increased ATO-induced cell death and apoptosis. siRNA-mediated attenuation of the expression of the inducible isoform of HSP70 (HSP70i) or HSP90{alpha}/{beta} also enhanced ATO-induced apoptosis. In addition, cotreatment with ATO and 17-DMAG or KNK437 significantly increased ATO-induced mitotic arrest and ATO-induced BUBR1 phosphorylation and PDS1 accumulation. Cotreatment also significantly increased the percentage of mitotic cells with abnormal mitotic spindles and promoted metaphase arrest as compared to ATO treatment alone. These results indicated that 17-DMAG or KNK437 may enhance ATO cytotoxicity by potentiating mitotic arrest and mitotic apoptosis possibly through increased activation of the spindle checkpoint.

  19. Purification of 70S ribosomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-01

    Here we describe the further purification of prokaryotic ribosomal particles obtained after the centrifugation of a crude cell lysate through a sucrose cushion. In this final purification step, a fraction containing ribosomes, ribosomal subunits, and polysomes is centrifuged through a 7%-30% (w/w) linear sucrose gradient to isolate tight couple 70S ribosomes, as well as dissociated 30S and 50S subunits. The tight couples fraction, or translationally active ribosome fraction, is composed of intact vacant ribosomes that can be used in cell-free translation systems.

  20. Cyclooxygenase I and II inhibitors distinctly enhance hippocampal- and cortex-dependent cognitive functions in mice.

    PubMed

    Syed, Huma; Ikram, Muhammad Faisal; Yaqinuddin, Ahmed; Ahmed, Touqeer

    2015-11-01

    Cyclooxygenase (COX) enzymes are expressed in the brain; however, their role in hippocampus-dependent and cortex-dependent cognitive functions remains to be fully elucidated. The aim of the present study was to comparatively investigate the effects of piroxicam, a selective COX-I inhibitor, and celecoxib, a selective COX‑II inhibitor, on cognitive functions in an AlCl3‑induced neurotoxicity mouse model to understand the specific role of each COX enzyme in the hippocampus and cortex. The AlCl3 (250 mg/kg) was administered to the mice in drinking water and the drugs were administered in feed for 30 days. Assessments of memory, including a Morris water maze, social behavior and nesting behavior were performed in control and treated mice. The RNA expression of the COX enzymes were analyzed using reverse transcription‑quantitative polymerase chain reaction analysis. An ex‑vivo 2,2‑Diphenyl‑1‑picrylhydrazyl assay was performed in the hippocampus and cortex. Following 30 days of treatment with thedrugs, the mice in the celecoxib‑ and piroxicam‑treated groups exhibited enhanced learning (6.84 ± 0.76 and 9.20 ± 1.08, respectively), compared with the AlCl3‑induced neurotoxicity group (21.14 ± 0.76) on the fifth day of the Morris water maze test. Celecoxib treatment improved social affiliation in the AlCl3‑induced neurotoxicity group, the results of which were superior to piroxicam. Piroxicam led to better improvement in nesting score in the AlCl3‑induced neurotoxicity group. Both drugs decreased the expression levels of COX‑I and COX‑II in the hippocampus and cortex, and rescued oxidative stress levels. These findings suggested that each drug distinctly affected cognitive functions, highlighting the distinctive roles of COX-I and COX-II in learning and memory.

  1. Probenecid Treatment Enhances Retinal and Brain Delivery of N-4-Benzoylaminophenylsulfonylglycine, An Anionic Aldose Reductase Inhibitor

    PubMed Central

    Sunkara, Gangadhar; Ayalasomayajula, Surya P.; DeRuiter, Jack; Kompella, Uday B.

    2009-01-01

    Anion efflux transporters are expected to minimize target tissue delivery of N-[4-(benzoylaminophenyl)sulfonyl]glycine (BAPSG), a novel carboxylic acid aldose reductase inhibitor, which exists as a monocarboxylate anion at physiological conditions. Therefore, the objective of this study was to determine whether BAPSG delivery to various eye tissues including the retina and the brain can be enhanced by probenecid, a competitive inhibitor of anion transporters. To determine the influence of probenecid on eye and brain distribution of BAPSG, probenecid was administered intraperitoneally (120 mg/kg body weight; i.p.) 20 minutes prior to BAPSG (50 mg/kg; i.p.) administration. Drug disposition in various eye tissues including the retina and the brain was determined at 15 min, 1, 2 and 4 hr after BAPSG dose in male Sprauge-Dawley rats. To determine whether probenecid alters plasma clearance of BAPSG, influence of probenecid (120 mg/kg; i.p.) on the plasma pharmacokinetics of intravenously administered BAPSG (15 mg/kg) was studied as well. Finally, the effect of probenecid co-administration on the ocular tissue distribution of BAPSG was assessed in rabbits following topical (eye drop) administration. Following pretreatment with probenecid in the rat study, retinal delivery at 1 hr was increased by about 11 fold (2580 vs 244 ng/gm; p<0.05). Further, following probenecid pretreatment, significant BAPSG levels were detectable in the brain (45 ± 20 ng/gm) at 1 hr, unlike controls where the drug was not detectable. Plasma concentrations, plasma elimination half-life, and total body clearance of intravenously administered BAPSG were not altered by i.p. probenecid pretreatment. In the topical dosing study, a significant decline in BAPSG delivery was observed in the iris-ciliary body but no significant changes were observed in other tissues of the anterior segment of the eye including tears. Thus, inhibition of anion transporters is a useful approach to elevate retinal and brain

  2. Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

    PubMed

    Tsugawa, Daisuke; Oya, Yuki; Masuzaki, Ryota; Ray, Kevin; Engers, Darren W; Dib, Martin; Do, Nhue; Kuramitsu, Kaori; Ho, Karen; Frist, Audrey; Yu, Paul B; Bloch, Kenneth D; Lindsley, Craig W; Hopkins, Corey R; Hong, Charles C; Karp, Seth J

    2014-12-01

    Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

  3. Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

    PubMed

    Tsugawa, Daisuke; Oya, Yuki; Masuzaki, Ryota; Ray, Kevin; Engers, Darren W; Dib, Martin; Do, Nhue; Kuramitsu, Kaori; Ho, Karen; Frist, Audrey; Yu, Paul B; Bloch, Kenneth D; Lindsley, Craig W; Hopkins, Corey R; Hong, Charles C; Karp, Seth J

    2014-12-01

    Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease. PMID:25271257

  4. The PKD Inhibitor CID755673 Enhances Cardiac Function in Diabetic db/db Mice

    PubMed Central

    Venardos, Kylie; De Jong, Kirstie A.; Elkamie, Mansour; Connor, Timothy; McGee, Sean L.

    2015-01-01

    The development of diabetic cardiomyopathy is a key contributor to heart failure and mortality in obesity and type 2 diabetes (T2D). Current therapeutic interventions for T2D have limited impact on the development of diabetic cardiomyopathy. Clearly, new therapies are urgently needed. A potential therapeutic target is protein kinase D (PKD), which is activated by metabolic insults and implicated in the regulation of cardiac metabolism, contractility and hypertrophy. We therefore hypothesised that PKD inhibition would enhance cardiac function in T2D mice. We first validated the obese and T2D db/db mouse as a model of early stage diabetic cardiomyopathy, which was characterised by both diastolic and systolic dysfunction, without overt alterations in left ventricular morphology. These functional characteristics were also associated with increased PKD2 phosphorylation in the fed state and a gene expression signature characteristic of PKD activation. Acute administration of the PKD inhibitor CID755673 to normal mice reduced both PKD1 and 2 phosphorylation in a time and dose-dependent manner. Chronic CID755673 administration to T2D db/db mice for two weeks reduced expression of the gene expression signature of PKD activation, enhanced indices of both diastolic and systolic left ventricular function and was associated with reduced heart weight. These alterations in cardiac function were independent of changes in glucose homeostasis, insulin action and body composition. These findings suggest that PKD inhibition could be an effective strategy to enhance heart function in obese and diabetic patients and provide an impetus for further mechanistic investigations into the role of PKD in diabetic cardiomyopathy. PMID:25798941

  5. Crystallography of ribosomal particles

    NASA Astrophysics Data System (ADS)

    Yonath, A.; Frolow, F.; Shoham, M.; Müssig, J.; Makowski, I.; Glotz, C.; Jahn, W.; Weinstein, S.; Wittmann, H. G.

    1988-07-01

    Several forms of three-dimensional crystals and two-dimensional sheets of intact ribosomes and their subunits have been obtained as a result of: (a) an extensive systematic investigation of the parameters involved in crystallization, (b) a development of an experimental procedure for controlling the volumes of the crystallization droplets, (c) a study of the nucleation process, and (d) introducing a delicate seeding procedure coupled with variations in the ratios of mono- and divalent ions in the crystallization medium. In all cases only biologically active particles could be crystallized, and the crystalline material retains its integrity and activity. Crystallographic data have been collected from crystals of 50S ribosomal subunits, using synchrotron radiation at temperatures between + 19 and - 180°C. Although at 4°C the higher resolution reflections decay within minutes in the synchrotron beam, at cryo-temperature there was hardly any radiation damage, and a complete set of data to about 6Åresolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50S ribosomal subunits from a mutant of B. stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with in the native ones. Models, aimed to be used for low resolution phasing, have been reconstructed from two-dimensional sheets of 70S ribosomes and 50S subunits at 47 and 30Å, respectively. These models show the overall structure of these particles, the contact areas between the large and small subunits, the space where protein synthesis might take place and a tunnel which may provide the path for the nascent protein chain.

  6. Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight.

    PubMed

    Kim, Ju-Kon; Jang, In-Cheol; Wu, Ray; Zuo, Wei-Neng; Boston, Rebecca S; Lee, Yong-Hwan; Ahn, Il-Pyung; Nahm, Baek Hie

    2003-08-01

    Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression.

  7. An HDAC inhibitor enhances cancer therapeutic efficiency of RNA polymerase III promoter-driven IDO shRNA.

    PubMed

    Yen, M-C; Weng, T-Y; Chen, Y-L; Lin, C-C; Chen, C-Y; Wang, C-Y; Chao, H-L; Chen, C-S; Lai, M-D

    2013-06-01

    Histone deacetylase (HDAC) inhibitors are used in treating certain human malignancies. Our laboratories demonstrated their capability in enhancing antitumor effect of DNA vaccine driven by an RNA polymerase II (RNA pol II) promoter. However, it is unknown whether HDAC inhibitors enhance the therapeutic short hairpin RNA (shRNA) expressed by an RNA polymerase III (RNA pol III) promoter. We investigated whether HDAC inhibitors augmented antitumor effect of indoleamine 2,3 dioxygenase (IDO) shRNA. HDAC inhibitor OSU-HDAC42 and suberoylanilide hydroxamic acid enhanced RNA pol III-driven U6 and H1 promoter activity in three different cell types in vitro: 293, NIH3T3 and dendritic cell line DC2.4. Subcutaneous injection of OSU-HDAC42 enhanced U6 and H1 promoter activity on abdominal skin of mice in vivo. Combination of IDO shRNA and OSU-HDAC42 increased antitumor effect of IDO shRNA in MBT-2 murine bladder tumor model. IDO shRNA induced tumor-infiltrating CD8⁺ and CD4⁺ T cells, whereas OSU-HDAC42 treatment induced tumor-infiltrating CD4⁺ T cells. Combination of OSU-HDAC42 and IDO shRNA further induced tumor-infiltrating natural killer cells and enhanced interferon-γ in lymphocytes, but suppressed interleukin (IL)-4 expression of lymphocytes. In addition, OSU-HDAC42 treatment did not alter mRNA expression of IL-12 and tumor necrosis factor-α. In conclusion, HDAC inhibitor OSU-HDAC42 may serve as adjuvant of the therapeutic shRNA expressed by an RNA pol III promoter.

  8. Inhibitors of poly(ADP-ribose) synthesis enhance X-ray killing of log-phase Chinese hamster cells

    SciTech Connect

    Ben-Hur, E.; Utsumi, H.; Elkind, M.M.

    1984-03-01

    Postirradiation incubation of V79 Chinese hamster cells with inhibitors of poly(ADP-ribose) synthesis was found to potentiate the killing of cells by X rays. Potentiation increased with incubation time and with concentration of the inhibitor. Preirradiation incubation had only a small effect. The enhanced response correlated well with the known extent of the inhibition of poly(ADP-ribose) synthesis. A radiation-sensitive line, V79-AL162/S-10, was affected to a lesser extent than the normal cells. Cells repaired the radiation damage with which the inhibitors interacted within 1 hr, a process that has similar kinetics to what is observed when a postirradiation treatment with hypertonic buffer is used. However, the sectors of damage affected by inhibitors of poly(ADP-ribose) synthesis and hypertonic buffer do not entirely overlap. The inhibitor nicotinamide enhanced the killing mainly of late S-phase cells and did not affect cells at the G/sub 1//S border. It is concluded that the repair process(es) involving poly(ADP-ribose) synthesis is important for cell survival in repair-competent cells and that the radiation-sensitive cells that were examined are partially deficient in a repair pathway in which poly(ADP-ribose) participates.

  9. Investigate the Metabolic Reprogramming of Saccharomyces cerevisiae for Enhanced Resistance to Mixed Fermentation Inhibitors via 13C Metabolic Flux Analysis.

    PubMed

    Guo, Weihua; Chen, Yingying; Wei, Na; Feng, Xueyang

    2016-01-01

    The fermentation inhibitors from the pretreatment of lignocellulosic materials, e.g., acetic acid and furfural, are notorious due to their negative effects on the cell growth and chemical production. However, the metabolic reprogramming of the cells under these stress conditions, especially metabolic response for resistance to mixed inhibitors, has not been systematically investigated and remains mysterious. Therefore, in this study, 13C metabolic flux analysis (13C-MFA), a powerful tool to elucidate the intracellular carbon flux distributions, has been applied to two Saccharomyces cerevisiae strains with different tolerances to the inhibitors under acetic acid, furfural, and mixed (i.e., acetic acid and furfural) stress conditions to unravel the key metabolic responses. By analyzing the intracellular carbon fluxes as well as the energy and cofactor utilization under different conditions, we uncovered varied metabolic responses to different inhibitors. Under acetate stress, ATP and NADH production was slightly impaired, while NADPH tended towards overproduction. Under furfural stress, ATP and cofactors (including both NADH and NADPH) tended to be overproduced. However, under dual-stress condition, production of ATP and cofactors was severely impaired due to synergistic stress caused by the simultaneous addition of two fermentation inhibitors. Such phenomenon indicated the pivotal role of the energy and cofactor utilization in resisting the mixed inhibitors of acetic acid and furfural. Based on the discoveries, valuable insights are provided to improve the tolerance of S. cerevisiae strain and further enhance lignocellulosic fermentation.

  10. Investigate the Metabolic Reprogramming of Saccharomyces cerevisiae for Enhanced Resistance to Mixed Fermentation Inhibitors via 13C Metabolic Flux Analysis

    PubMed Central

    Guo, Weihua; Chen, Yingying; Wei, Na; Feng, Xueyang

    2016-01-01

    The fermentation inhibitors from the pretreatment of lignocellulosic materials, e.g., acetic acid and furfural, are notorious due to their negative effects on the cell growth and chemical production. However, the metabolic reprogramming of the cells under these stress conditions, especially metabolic response for resistance to mixed inhibitors, has not been systematically investigated and remains mysterious. Therefore, in this study, 13C metabolic flux analysis (13C-MFA), a powerful tool to elucidate the intracellular carbon flux distributions, has been applied to two Saccharomyces cerevisiae strains with different tolerances to the inhibitors under acetic acid, furfural, and mixed (i.e., acetic acid and furfural) stress conditions to unravel the key metabolic responses. By analyzing the intracellular carbon fluxes as well as the energy and cofactor utilization under different conditions, we uncovered varied metabolic responses to different inhibitors. Under acetate stress, ATP and NADH production was slightly impaired, while NADPH tended towards overproduction. Under furfural stress, ATP and cofactors (including both NADH and NADPH) tended to be overproduced. However, under dual-stress condition, production of ATP and cofactors was severely impaired due to synergistic stress caused by the simultaneous addition of two fermentation inhibitors. Such phenomenon indicated the pivotal role of the energy and cofactor utilization in resisting the mixed inhibitors of acetic acid and furfural. Based on the discoveries, valuable insights are provided to improve the tolerance of S. cerevisiae strain and further enhance lignocellulosic fermentation. PMID:27532329

  11. Investigate the Metabolic Reprogramming of Saccharomyces cerevisiae for Enhanced Resistance to Mixed Fermentation Inhibitors via 13C Metabolic Flux Analysis.

    PubMed

    Guo, Weihua; Chen, Yingying; Wei, Na; Feng, Xueyang

    2016-01-01

    The fermentation inhibitors from the pretreatment of lignocellulosic materials, e.g., acetic acid and furfural, are notorious due to their negative effects on the cell growth and chemical production. However, the metabolic reprogramming of the cells under these stress conditions, especially metabolic response for resistance to mixed inhibitors, has not been systematically investigated and remains mysterious. Therefore, in this study, 13C metabolic flux analysis (13C-MFA), a powerful tool to elucidate the intracellular carbon flux distributions, has been applied to two Saccharomyces cerevisiae strains with different tolerances to the inhibitors under acetic acid, furfural, and mixed (i.e., acetic acid and furfural) stress conditions to unravel the key metabolic responses. By analyzing the intracellular carbon fluxes as well as the energy and cofactor utilization under different conditions, we uncovered varied metabolic responses to different inhibitors. Under acetate stress, ATP and NADH production was slightly impaired, while NADPH tended towards overproduction. Under furfural stress, ATP and cofactors (including both NADH and NADPH) tended to be overproduced. However, under dual-stress condition, production of ATP and cofactors was severely impaired due to synergistic stress caused by the simultaneous addition of two fermentation inhibitors. Such phenomenon indicated the pivotal role of the energy and cofactor utilization in resisting the mixed inhibitors of acetic acid and furfural. Based on the discoveries, valuable insights are provided to improve the tolerance of S. cerevisiae strain and further enhance lignocellulosic fermentation. PMID:27532329

  12. Combination of verteporfin-PDT and PI3K inhibitors enhances cell growth inhibition and apoptosis in endothelial cells

    NASA Astrophysics Data System (ADS)

    Kraus, Daniel; Chen, Bin

    2016-03-01

    Vascular targeted photodynamic therapy is a promising cancer treatment modality by ablating tumor vasculature. The effectiveness of this treatment is often compromised by regrowth of endothelial cells, which causes tumor recurrence. In this preliminary report, we showed that activated PI3K signaling was involved in endothelial cell regrowth after PDT with verteporfin and combination between verteporfin-PDT and PI3K pathway inhibitor BEZ235 induced more cell apoptosis and greater inhibition in cell proliferation. These results suggest that rational combination of verteporfin-PDT and PI3K inhibitors result in enhanced treatment outcomes.

  13. Expanding the ribosomal universe.

    PubMed

    Dinman, Jonathan D; Kinzy, Terri Goss

    2009-12-01

    In this issue of Structure, Taylor et al. (2009) present the most complete model of an eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery.

  14. Ribosomal Antibiotics: Contemporary Challenges.

    PubMed

    Auerbach-Nevo, Tamar; Baram, David; Bashan, Anat; Belousoff, Matthew; Breiner, Elinor; Davidovich, Chen; Cimicata, Giuseppe; Eyal, Zohar; Halfon, Yehuda; Krupkin, Miri; Matzov, Donna; Metz, Markus; Rufayda, Mruwat; Peretz, Moshe; Pick, Ophir; Pyetan, Erez; Rozenberg, Haim; Shalev-Benami, Moran; Wekselman, Itai; Zarivach, Raz; Zimmerman, Ella; Assis, Nofar; Bloch, Joel; Israeli, Hadar; Kalaora, Rinat; Lim, Lisha; Sade-Falk, Ofir; Shapira, Tal; Taha-Salaime, Leena; Tang, Hua; Yonath, Ada

    2016-06-29

    Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of "pathogen-specific antibiotics," in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification.

  15. 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

  16. Ribosomal Antibiotics: Contemporary Challenges.

    PubMed

    Auerbach-Nevo, Tamar; Baram, David; Bashan, Anat; Belousoff, Matthew; Breiner, Elinor; Davidovich, Chen; Cimicata, Giuseppe; Eyal, Zohar; Halfon, Yehuda; Krupkin, Miri; Matzov, Donna; Metz, Markus; Rufayda, Mruwat; Peretz, Moshe; Pick, Ophir; Pyetan, Erez; Rozenberg, Haim; Shalev-Benami, Moran; Wekselman, Itai; Zarivach, Raz; Zimmerman, Ella; Assis, Nofar; Bloch, Joel; Israeli, Hadar; Kalaora, Rinat; Lim, Lisha; Sade-Falk, Ofir; Shapira, Tal; Taha-Salaime, Leena; Tang, Hua; Yonath, Ada

    2016-01-01

    Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of "pathogen-specific antibiotics," in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification. PMID:27367739

  17. Ribosomal Antibiotics: Contemporary Challenges

    PubMed Central

    Auerbach-Nevo, Tamar; Baram, David; Bashan, Anat; Belousoff, Matthew; Breiner, Elinor; Davidovich, Chen; Cimicata, Giuseppe; Eyal, Zohar; Halfon, Yehuda; Krupkin, Miri; Matzov, Donna; Metz, Markus; Rufayda, Mruwat; Peretz, Moshe; Pick, Ophir; Pyetan, Erez; Rozenberg, Haim; Shalev-Benami, Moran; Wekselman, Itai; Zarivach, Raz; Zimmerman, Ella; Assis, Nofar; Bloch, Joel; Israeli, Hadar; Kalaora, Rinat; Lim, Lisha; Sade-Falk, Ofir; Shapira, Tal; Taha-Salaime, Leena; Tang, Hua; Yonath, Ada

    2016-01-01

    Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of “pathogen-specific antibiotics,” in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification. PMID:27367739

  18. Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation*

    PubMed Central

    Fernández-Pevida, Antonio; Rodríguez-Galán, Olga; Díaz-Quintana, Antonio; Kressler, Dieter; de la Cruz, Jesús

    2012-01-01

    Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process. PMID:22995916

  19. Constructing ribosomes along the Danube

    PubMed Central

    Warner, Jonathan R.

    2010-01-01

    The EMBO Conference on Ribosome Synthesis held last summer explored the latest breakthroughs in ribosome assembly and how it affects disease. Both of these topics have recently seen important advances that enlighten how almost 200 proteins cooperate to produce a ribosome and how the cell responds to a malfunction in this process. PMID:20010797

  20. Aminopeptidase N (APN)/CD13 inhibitor, Ubenimex, enhances radiation sensitivity in human cervical cancer

    PubMed Central

    Tsukamoto, Hirohisa; Shibata, Kiyosumi; Kajiyama, Hiroaki; Terauchi, Mikio; Nawa, Akihiro; Kikkawa, Fumitaka

    2008-01-01

    Background Radiotherapy can be used to treat all stages of cervical cancer. For improving local control via radiotherapy, it is important to use additional antitumor agents. Aminopeptidase N (APN)/CD13, a 150-kDa metalloproteinase, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression in several human malignancies. Methods We investigated whether the suppression of APN/CD13 using Ubenimex, an inhibitor of APN/CD13 activity, may affect tumor radiosensitivity in cervical cancer cells both in vitro and in vivo. Cell surface APN/CD13 activity in HeLa cells was calculated using alanine-p-nitroanilido as a substrate. For colony formation assays, single-dose radiation and/or Ubenimex were administered to each dish of HeLa cells, and these dishes were cultured for 14 days. Molecular changes of apoptosis were determined by Western blot. Apoptosis was evaluated by Annexin-V PI staining (flow cytometry analysis) and the Tunel method. Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice. Results We demonstrated that Ubenimex enhanced the effectiveness of radiotherapy, acting as a radiosensitizer both in vitro and in vivo. In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells. Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups. We also showed that ubenimex enhanced radiation-induced apoptosis in vitro and in vivo. Conclusion Although further studies are needed, this report suggests that Ubeniemx acts as a radiosensitizer in cervical cancer treatment, and that the inhibition of APN/CD13 activity may represent a new approach for improving the therapeutic efficacy of radiotherapy for uterine cervical cancer. PMID:18366676

  1. Knockdown of the Cell Cycle Inhibitor p21 Enhances Cartilage Formation by Induced Pluripotent Stem Cells

    PubMed Central

    Diekman, Brian O.; Thakore, Pratiksha I.; O'Connor, Shannon K.; Willard, Vincent P.; Brunger, Jonathan M.; Christoforou, Nicolas; Leong, Kam W.

    2015-01-01

    The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded ∼150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering. PMID:25517798

  2. Knockdown of the cell cycle inhibitor p21 enhances cartilage formation by induced pluripotent stem cells.

    PubMed

    Diekman, Brian O; Thakore, Pratiksha I; O'Connor, Shannon K; Willard, Vincent P; Brunger, Jonathan M; Christoforou, Nicolas; Leong, Kam W; Gersbach, Charles A; Guilak, Farshid

    2015-04-01

    The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded ∼150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering.

  3. The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer

    PubMed Central

    Diaz, R; Nguewa, P A; Diaz-Gonzalez, J A; Hamel, E; Gonzalez-Moreno, O; Catena, R; Serrano, D; Redrado, M; Sherris, D; Calvo, A

    2009-01-01

    Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 μM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg−1 P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer. PMID:19240717

  4. Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.

    PubMed

    Arai, Satoko; Kitada, Kento; Yamazaki, Tomoko; Takai, Ryosuke; Zhang, Xizhong; Tsugawa, Yoji; Sugisawa, Ryoichi; Matsumoto, Ayaka; Mori, Mayumi; Yoshihara, Yasunori; Doi, Kent; Maehara, Natsumi; Kusunoki, Shunsuke; Takahata, Akiko; Noiri, Eisei; Suzuki, Yusuke; Yahagi, Naoki; Nishiyama, Akira; Gunaratnam, Lakshman; Takano, Tomoko; Miyazaki, Toru

    2016-02-01

    Acute kidney injury (AKI) is associated with prolonged hospitalization and high mortality, and it predisposes individuals to chronic kidney disease. To date, no effective AKI treatments have been established. Here we show that the apoptosis inhibitor of macrophage (AIM) protein on intraluminal debris interacts with kidney injury molecule (KIM)-1 and promotes recovery from AKI. During AKI, the concentration of AIM increases in the urine, and AIM accumulates on necrotic cell debris within the kidney proximal tubules. The AIM present in this cellular debris binds to KIM-1, which is expressed on injured tubular epithelial cells, and enhances the phagocytic removal of the debris by the epithelial cells, thus contributing to kidney tissue repair. When subjected to ischemia-reperfusion (IR)-induced AKI, AIM-deficient mice exhibited abrogated debris clearance and persistent renal inflammation, resulting in higher mortality than wild-type (WT) mice due to progressive renal dysfunction. Treatment of mice with IR-induced AKI using recombinant AIM resulted in the removal of the debris, thereby ameliorating renal pathology. We observed this effect in both AIM-deficient and WT mice, but not in KIM-1-deficient mice. Our findings provide a basis for the development of potentially novel therapies for AKI. PMID:26726878

  5. Sm-like protein enhanced tolerance of recombinant Saccharomyces cerevisiae to inhibitors in hemicellulosic hydrolysate.

    PubMed

    Gao, Lan; Xia, Liming

    2012-11-01

    A current challenge of the cellulosic ethanol industry is to improve the resistance of inhibitors present in biomass hydrolysates. RNA-binding protein gene lsm6 was cloned from industrial Saccharomyces cerevisiae ZU-E8, which is able to conferment glucose and xylose, and transformed into ZU-E8 via expression vector pRS426. The positive transformant ZU-910 with over-expressing lsm6 was identified on the culture plates using high concentration of acetate and re-screened by fermentation test. Fermentation by the recombinants was performed in a medium containing 80 g/L xylose and 2 g/L acetic acid or 20 g/L NH(4)Ac/NaAc. After 96 h shaking-flask fermentation, ZU-910 utilized 90.2% xylose with an ethanol yield of 26.9 g/L, which was 8.5- and 10-fold higher than ZU-E8. Further, in the corn stover hemicellulosic hydrolysate fermentation, both the xylose conversion and ethanol production by ZU-910 was larger by 50% and 40% than ZU-E8. ZU-910 has also enhanced tolerance against furfural and SO(4)(2-).

  6. Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.

    PubMed

    Arai, Satoko; Kitada, Kento; Yamazaki, Tomoko; Takai, Ryosuke; Zhang, Xizhong; Tsugawa, Yoji; Sugisawa, Ryoichi; Matsumoto, Ayaka; Mori, Mayumi; Yoshihara, Yasunori; Doi, Kent; Maehara, Natsumi; Kusunoki, Shunsuke; Takahata, Akiko; Noiri, Eisei; Suzuki, Yusuke; Yahagi, Naoki; Nishiyama, Akira; Gunaratnam, Lakshman; Takano, Tomoko; Miyazaki, Toru

    2016-02-01

    Acute kidney injury (AKI) is associated with prolonged hospitalization and high mortality, and it predisposes individuals to chronic kidney disease. To date, no effective AKI treatments have been established. Here we show that the apoptosis inhibitor of macrophage (AIM) protein on intraluminal debris interacts with kidney injury molecule (KIM)-1 and promotes recovery from AKI. During AKI, the concentration of AIM increases in the urine, and AIM accumulates on necrotic cell debris within the kidney proximal tubules. The AIM present in this cellular debris binds to KIM-1, which is expressed on injured tubular epithelial cells, and enhances the phagocytic removal of the debris by the epithelial cells, thus contributing to kidney tissue repair. When subjected to ischemia-reperfusion (IR)-induced AKI, AIM-deficient mice exhibited abrogated debris clearance and persistent renal inflammation, resulting in higher mortality than wild-type (WT) mice due to progressive renal dysfunction. Treatment of mice with IR-induced AKI using recombinant AIM resulted in the removal of the debris, thereby ameliorating renal pathology. We observed this effect in both AIM-deficient and WT mice, but not in KIM-1-deficient mice. Our findings provide a basis for the development of potentially novel therapies for AKI.

  7. The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin

    PubMed Central

    Carcaboso, Angel M.; Herrero-Martín, David; García-Macías, María del Carmen; Sevillano, Vicky; Alonso, Diego; Pascual-Pasto, Guillem; San-Segundo, Laura; Vila-Ubach, Monica; Rodrigues, Telmo; Fraile, Susana; Teodosio, Cristina; Mayo-Iscar, Agustín; Aracil, Miguel; Galmarini, Carlos María; Tirado, Oscar M.; Mora, Jaume; de Álava, Enrique

    2015-01-01

    Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting. PMID:26056084

  8. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

    PubMed Central

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  9. Sequential application of a cytotoxic nanoparticle and a PI3K inhibitor enhances antitumor efficacy

    PubMed Central

    Pandey, Ambarish; Goldman, Aaron; Sarangi, Sasmit; Sengupta, Poulomi; Phipps, Colin; Kopparam, Jawahar; Oh, Michael; Basu, Sudipta; Kohandel, Mohammad; Sengupta, Shiladitya

    2013-01-01

    Nanomedicines that preferentially deploy cytotoxic agents to tumors, and molecular targeted therapeutics that inhibit specific aberrant oncogenic drivers are emerging as the new paradigm for the management of cancer. While combination therapies are a mainstay of cancer chemotherapy, few studies have addressed the combination of nanomedicines and molecular targeted therapeutics. Furthermore, limited knowledge exists on the impact of sequencing of such therapeutics and nanomedicines on the antitumor outcome. Here we engineered a supramolecular cis-platinum nanoparticle, which induced apoptosis in breast cancer cells but also elicited pro-survival signaling via an epidermal growth factor receptor-phosphatidylinositol 3 kinase (PI3K) pathway. A combination of mathematical modeling and in vitro and in vivo validation using a pharmacological inhibitor of PI3K, PI828, demonstrate that administration of PI828 following treatment with the supramolecular cis-platinum nanoparticle results in enhanced antitumor efficacy in breast cancer as compared with when the sequence is reversed or when the two treatments are administered simultaneously. This study addresses, for the first time, the impact of drug sequencing in the case of a combination of a nanomedicine and a targeted therapeutic. Furthermore, our results indicate that a rational combination of cis-platinum nanoparticles and a PI3K-targeted therapeutic can emerge as a potential therapy for breast cancer. PMID:24121494

  10. A 'garbage can' for ribosomes: how eukaryotes degrade their ribosomes.

    PubMed

    Lafontaine, Denis L J

    2010-05-01

    Ribosome synthesis is a major metabolic activity that involves hundreds of individual reactions, each of which is error-prone. Ribosomal insults occur in cis (alteration in rRNA sequences) and in trans (failure to bind to, or loss of, an assembly factor or ribosomal protein). In addition, specific growth conditions, such as starvation, require that excess ribosomes are turned over efficiently. Recent work indicates that cells evolved multiple strategies to recognize specifically, and target for clearance, ribosomes that are structurally and/or functionally deficient, as well as in excess. This surveillance is active at every step of the ribosome synthesis pathway and on mature ribosomes, involves nearly entirely different mechanisms for the small and large subunits, and requires specialized subcellular organelles. PMID:20097077

  11. The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology

    PubMed Central

    Wandrey, Franziska; Montellese, Christian; Koos, Krisztian; Badertscher, Lukas; Bammert, Lukas; Cook, Atlanta G.; Zemp, Ivo; Horvath, Peter

    2015-01-01

    The interleukin enhancer binding factors ILF2 (NF45) and ILF3 (NF90/NF110) have been implicated in various cellular pathways, such as transcription, microRNA (miRNA) processing, DNA repair, and translation, in mammalian cells. Using tandem affinity purification, we identified human NF45 and NF90 as components of precursors to 60S (pre-60S) ribosomal subunits. NF45 and NF90 are enriched in nucleoli and cosediment with pre-60S ribosomal particles in density gradient analysis. We show that association of the NF45/NF90 heterodimer with pre-60S ribosomal particles requires the double-stranded RNA binding domains of NF90, while depletion of NF45 and NF90 by RNA interference leads to a defect in 60S biogenesis. Nucleoli of cells depleted of NF45 and NF90 have altered morphology and display a characteristic spherical shape. These effects are not due to impaired rRNA transcription or processing of the precursors to 28S rRNA. Consistent with a role of the NF45/NF90 heterodimer in nucleolar steps of 60S subunit biogenesis, downregulation of NF45 and NF90 leads to a p53 response, accompanied by induction of the cyclin-dependent kinase inhibitor p21/CIP1, which can be counteracted by depletion of RPL11. Together, these data indicate that NF45 and NF90 are novel higher-eukaryote-specific factors required for the maturation of 60S ribosomal subunits. PMID:26240280

  12. Combined Treatment with a BACE Inhibitor and Anti-Aβ Antibody Gantenerumab Enhances Amyloid Reduction in APPLondon Mice

    PubMed Central

    Ozmen, Laurence; Caruso, Antonello; Narquizian, Robert; Hilpert, Hans; Jacobsen, Bjoern; Terwel, Dick; Tanghe, An

    2014-01-01

    Therapeutic approaches for prevention or reduction of amyloidosis are currently a main objective in basic and clinical research on Alzheimer‘s disease. Among the agents explored in clinical trials are anti-Aβ peptide antibodies and secretase inhibitors. Most anti-Aβ antibodies are considered to act via inhibition of amyloidosis and enhanced clearance of existing amyloid, although secretase inhibitors reduce the de novo production of Aβ. Limited information is currently available on the efficacy and potential advantages of combinatorial antiamyloid treatment. We performed a chronic study in APPLondon transgenic mice that received treatment with anti-Aβ antibody gantenerumab and BACE inhibitor RO5508887, either as mono- or combination treatment. Treatment aimed to evaluate efficacy on amyloid progression, similar to preexisting amyloidosis as present in Alzheimer's disease patients. Mono-treatments with either compound caused a dose-dependent reduction of total brain Aβ and amyloid burden. Combination treatment with both compounds significantly enhanced the antiamyloid effect. The observed combination effect was most pronounced for lowering of amyloid plaque load and plaque number, which suggests effective inhibition of de novo plaque formation. Moreover, significantly enhanced clearance of pre-existing amyloid plaques was observed when gantenerumab was coadministered with RO5508887. BACE inhibition led to a significant time- and dose-dependent decrease in CSF Aβ, which was not observed for gantenerumab treatment. Our results demonstrate that combining these two antiamyloid agents enhances overall efficacy and suggests that combination treatments may be of clinical relevance. PMID:25164658

  13. Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.

    PubMed

    Bhargava, Ajay; Pelech, Steven; Woodard, Ben; Kerwin, John; Maherali, Nimet

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAF(WT) tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

  14. Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

    PubMed Central

    Bhargava, Ajay; Pelech, Steven; Woodard, Ben; Kerwin, John; Maherali, Nimet

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAFWT tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.09976.001 PMID:26882073

  15. The ATRA-induced differentiation of medulloblastoma cells is enhanced with LOX/COX inhibitors: an analysis of gene expression

    PubMed Central

    2014-01-01

    Background A detailed analysis of the expression of 440 cancer-related genes was performed after the combined treatment of medulloblastoma cells with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). The combinations of retinoids and celecoxib as a COX-2 inhibitor were reported to be effective in some regimens of metronomic therapy of relapsed solid tumors with poor prognosis. Our previous findings on neuroblastoma cells using expression profiling showed that LOX/COX inhibitors have the capability of enhancing the differentiating action of ATRA. Presented study focused on the continuation of our previous work to confirm the possibility of enhancing ATRA-induced cell differentiation in these cell lines via the application of LOX/COX inhibitors. This study provides more detailed information concerning the mechanisms of the enhancement of the ATRA-induced differentiation of medulloblastoma cells. Methods The Daoy and D283 Med medulloblastoma cell lines were chosen for this study. Caffeic acid (an inhibitor of 5-LOX) and celecoxib (an inhibitor on COX-2) were used in combined treatment with ATRA. The expression profiling was performed using Human Cancer Oligo GEArray membranes, and the most promising results were verified using RT-PCR. Results The expression profiling of the selected cancer-related genes clearly confirmed that the differentiating effects of ATRA should be enhanced via its combined administration with caffeic acid or celecoxib. This effect was detected in both cell lines. An increased expression of the genes that encoded the proteins participating in induced differentiation and cytoskeleton remodeling was detected in both cell lines in a concentration-dependent manner. This effect was also observed for the CDKN1A gene encoding the p21 protein, which is an important regulator of the cell cycle, and for the genes encoding proteins that are associated with proteasome activity. Furthermore, our results showed

  16. Ribosomal Database Project II

    DOE Data Explorer

    The Ribosomal Database Project (RDP) provides ribosome related data and services to the scientific community, including online data analysis and aligned and annotated Bacterial small-subunit 16S rRNA sequences. As of March 2008, RDP Release 10 is available and currently (August 2009) contains 1,074,075 aligned 16S rRNA sequences. Data that can be downloaded include zipped GenBank and FASTA alignment files, a histogram (in Excel) of the number of RDP sequences spanning each base position, data in the Functional Gene Pipeline Repository, and various user submitted data. The RDP-II website also provides numerous analysis tools.[From the RDP-II home page at http://rdp.cme.msu.edu/index.jsp

  17. Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

    PubMed Central

    Coelho, Tatiane; Machado, Diana; Couto, Isabel; Maschmann, Raquel; Ramos, Daniela; von Groll, Andrea; Rossetti, Maria L.; Silva, Pedro A.; Viveiros, Miguel

    2015-01-01

    Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates. PMID:25972842

  18. A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

    PubMed Central

    Su, Wen-Chi; Hsu, Shih-Feng; Lee, Yi-Yuan; Jeng, King-Song

    2015-01-01

    ABSTRACT Influenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals. IMPORTANCE Influenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we

  19. The mTOR inhibitor Everolimus synergizes with the PI3K inhibitor GDC0941 to enhance anti-tumor efficacy in uveal melanoma

    PubMed Central

    Amirouchene-Angelozzi, Nabil; Frisch-Dit-Leitz, Estelle; Carita, Guillaume; Dahmani, Ahmed; Raymondie, Chloé; Liot, Géraldine; Gentien, David; Némati, Fariba; Decaudin, Didier

    2016-01-01

    Uveal melanoma (UM) is the most frequent malignant ocular tumor in adults. While the primary tumor is efficiently treated by surgery and/or radiotherapy, about one third of UM patients develop metastases, for which no effective treatment is currently available. The PKC, MAPK and PI3K/AKT/mTOR signaling cascades have been shown to be associated with tumor growth. However, none of the compounds against those pathways results in tumor regression when used as single agents. To identify more effective therapeutic strategies for UM patients, we performed a combination screen using seven targeted agents inhibiting PKC, MEK, AKT, PI3K and mTOR in a panel of ten UM cell lines, representative of the UM disease. We identified a strong synergy between the mTOR inhibitor Everolimus and the PI3K inhibitor GDC0941. This combination resulted in an increase in apoptosis in several UM cell lines compared to monotherapies and enhanced the anti-tumor effect of each single agent in two patient-derived xenografts. Furthermore, we showed that the synergism between the two drugs was associated with the relief by GDC0491 of a reactivation of AKT induced by Everolimus. Altogether, our results highlight a novel and effective combination strategy, which could be beneficial for UM patients. PMID:26988753

  20. Organization of ribosomes and nucleoids in Escherichia coli cells during growth and in quiescence.

    PubMed

    Chai, Qian; Singh, Bhupender; Peisker, Kristin; Metzendorf, Nicole; Ge, Xueliang; Dasgupta, Santanu; Sanyal, Suparna

    2014-04-18

    We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

  1. Enhanced endogenous thrombolysis induced by a specific factor Xa inhibitor, DX-9065a, evaluated in a rat arterial thrombolysis model in vivo.

    PubMed

    Hashimoto, Masaru; Onobayashi, Yuko; Oiwa, Kazuhiro; Giddings, John C; Yamamoto, Junichiro

    2002-04-15

    We have previously established an animal model to investigate mechanisms of arterial thrombolysis in vivo and have demonstrated that endogenous thrombolysis, mediated by thrombin-activatable fibrinolysis inhibitor, is enhanced by administration of specific thrombin inhibitors. The aim of the present study was to evaluate the effects of a synthetic and specific factor Xa inhibitor, DX-9065a, on endogenous fibrinolysis. Mural thrombi were formed in rat mesenteric arterioles by helium-neon laser irradiation in the presence of Evans blue. Thrombolysis was continuously monitored by video microscopy and was quantified using image analysis software. Oral and intravenous administration of DX-9065a enhanced endogenous thrombolysis in vivo. The mechanisms require additional investigation using other experimental systems, but nevertheless, the present results extended our previous findings and further suggested that the enhanced fibrinolysis might be due to depressed activity thrombin-activatable fibrinolysis inhibitor. The synthetic factor Xa inhibitor could provide the basis for a useful thrombolytic agent. PMID:12182917

  2. Fluorescence bimolecular complementation enables facile detection of ribosome assembly defects in Escherichia coli.

    PubMed

    Sharma, Himanshu; Anand, Baskaran

    2016-09-01

    Assembly factors promote the otherwise non-spontaneous maturation of ribosome under physiological conditions inside the cell. Systematic identification and characterization of candidate assembly factors are fraught with bottlenecks due to lack of facile assay system to capture assembly defects. Here, we show that bimolecular fluorescence complementation (BiFC) allows detection of assembly defects that are induced by the loss of assembly factors. The fusion of N and C-terminal fragments of Venus fluorescent protein to the ribosomal proteins uS13 and uL5, respectively, in Escherichia coli facilitated the incorporation of the tagged uS13 and uL5 onto the respective ribosomal subunits. When the ribosomal subunits associated to form the 70S particle, the complementary fragments of Venus were brought into proximity and rendered the Venus fluorescent. Assembly defects that inhibit the subunits association were provoked by either the loss of the known assembly factors such as RsgA and SrmB or the presence of small molecule inhibitors of ribosome maturation such as Lamotrigine and several ribosome-targeting antibiotics and these showed abrogation of the fluorescence complementation. This suggests that BiFC can be employed as a surrogate measure to detect ribosome assembly defects proficiently by circumventing the otherwise cumbersome procedures. BiFC thus offers a facile platform not only for systematic screening to validate potential assembly factors but also to discover novel small molecule inhibitors of ribosome assembly toward mapping the complex assembly landscape of ribosome.

  3. Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors

    PubMed Central

    Du, Yi; Yamaguchi, Hirohito; Wei, Yongkun; Hsu, Jennifer L.; Wang, Hung-Ling; Hsu, Yi-Hsin; Lin, Wan-Chi; Yu, Wen-Hsuan; Leonard, Paul G.; Lee, Gilbert R.; Chen, Mei-Kuang; Nakai, Katsuya; Hsu, Ming-Chuan; Chen, Chun-Te; Sun, Ye; Wu, Yun; Chang, Wei-Chao; Huang, Wen-Chien; Liu, Chien-Liang; Chang, Yuan-Ching; Chen, Chung-Hsuan; Park, Morag; Jones, Philip; Hortobagyi, Gabriel N.; Hung, Mien-Chie

    2016-01-01

    Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials1. One PARP inhibitor, olaparib (Lynparza™, AstraZeneca), was recently approved by the FDA to treat ovarian cancer with BRCA mutations. BRCA1 and BRCA2 play essential roles in repairing DNA double strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition2,3. Here we show that receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907. Phosphorylation of PARP1 Tyr907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. Combining c-Met and PARP1 inhibitors synergized to suppress growth of breast cancer cells in vitro and xenograft tumor models. Similar synergistic effects were observed in a lung cancer xenograft tumor model. These results suggest that PARP1 pTyr907 abundance may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients bearing tumors with high c-Met expression who do not respond to PARP inhibition alone. PMID:26779812

  4. Endoplasmic reticulum ribosome-binding protein 1 (RRBP1) overexpression is frequently found in lung cancer patients and alleviates intracellular stress-induced apoptosis through the enhancement of GRP78.

    PubMed

    Tsai, H-Y; Yang, Y-F; Wu, A T; Yang, C-J; Liu, Y-P; Jan, Y-H; Lee, C-H; Hsiao, Y-W; Yeh, C-T; Shen, C-N; Lu, P-J; Huang, M-S; Hsiao, M

    2013-10-10

    Lung cancer is the leading cause of cancer deaths and is the most occurring malignancy worldwide. Unraveling the molecular mechanisms involved in lung tumorigenesis will greatly improve therapy. During early tumorigenesis, rapid proliferating tumor cells require increased activity of endoplasmic reticulum (ER) for protein synthesis, folding and secretion, thereby are subjected to ER stress. Ribosome-binding protein 1 (RRBP1) was originally identified as a ribosome-binding protein located on the rough ER and associated with unfolding protein response (UPR). In this report, we investigated the role of RRBP1 in lung cancer. RRBP1 was highly expressed in lung cancer tissue, as compared with adjacent normal tissues as assessed by immunohistochemistry (IHC) using lung cancer tissue array (n=87). Knockdown of RRBP1 by short-hairpin RNAs caused ER stress and significantly reduced cell viability and tumorigenicity. This effect was associated with a significant reduction in the expression of glucose-regulated protein 78 (GRP78). UPR regulator GRP78, an anti-apoptotic protein that is widely upregulated in cancer, has a critical role in chemotherapy resistance in some cancers. According to our results, cells with a higher level of RRBP1 were more resistant to ER stress. Ectopic expression of RRBP1 alleviated apoptosis that was induced by the ER-stress agent tunicamycin, 2-deoxy-D-glucose (2DG) or doxorubicin via enhancing GRP78 protein expression. A strong correlation was observed between the expression of RRBP1 and GRP78 in tumor biopsies using the database GSE10072. Our results also indicated that RRBP1 may involve in the regulation of mRNA stability of UPR components including ATF6 and GRP78. Taken together, RRBP1 could alleviate ER stress and help cancer cell survive. RRBP1 is critical for tumor cell survival, which may make it a useful target in lung cancer treatment and a candidate for the development of new targeted therapeutics.

  5. Closing escape routes: inhibition of IL-8 signaling enhances the anti-tumor efficacy of PI3K inhibitors.

    PubMed

    Juvekar, Ashish; Wulf, Gerburg M

    2013-04-08

    The phosphoinositide 3-kinase (PI3K) pathway serves as a relay where signals that emanate from the cell membrane are received and converted into intracellular signals that promote proliferation and survival. Inhibitors of PI3K hold promise for the treatment of breast cancer because activation of this pathway is highly prevalent. However, as is increasingly observed with inhibitors of cell signaling, there appear to be mechanisms of primary and secondary resistance. Britschgi and colleagues report that compensatory activation of the IL-8 signaling axis is a mechanism of primary resistance to PI3K inhibitors in some triple-negative breast cancers. In a set of experiments that carefully emulate the clinical scenario in a mouse model, they show that simultaneous inhibition of Janus kinase 2 enhances the efficacy of PI3K/mammalian target of rapamycin inhibition. Their paper lends further support to the concept that successful design of treatments with signal transduction inhibitors must anticipate potential escape routes - and include agents to simultaneously block them.

  6. HDAC inhibitors enhance the lethality of low dose salinomycin in parental and stem-like GBM cells.

    PubMed

    Booth, Laurence; Roberts, Jane L; Conley, Adam; Cruickshanks, Nichola; Ridder, Thomas; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2014-03-01

    The present studies determined whether the antibiotic salinomycin interacted with HDAC inhibitors to kill primary human GBM cells. Regardless of PTEN, ERBB1, or p53 mutational status salinomycin interacted with HDAC inhibitors in a synergistic fashion to kill GBM cells. Inhibition of CD95/Caspase 8 or of CD95/RIP-1/AIF signaling suppressed killing by the drug combination. Salinomycin increased the levels of autophagosomes that correlated with increased p62 and LC3II levels; valproate co-treatment correlated with reduced LC3II and p62 expression, and increased caspase 3 cleavage. Molecular inhibition of autophagosome formation was protective against drug exposure. The drug combination enhanced eIF2α phosphorylation and decreased expression of MCL-1 and phosphorylation of mTOR and p70 S6K. Activation of p70 S6K or mTOR promoted cell survival in the face of combined drug exposure. Overexpression of BCL-XL or c-FLIP-s was protective. Collectively our data demonstrate that the lethality of low nanomolar concentrations of salinomycin are enhanced by HDAC inhibitors in GBM cells and that increased death receptor signaling together with reduced mitochondrial function are causal in the combinatorial drug necro-apoptotic killing effect.

  7. Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response

    PubMed Central

    Liu, Ming-Kun; Lin, Jhe-Jhih; Chen, Chung-Yung; Kuo, Szu-Cheng; Wang, Yu-Ming; Chan, Hong-Lin; Wu, Tzong Yuan

    2016-01-01

    BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy. PMID:27314325

  8. [Inhibitors of neuronal and inducible NO-syntases enhance the effect of short-term adaptation to hypoxia in rats of Krushinsky-Molodkina strain].

    PubMed

    Krushinskiĭ, A L; Kuzenkov, V S; D'iakonova, V E; Reutov, V P

    2015-01-01

    We found that selective inhibitors of neuronal and inducible NOS (7-nitroindazole and aminoguanidine) significantly enhance the protective effect of short-term adaptation to hypoxia on the development of stress lesions in rats Krushinsky-Molodkina.

  9. Blockade of the ERK pathway enhances the therapeutic efficacy of the histone deacetylase inhibitor MS-275 in human tumor xenograft models

    SciTech Connect

    Sakamoto, Toshiaki; Ozaki, Kei-ichi; Fujio, Kohsuke; Kajikawa, Shu-hei; Uesato, Shin-ichi; Watanabe, Kazushi; Tanimura, Susumu; Koji, Takehiko; Kohno, Michiaki

    2013-04-19

    Highlights: •Blockade of the ERK pathway enhances the anticancer efficacy of HDAC inhibitors. •MEK inhibitors sensitize human tumor xenografts to HDAC inhibitor cytotoxicity. •Such the enhanced efficacy is achieved by a transient blockade of the ERK pathway. •This drug combination provides a promising therapeutic strategy for cancer patients. -- Abstract: The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.

  10. Isolation of ribosomes by chromatography.

    PubMed

    Maguire, Bruce A

    2015-04-01

    Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.

  11. Exploring Ribosome Positioning on Translating Transcripts with Ribosome Profiling.

    PubMed

    Spealman, Pieter; Wang, Hao; May, Gemma; Kingsford, Carl; McManus, C Joel

    2016-01-01

    Recent technological advances (e.g., microarrays and massively parallel sequencing) have facilitated genome-wide measurement of many aspects of gene regulation. Ribosome profiling is a high-throughput sequencing method used to measure gene expression at the level of translation. This is accomplished by quantifying both the number of translating ribosomes and their locations on mRNA transcripts. The inventors of this approach have published several methods papers detailing its implementation and addressing the basics of ribosome profiling data analysis. Here we describe our lab's procedure, which differs in some respects from those published previously. In addition, we describe a data analysis pipeline, Ribomap, for ribosome profiling data. Ribomap allocates sequence reads to alternative mRNA isoforms, normalizes sequencing bias along transcripts using RNA-seq data, and outputs count vectors of per-codon ribosome occupancy for each transcript.

  12. Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production.

    PubMed

    Ye, Junli; Jiang, Zhongxin; Chen, Xuehong; Liu, Mengyang; Li, Jing; Liu, Na

    2016-01-15

    Reactive oxygen species (ROS) are believed to be mediators of excessive microglial activation, yet the resources and mechanism are not fully understood. Here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of electron transport chain (ETC), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, and NaN3 to induce mitochondrial ROS production and we observed the role of mitochondrial ROS in microglial activation. Our results showed that ETC inhibitors resulted in significant changes in cell viability, microglial morphology, cell cycle arrest and mitochondrial ROS production in a dose-dependent manner in both primary cultural microglia and BV-2 cell lines. Moreover, ETC inhibitors, especially rotenone and antimycin A stimulated secretion of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) by microglia with marked activation of mitogen-activated proteinkinases (MAPKs) and nuclear factor κB (NF-κB), which could be blocked by specific inhibitors of MAPK and NF-κB and mitochondrial antioxidants, Mito-TEMPO. Taken together, our results demonstrated that inhibition of mitochondrial respiratory chain in microglia led to production of mitochondrial ROS and therefore may activate MAPK/NF-кB dependent inflammatory cytokines release in microglia, which indicated that mitochondrial-derived ROS were contributed to microglial activation.

  13. The glycolytic inhibitor 2-deoxy-D-glucose enhances the efficacy of etoposide in ehrlich ascites tumor-bearing mice.

    PubMed

    Gupta, Seema; Mathur, Rohit; Dwarakanath, B S

    2005-01-01

    Earlier studies have shown that 2-deoxy-D-glucose (2-DG), a glucose analogue and inhibitor of glycolytic ATP production significantly enhances the cytotoxic effects of anticancer agents like topoisomerase inhibitors (etoposide and camptothecin) and a radiomimetic drug (bleomycin) in established human tumor cell lines. Therefore, combination of 2-DG and DNA damage causing cytotoxic agents could be very useful in enhancing local tumor control. The purpose of the present studies was to investigate the therapeutic effects of etoposide and 2-DG in Ehrlich ascites tumor (EAT) bearing mice, grown as solid tumor as well as in the ascites form. Cell growth, cell cycle perturbations (flow cytometry), cytogenetic damage (micronuclei assay) and apoptosis (DNA content, morphological changes) were studied as parameters of cellular response, while delay in tumor growth and cure rate (tumor free survival) were evaluated as parameters of systemic response. Body weight and general condition as well as the damage to bone marrow and spleen was monitored to evaluate normal tissue toxicity. Intraperitoneal administration of etoposide (30 mg/Kg b. wt.) resulted in significant tumor growth delay and cure (approximately 11%) only in subcutaneous tumors leading to local tumor control. When etoposide was combined with 2-DG (2 g/Kg b. wt.; i.v./i.p.; 4 h after etoposide injection), these effects were further enhanced resulting in a cure rate of approximately 22% in case of subcutaneous tumors and 20% in ascites bearing mice. Analysis of cells obtained from ascitic fluid as well as solid tumors during follow up clearly showed that etoposide induced cell death was mainly due to apoptosis, which was enhanced further by 2-DG. Although, there was a significant level of toxicity revealed by reduced animal survival, decrease in body weight and damage to sensitive organ status like spleen and bone marrow at 60 mg/Kg b. wt. of etoposide, it was not significant at 30 mg/Kg b.wt. 2-DG, however, did not

  14. PI3K Inhibitor Combined With Chemotherapy Can Enhance the Apoptosis of Neuroblastoma Cells In Vitro and In Vivo.

    PubMed

    Geng, Xianjie; Xie, Lingling; Xing, Hongshun

    2016-10-01

    Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is a novel poor prognostic indicator of neuroblastoma (NB), and the positive effects of chemotherapy on NB have been confirmed. In this study, we investigated the effect of small molecule PI3K inhibitor PI103 on chemosensitivity. The PI3K inhibitor cooperates with doxorubicin to synergistically induce apoptosis and to reduce tumor growth of NB in in vitro and in vivo models. Human NB cells, SH-SY5Y and SK-N-BE(2), were treated with PI103 combined doxorubicin-enhanced Bid cleavage, activated Bax, and caspase 3. Activation of caspase 3 was also observed in xenografts of NB in nude mice upon combination of doxorubicin with the specific PI3K inhibitor PI103. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Both PI103 and doxorubicin inhibited growth of NB in vitro and PI103 induced a G1 arrest of NB cells. PI103 combined doxorubicin significantly inhibits the growth of established NB tumors, induced apoptosis of tumor cells, and improved the survival of mice in vivo Taken together, our findings suggest that PI3K inhibition seems to be a promising option to sensitize tumor cells for chemotherapy in NB, which may be effective in the treatment of NBs.

  15. PI3K Inhibitor Combined With Chemotherapy Can Enhance the Apoptosis of Neuroblastoma Cells In Vitro and In Vivo.

    PubMed

    Geng, Xianjie; Xie, Lingling; Xing, Hongshun

    2016-10-01

    Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is a novel poor prognostic indicator of neuroblastoma (NB), and the positive effects of chemotherapy on NB have been confirmed. In this study, we investigated the effect of small molecule PI3K inhibitor PI103 on chemosensitivity. The PI3K inhibitor cooperates with doxorubicin to synergistically induce apoptosis and to reduce tumor growth of NB in in vitro and in vivo models. Human NB cells, SH-SY5Y and SK-N-BE(2), were treated with PI103 combined doxorubicin-enhanced Bid cleavage, activated Bax, and caspase 3. Activation of caspase 3 was also observed in xenografts of NB in nude mice upon combination of doxorubicin with the specific PI3K inhibitor PI103. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Both PI103 and doxorubicin inhibited growth of NB in vitro and PI103 induced a G1 arrest of NB cells. PI103 combined doxorubicin significantly inhibits the growth of established NB tumors, induced apoptosis of tumor cells, and improved the survival of mice in vivo Taken together, our findings suggest that PI3K inhibition seems to be a promising option to sensitize tumor cells for chemotherapy in NB, which may be effective in the treatment of NBs. PMID:26224681

  16. Sites of synthesis of chloroplast ribosomal proteins in Chlamydomonas

    PubMed Central

    1983-01-01

    Cells of Chlamydomonas reinhardtii were pulse-labeled in vivo in the presence of inhibitors of cytoplasmic (anisomycin) or chloroplast (lincomycin) protein synthesis to ascertain the sites of synthesis of chloroplast ribosomal proteins. Fluorographs of the labeled proteins, resolved on two-dimensional (2-D) charge/SDS and one-dimensional (1-D) SDS-urea gradient gels, demonstrated that five to six of the large subunit proteins are products of chloroplast protein synthesis while 26 to 27 of the large subunit proteins are synthesized on cytoplasmic ribosomes. Similarly, 14 of 31 small subunit proteins are products of chloroplast protein synthesis, while the remainder are synthesized in the cytoplasm. The 20 ribosomal proteins shown to be made in the chloroplast of Chlamydomonas more than double the number of proteins known to be synthesized in the chloroplast of this alga. PMID:6841455

  17. Ribosomal Initiation Complex Assembly within the Wild-Strain of Coxsackievirus B3 and Live-Attenuated Sabin3-like IRESes during the Initiation of Translation

    PubMed Central

    Souii, Amira; M’hadheb-Gharbi, Manel Ben; Sargueil, Bruno; Brossard, Audrey; Chamond, Nathalie; Aouni, Mahjoub; Gharbi, Jawhar

    2013-01-01

    Coxsackievirus B3 (CVB3) is an enterovirus of the family of Picornaviridae. The Group B coxsackieviruses include six serotypes (B1 to B6) that cause a variety of human diseases, including myocarditis, meningitis, and diabetes. Among the group B, the B3 strain is mostly studied for its cardiovirulence and its ability to cause acute and persistent infections. Translation initiation of CVB3 RNA has been shown to be mediated by a highly ordered structure of the 5′-untranslated region (5′UTR), which harbors an internal ribosome entry site (IRES). Translation initiation is a complex process in which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of the mRNA. We have previously addressed the question of whether the attenuating mutations of domain V of the poliovirus IRES were specific for a given genomic context or whether they could be transposed and extrapolated to a genomic related virus, i.e., CVB3 wild-type strain. In this context, we have described that Sabin3-like mutation (U473→C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we analyzed the efficiency of formation of ribosomal initiation complexes 48S and 80S through 10%–30% and 10%–50% sucrose gradients using rabbit reticulocyte lysates (RRLs) and stage-specific translation inhibitors: 5′-Guanylyl-imidodiphosphate (GMP-PNP) and Cycloheximide (CHX), respectively. We demonstrated that the interaction of 48S and 80S ribosomal complexes within the mutant CVB3 RNA was abolished compared with the wild-type RNA by ribosome assembly analysis. Taken together, it is possible that the mutant RNA was unable to interact with some trans-acting factors critical for enhanced IRES function. PMID:23439549

  18. [ENHANCEMENT OF AGROBACTERIAL TRANSFORMATION OF PLANTS USING PROTEIN KINASE INHIBITORS TRIFLUOPERAZINE AND GENISTEIN].

    PubMed

    Yemets, A I; Fedorchuk, V V; Blume, Ya B

    2016-01-01

    The effect of different concentrations of protein tyrosine kinase inhibitor, genistein and serine/threonine protein kinase inhibitor, trifluoperazine, on the frequency of Agrobacterium-mediated transformation of leaf explants of N. tabacum was investigated. The influence of different concentrations of trifluoperazine in the range from 10 to 300 μM was investigated. It was found that 10 μM trifluoperazine provoked the increase of the frequency of agrobacterial transformation of tobacco leaf disks on 25%. In parallel, the influence of different concentrations of genistein in the range from 10 to 100 μM was investigated. It was found 100 μM genistein provoked the increase of the frequency of agrobacterial transformation of tobacco leaf disks on 12%.

  19. Enhanced bioavailability of subcutaneously injected insulin by pretreatment with ointment containing protease inhibitors

    SciTech Connect

    Takeyama, M.; Ishida, T.; Kokubu, N.; Komada, F.; Iwakawa, S.; Okumura, K.; Hori, R. )

    1991-01-01

    The present study was undertaken to develop an ointment preparation containing a protease inhibitor for stabilizing subcutaneously injected insulin. The ointment containing the protease inhibitor, gabexate mesilate or nafamostat mesilate, was applied to the skin around the insulin injection site. Three results were obtained. First, gabexate and nafamostat inhibited insulin degradation in subcutaneous tissue homogenates in vitro. Second, after application of gabexate or nafamostat ointment, an appreciable amount of gabexate or nafamostat appeared in the subcutaneous tissue of rats or hairless mice and their concentrations were comparable to those seen in the in vitro experiment. Third, insulin degradation at the subcutaneous injection site in the rat was depressed after pretreatment with gabexate or nafamostat ointment. Pretreatment with gabexate or nafamostat ointment increased the plasma immunoreactive insulin (IRI) levels and the hypoglycermic effect of insulin in healthy volunteers. These results indicate that gabexate or nafamostat ointments stabilize subcutaneously injected insulin.

  20. [ENHANCEMENT OF AGROBACTERIAL TRANSFORMATION OF PLANTS USING PROTEIN KINASE INHIBITORS TRIFLUOPERAZINE AND GENISTEIN].

    PubMed

    Yemets, A I; Fedorchuk, V V; Blume, Ya B

    2016-01-01

    The effect of different concentrations of protein tyrosine kinase inhibitor, genistein and serine/threonine protein kinase inhibitor, trifluoperazine, on the frequency of Agrobacterium-mediated transformation of leaf explants of N. tabacum was investigated. The influence of different concentrations of trifluoperazine in the range from 10 to 300 μM was investigated. It was found that 10 μM trifluoperazine provoked the increase of the frequency of agrobacterial transformation of tobacco leaf disks on 25%. In parallel, the influence of different concentrations of genistein in the range from 10 to 100 μM was investigated. It was found 100 μM genistein provoked the increase of the frequency of agrobacterial transformation of tobacco leaf disks on 12%. PMID:27266180

  1. The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing

    PubMed Central

    Tesori, Valentina; Piscaglia, Anna Chiara; Samengo, Daniela; Barba, Marta; Bernardini, Camilla; Scatena, Roberto; Pontoglio, Alessandro; Castellini, Laura; Spelbrink, Johannes N.; Maulucci, Giuseppe; Puglisi, Maria Ausiliatrice; Pani, Giovambattista; Gasbarrini, Antonio

    2015-01-01

    Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5–5 μM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies. PMID:25779766

  2. The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

    PubMed

    Tesori, Valentina; Piscaglia, Anna Chiara; Samengo, Daniela; Barba, Marta; Bernardini, Camilla; Scatena, Roberto; Pontoglio, Alessandro; Castellini, Laura; Spelbrink, Johannes N; Maulucci, Giuseppe; Puglisi, Maria Ausiliatrice; Pani, Giovambattista; Gasbarrini, Antonio

    2015-03-17

    Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5-5 μM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.

  3. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    PubMed

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway.

  4. MiR-21 enhances melanoma invasiveness via inhibition of tissue inhibitor of metalloproteinases 3 expression: in vivo effects of MiR-21 inhibitor.

    PubMed

    Martin del Campo, Sara E; Latchana, Nicholas; Levine, Kala M; Grignol, Valerie P; Fairchild, Ene T; Jaime-Ramirez, Alena Cristina; Dao, Thao-Vi; Karpa, Volodymyr I; Carson, Mary; Ganju, Akaansha; Chan, Anthony N; Carson, William E

    2015-01-01

    Metastatic melanoma is the most aggressive form of this cancer. It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma. Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity. It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma. It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21. Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls. This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39. The proliferation and migration of miR-21 over-expressing cell lines was not affected. Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression. Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice. Intra-tumoral injections of anti-miR-21 produced similar effects. This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.

  5. RT-21Mre11-Rad50-Nbs1 COMPLEX INHIBITOR MIRIN ENHANCES RADIOSENSITIVITY IN HUMAN GLIOBLASTOMA CELLS

    PubMed Central

    Mishima, Kazuhiko; Mishima-Kaneko, Masayo; Saya, Hideyuki; Ishimaru, Naozumi; Yamada, Kouichi; Fukada, Junichi; Nishikawa, Ryo; Kawata, Tetsuya

    2014-01-01

    PURPOSE: Radiation therapy plays a central part in the treatment of glioblastoma, however, it is not curative due to the high tumor radioresistance. Therefore, increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma. The Mre11, Rad 50 and Nbs1 proteins form a complex (MRN) that has a critical role in DNA damage detection and signaling. Because defects in MRN enhance radiosensitivity, it has been proposed that small molecule inhibitors targeted to these proteins might be used as radiosensitizers. Here, we investigated the effects of the MRN complex inhibitor, Mirin, on radiation response of human glioma cells. MATERIALS AND METHODS: Glioma cell lines (U251, LN229 and LN428) were irradiated with and without Mirin and then clonogenicity, apoptosis, and cell cycle change were examined. Western blot analysis was performed to determine the relative potency of Mirin to inhibit the radioresistance, through the signaling activity of AKT. We also examined the levels of H2AX phosphorylation (γH2AX), which is a marker of DNA double-strand breaks (DSBs) using Western blot. RESULTS: Glioblastoma cells pretreated with Mirin demonstrated an enhanced sensitivity to radiation. FACS analysis revealed that Mirin and radiation caused the glioma cells to accumulate in the G2/M-phase of the cell cycle and the combination of these two treatments further increased the G2/M fraction of the glioma cells. Mirin significantly enhanced radiation-induced apoptotic cell death. Also, Mirin blocked the basal and increase of radiation-induced AKT phosphorylation. We observed that the combination of Mirin and radiation increased persistence of γH2AX at 24 h suggesting the inhibition of DNA DSBs repair. CONCLUSION: These results indicate that Mirin can effectively enhance glioma cell radiosensitivity. It suggests that Mirin is a potent radiosensitizer for treating glioma cells.

  6. Targeting tumor-associated immune suppression with selective protein kinase A type I (PKAI) inhibitors may enhance cancer immunotherapy.

    PubMed

    Hussain, Muzammal; Shah, Zahir; Abbas, Nasir; Javeed, Aqeel; Mukhtar, Muhammad Mahmood; Zhang, Jiancun

    2016-01-01

    Despite the tremendous progress in last few years, the cancer immunotherapy has not yet improved disease-free because of the tumor-associated immune suppression being a major barrier. Novel trends to enhance cancer immunotherapy aims at harnessing the therapeutic manipulation of signaling pathways mediating the tumor-associated immune suppression, with the general aims of: (a) reversing the tumor immune suppression; (b) enhancing the innate and adaptive components of anti-tumor immunosurveillance, and (c) protecting immune cells from the suppressive effects of T regulatory cells (Tregs) and the tumor-derived immunoinhibitory mediators. A particular striking example in this context is the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A type I (PKAI) pathway. Oncogenic cAMP/PKAI signaling has long been implicated in the initiation and progression of several human cancers. Emerging data indicate that cAMP/PKAI signaling also contributes to tumor- and Tregs-derived suppression of innate and adaptive arms of anti-tumor immunosurveillance. Therapeutically, selective PKAI inhibitors have been developed which have shown promising anti-cancer activity in pre-clinical and clinical settings. Rp-8-Br-cAMPS is a selective PKAI antagonist that is widely used as a biochemical tool in signal transduction research. Collateral data indicate that Rp-8-Br-cAMPS has shown immune-rescuing potential in terms of enhancing the innate and adaptive anti-tumor immunity, as well as protecting adaptive T cells from the suppressive effects of Tregs. Therefore, this proposal specifically implicates that combining selective PKAI antagonists/inhibitors with cancer immunotherapy may have multifaceted benefits, such as rescuing the endogenous anti-tumor immunity, enhancing the efficacy of cancer immunotherapy, and direct anti-cancer effects.

  7. Chemosensitizing effect of podophyllotoxin acetate on topoisomerase inhibitors leads to synergistic enhancement of lung cancer cell apoptosis

    PubMed Central

    HONG, WAN GI; CHO, JEONG HYUN; HWANG, SANG-GU; LEE, EUNAH; LEE, JAESEOK; KIM, JONG-IL; UM, HONG-DUCK; PARK, JONG KUK

    2016-01-01

    Podophyllotoxin acetate (PA) acts as a radiosensitizer against non-small cell lung cancer (NSCLC) in vitro and in vivo. In this study, we examined its potential role as a chemosensitizer in conjunction with the topoisomerase inhibitors etoposide (Eto) and camptothecin (Cpt). The effects of combinations of PA and Eto/Cpt were examined with CompuSyn software in two NSCLC cell lines, A549 and NCI-H1299. Combination index (CI) values indicated synergistic effects of PA and the topoisomerase inhibitors. The intracellular mechanism underlying synergism was further determined using propidium iodide uptake, immunoblotting and electrophoretic mobility shift assay (EMSA). Combination of PA with Eto/Cpt promoted disruption of the dynamics of actin filaments, leading to subsequent enhancement of apoptotic cell death via induction of caspase-3, -8, and -9, accompanied by increased phosphorylation of p38. Conversely, suppression of p38 phosphorylation blocked the apoptotic effect of the drug combinations. Notably, CREB-1, a transcription factor, was constitutively activated in both cell types, and synergistically inhibited upon combination treatment. Our results collectively indicate that PA functions as a chemosensitizer by enhancing apoptosis through activation of the p38/caspase axis and suppression of CREB-1. PMID:27035096

  8. Targeting TORC1/2 enhances sensitivity to EGFR inhibitors in head and neck cancer preclinical models.

    PubMed

    Cassell, Andre; Freilino, Maria L; Lee, Jessica; Barr, Sharon; Wang, Lin; Panahandeh, Mary C; Thomas, Sufi M; Grandis, Jennifer R

    2012-11-01

    Head and neck squamous cell carcinoma (HNSCC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) where treatments targeting EGFR have met with limited clinical success. Elucidation of the key downstream-pathways that remain activated in the setting of EGFR blockade may reveal new therapeutic targets. The present study was undertaken to test the hypothesis that inhibition of the mammalian target of rapamycin (mTOR) complex would enhance the effects of EGFR blockade in HNSCC preclinical models. Treatment of HNSCC cell lines with the newly developed TORC1/TORC2 inhibitor OSI-027/ASP4876 resulted in dose-dependent inhibition of proliferation with abrogation of phosphorylation of known downstream targets including phospho-AKT (Ser473), phospho-4E-BP1, phospho-p70s6K, and phospho-PRAS40. Furthermore, combined treatment with OSI-027 and erlotinib resulted in enhanced biochemical effects and synergistic growth inhibition in vitro. Treatment of mice bearing HNSCC xenografts with a combination of the Food and Drug Administration (FDA)-approved EGFR inhibitor cetuximab and OSI-027 demonstrated a significant reduction of tumor volumes compared with either treatment alone. These findings suggest that TORC1/TORC2 inhibition in conjunction with EGFR blockade represents a plausible therapeutic strategy for HNSCC. PMID:23226094

  9. Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells.

    PubMed

    Hibino, S; Saito, Y; Muramatsu, T; Otani, A; Kasai, Y; Kimura, M; Saito, H

    2014-01-01

    Enhancer of zeste homolog 2 (EZH2) enhances tumorigenesis and is commonly overexpressed in several types of cancer. To investigate the anticancer effects of EZH2 inhibitors, microRNA (miRNA) expression profiles were examined in gastric and liver cancer cells treated with suberoylanilide hydroxamic acid (SAHA) and 3-deazaneplanocin A (DZNep). We confirmed that SAHA and DZNep suppressed EZH2 expression in AGS and HepG2 cells and inhibited their proliferation. The results of microarray analyses demonstrated that miR-1246 was commonly upregulated in cancer cells by treatment with SAHA and DZNep. MiR-302a and miR-4448 were markedly upregulated by treatment with SAHA and DZNep, respectively. DYRK1A, CDK2, BMI-1 and Girdin, which are targets of miR-1246, miR-302a and miR-4448, were suppressed by treatment with SAHA and DZNep, leading to apoptosis, cell cycle arrest and reduced migration of AGS and HepG2 cells. ChIP assay revealed that SAHA and DZNep inhibited the binding of EZH2 to the promoter regions of miR-1246, miR-302a and miR-4448. These findings suggest that EZH2 inhibitors such as SAHA and DZNep exert multiple anticancer effects through activation of tumor-suppressor miRNAs. PMID:24861464

  10. Discovery of a small molecule that inhibits bacterial ribosome biogenesis

    PubMed Central

    Stokes, Jonathan M; Davis, Joseph H; Mangat, Chand S; Williamson, James R; Brown, Eric D

    2014-01-01

    While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15°C. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly. DOI: http://dx.doi.org/10.7554/eLife.03574.001 PMID:25233066

  11. A functional interaction between ribosomal proteins S7 and S11 within the bacterial ribosome.

    PubMed

    Robert, Francis; Brakier-Gingras, Léa

    2003-11-01

    In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.

  12. Polynucleotide kinase as a potential target for enhancing cytotoxicity by ionizing radiation and topoisomerase I inhibitors

    PubMed Central

    Bernstein, N. K.; Karimi-Busheri, F.; Rasouli-Nia, A.; Mani, R.; Dianov, G.; Glover, J. N. M.; Weinfeld, M.

    2010-01-01

    The cytotoxicity of many antineoplastic agents is due to their capacity to damage DNA and there is evidence indicating that DNA repair contributes to the cellular resistance to such agents. DNA strand breaks constitute a significant proportion of the lesions generated by a broad range of genotoxic agents, either directly, or during the course of DNA repair. Strand breaks that are caused by many agents including ionizing radiation, topoisomerase I inhibitors, and DNA repair glycosylases such as NEIL1 and NEIL2, often contain 5’-hydroxyl and/or 3’-phosphate termini. These ends must be converted to 5’-phosphate and 3’-hydroxyl termini in order to allow DNA polymerases and ligases to catalyze repair synthesis and strand rejoining. A key enzyme involved in this end-processing is polynucleotide kinase (PNK), which possesses two enzyme activities, a DNA 5’-kinase activity and a 3’-phosphatase activity. PNK participates in the single-strand break repair pathway and the non-homologous end joining pathway for double-strand break repair. RNAi-mediated down-regulation of PNK renders cells more sensitive to ionizing radiation and camptothecin, a topoisomerase I inhibitor. Structural analysis of PNK revealed the protein is composed of three domains, the kinase domain at the C-terminus, the phosphatase domain in the centre and a forkhead associated (FHA) domain at the N-terminus. The FHA domain plays a critical role in the binding of PNK to other DNA repair proteins. Thus each PNK domain may be a suitable target for small molecule inhibition to effectively reduce resistance to ionizing radiation and topoisomerase I inhibitors. PMID:18473721

  13. Immune checkpoint inhibitors enhance cytotoxicity of cytokine-induced killer cells against human myeloid leukaemic blasts.

    PubMed

    Poh, Su Li; Linn, Yeh Ching

    2016-05-01

    We studied whether blockade of inhibitory receptors on cytokine-induced killer (CIK) cells by immune checkpoint inhibitors could increase its anti-tumour potency against haematological malignancies. CIK cultures were generated from seven normal donors and nine patients with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) or multiple myeloma (MM). The inhibitory receptors B and T lymphocyte attenuator, CD200 receptor, lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and mucin-domain-containing-3 (TIM-3) were present at variable percentages in most CIK cultures, while cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1) and killer cell immunoglobulin-like receptors (KIR2DL1/2/3) were expressed at low level in most cultures. Without blockade, myeloid leukaemia cells were susceptible to autologous and allogeneic CIK-mediated cytotoxicity. Blockade of KIR, LAG-3, PD-1 and TIM-3 but not CTLA-4 resulted in remarkable increase in killing against these targets, even in those with poor baseline cytotoxicity. ALL and MM targets were resistant to CIK-mediated cytotoxicity, and blockade of receptors did not increase cytotoxicity to a meaningful extent. Combination of inhibitors against two receptors did not further increase cytotoxicity. Interestingly, potentiation of CIK killing by blocking antibodies was not predicted by expression of receptors on CIK and their respective ligands on the targets. Compared to un-activated T and NK cells, blockade potentiated the cytotoxicity of CIK cells to a greater degree and at a lower E:T ratio, but without significant increase in cytotoxicity against normal white cell. Our findings provide the basis for clinical trial combining autologous CIK cells with checkpoint inhibitors for patients with AML.

  14. Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution

    SciTech Connect

    Derewenda, Urszula; Artamonov, Mykhaylo; Szukalska, Gabriela; Utepbergenov, Darkhan; Olekhnovich, Natalya; Parikh, Hardik I.; Kellogg, Glen E.; Somlyo, Avril V.; Derewenda, Zygmunt S.

    2013-02-01

    The crystal structure of quercitrin, a naturally occurring flavonol glycoside, has been determined in a complex with the N-terminal kinase domain of murine RSK2. The structure revealed that quercitrin inhibits the RSK2 kinase in the same fashion as another known inhibitor, SL0101. Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45–346) of mouse RSK2, or RSK2{sup NTKD}, has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3′′,4′′-di-O-acetyl-α-l-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-l-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2{sup NTKD} with a dissociation constant (K{sub d}) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K{sub d} for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca{sup 2+}.

  15. MuLV IN Mutants Responsive to HDAC Inhibitors Enhance Transcription from Unintegrated Retroviral DNA

    PubMed Central

    Schneider, William M.; Wu, Dai-tze; Amin, Vaibhav; Aiyer, Sriram; Roth, Monica J.

    2012-01-01

    For Moloney murine leukemia virus (M-MuLV), sustained viral infections require expression from an integrated provirus. For many applications, non-integrating retroviral vectors have been utilized to avoid the unwanted effects of integration, however, the level of expression from unintegrated DNA is significantly less than that of integrated provirus. We find that unintegrated DNA expression can be increased in the presence of HDAC inhibitors, such as TSA, when applied in combination with integrase (IN) mutations. These mutants include an active site mutation as well as catalytically active INs bearing mutations of K376 in the MuLV C-terminal domain of IN. MuLV IN K376 is homologous to K266 in HIV-1 IN, a known substrate for acetylation. The MuLV IN protein is acetylated by p300 in vitro, however, the effect of HDAC inhibitors on gene expression from unintegrated DNA is not dependent on the acetylation state of MuLV IN K376. PMID:22365328

  16. Targeting radioresistant breast cancer cells by single agent CHK1 inhibitor via enhancing replication stress

    PubMed Central

    Du, Zhanwen; Gao, Jinnan; Yang, Shuming; Gorityala, Shashank; Xiong, Xiahui; Deng, Ou; Ma, Zhefu; Yan, Chunhong; Susana, Gonzalo; Xu, Yan; Zhang, Junran

    2016-01-01

    Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher levels of RS in RBCC, compared to the parental cells. The mechanisms by which CHK1 inhibition led to specific increase of RS in RBCC were related to the interruptions in the replication fork dynamics and the homologous recombination (HR). In summary, RBCC activate oncogenic pathways and thus depend upon mechanisms controlled by CHK1 signaling to maintain RS under control for survival. Our study provided the first example where upregulating RS by CHK1 inhibitor contributes to the specific killing of RBCC, and highlight the importance of the CHK1 as a potential target for treatment of radioresistant cancer cells. PMID:27167194

  17. The Akt inhibitor MK-2206 enhances the cytotoxicity of paclitaxel (Taxol) and cisplatin in ovarian cancer cells.

    PubMed

    Lin, Ying-Hsi; Chen, Bert Yu-Hung; Lai, Wei-Ting; Wu, Shao-Fu; Guh, Jih-Hwa; Cheng, Ann-Lii; Hsu, Lih-Ching

    2015-01-01

    Abnormalities in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway are commonly observed in human cancers and contribute to chemotherapy resistance. Combination therapy, involving the use of molecular targeted agents and traditional cytotoxic drugs, may represent a promising strategy to lower resistance and enhance cytotoxicity. Here, we demonstrate the efficacy of an Akt inhibitor, MK-2206, in increasing the cytotoxic effect of either paclitaxel (Taxol) or cisplatin against the ovarian cancer cell lines SKOV3 (with constitutively active Akt) and ES2 (with inactive Akt). Sequential treatment of Taxol or cisplatin, followed by MK-2206, induced a synergistic inhibition of cell proliferation and effectively promoted cell death, either by inhibiting the phosphorylation of Akt and its downstream effectors 4E-BP1 and p70S6K in SKOV3 cells or by restoring p53 levels, which were downregulated after Taxol or cisplatin treatment, in ES2 cells. Combination treatment also downregulated the pro-survival protein Bcl-2 in both SKOV3 and ES2 cells, which may have contributed to cell death. In addition, we discovered that Taxol/MK-2206 or cisplatin/MK-2206 combination treatment resulted in significant enhancement of intracellular reactive oxygen species (ROS) induced by MK-2206, in both SKOV3 and ES2 cells; however, MK-2206-induced growth inhibition was reversed by a ROS scavenger only in ES2 cells. MK-2206 also suppressed DNA repair, particularly in SKOV3 cells. Taken together, our results demonstrate that the Akt inhibitor MK-2206 enhances the efficacy of cytotoxic agents in both Akt-active and Akt-inactive ovarian cancer cells but through different mechanisms.

  18. The Ribosomal Database Project (RDP).

    PubMed Central

    Maidak, B L; Olsen, G J; Larsen, N; Overbeek, R; McCaughey, M J; Woese, C R

    1996-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and World Wide Web (WWW)(http://rdpwww.life.uiuc.edu/). The electronic mail and WWW servers provide ribosomal probe checking, screening for possible chimeric rRNA sequences, automated alignment and approximate phylogenetic placement of user-submitted sequences on an existing phylogenetic tree. PMID:8594608

  19. The Ribosomal Database Project (RDP).

    PubMed

    Maidak, B L; Olsen, G J; Larsen, N; Overbeek, R; McCaughey, M J; Woese, C R

    1996-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and World Wide Web (WWW)(http://rdpwww.life.uiuc.edu/). The electronic mail and WWW servers provide ribosomal probe checking, screening for possible chimeric rRNA sequences, automated alignment and approximate phylogenetic placement of user-submitted sequences on an existing phylogenetic tree.

  20. Nucleocytoplasmic transport of ribosomes in a eukaryotic system: Is there a facilitated transport process

    SciTech Connect

    Khanna-Gupta, A.; Ware, V.C. )

    1989-03-01

    The authors have examined the kinetics of the process by which ribosomes are exported from the nucleus to the cytoplasm using Xenopus laevis oocytes microinjected into the germinal vesicle with radiolabeled ribosomes or ribosomal subunits from X. laevis, Tetrahymena thermophila, or Escherichia coli. Microinjected eukaryotic mature ribosomes are redistributed into the oocyte cytoplasm by an apparent carrier-mediated transport process that exhibits saturation kinetics as increasing amounts of ribosomes are injected. T. thermophila ribosomes are competent to traverse the Xenopus nuclear envelope, suggesting that the basic mechanism underlying ribosome transport is evolutionarily conserved. Microinjected E. coli ribosomes are not transported in this system, indicating that prokaryotic ribosomes lack the signals required for transport. Surprisingly, coinjected small (40S) and large (60S) subunits from T. thermophila are transported significantly faster than individual subunits. These observations support a facilitated transport model for the translocation of ribosomal subunits as separate units across the nuclear envelope whereby the transport rate of 60S or 40S subunits is enhanced by the presence of the partner subunit. Although the basic features of the transport mechanism have been preserved through evolution, other aspects of the process may be mediated through species-specific interactions. They hypothesize that a species-specific nuclear 40S-60S subunit association may expedite the transport of individual subunits across the nuclear envelope.

  1. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    SciTech Connect

    Honma, Yuichi; Harada, Masaru

    2013-08-15

    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation.

  2. Ribosome dynamics and the evolutionary history of ribosomes

    NASA Astrophysics Data System (ADS)

    Fox, George E.; Paci, Maxim; Tran, Quyen; Petrov, Anton S.; Williams, Loren D.

    2015-09-01

    The ribosome is a dynamic nanomachine responsible for coded protein synthesis. Its major subsystems were essentially in place at the time of the last universal common ancestor (LUCA). Ribosome evolutionary history thus potentially provides a window into the pre- LUCA world. This history begins with the origins of the peptidyl transferase center where the actual peptide is synthesized and then continues over an extended timeframe as additional functional centers including the GTPase center are added. The large ribosomal RNAs (rRNAs) have grown over time by an accretion process and a model exists that proposes a relative age of each accreted element. We have compared atomic resolution ribosome structures before and after EF-G bound GTP hydrolysis and thereby identified the location of 23 pivot points in the large rRNAs that facilitate ribosome dynamics. Pivots in small subunit helices h28 and h44 appear to be especially central to the process and according to the accretion model significantly older than the other helices containing pivots. Overall, the results suggest that ribosomal dynamics occurred in two phases. In the first phase, an inherently mobile h28/h44 combination provided the flexibility needed to create a dynamic ribosome that was essentially a Brownian machine. This addition likely made coded peptide synthesis possible by facilitating movement of a primitive mRNA. During the second phase, addition of pivoting elements and the creation of a factor binding site allowed the regulation of the inherent motion created by h28/h44. All of these events likely occurred before LUCA.

  3. Radiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2 inhibitor

    PubMed Central

    Paquette, B; Therriault, H; Desmarais, G; Wagner, R; Royer, R; Bujold, R

    2011-01-01

    Background: Recent evidences support that radiation can promote the invasion of cancer cells. As interactions between cancer cells and surrounding stromal cells can have an important role in tumour progression, we determined whether an irradiation to fibroblasts can enhance the invasiveness of breast cancer cells. The role of cyclooxygenase-2 (COX-2), an inflammatory enzyme frequently induced by radiotherapy, was investigated. Methods: Irradiated 3T3 fibroblasts were plated in the lower compartment of invasion chambers and used as chemoattractant for non-irradiated human breast cancer cell MDA-MB-231, which are oestrogen receptor negative (ER(−)) and the oestrogen receptor positive (ER(+)) MCF-7 cells. Stimulation of COX-2 expression in irradiated 3T3 cells was measured by a semi-quantitative qPCR and western blot. Capacity of the major product of COX-2, the prostaglandin E2 (PGE2), to stimulate the production of the matrix metalloproteinase-2 (MMP-2) and cancer cell invasion were assessed with a zymography gel and invasion chambers. Results: Irradiation (5 Gy) of 3T3 fibroblasts increased COX-2 expression and enhanced by 5.8-fold the invasiveness of non-irradiated MDA-MB-231 cells, while their migration was not modified. Addition of the COX-2 inhibitor NS-398 completely prevented radiation-enhancement of cancer cell invasion. Further supporting the potential role of COX-2, addition of PGE2 has increased cancer cell invasion and release of MMP-2 from the MDA-MB-231 cells. This effect of radiation was dependant on the expression of membrane type 1 (MT1)–MMP, which is required to activate the MMP-2, but was not associated with the ER status. Although irradiated fibroblasts stimulated the invasiveness of MDA-MB-231 ER(−) cells, no enhancement was measured with the ER(+) cell line MCF-7. Conclusions: Radiation-enhancement of breast cancer cell invasion induced by irradiated 3T3 fibroblasts is not dependant on the ER status, but rather the expression of MT1

  4. Targeted delivery of the hydroxylase inhibitor DMOG provides enhanced efficacy with reduced systemic exposure in a murine model of colitis.

    PubMed

    Tambuwala, Murtaza M; Manresa, Mario C; Cummins, Eoin P; Aversa, Vincenzo; Coulter, Ivan S; Taylor, Cormac T

    2015-11-10

    Targeting hypoxia-sensitive pathways has recently been proposed as a new therapeutic approach to the treatment of intestinal inflammation. HIF-hydroxylases are enzymes which confer hypoxic-sensitivity upon the hypoxia-inducible factor (HIF), a major regulator of the adaptive response to hypoxia. Previous studies have shown that systemic (intraperitoneal) administration of hydroxylase inhibitors such as dimethyloxalylglycine (DMOG) is profoundly protective in multiple models of colitis, however the therapeutic potential of this approach is limited due to potential side-effects associated with systemic drug exposure and the fact that orally delivered DMOG is ineffective (likely due to drug inactivation by gastric acid). In order to overcome these issues, we formulated DMOG in a liquid emulsion drug delivery system which, when coated with specific polymer coatings, permits oral delivery of a reduced dose which is released locally throughout the colon. This colon-targeted DMOG formulation demonstrated increased relative colonic bioactivity with reduced systemic exposure and provided a similar degree of protection to systemic (intraperitoneal) administration at a 40-fold lower dose in DSS-induced colitis. In summary, targeted delivery of DMOG to the colon provides local protection resulting in enhanced efficacy with reduced systemic exposure in the treatment of colitis. This novel approach to targeting hydroxylase inhibitors to specific diseased regions of the GI tract may improve it's potential as a new therapeutic in inflammatory bowel diseases such as ulcerative colitis.

  5. Specialized yeast ribosomes: a customized tool for selective mRNA translation.

    PubMed

    Bauer, Johann W; Brandl, Clemens; Haubenreisser, Olaf; Wimmer, Bjoern; Weber, Manuela; Karl, Thomas; Klausegger, Alfred; Breitenbach, Michael; Hintner, Helmut; von der Haar, Tobias; Tuite, Mick F; Breitenbach-Koller, Lore

    2013-01-01

    Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered 'specialized' ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.

  6. Cognitive enhancing effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers on learning and memory

    PubMed Central

    Nade, V. S.; Kawale, L. A.; Valte, K. D.; Shendye, N. V.

    2015-01-01

    Objective: The present study was designed to investigate cognitive enhancing property of angiotensin-converting enzymes inhibitors (ACEI) and angiotensin receptor blockers (ARBs) in rats. Materials and Methods: The elevated plus maze (EPM), passive avoidance test (PAT), and water maze test (WMT) were used to assess cognitive enhancing activity in young and aged rats. Ramipril (10 mg/kg, p.o.), perindopril (10 mg/kg, i.p), losartan (20 mg/kg, i.p), and valsartan (20 mg/kg, p.o) were administered to assess their effect on learning and memory. Scopolamine (1 mg/kg, i.p) was used to impair cognitive function. Piracetam (200 mg/kg, i.p) was used as reference drug. Results: All the treatments significantly attenuated amnesia induced by aging and scopolamine. In EPM, aged and scopolamine-treated rats showed an increase in transfer latency (TL) whereas, ACEI and ARBs showed a significant decrease in TL. Treatment with ACEI and ARBs significantly increased step down latencies and decreased latency to reach the platform in target quadrant in young, aged and scopolamine-treated animals in PAT and WMT, respectively. The treatments inhibited acetylcholinesterase (AChE) enzyme in the brain. Similarly, all the treatments attenuated scopolamine-induced lipid peroxidation and normalize antioxidant enzymes. Conclusion: The results suggest that the cognitive enhancing effect of ACEI and ARBs may be due to inhibition of AChE or by regulation of antioxidant system or increase in formation of angiotensin IV. PMID:26069362

  7. Comparison of permeation enhancing strategies for an oral factor Xa inhibitor using the Caco-2 cell monolayer model.

    PubMed

    Bruesewitz, Carsten; Funke, Adrian; Kuhland, Urte; Wagner, Torsten; Lipp, Ralph

    2006-10-01

    FXai, a direct inhibitor of the clotting factor Xa, provides high water solubility but poor membrane permeability due to multiple sites of ionization and a molecular weight exceeding 500 Da, making it a Class III drug according to the Biopharmaceutics Classification System. To overcome the ionization problem and increase the transcellular permeability, various ester and hydroxyamidine prodrugs exhibiting a reduced number of ionization sites were studied in the Caco-2 monolayer model for intestinal permeation. Alternatively, the potential transcellular permeation enhancement of Imwitor 742 and the potential paracellular enhancement of three chitosan formulations were investigated in the same model. FXai has an apparent permeability (P(app)) of about 1 nm/s, which is generally regarded as very low. The butylester-hydroxyamidine double-prodrug was found to provide a markedly increased permeability (40.4 nm/s) as did the co-application of chitosan (43.3 nm/s). Other prodrugs slightly increased permeability (1.3-9.2 nm/s) but were inferior to the previous attempts to enhance permeability while the Imwitor admixture showed no effect (1.1 nm/s). Moreover, a bioactivating metabolism towards the hydroxyamidine mono-prodrug was detected in the Caco-2 cell permeation model. Although esterases were overexpressed and mainly located apically, an acceptable permeation was reached. In addition, the prodrugs triggered an efflux system that is not inhibited by verapamil but by quinidine, suggesting the involvement of an organic cation transporter.

  8. RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel.

    PubMed

    Yoshida, Hideji; Yamamoto, Hiroshi; Uchiumi, Toshio; Wada, Akira

    2004-04-01

    In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S ribosomes under stress conditions. The 100S ribosome, a dimer of 70S ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S ribosomes. During the stationary phase, most of the 70S ribosomes turn to 100S ribosomes, which have lost translational activity. This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase. If stationary phase cells are transferred to fresh medium, the 100S ribosomes immediately go back to active 70S ribosomes, showing that inactive 100S <--> active 70S interconversion is a major system regulating translation activity in stationary phase cells. To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E. coli were examined by a chemical probing method using dimethyl sulphate (DMS). As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced. Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity. We conclude that RMF inactivates ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel. It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.

  9. A role for the extended amygdala in the fear-enhancing effects of acute selective serotonin reuptake inhibitor treatment.

    PubMed

    Ravinder, S; Burghardt, N S; Brodsky, R; Bauer, E P; Chattarji, S

    2013-01-01

    Selective serotonin reuptake inhibitors (SSRIs) are reported to exacerbate symptoms of anxiety when treatment is initiated. These clinical findings have been extended to animal models wherein SSRIs also potentiate anxiety and fear learning, which depend on the amygdala. Yet, little is known about the role of specific amygdalar circuits in these acute effects of SSRIs. Here, we first confirmed that a single injection of fluoxetine 1 h before auditory fear conditioning potentiated fear memory in rats. To probe the neural substrates underlying this enhancement, we analyzed the expression patterns of the immediate early gene, Arc (activity-regulated cytoskeleton-associated protein). Consistent with previous reports, fear conditioning induced Arc protein expression in the lateral and basal amygdala. However, this was not enhanced further by pre-treatment with fluoxetine. Instead, fluoxetine significantly enhanced expression of Arc in the central amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). Next, we tested whether direct targeted infusions of fluoxetine into the CeA, or BNST, leads to the same fear-potentiating effect. Strikingly, direct infusion of fluoxetine into the BNST, but not the CeA, was sufficient to enhance fear memory. Moreover, this behavioral effect was also accompanied by robust Arc expression in the CeA, similar to the systemic injection. Our results identify a novel role for the BNST in the acute fear-enhancing effects of SSRIs. These findings highlight the need to look beyond the traditional focus on input nuclei of the amygdala and add to accumulating evidence implicating these microcircuits in gating fear and anxiety. PMID:23321806

  10. [Ribosomal RNA Evolution

    NASA Technical Reports Server (NTRS)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  11. Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

    PubMed Central

    Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

    2014-01-01

    In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

  12. The Histone Deacetylase Inhibitor Valproic Acid Enhances Acquisition, Extinction, and Reconsolidation of Conditioned Fear

    ERIC Educational Resources Information Center

    Bredy, Timothy W.; Barad, Mark

    2008-01-01

    Histone modifications contribute to the epigenetic regulation of gene expression, a process now recognized to be important for the consolidation of long-term memory. Valproic acid (VPA), used for many years as an anticonvulsant and a mood stabilizer, has effects on learning and memory and enhances the extinction of conditioned fear through its…

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

    PubMed

    Poirot, Olivier; Timsit, Youri

    2016-01-01

    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. PMID:27225526

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

    PubMed Central

    Poirot, Olivier; Timsit, Youri

    2016-01-01

    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. PMID:27225526

  15. Enhancement of the efficiency of photodynamic therapy by combination with the microtubule inhibitor vincristine

    NASA Astrophysics Data System (ADS)

    Ma, Li Wei; Berg, Kristian; Danielsen, Havard E.; Iani, Vladimir; Moan, Johan

    1996-01-01

    Combination effects of photodynamic therapy (PDT) with meso-tetra (di-adjacent- sulfonatophenyl) porphine (TPPS2a) and the microtubule (MT) inhibitor, vincristine (VCR), were studied in the CaD2 mouse tumor model in mice. A synergistic effect was found when VCR, at an almost nontoxic dose (1 mg/kg), was injected i.p. into the mice 6 hr before PDT. The data on mitotic index show a 4 - 5 fold accumulation of the cells in mitosis 6 hr after injection of VCR into the mice. Cell cycle and ploidy distributions in tumor tissues were determined by means of image analysis with measurement of integrated optical density after Feulgen reaction on monolayers. Ploidy distribution of the tumors was not significantly changed 6 and 12 hr after administration of VCR only, while an increasing aneuploidy was observed 24 and 48 hr after VCR treatment. No prominent changes of the cell cycle and ploidy distributions were found in the tumor tissues after PDT or PDT combined with VCR.

  16. Gold nanoparticles enhance the effect of tyrosine kinase inhibitors in acute myeloid leukemia therapy

    PubMed Central

    Petrushev, Bobe; Boca, Sanda; Simon, Timea; Berce, Cristian; Frinc, Ioana; Dima, Delia; Selicean, Sonia; Gafencu, Grigore-Aristide; Tanase, Alina; Zdrenghea, Mihnea; Florea, Adrian; Suarasan, Sorina; Dima, Liana; Stanciu, Raluca; Jurj, Ancuta; Buzoianu, Anca; Cucuianu, Andrei; Astilean, Simion; Irimie, Alexandru; Tomuleasa, Ciprian; Berindan-Neagoe, Ioana

    2016-01-01

    Background and aims Every year, in Europe, acute myeloid leukemia (AML) is diagnosed in thousands of adults. For most subtypes of AML, the backbone of treatment was introduced nearly 40 years ago as a combination of cytosine arabinoside with an anthracycline. This therapy is still the worldwide standard of care. Two-thirds of patients achieve complete remission, although most of them ultimately relapse. Since the FLT3 mutation is the most frequent, it serves as a key molecular target for tyrosine kinase inhibitors (TKIs) that inhibit FLT3 kinase. In this study, we report the conjugation of TKIs onto spherical gold nanoparticles. Materials and methods The internalization of TKI-nanocarriers was proved by the strongly scattered light from gold nanoparticles and was correlated with the results obtained by transmission electron microscopy and dark-field microscopy. The therapeutic effect of the newly designed drugs was investigated by several methods including cell counting assay as well as the MTT assay. Results We report the newly described bioconjugates to be superior when compared with the drug alone, with data confirmed by state-of-the-art analyses of internalization, cell biology, gene analysis for FLT3-IDT gene, and Western blotting to assess degradation of the FLT3 protein. Conclusion The effective transmembrane delivery and increased efficacy validate its use as a potential therapeutic. PMID:26929621

  17. Serine protease inhibitors block priming of monocytes for enhanced release of superoxide.

    PubMed Central

    Megyeri, P; Pabst, K M; Pabst, M J

    1995-01-01

    Monocytes freshly isolated from human blood produced large amounts of superoxide when triggered by phorbol ester. After monocytes were cultured for 18-24 hr in endotoxin-free, non-adherent conditions, they produced low amounts of superoxide. Addition of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), or platelet-activating factor (PAF) at the beginning of culture 'primed' the monocytes, causing them to maintain a high superoxide response for at least 96 hr. Also, in response to LPS, monocytes secreted TNF-alpha. The ability of LPS, IFN-gamma, TNF-alpha or PAF to maintain the high superoxide response was blocked by addition of inhibitors of serine proteases, either 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) or 3,4-dichloroisocoumarin. AEBSF was most effective at 200 microns, and required 6 hr for maximum effect. AEBSF did not affect phorbol-triggered superoxide release by unprimed monocytes. AEBSF did not affect cell viability, nor did it interfere with the TNF-alpha secretion in response to LPS. An analogue of AEBSF that lacked ability to inhibit proteases did not affect monocyte responses. 3,4-Dichloroisocoumarin blocked priming at a low concentration, 1 microM. We conclude that activity of a monocyte serine protease is required to maintain the high superoxide response in monocytes primed with LPS, IFN-gamma, TNF-alpha, or PAF. PMID:8567031

  18. Enhanced permeation, leaf retention, and plant protease inhibitor activity with bicontinuous microemulsions.

    PubMed

    Tamhane, Vaijayanti A; Dhaware, Deepika G; Khandelwal, Neha; Giri, Ashok P; Panchagnula, Venkateswarlu

    2012-10-01

    Bicontinuous microemulsions (BCMEs) have excellent solubulizing properties along with low interfacial tension and aqueous content that can be controlled. In this work, water soluble plant protease inhibitor (PI), well characterized for its activity against insect pests, was incorporated into a BCME system and explored for permeation on hydrophobic leaf surfaces and protease inhibition activity. The bicontinuous nature of the microemulsion containing water:2-propanol:1-butanol (55:35:10 w/w) was characterized using conductivity and self-diffusion coefficient measurements. The PI was soluble in the water-rich bicontinuous domains, stable in the microemulsions, and protease inhibition activity was retained for a prolonged duration. The microemulsions ensured greater wettability and a wider spread of the PI on hydrophobic leaf surfaces as revealed by contact angle measurements. Significantly, trypsin inhibition activity assays of the PI recovered from the leaves after delivery from the microemulsion indicated a significant increase in the PI retention on the leaf. This BCME enabled greater leaf permeation and retention of the PI can be attributed to a temporary disruption of the waxy leaf surface followed by self-repair without causing any long term damage to the plant.

  19. Enhancement of cellular uptake, transport and oral absorption of protease inhibitor saquinavir by nanocrystal formulation

    PubMed Central

    He, Yuan; Xia, Deng-ning; Li, Qiu-xia; Tao, Jin-song; Gan, Yong; Wang, Chi

    2015-01-01

    Aim: Saquinavir (SQV) is the first protease inhibitor for the treatment of HIV infection, but with poor solubility. The aim of this study was to prepare a colloidal nanocrystal suspension for improving the oral absorption of SQV. Methods: SQV nanocrystals were prepared using anti-solvent precipitation–high pressure homogenization method. The nanocrystals were characterized by a Zetasizer and transmission electron microscopy (TEM). Their dissolution, cellular uptake and transport across the human colorectal adenocarcinoma cell line (Caco-2) monolayer were investigated. Bioimaging of ex vivo intestinal sections of rats was conducted with confocal laser scanning microscopy. Pharmacokinetic analysis was performed in rats administered nanocrystal SQV suspension (50 mg/kg, ig), and the plasma SQV concentrations were measured with HPLC. Results: The SQV nanocrystals were approximately 200 nm in diameter, with a uniform size distribution. The nanocrystals had a rod-like shape under TEM. The dissolution, cellular uptake, and transport across a Caco-2 monolayer of the nanocrystal formulation were significantly improved compared to those of the coarse crystals. The ex vivo intestinal section study revealed that the fluorescently labeled nanocrystals were located in the lamina propria and the epithelium of the duodenum and jejunum. Pharmacokinetic study showed that the maximal plasma concentration (Cmax) was 2.16-fold of that for coarse crystalline SQV suspension, whereas the area under the curve (AUC) of nanocrystal SQV suspension was 1.95-fold of that for coarse crystalline SQV suspension. Conclusion: The nanocrystal drug delivery system significantly improves the oral absorption of saquinavir. PMID:26256404

  20. Nanoemulsion-based gel formulations of COX-2 inhibitors for enhanced efficacy in inflammatory conditions

    NASA Astrophysics Data System (ADS)

    Lala, R. R.; Awari, N. G.

    2013-01-01

    In the present study, we have investigated the potential of a nanoemulsion (thermodynamically stable transparent dispersions of oil and water having a droplet size <200 nm) formulation for the topical delivery of COX-2 inhibitors using etoricoxib as a model drug. Various oil-in-water nanoemulsions were prepared by the spontaneous emulsification method. The nanoemulsion area was identified by constructing pseudo-ternary phase diagrams. The prepared nanoemulsions were subjected to thermodynamic stability testing. Those that passed these tests were characterized for viscosity, droplet size and differential scanning calorimetry. Topical permeation of etoricoxib through porcine abdominal skin was estimated using the Franz diffusion cell. The ex vivo skin permeation profile of optimized formulations was compared with that of etoricoxib conventional gel. A significant increase in permeability was observed in optimized nanoemulsion formulations consisting of 2 % w/w of etoricoxib, 20 % w/w of Triacetin, 38 % w/w of a surfactant mixture (Cremophor RH 40:Transcutol P), and 42 % w/w of water. The anti-inflammatory effects of this formulation on carrageenan-induced paw edema in rats showed a significant increase in the percent inhibition value (84.61 % with the nanoemulsion gel and 92.30 % with the nanoemulsion) as compared with the conventional gel (69.23 %) after 6 h when compared with etoricoxib conventional gel. These results suggest that nanoemulsions can serve as potential vehicles for improved transdermal delivery of anti-inflammatory agents such as etoricoxib.

  1. Use of lipolanthionine peptide, a toll-like receptor 2 inhibitor, enhances transdermal delivery efficiency

    PubMed Central

    CHEN, BIN; LIU, DA-LIE; PAN, WEN-YAN; YANG, XIAO-HUI; SHOU, JIA-BAO; WU, JU-HUA; MAO, QING-LONG; WANG, JIA

    2014-01-01

    The transdermal delivery system (TDS) is able to obtain a systemic therapeutic effect by administration through the skin, which has low side effects and is able to maintain a sustained blood concentration. However, due to the barrier presented by the stratum corneum, numerous drugs have poor percutaneous permeability. Therefore, the improvement of skin permeability is key to TDS. The main method of promoting transdermal absorption is through the usage of penetration enhancers. Dimethyl sulfoxide (DMSO) is a commonly used penetration enhancer, which has anti-inflammatory analgesic effects and is able to penetrate the skin. Retinoic acid (RA) and lipolanthionine peptide (LP) may also benefit the permeation efficiency of TDS. Therefore, the present study examined the function of DMSO, RA and LP as penetration enhancers in TDS. Firstly, the optimum concentration of DMSO was confirmed by detecting the expression of the LacZ gene in vitro. Secondly, different combinations of LP, RA and DMSO were applied to mouse skin to analyze the penetration enhancer combination with the greatest efficacy. All the animals were divided into five groups: The RA + LP + DMSO + pORF-LacZ group, the RA + DMSO + pORF-LacZ group, the LP + DMSO + pORF-LacZ group, the DMSO + pORF-LacZ group and the control group. Skin was soaked in combinations of LP, RA and DMSO for seven days and then the pORF-LacZ plasmids were daubed onto the skin once daily three days. On the 11th day, all the animals were sacrificed by cervical dislocation and the skin and blood samples were collected. The blood samples were used to detect the expression of the LacZ gene by quantitative polymerase chain reaction and the skin samples were used to detect the expression of claudin-4 and zonula occluden-1 (ZO-1) proteins by immunohistochemistry and western blot analysis. The results demonstrated that the combination of LP, RA and DMSO exhibited the greatest transdermal delivery efficiency, which verified that RA and LP were

  2. The Drug Diazaborine Blocks Ribosome Biogenesis by Inhibiting the AAA-ATPase Drg1*

    PubMed Central

    Loibl, Mathias; Klein, Isabella; Prattes, Michael; Schmidt, Claudia; Kappel, Lisa; Zisser, Gertrude; Gungl, Anna; Krieger, Elmar; Pertschy, Brigitte; Bergler, Helmut

    2014-01-01

    The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation. PMID:24371142

  3. Enhancement of Peripheral Nerve Regrowth by the Purine Nucleoside Analog and Cell Cycle Inhibitor, Roscovitine

    PubMed Central

    Law, Vincent; Dong, Sophie; Rosales, Jesusa L.; Jeong, Myung-Yung; Zochodne, Douglas; Lee, Ki-Young

    2016-01-01

    Peripheral nerve regeneration is a slow process that can be associated with limited outcomes and thus a search for novel and effective therapy for peripheral nerve injury and disease is crucial. Here, we found that roscovitine, a synthetic purine nucleoside analog, enhances neurite outgrowth in neuronal-like PC12 cells. Furthermore, ex vivo analysis of pre-injured adult rat dorsal root ganglion (DRG) neurons showed that roscovitine enhances neurite regrowth in these cells. Likewise, in vivo transected sciatic nerves in rats locally perfused with roscovitine had augmented repopulation of new myelinated axons beyond the transection zone. By mass spectrometry, we found that roscovitine interacts with tubulin and actin. It interacts directly with tubulin and causes a dose-dependent induction of tubulin polymerization as well as enhances Guanosine-5′-triphosphate (GTP)-dependent tubulin polymerization. Conversely, roscovitine interacts indirectly with actin and counteracts the inhibitory effect of cyclin-dependent kinases 5 (Cdk5) on Actin-Related Proteins 2/3 (Arp2/3)-dependent actin polymerization, and thus, causes actin polymerization. Moreover, in the presence of neurotrophic factors such as nerve growth factor (NGF), roscovitine-enhanced neurite outgrowth is mediated by increased activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways. Since microtubule and F-actin dynamics are critical for axonal regrowth, the ability of roscovitine to activate the ERK1/2 and p38 MAPK pathways and support polymerization of tubulin and actin indicate a major role for this purine nucleoside analog in the promotion of axonal regeneration. Together, our findings demonstrate a therapeutic potential for the purine nucleoside analog, roscovitine, in peripheral nerve injury. PMID:27799897

  4. MRE11-RAD50-NBS1 COMPLEX INHIBITOR MIRIN ENHANCES RADIOSENSITIVITY IN HUMAN GLIOBLASTOMA CELLS

    PubMed Central

    Mishima, Kazuhiko; Mishima-Kaneko, Masayo; Kawata, Tetsuya; Saya, Hideyuki; Ishimaru, Naozumi; Yamada, Kouichi; Nishikawa, Ryo; Shigematsu, Naoyuki

    2014-01-01

    BACKGROUND: (blind field) METHODS: Glioma cell lines (U251, LN229 and LN428) were irradiated with and without Mirin and then clonogenicity, apoptosis, and cell cycle change were examined. Western blot analysis was performed to determine the relative potency of Mirin to inhibit the radioresistance, through the signaling activity of AKT. We also examined the levels of H2AX phosphorylation (γH2AX), which is a marker of DNA double-strand breaks (DSBs) using Western blot. RESULTS: Glioblastoma cells pretreated with Mirin demonstrated an enhanced sensitivity to radiation. FACS analysis revealed that Mirin and radiation caused the glioma cells to accumulate in the G2/M-phase of the cell cycle and the combination of these two treatments further increased the G2/M fraction of the glioma cells. Mirin significantly enhanced radiation-induced apoptotic cell death. Also, Mirin blocked the basal and increase of radiation-induced AKT phosphorylation. We observed that the combination of Mirin and radiation increased persistence of γH2AX at 24 h suggesting the inhibition of DNA DSBs repair. CONCLUSIONS: These results indicate that Mirin can effectively enhance glioma cell radiosensitivity. It suggests that Mirin is a potent radiosensitizer for treating glioma cells. SECONDARY CATEGORY: n/a.

  5. Selective enhancement of the uptake and bioactivity of a TAT-conjugated peptide inhibitor of glycogen synthase kinase-3.

    PubMed

    Manceur, Aziza P; Driscoll, Brandon D; Sun, Wei; Audet, Julie

    2009-03-01

    The use of cell-penetrating peptides as transduction vectors is a promising approach to deliver peptides and proteins into cells. However, the uptake and bioavailability of trans-activating transcriptor (TAT)-conjugated molecules vary depending on the cell type and the cargo. This study aimed to determine whether a low-voltage electrical pulse can enhance the TAT-mediated delivery of peptide cargoes in different cell types. In TF-1 and mouse embryonic stem cells, the uptake of a novel detachable TAT-conjugated glycogen synthase kinase-3 (GSK-3) peptide inhibitor was enhanced by an order of magnitude without affecting the cell viability. A similar increase in uptake was achieved in primary mouse bone marrow cells while maintaining >80% of their viability. Interestingly, under these low-voltage conditions, the uptake of a control peptide not conjugated to TAT was not significantly increased. A T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay was also used to assess the bioactivity of the TAT construct. The results indicated that cells loaded with a low-voltage electrical pulse had a twofold increase in TCF/LEF activity, which was equivalent to a level of GSK-3 inhibition similar to that of cells treated with 20 mmol/l lithium or 500 nmol/l (2'Z,3'E)-6-bromoindirubin-3'-oxime. These results demonstrate the usefulness of low-voltage electrical pulses to enhance the uptake and bioactivity of TAT-conjugated molecules in different cell types. PMID:19107119

  6. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  7. The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis.

    PubMed

    Shirazi, F; Kontoyiannis, D P

    2013-09-01

    The calcineurin pathway regulates antifungal drug resistance and the virulence of several major human-pathogenic fungi, including the recalcitrant Mucorales. We hypothesized that the fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) become fungicidal in the setting of the calcineurin inhibitor tacrolimus (TCR) and that such an effect is mediated through apoptosis. Fungicidal activity and apoptosis were studied using standard microbiological techniques and hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting intracellular Ca(2+), phosphatidylserine (PS) externalization, DNA fragmentation, plasma membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, caspase-like activity, ATP, and cytochrome c release. MICs for PCZ and ICZ alone were significantly higher (8 to 128 μg/ml) than those of PCZ or ICZ plus TCR (0.25 to 4 μg/ml) for Rhizopus oryzae, Cunninghamella bertholletiae, and Mucor circinelloides. Both PCZ and ICZ in combination with TCR became fungicidal, and their activity was mediated through increased apoptotic cell death of R. oryzae (10 to 50%), C. bertholletiae (5 to 50%), and M. circinelloides (5 to 55%) germlings, with morphological apoptotic changes characterized by externalization of PS, nuclear condensation, and DNA fragmentation. Moreover, activation of the caspase-like activity was correlated with cell death induced by TCR plus PCZ or ICZ. These changes correlated with elevated intracellular Ca(2+) and ROS levels and disturbance of mitochondrial potential. We found that PCZ or ICZ in combination with TCR renders Mucorales sensitive to triazoles via apoptotic death. These observations could serve as a new paradigm for the development of new therapeutic strategies. PMID:23851337

  8. Herpes simplex virus downregulation of secretory leukocyte protease inhibitor enhances human papillomavirus type 16 infection.

    PubMed

    Skeate, Joseph G; Porras, Tania B; Woodham, Andrew W; Jang, Julie K; Taylor, Julia R; Brand, Heike E; Kelly, Thomas J; Jung, Jae U; Da Silva, Diane M; Yuan, Weiming; Kast, W Martin

    2016-02-01

    Herpes simplex virus (HSV) was originally implicated in the aetiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiological link between HSV and HPV-associated cancers persists. The annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here, we show that in vitro infection of human keratinocytes with HSV-1 or HSV-2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70% reduction of SLPI mRNA and a >60% decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (two- and 2.5-fold, respectively) in HSV-1- and HSV-2-infected human keratinocyte cell cultures compared with uninfected cells, whereas exogenously added SLPI reversed this effect. Using a SiMPull (single-molecule pulldown) assay, we demonstrated that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggested that ongoing HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the aetiological link between HSV and HPV-associated cancers.

  9. Plasminogen activator inhibitor-1 enhances radioresistance and aggressiveness of non-small cell lung cancer cells

    PubMed Central

    Youn, HyeSook; Kim, Joong Sun; Youn, BuHyun

    2016-01-01

    Acquired resistance of tumor cells during treatment limits the clinical efficacy of radiotherapy. Recent studies to investigate acquired resistance under treatment have focused on intercellular communication because it promotes survival and aggressiveness of tumor cells, causing therapy failure and tumor relapse. Accordingly, a better understanding of the functional communication between subpopulations of cells within a tumor is essential to development of effective cancer treatment strategies. Here, we found that conditioned media (CM) from radioresistant non-small cell lung cancer (NSCLC) cells increased survival of radiosensitive cells. Comparative proteomics analysis revealed plasminogen activator inhibitor-1 (PAI-1) as a key molecule in the secretome that acts as an extracellular signaling trigger to strengthen resistance to radiation. Our results revealed that expression and secretion of PAI-1 in radioresistant cells was increased by radiation-induced transcription factors, including p53, HIF-1α, and Smad3. When CM from radioresistant cells was applied to radiosensitive cells, extracellular PAI-1 activated the AKT and ERK1/2 signaling pathway and inhibited caspase-3 activity. Our study also proposed that PAI-1 activates the signaling pathway in radiosensitive cells via extracellular interaction with its binding partners, not clathrin-mediated endocytosis. Furthermore, secreted PAI-1 increased cell migration capacity and expression of EMT markers in vitro and in vivo. Taken together, our findings demonstrate that PAI-1 secreted from radioresistant NSCLC cells reduced radiosensitivity of nearby cells in a paracrine manner, indicating that functional inhibition of PAI-1 signaling has therapeutic potential because it prevents sensitive cells from acquiring radioresistance. PMID:27004408

  10. Aurora inhibitor MLN8237 in combination with docetaxel enhances apoptosis and anti-tumor activity in mantle cell lymphoma.

    PubMed

    Qi, Wenqing; Cooke, Laurence S; Liu, Xiaobing; Rimsza, Lisa; Roe, Denise J; Manziolli, Ann; Persky, Daniel O; Miller, Thomas P; Mahadevan, Daruka

    2011-04-01

    Auroras (A and B) are oncogenic serine/threonine kinases that play key roles in the mitotic phase of the eukaryotic cell cycle. Analysis of the leukemia lymphoma molecular profiling project (LLMPP) database indicates Aurora over-expression correlates with poor prognosis. A tissue microarray (TMA) composed of 20 paired mantle cell lymphoma (MCL) patients demonstrated >75% of patients had high levels Aurora expression. Aurora A and B were also found elevated in 13 aggressive B-NHL cell lines. MLN8237, an Aurora inhibitor induced G2/M arrest with polyploidy and abrogated Aurora A and histone-H3 phosphorylation. MLN8237 inhibited aggressive B-NHL cell proliferation at an IC(50) of 10-50 nM and induced apoptosis in a dose- and time-dependent manner. Low dose combinations of MLN8237+docetaxel enhanced apoptosis by ~3-4-fold in cell culture compared to single agents respectively. A mouse xenograft model of MCL demonstrated that MLN8237 (10 or 30 mg/kg) or docetaxel (10mg/kg) alone had modest anti-tumor activity. However, MLN8237 plus docetaxel demonstrated a statistically significant tumor growth inhibition and enhanced survival compared to single agent therapy. Together, our results suggest that MLN8237 plus docetaxel may represent a novel therapeutic strategy that could be evaluated in early phase trials in relapsed/refractory aggressive B-cell NHL. PMID:21291867

  11. Enhancement of human sodium iodide symporter gene therapy for breast cancer by HDAC inhibitor mediated transcriptional modulation.

    PubMed

    Kelkar, Madhura G; Senthilkumar, Kalimuthu; Jadhav, Smita; Gupta, Sudeep; Ahn, Beyong-Cheol; De, Abhijit

    2016-01-18

    The aberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) has raised the possibility of using targeted radioiodide therapy. Here we investigate modulation of endogenous, functional NIS expression by histone deacetylase inhibitors (HDACi) in vitro and in vivo. Luciferase reporter based initial screening of six different HDACi shows 2-10 fold enhancement of NIS promoter activity in majority of the cell types tested. As a result of drug treatment, endogenous NIS transcript and protein shows profound induction in BC cells. To get an insight on the mechanism of such transcriptional activation, role of Stat4, CREB and other transcription factors are revealed by transcription factor profiling array. Further, NIS-mediated intracellular iodide uptake also enhances substantially (p < 0.05) signifying functional relevance of the transcriptional modulation strategy. Gamma camera imaging confirms 30% higher uptake in VPA or NaB treated BC tumor xenograft. Corroborating with such functional impact of NIS, significant reduction in cell survival (p < 0.005) is observed in VPA, NaB or CI994 drug and (131)I combination treatment in vivo indicating effective radioablation. Thus, for the first time this study reveals the mechanistic basis and demonstrates functional relevance of HDACi pre-treatment strategy in elevating NIS gene therapy approach for BC management in clinic.

  12. Enhancement of human sodium iodide symporter gene therapy for breast cancer by HDAC inhibitor mediated transcriptional modulation

    PubMed Central

    Kelkar, Madhura G.; Senthilkumar, Kalimuthu; Jadhav, Smita; Gupta, Sudeep; Ahn, Beyong-Cheol; De, Abhijit

    2016-01-01

    The aberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) has raised the possibility of using targeted radioiodide therapy. Here we investigate modulation of endogenous, functional NIS expression by histone deacetylase inhibitors (HDACi) in vitro and in vivo. Luciferase reporter based initial screening of six different HDACi shows 2–10 fold enhancement of NIS promoter activity in majority of the cell types tested. As a result of drug treatment, endogenous NIS transcript and protein shows profound induction in BC cells. To get an insight on the mechanism of such transcriptional activation, role of Stat4, CREB and other transcription factors are revealed by transcription factor profiling array. Further, NIS-mediated intracellular iodide uptake also enhances substantially (p < 0.05) signifying functional relevance of the transcriptional modulation strategy. Gamma camera imaging confirms 30% higher uptake in VPA or NaB treated BC tumor xenograft. Corroborating with such functional impact of NIS, significant reduction in cell survival (p < 0.005) is observed in VPA, NaB or CI994 drug and 131I combination treatment in vivo indicating effective radioablation. Thus, for the first time this study reveals the mechanistic basis and demonstrates functional relevance of HDACi pre-treatment strategy in elevating NIS gene therapy approach for BC management in clinic. PMID:26777440

  13. Aurora inhibitor MLN8237 in combination with docetaxel enhances apoptosis and anti-tumor activity in mantle cell lymphoma.

    PubMed

    Qi, Wenqing; Cooke, Laurence S; Liu, Xiaobing; Rimsza, Lisa; Roe, Denise J; Manziolli, Ann; Persky, Daniel O; Miller, Thomas P; Mahadevan, Daruka

    2011-04-01

    Auroras (A and B) are oncogenic serine/threonine kinases that play key roles in the mitotic phase of the eukaryotic cell cycle. Analysis of the leukemia lymphoma molecular profiling project (LLMPP) database indicates Aurora over-expression correlates with poor prognosis. A tissue microarray (TMA) composed of 20 paired mantle cell lymphoma (MCL) patients demonstrated >75% of patients had high levels Aurora expression. Aurora A and B were also found elevated in 13 aggressive B-NHL cell lines. MLN8237, an Aurora inhibitor induced G2/M arrest with polyploidy and abrogated Aurora A and histone-H3 phosphorylation. MLN8237 inhibited aggressive B-NHL cell proliferation at an IC(50) of 10-50 nM and induced apoptosis in a dose- and time-dependent manner. Low dose combinations of MLN8237+docetaxel enhanced apoptosis by ~3-4-fold in cell culture compared to single agents respectively. A mouse xenograft model of MCL demonstrated that MLN8237 (10 or 30 mg/kg) or docetaxel (10mg/kg) alone had modest anti-tumor activity. However, MLN8237 plus docetaxel demonstrated a statistically significant tumor growth inhibition and enhanced survival compared to single agent therapy. Together, our results suggest that MLN8237 plus docetaxel may represent a novel therapeutic strategy that could be evaluated in early phase trials in relapsed/refractory aggressive B-cell NHL.

  14. Fluorescence bimolecular complementation enables facile detection of ribosome assembly defects in Escherichia coli.

    PubMed

    Sharma, Himanshu; Anand, Baskaran

    2016-09-01

    Assembly factors promote the otherwise non-spontaneous maturation of ribosome under physiological conditions inside the cell. Systematic identification and characterization of candidate assembly factors are fraught with bottlenecks due to lack of facile assay system to capture assembly defects. Here, we show that bimolecular fluorescence complementation (BiFC) allows detection of assembly defects that are induced by the loss of assembly factors. The fusion of N and C-terminal fragments of Venus fluorescent protein to the ribosomal proteins uS13 and uL5, respectively, in Escherichia coli facilitated the incorporation of the tagged uS13 and uL5 onto the respective ribosomal subunits. When the ribosomal subunits associated to form the 70S particle, the complementary fragments of Venus were brought into proximity and rendered the Venus fluorescent. Assembly defects that inhibit the subunits association were provoked by either the loss of the known assembly factors such as RsgA and SrmB or the presence of small molecule inhibitors of ribosome maturation such as Lamotrigine and several ribosome-targeting antibiotics and these showed abrogation of the fluorescence complementation. This suggests that BiFC can be employed as a surrogate measure to detect ribosome assembly defects proficiently by circumventing the otherwise cumbersome procedures. BiFC thus offers a facile platform not only for systematic screening to validate potential assembly factors but also to discover novel small molecule inhibitors of ribosome assembly toward mapping the complex assembly landscape of ribosome. PMID:27388791

  15. Profiling of Mycoplasma gallisepticum Ribosomes.

    PubMed

    Fisunov, G Y; Evsyutina, D V; Arzamasov, A A; Butenko, I O; Govorun, V M

    2015-01-01

    The development of high-throughput technologies is increasingly resulting in identification of numerous cases of low correlation between mRNA and the protein level in cells. These controversial observations were made on various bacteria, such as E. coli, Desulfovibrio vulgaris, and Lactococcus lactis. Thus, it is important to develop technologies, including high-throughput techniques, aimed at studying gene expression regulation at the level of translation. In the current study, we performed proteomic profiling of M. gallisepticum ribosomes and identified high abundant noncanonical proteins. We found that binding of mRNAs to ribosomes is mainly determined by two parameters: (1) abundance of mRNA itself and (2) complimentary interactions between the 3' end of 16S rRNA and the ribosome binding site in the 5'-untranslated region of mRNA. PMID:26798497

  16. The ABC of Ribosome-Related Antibiotic Resistance.

    PubMed

    Wilson, Daniel N

    2016-01-01

    The increase in multidrug-resistant pathogenic bacteria is limiting the utility of our current arsenal of antimicrobial agents. Mechanistically understanding how bacteria obtain antibiotic resistance is a critical first step to the development of improved inhibitors. One common mechanism for bacteria to obtain antibiotic resistance is by employing ATP-binding cassette (ABC) transporters to actively pump the drug from the cell. The ABC-F family includes proteins conferring resistance to a variety of clinically important ribosome-targeting antibiotics; however, controversy remains as to whether resistance is conferred via efflux like other ABC transporters or whether another mechanism, such as ribosome protection, is at play. A recent study by Sharkey and coworkers (L. K. Sharkey, T. A. Edwards, and A. J. O'Neill, mBio 7:e01975-15, 2016, http://dx.doi.org/10.1128/mBio.01975-15) provides strong evidence that ABC-F proteins conferring antibiotic resistance utilize ribosome protection mechanisms, namely, by interacting with the ribosome and displacing the drug from its binding site, thus revealing a novel role for ABC-F proteins in antibiotic resistance. PMID:27143393

  17. The ABC of Ribosome-Related Antibiotic Resistance.

    PubMed

    Wilson, Daniel N

    2016-05-03

    The increase in multidrug-resistant pathogenic bacteria is limiting the utility of our current arsenal of antimicrobial agents. Mechanistically understanding how bacteria obtain antibiotic resistance is a critical first step to the development of improved inhibitors. One common mechanism for bacteria to obtain antibiotic resistance is by employing ATP-binding cassette (ABC) transporters to actively pump the drug from the cell. The ABC-F family includes proteins conferring resistance to a variety of clinically important ribosome-targeting antibiotics; however, controversy remains as to whether resistance is conferred via efflux like other ABC transporters or whether another mechanism, such as ribosome protection, is at play. A recent study by Sharkey and coworkers (L. K. Sharkey, T. A. Edwards, and A. J. O'Neill, mBio 7:e01975-15, 2016, http://dx.doi.org/10.1128/mBio.01975-15) provides strong evidence that ABC-F proteins conferring antibiotic resistance utilize ribosome protection mechanisms, namely, by interacting with the ribosome and displacing the drug from its binding site, thus revealing a novel role for ABC-F proteins in antibiotic resistance.

  18. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development.

    PubMed

    Nogueira, D; Ron-El, R; Friedler, S; Schachter, M; Raziel, A; Cortvrindt, R; Smitz, J

    2006-01-01

    Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

  19. An insect growth inhibitor--lufenuron--enhances albendazole activity against hydatid cyst.

    PubMed

    Breijo, Martín; Isnardi, Fernanda; Brauer, Mónica; Schenker, Rudolf; Ferrari, Mariana; Ferreira, Ana M

    2011-09-27

    The aim of this work was to evaluate the potential of lufenuron, a benzylphenylurea with ability to interfere with the formation of insect exoskeleton, as a therapeutic drug for larval echinococcosis (hydatid disease). For this purpose lufenuron, alone or in combination with albendazole, was administered to CD1 mice bearing Echinococcus granulosus hydatid cysts in the peritoneal cavity. Neither of the drugs alone was able to exert parasiticidal effects. However, in combination with albendazole, lufenuron reduced the growth of cysts (30-40% in cyst diameter respect to control, p<0.05). This effect was associated with ultrastructural alterations of the hydatid cyst wall and a reduction of the content of myo-inositol-hexakisphosphate, the major component of the electron dense granules of the laminated layer. Overall, this work provides evidence that lufenuron could represent a useful compound for the use in chemotherapy against larval echinococcosis, by enhancing albendazole parasiticidal activity. PMID:21592667

  20. Benzoxazole and benzothiazole amides as novel pharmacokinetic enhancers of HIV protease inhibitors.

    PubMed

    Jonckers, Tim H M; Rouan, Marie-Claude; Haché, Geerwin; Schepens, Wim; Hallenberger, Sabine; Baumeister, Judith; Sasaki, Jennifer C

    2012-08-01

    A new class of benzoxazole and benzothiazole amide derivatives exhibiting potent CYP3A4 inhibiting properties was identified. Extensive lead optimization was aimed at improving the CYP3A4 inhibitory properties as well as overall ADME profile of these amide derivatives. This led to the identification of thiazol-5-ylmethyl (2S,3R)-4-(2-(ethyl(methyl)amino)-N-isobutylbenzo[d]oxazole-6-carboxamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (C1) as a lead candidate for this class. This compound together with structurally similar analogues demonstrated excellent 'boosting' properties when tested in dogs. These findings warrant further evaluation of their properties in an effort to identify valuable alternatives to Ritonavir as pharmacokinetic enhancers.

  1. Tumor-Priming Smoothened Inhibitor Enhances Deposition and Efficacy of Cytotoxic Nanoparticles in a Pancreatic Cancer Model.

    PubMed

    Roy Chaudhuri, Tista; Straubinger, Ninfa L; Pitoniak, Rosemarie F; Hylander, Bonnie L; Repasky, Elizabeth A; Ma, Wen Wee; Straubinger, Robert M

    2016-01-01

    Most pancreatic adenocarcinoma patients present with unresectable disease and benefit little from chemotherapy. Poor tumor perfusion and vascular permeability limit drug deposition. Previous work showed that Smoothened inhibitors of hedgehog signaling (sHHI) promote neovascularization in spontaneous mouse models of pancreatic cancer (PaCA) and enhance tumor permeability to low-molecular weight compounds. Here, we tested the hypothesis that sHHI can enhance tumor deposition and efficacy of drug-containing nanoparticles consisting of 80 to 100 nm sterically-stabilized liposomes (SSL) containing doxorubicin (SSL-DXR). SCID mice bearing low-passage patient-derived PaCA xenografts (PDX) were pretreated p.o. for 10 days with 40 mg/kg/d NVP-LDE225 (erismodegib), followed by i.v. SSL-DXR. Microvessel density, permeability, perfusion, and morphology were compared with untreated controls, as was SSL deposition and therapeutic efficacy. The sHHI alone affected tumor growth minimally, but markedly increased extravasation of nanoparticles into adenocarcinoma cell-enriched regions of the tumor. Immunostaining showed that sHHI treatment decreased pericyte coverage (α-SMA(+)) of CD31(+) vascular endothelium structures, and increased the abundance of endothelium-poor (CD31(-)) basement membrane structures (collagen IV(+)), suggesting increased immature microvessels. SSL-DXR (15 mg/kg) administered after sHHI pretreatment arrested tumor volume progression and decreased tumor perfusion/permeability, suggesting an initial vascular pruning response. Compared with controls, one cycle of 10-day sHHI pretreatment followed by 6 mg/kg SSL-DXR doubled median tumor progression time. Three cycles of treatment with sHHI and SSL-DXR, with a 10-day between-cycle drug holiday, nearly tripled median tumor progression time. Based upon these data, short-term sHHI treatment sequenced with nanoparticulate drug carriers constitutes a potential strategy to enhance efficacy of pancreatic cancer therapy

  2. Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in A549 cells.

    PubMed

    Li, Peng; Zhao, Qing-Li; Jawaid, Paras; Rehman, Mati Ur; Sakurai, Hiroaki; Kondo, Takashi

    2016-09-01

    KRAS mutant lung cancers have long been considered as untreatable with drugs. Transforming growth factor-β-activated kinase 1 (TAK1) appears to play an anti-apoptotic role in response to multiple stresses and has been reported to be a responsive kinase that regulates cell survival in KRAS-dependent cells. In this study, in order to find a useful approach to treat KRAS mutant lung cancer, we focused on the combined effects of 5Z-7-oxozeaenol, a TAK1 inhibitor, with hyperthermia (HT) in KRAS mutant lung cancer cell line A549. Annexin V-FITC/PI assay, cell cycle analysis, and colony formation assay revealed a significant enhancement in apoptosis induced by HT treatment, when the cells were pre-incubated with 5Z-7-oxozeaenol in a dose-dependent manner. The enhanced apoptosis by 5Z-7-oxozeaenol was accompanied by a significant increase in reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential (MMP). In addition, western blot showed that 5Z-7-oxozeaenol enhanced HT-induced expressions of cleaved caspase-3, cleaved caspase-8, and HSP70 and decreased HT-induced expressions of Bcl-2, p-p38, p-JNK, and LC3. Moreover, 5Z-7-oxozeaenol pre-treatment resulted in a marked elevation of intracellular calcium level which might be associated with endoplasmic reticulum (ER) stress-related pathway. Taken together, our data provides further insights of the mechanism of action of 5Z-7-oxozeaenol and HT treatment, and their potential application as a novel approache to treat patients with KRAS mutant lung cancer.

  3. Enhancement of glioblastoma radioresponse by a selective COX-2 inhibitor celecoxib: Inhibition of tumor angiogenesis with extensive tumor necrosis

    SciTech Connect

    Kang, Khong Bee . E-mail: dmskkb@nccs.com.sg; Wang, Ting Ting; Woon, Chow Thai; Cheah, Elizabeth S.; Moore, Xiao Lei; Zhu Congju; Wong, Meng Cheong

    2007-03-01

    Purpose: Toward improved glioblastoma multiforme treatment, we determined whether celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, could enhance glioblastoma radiosensitivity by inducing tumor necrosis and inhibiting tumor angiogenesis. Methods and Materials: U-87MG cells treated with celecoxib, irradiation, or both were assayed for clonogenic survival and angiogenic factor protein analysis (angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor [VEGF]). In vivo, survival of mice intracranially implanted with U-87MG cells and treated with celecoxib and/or irradiation was monitored. Isolated tumors were assessed for tumor necrosis and tumor microvascular density by von Williebrand's factor (vWF) immunohistochemical staining. Results: Celecoxib (4 and 30 {mu}M; 24, 48, and 72 h) enhanced U-87MG cell radiosensitivity by significantly reducing clonogenic survival of irradiated cells. Angiopoietin-1 and VEGF proteins were decreased, whereas angiopoietin-2 expression increased after 72 h of celecoxib alone and when combined with irradiation. In vivo, median survival of control mice intracranially implanted with U-87MG cells was 18 days. Celecoxib (100 mg/kg/day, 2 weeks) significantly extended median survival of irradiated mice (24 Gy total) from 34 to 41 days, with extensive tumor necrosis [24.5 {+-} 8.6% of tumor region, compared with irradiation alone (2.7 {+-} 1.8%)]. Tumor microvascular density was significantly reduced in combined celecoxib and irradiated tumors (52.5 {+-} 2.9 microvessels per mm{sup 2} tumor region), compared with irradiated tumors alone (65.4 {+-} 4.0 microvessels per mm{sup 2}). Conclusion: Celecoxib significantly enhanced glioblastoma radiosensitivity, reduced clonogenic survival, and prolonged survival of glioblastoma-implanted mice by inhibition of tumor angiogenesis with extensive tumor necr0010os.

  4. Tumor-Priming Smoothened Inhibitor Enhances Deposition and Efficacy of Cytotoxic Nanoparticles in a Pancreatic Cancer Model.

    PubMed

    Roy Chaudhuri, Tista; Straubinger, Ninfa L; Pitoniak, Rosemarie F; Hylander, Bonnie L; Repasky, Elizabeth A; Ma, Wen Wee; Straubinger, Robert M

    2016-01-01

    Most pancreatic adenocarcinoma patients present with unresectable disease and benefit little from chemotherapy. Poor tumor perfusion and vascular permeability limit drug deposition. Previous work showed that Smoothened inhibitors of hedgehog signaling (sHHI) promote neovascularization in spontaneous mouse models of pancreatic cancer (PaCA) and enhance tumor permeability to low-molecular weight compounds. Here, we tested the hypothesis that sHHI can enhance tumor deposition and efficacy of drug-containing nanoparticles consisting of 80 to 100 nm sterically-stabilized liposomes (SSL) containing doxorubicin (SSL-DXR). SCID mice bearing low-passage patient-derived PaCA xenografts (PDX) were pretreated p.o. for 10 days with 40 mg/kg/d NVP-LDE225 (erismodegib), followed by i.v. SSL-DXR. Microvessel density, permeability, perfusion, and morphology were compared with untreated controls, as was SSL deposition and therapeutic efficacy. The sHHI alone affected tumor growth minimally, but markedly increased extravasation of nanoparticles into adenocarcinoma cell-enriched regions of the tumor. Immunostaining showed that sHHI treatment decreased pericyte coverage (α-SMA(+)) of CD31(+) vascular endothelium structures, and increased the abundance of endothelium-poor (CD31(-)) basement membrane structures (collagen IV(+)), suggesting increased immature microvessels. SSL-DXR (15 mg/kg) administered after sHHI pretreatment arrested tumor volume progression and decreased tumor perfusion/permeability, suggesting an initial vascular pruning response. Compared with controls, one cycle of 10-day sHHI pretreatment followed by 6 mg/kg SSL-DXR doubled median tumor progression time. Three cycles of treatment with sHHI and SSL-DXR, with a 10-day between-cycle drug holiday, nearly tripled median tumor progression time. Based upon these data, short-term sHHI treatment sequenced with nanoparticulate drug carriers constitutes a potential strategy to enhance efficacy of pancreatic cancer therapy.

  5. Sildenafil, a selective phosphodiesterase type 5 inhibitor, enhances memory reconsolidation of an inhibitory avoidance task in mice.

    PubMed

    Boccia, M M; Blake, M G; Krawczyk, M C; Baratti, C M

    2011-07-01

    Intracellular levels of the second messengers cAMP and cGMP are maintained through a balance between production, carried out by adenyl cyclase (AC) and guanylyl cyclase (GC), and degradation, carried out by phosphodiesterases (PDEs). Recently, PDEs have gained increased attention as potential new targets for cognition enhancement, with particular reference to phosphodiesterase type 5 (PDE5A). It is accepted that once consolidation is completed memory becomes permanent, but it has also been suggested that reactivation (memory retrieval) of the original memory makes it sensitive to the same treatments that affect memory consolidation when given after training. This new period of sensitivity coined the term reconsolidation. Sildenafil (1, 3, and 10mg/kg, ip), a cGMP-PDE5 inhibitor, facilitated retention performance of a one-trial step-through inhibitory avoidance task, when administered to CF-1 male mice immediately after retrieval. The effects of sildenafil (1mg/kg, ip) were time-dependent, long-lasting and inversely correlated with memory age. The administration of sildenafil (1mg/kg, ip) 30 min prior to the 2nd retention test did not affect retention of mice given post-retrieval injections of either vehicle or sildenafil (1mg/kg, ip). Finally, an enhancement of retention was also observed in CF-1 female mice receiving sildenafil (1mg/kg, ip) immediately, but not 180 min after retrieval. In the present paper we reported for the first time that systemic administration of sildenafil after memory reactivation enhances retention performance of the original learning. Our results indirectly point out cGMP, a component of the NO/cGMP/PKG pathway, as a necessary factor for memory reconsolidation.

  6. Interaction of neomycin with ribosomes and ribosomal ribonucleic acid.

    PubMed

    Dahlberg, A E; Horodyski, F; Keller, P

    1978-02-01

    Neomycin binds ribosomes and ribosomal ribonucleic acid (rRNA) in vivo and in vitro producing changes detectable by increases in gel electrophoretic mobility. These changes were observed in gels that contain ethylenediaminetetraacetic acid or no added magnesium ion. The progressive increase in gel electrophoretic mobility with increasing antibiotic concentrations suggests that neomycin is binding at multiple sites on RNA. The binding was reversible but sufficiently stable to survive dialysis and electrophoresis. It is proposed that bound neomycin stabilizes the ribosome and RNA structures, restricting the unfolding of the particles during electrophoresis and thus allowing for a more rapid migration in the gel. Gentamicin produced an effect similar to that of neomycin. Paromomycin, differing from neomycin by only one amino group, had considerably less effect on ribosome and rRNA mobilities. The binding of neomycin to rRNA improved the linearity of the plot of log molecular weight versus mobility and thus may be of benefit in providing a more accurate estimation of molecular weights of large RNAs.

  7. Further characterization of ribosome binding to thylakoid membranes. [Pisum sativum

    SciTech Connect

    Hurewitz, J.; Jagendorf, A.T.

    1987-05-01

    Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo. With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation, and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of (/sup 3/H)leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.

  8. Taurine enhances antinociception produced by a COX-2 inhibitor in an inflammatory pain model.

    PubMed

    de Rienzo-Madero, Beatriz; Coffeen, Ulises; Simón-Arceo, Karina; Mercado, Francisco; Jaimes, Orlando; Magis-Weinberg, Lucía; Contreras, Bernardo; Pellicer, Francisco

    2013-06-01

    The temporal activation of the sensory systems, especially in pain, determines intermediate states that define the future of the response to sensory stimulation. In this work, we interfere pharmacologically with those states that produce peripheral and central sensitisation after an acute inflammatory process, inhibiting at the periphery the COX-2 with celecoxib and using taurine (glycine A receptor agonist) for central pain relief. We tested the paw withdrawal reflex latencies to thermo- and mechanonociception after the induction of an acute inflammatory process with carrageenan. Celecoxib at low doses [0.13 and 1.3 mg/kg, intraperitoneal (i.p.)] in combination with taurine (300 mg/kg, i.p.) produces a decrease of the nociceptive response in thermo- and mechanonociception, as compared with the effect of both drugs alone. We propose that the enhancement of the analgesic effect of celecoxib in combination with taurine could be due the simultaneous action of these drugs at both, peripheral and central levels.

  9. Design of a RANK-Mimetic Peptide Inhibitor of Osteoclastogenesis with Enhanced RANKL-Binding Affinity.

    PubMed

    Hur, Jeonghwan; Ghosh, Ambarnil; Kim, Kabsun; Ta, Hai Minh; Kim, Hyunju; Kim, Nacksung; Hwang, Hye-Yeon; Kim, Kyeong Kyu

    2016-04-30

    The receptor activator of nuclear factor κB (RANK) and its ligand RANKL are key regulators of osteoclastogenesis and well-recognized targets in developing treatments for bone disorders associated with excessive bone resorption, such as osteoporosis. Our previous work on the structure of the RANK-RANKL complex revealed that Loop3 of RANK, specifically the non-canonical disulfide bond at the tip, performs a crucial role in specific recognition of RANKL. It also demonstrated that peptide mimics of Loop3 were capable of interfering with the function of RANKL in osteoclastogenesis. Here, we reported the structure-based design of a smaller peptide with enhanced inhibitory efficiency. The kinetic analysis and osteoclast differentiation assay showed that in addition to the sharp turn induced by the disulfide bond, two consecutive arginine residues were also important for binding to RANKL and inhibiting osteoclastogenesis. Docking and molecular dynamics simulations proposed the binding mode of the peptide to the RANKL trimer, showing that the arginine residues provide electrostatic interactions with RANKL and contribute to stabilizing the complex. These findings provided useful information for the rational design of therapeutics for bone diseases associated with RANK/RANKL function.

  10. Design of a RANK-Mimetic Peptide Inhibitor of Osteoclastogenesis with Enhanced RANKL-Binding Affinity

    PubMed Central

    Hur, Jeonghwan; Ghosh, Ambarnil; Kim, Kabsun; Ta, Hai Minh; Kim, Hyunju; Kim, Nacksung; Hwang, Hye-Yeon; Kim, Kyeong Kyu

    2016-01-01

    The receptor activator of nuclear factor κB (RANK) and its ligand RANKL are key regulators of osteoclastogenesis and well-recognized targets in developing treatments for bone disorders associated with excessive bone resorption, such as osteoporosis. Our previous work on the structure of the RANK-RANKL complex revealed that Loop3 of RANK, specifically the non-canonical disulfide bond at the tip, performs a crucial role in specific recognition of RANKL. It also demonstrated that peptide mimics of Loop3 were capable of interfering with the function of RANKL in osteoclastogenesis. Here, we reported the structure-based design of a smaller peptide with enhanced inhibitory efficiency. The kinetic analysis and osteoclast differentiation assay showed that in addition to the sharp turn induced by the disulfide bond, two consecutive arginine residues were also important for binding to RANKL and inhibiting osteoclastogenesis. Docking and molecular dynamics simulations proposed the binding mode of the peptide to the RANKL trimer, showing that the arginine residues provide electrostatic interactions with RANKL and contribute to stabilizing the complex. These findings provided useful information for the rational design of therapeutics for bone diseases associated with RANK/RANKL function. PMID:26923188

  11. Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae.

    PubMed

    Chiabudini, Marco; Tais, Arlette; Zhang, Ying; Hayashi, Sachiko; Wölfle, Tina; Fitzke, Edith; Rospert, Sabine

    2014-11-01

    Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

  12. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  13. All Ribosomes Are Created Equal. Really?

    PubMed

    Preiss, Thomas

    2016-02-01

    Ribosomes are generally thought of as molecular machines with a constitutive rather than regulatory role during protein synthesis. A study by Slavov et al.[1] now shows that ribosomes of distinct composition and functionality exist within eukaryotic cells, giving credence to the concept of 'specialized' ribosomes.

  14. Mutations of highly conserved bases in the peptidyltransferase center induce compensatory rearrangements in yeast ribosomes.

    PubMed

    Rakauskaite, Rasa; Dinman, Jonathan D

    2011-05-01

    Molecular dynamics simulation identified three highly conserved rRNA bases in the large subunit of the ribosome that form a three-dimensional (3D) "gate" that induces pausing of the aa-tRNA acceptor stem during accommodation into the A-site. A nearby fourth base contacting the "tryptophan finger" of yeast protein L3, which is involved in the coordinating elongation factor recruitment to the ribosome with peptidyltransfer, is also implicated in this process. To better understand the functional importance of these bases, single base substitutions as well as deletions at all four positions were constructed and expressed as the sole forms of ribosomes in yeast Saccharomyces cerevisiae. None of the mutants had strong effects on cell growth, translational fidelity, or on the interactions between ribosomes and tRNAs. However, the mutants did promote strong effects on cell growth in the presence of translational inhibitors, and differences in viability between yeast and Escherichia coli mutants at homologous positions suggest new targets for antibacterial therapeutics. Mutant ribosomes also promoted changes in 25S rRNA structure, all localized to the core of peptidyltransferase center (i.e., the proto-ribosome area). We suggest that a certain degree of structural plasticity is built into the ribosome, enabling it to ensure accurate translation of the genetic code while providing it with the flexibility to adapt and evolve.

  15. Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6.

    PubMed

    Klinge, Sebastian; Voigts-Hoffmann, Felix; Leibundgut, Marc; Arpagaus, Sofia; Ban, Nenad

    2011-11-18

    Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.

  16. Calcium-dependent interaction of calmodulin with human 80S ribosomes and polyribosomes.

    PubMed

    Behnen, Petra; Davis, Elizabeth; Delaney, Erin; Frohm, Birgitta; Bauer, Mikael; Cedervall, Tommy; O'Connell, David; Åkerfeldt, Karin S; Linse, Sara

    2012-08-28

    Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.

  17. The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA.

    PubMed

    Ganapathi, Karthik A; Austin, Karyn M; Lee, Chung-Sheng; Dias, Anusha; Malsch, Maggie M; Reed, Robin; Shimamura, Akiko

    2007-09-01

    Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large ribosomal subunit in sucrose gradients and coprecipitates with 28S ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S ribosomal subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome.

  18. The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA

    PubMed Central

    Ganapathi, Karthik A.; Austin, Karyn M.; Lee, Chung-Sheng; Dias, Anusha; Malsch, Maggie M.; Reed, Robin

    2007-01-01

    Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large ribosomal subunit in sucrose gradients and coprecipitates with 28S ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S ribosomal subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome. PMID:17475909

  19. Decitabine priming enhances the antileukemic effects of exportin 1 (XPO1) selective inhibitor selinexor in acute myeloid leukemia.

    PubMed

    Ranganathan, Parvathi; Yu, Xueyan; Santhanam, Ramasamy; Hofstetter, Jessica; Walker, Alison; Walsh, Katherine; Bhatnagar, Bhavana; Klisovic, Rebecca; Vasu, Sumithira; Phelps, Mitch A; Devine, Steven; Shacham, Sharon; Kauffman, Michael; Marcucci, Guido; Blum, William; Garzon, Ramiro

    2015-04-23

    The prognosis of acute myeloid leukemia (AML) is poor, highlighting the need for novel treatments. Hypomethylating agents, including decitabine are used to treat elderly AML patients with relative success. Targeting nuclear export receptor (exportin 1 [XPO1]) is a novel approach to restore tumor suppressor (TS) function in AML. Here, we show that sequential treatment of AML blasts with decitabine followed by selinexor (XPO1 inhibitor) enhances the antileukemic effects of selinexor. These effects could be mediated by the re-expression of a subset of TSs (CDKN1A and FOXO3A) that are epigenetically silenced via DNA methylation, and cytoplasmic-nuclear trafficking is regulated by XPO1. We observed a significant upregulation of CDKN1A and FOXO3A in decitabine- versus control-treated cells. Sequential treatment of decitabine followed by selinexor in an MV4-11 xenograft model significantly improved survival compared with selinexor alone. On the basis of these preclinical results, a phase 1 clinical trial of decitabine followed by selinexor in elderly patients with AML has been initiated.

  20. Avasimibe, an ACAT inhibitor, enhances the lipid lowering effect of atorvastatin in subjects with homozygous familial hypercholesterolemia.

    PubMed

    Raal, Frederick J; Marais, A David; Klepack, Ellen; Lovalvo, Jennifer; McLain, Richard; Heinonen, Therese

    2003-12-01

    This study assessed the efficacy and safety of avasimibe (CI-1011), an inhibitor of acyl coenzyme A-cholesterol acyltransferase (ACAT) in subjects with homozygous familial hypercholesterolemia (HoFH). Twenty seven subjects were enrolled in a double-blind, randomized, 3-sequence crossover trial of atorvastatin 80 mg QD, avasimibe 750 mg QD, and the combined treatment of atorvastatin 80 mg QD and avasimibe 750 mg QD after a washout period of 4 weeks. Each treatment period was administered over 6 weeks for a total of 18 weeks. There were no significant lipid changes resulting from the administration of avasimibe monotherapy. Avasimibe in combination with atorvastatin resulted in a significantly better reduction of total cholesterol (TC) as compared to atorvastatin alone (-22% versus -18%) (P < 0.05). All other lipid changes were not statistically significant for combination therapy compared to atorvastatin monotherapy, however there were greater reductions in triglycerides (TG) (-24% versus -13%), low-density lipoprotein cholesterol (LDL-C) (-23% versus -19%), very low-density lipoprotein cholesterol (VLDL-C) (-24% versus -13%) and high-density lipoprotein cholesterol (HDL-C) (-11% versus -6%). Avasimibe may modestly enhance the lipid-reducing effect of atorvastatin by further inhibiting the production of intracellular cholesterol through mechanisms that appear to be compatible in this population. PMID:14644397

  1. Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI

    PubMed Central

    Puy, Cristina; Tucker, Erik I.; Ivanov, Ivan S.; Gailani, David; Smith, Stephanie A.; Morrissey, James H.; Gruber, András; McCarty, Owen J. T.

    2016-01-01

    Introduction Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Methods and Results Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Conclusions Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP. PMID:27764259

  2. Decitabine priming enhances the antileukemic effects of exportin 1 (XPO1) selective inhibitor selinexor in acute myeloid leukemia

    PubMed Central

    Ranganathan, Parvathi; Yu, Xueyan; Santhanam, Ramasamy; Hofstetter, Jessica; Walker, Alison; Walsh, Katherine; Bhatnagar, Bhavana; Klisovic, Rebecca; Vasu, Sumithira; Phelps, Mitch A.; Devine, Steven; Shacham, Sharon; Kauffman, Michael; Marcucci, Guido; Blum, William

    2015-01-01

    The prognosis of acute myeloid leukemia (AML) is poor, highlighting the need for novel treatments. Hypomethylating agents, including decitabine are used to treat elderly AML patients with relative success. Targeting nuclear export receptor (exportin 1 [XPO1]) is a novel approach to restore tumor suppressor (TS) function in AML. Here, we show that sequential treatment of AML blasts with decitabine followed by selinexor (XPO1 inhibitor) enhances the antileukemic effects of selinexor. These effects could be mediated by the re-expression of a subset of TSs (CDKN1A and FOXO3A) that are epigenetically silenced via DNA methylation, and cytoplasmic-nuclear trafficking is regulated by XPO1. We observed a significant upregulation of CDKN1A and FOXO3A in decitabine- versus control-treated cells. Sequential treatment of decitabine followed by selinexor in an MV4-11 xenograft model significantly improved survival compared with selinexor alone. On the basis of these preclinical results, a phase 1 clinical trial of decitabine followed by selinexor in elderly patients with AML has been initiated. PMID:25716206

  3. Histone Deacetylase Inhibitors Enhance CD4 T Cell Susceptibility to NK Cell Killing but Reduce NK Cell Function

    PubMed Central

    Pace, Matthew; Williams, James; Kurioka, Ayako; Gerry, Andrew B.; Jakobsen, Bent; Klenerman, Paul; Nwokolo, Nneka; Fox, Julie

    2016-01-01

    In the search for a cure for HIV-1 infection, histone deacetylase inhibitors (HDACi) are being investigated as activators of latently infected CD4 T cells to promote their targeting by cytotoxic T-lymphocytes (CTL). However, HDACi may also inhibit CTL function, suggesting different immunotherapy approaches may need to be explored. Here, we study the impact of different HDACi on both Natural Killer (NK) and CTL targeting of HIV-1 infected cells. We found HDACi down-regulated HLA class I expression independently of HIV-1 Nef which, without significantly compromising CTL function, led to enhanced targeting by NK cells. HDACi-treated HIV-1-infected CD4 T cells were also more effectively cleared than untreated controls during NK co-culture. However, HDACi impaired NK function, reducing degranulation and killing capacity. Depending on the HDACi and dose, this impairment could counteract the benefit gained by treating infected target cells. These data suggest that following HDACi-induced HLA class I down-regulation NK cells kill HIV-1-infected cells, although HDACi-mediated NK cell inhibition may negate this effect. Our data emphasize the importance of studying the effects of potential interventions on both targets and effectors. PMID:27529554

  4. A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens

    NASA Astrophysics Data System (ADS)

    Baek, Kyung-Hwa; Zhang, Haiying; Lee, Bo Ryeong; Kwon, Young-Guen; Ha, Sang-Jun; Shin, Injae

    2015-12-01

    The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

  5. Cap-dependent translation is mediated by 'RNA looping' rather than 'ribosome scanning'.

    PubMed

    Jang, Sung Key; Paek, Ki Young

    2016-01-01

    The 40S ribosomal subunit cannot directly recognize the start codon of eukaryotic mRNAs. Instead, it recognizes the start codon after its association with the 5'-cap structure via translation initiation factors. Base-by-base inspection of the 5'UTR by a scanning ribosome is the generally accepted hypothesis of start codon selection. As part of an effort to confirm the underlying mechanism of start codon selection by the 40S ribosome, we investigated the role of eIF4G, which participates in the recruitment of 40S ribosomes to various translation enhancers, such as 5'-cap structure, poly(A) tail, and several internal ribosome entry sites. We found that an artificial translation factor composed of recombinant eIF4G fused with MS2 greatly enhanced translation of an upstream reporter gene when it was tethered to the 3'UTR. These data suggest that the 40S ribosome recruited to a translation enhancer can find the start codon by looping of the intervening RNA segment. The 'RNA-looping' hypothesis of translation start codon recognition was further supported by an analysis of the effect of 5'UTR length on translation efficiency and the mathematically predicted probability of RNA-loop-mediated interactions between the start codon and the 40S ribosome associated at the 5'-end. PMID:26515582

  6. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

    PubMed Central

    Perdigão, João; Couto, Isabel; Portugal, Isabel; Martins, Marta; Amaral, Leonard; Anes, Elsa; Viveiros, Miguel

    2016-01-01

    Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their

  7. Biofilm production by Zymomonas mobilis enhances ethanol production and tolerance to toxic inhibitors from rice bran hydrolysate.

    PubMed

    Todhanakasem, Tatsaporn; Sangsutthiseree, Atit; Areerat, Kamonchanok; Young, Glenn M; Thanonkeo, Pornthap

    2014-09-25

    Microorganisms play a significant role in bioethanol production from lignocellulosic material. A challenging problem in bioconversion of rice bran is the presence of toxic inhibitors in lignocellulosic acid hydrolysate. Various strains of Zymomonas mobilis (ZM4, TISTR 405, 548, 550 and 551) grown under biofilm or planktonic modes were used in this study to examine their potential for bioconversion of rice bran hydrolysate and ethanol production efficiencies. Z. mobilis readily formed bacterial attachment on plastic surfaces, but not on glass surfaces. Additionally, the biofilms formed on plastic surfaces steadily increased over time, while those formed on glass were speculated to cycle through accumulation and detachment phases. Microscopic analysis revealed that Z. mobilis ZM4 rapidly developed homogeneous biofilm structures within 24 hours, while other Z. mobilis strains developed heterogeneous biofilm structures. ZM4 biofilms were thicker and seemed to be more stable than other Z. mobilis strains. The percentage of live cells in biofilms was greater than that for planktonic cells (54.32 ± 7.10% vs. 28.69 ± 3.03%), suggesting that biofilms serve as a protective niche for growth of bacteria in the presence of toxic inhibitors in the rice bran hydrolysate. The metabolic activity of ZM4 grown as a biofilm was also higher than the same strain grown planktonically, as measured by ethanol production from rice bran hydrolysate (13.40 ± 2.43 g/L vs. 0.432 ± 0.29 g/L, with percent theoretical ethanol yields of 72.47 ± 6.13% and 3.71 ± 5.24% respectively). Strain TISTR 551 was also quite metabolically active, with ethanol production by biofilm and planktonically grown cells of 8.956 ± 4.06 g/L and 0.0846 ± 0.064 g/L (percent theoretical yields were 48.37 ± 16.64% and 2.046 ± 1.58%, respectively). This study illustrates the potential for enhancing ethanol production by utilizing bacterial biofilms in the bioconversion of a readily available and normally unusable

  8. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis.

    PubMed

    Machado, Diana; Pires, David; Perdigão, João; Couto, Isabel; Portugal, Isabel; Martins, Marta; Amaral, Leonard; Anes, Elsa; Viveiros, Miguel

    2016-01-01

    Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their

  9. Autophagy inhibition enhances colorectal cancer apoptosis induced by dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235

    PubMed Central

    YANG, XIAOYU; NIU, BINGXUAN; WANG, LIBO; CHEN, MEILING; KANG, XIAOCHUN; WANG, LUONAN; JI, YINGHUA; ZHONG, JIATENG

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway performs a central role in tumorigenesis and is constitutively activated in many malignancies. As a novel dual PI3K/mTOR inhibitor currently undergoing evaluation in a phase I/II clinical trial, NVP-BEZ235 indicates a significant antitumor efficacy in diverse solid tumors, including colorectal cancer (CRC). Autophagy is a catabolic process that maintains cellular homeostasis and reduces diverse stresses through lysosomal recycling of the unnecessary and damaged cell components. This process is also observed to antagonize the antitumor efficacy of PI3K/mTOR inhibitor agents such as NVP-BEZ235, via apoptosis inhibition. In the present study, we investigated anti-proliferative and apoptosis-inducing ability of NVP-BEZ235 in SW480 cells and the crosstalk between autophagy and apoptosis in SW480 cells treated with NVP-BEZ235 in combination with an autophagy inhibitor. The results revealed that, NVP-BEZ235 effectively inhibit the growth of SW480 cells by targeting the PI3K/mTOR signaling pathway and induced apoptosis. The inhibition of autophagy with 3-methyladenine or chloroquine inhibitors in combination with NVP-BEZ235 in SW480 cells enhanced the apoptotic rate as componets to NVP-BEZ235 alone. In conclusion, the findings provide a rationale for chemotherapy targeting the PI3K/mTOR signaling pathway presenting a potential therapeutic strategy to enhance the efficacy of dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with an autophagy inhibitor in CRC treatment and treatment of other tumors. PMID:27347108

  10. 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

    2015-10-01

    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.

  11. Alcoholic Liver Disease and the Mitochondrial Ribosome

    PubMed Central

    Cahill, Alan; Sykora, Peter

    2009-01-01

    Summary Chronic alcohol consumption has been shown to severely compromise mitochondrial protein synthesis. Hepatic mitochondria isolated from alcoholic animals contain decreased levels of respiratory complexes and display depressed respiration rates when compared to pair-fed controls. One underlying mechanism for this involves ethanol-elicited alterations in the structural and functional integrity of the mitochondrial ribosome. Ethanol feeding results in ribosomal changes that include decreased sedimentation rates, larger hydrodynamic volumes, increased levels of unassociated subunits and changes in the levels of specific ribosomal proteins. The methods presented in this chapter detail how to isolate mitochondrial ribosomes, determine ribosomal activity, separate ribosomes into nucleic acid and protein, and perform two-dimensional nonequilibrium pH gradient electrophoretic polyacrylamide gel electrophoresis to separate and subsequently identify mitochondrial ribosomal proteins. PMID:18369931

  12. The Synthesis of Ribosomes in E. coli

    PubMed Central

    Britten, R. J.; McCarthy, B. J.; Roberts, R. B.

    1962-01-01

    The incorporation of C14 leucine into the protein moiety of ribosomes has been studied as a sequel to the studies of ribosomal RNA synthesis. In contrast to the latter studies, labeled leucine is incorporated directly into 50S and 30S ribosomes without measurable delay by precursor stages. There is, however, evidence of some transfer of radioactivity from the 43S group of particles to the 50S. The inhibition of protein synthesis by chloramphenicol results in the accumulation of material similar to the eosome—the primary precursor in ribosome synthesis. There is also evidence for the synthesis of some neosome. The results of the studies of ribosomal RNA and protein synthesis are combined into a model of ribosome synthesis. Finally, consideration is made of the significance of these studies of ribosome synthesis for general problems of protein synthesis and information transfer. PMID:13873182

  13. Crystal structure of the eukaryotic ribosome.

    PubMed

    Ben-Shem, Adam; Jenner, Lasse; Yusupova, Gulnara; Yusupov, Marat

    2010-11-26

    Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

  14. 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.

  15. Pathways to Specialized Ribosomes: The Brussels Lecture.

    PubMed

    Dinman, Jonathan D

    2016-05-22

    "Specialized ribosomes" is a topic of intense debate and research whose provenance can be traced to the earliest days of molecular biology. Here, the history of this idea is reviewed, and critical literature in which the specialized ribosomes have come to be presently defined is discussed. An argument supporting the evolution of a variety of ribosomes with specialized functions as a consequence of selective pressures acting on a near-infinite set of possible ribosomes is presented, leading to a discussion of how this may also serve as a biological buffering mechanism. The possible relationship between specialized ribosomes and human health is explored. A set of criteria and possible approaches are also presented to help guide the definitive identification of "specialized" ribosomes, and this is followed by a discussion of how synthetic biology approaches might be used to create new types of special ribosomes.

  16. 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.

  17. [About the ribosomal biogenesis in human].

    PubMed

    Tafforeau, Lionel

    2015-01-01

    Ribosomes are cellular ribonucleoprotein particles required for a fundamental mechanism, translation of the genetic information into proteins. Ribosome biogenesis is a highly complex pathway involving many maturation steps: ribosomal RNA (rRNA) synthesis, rRNA processing, pre-rRNA modifications, its assembly with ribosomal proteins in the nuceolus, export of the subunit precursors to the nucleoplasm and the cytoplasm. Ribosome biogenesis has mainly being investigated in yeast during these last 25 years. However, recent works have shown that, despite many similarities between yeast and human ribosome structure and biogenesis, human pre-rRNA processing is far more complex than in yeast. In order to better understand diseases related to a malfunction in ribosome synthesis, the ribosomopathies, research should be conducted directly in human cells and animal models. PMID:26152166

  18. Pathways to Specialized Ribosomes: The Brussels Lecture.

    PubMed

    Dinman, Jonathan D

    2016-05-22

    "Specialized ribosomes" is a topic of intense debate and research whose provenance can be traced to the earliest days of molecular biology. Here, the history of this idea is reviewed, and critical literature in which the specialized ribosomes have come to be presently defined is discussed. An argument supporting the evolution of a variety of ribosomes with specialized functions as a consequence of selective pressures acting on a near-infinite set of possible ribosomes is presented, leading to a discussion of how this may also serve as a biological buffering mechanism. The possible relationship between specialized ribosomes and human health is explored. A set of criteria and possible approaches are also presented to help guide the definitive identification of "specialized" ribosomes, and this is followed by a discussion of how synthetic biology approaches might be used to create new types of special ribosomes. PMID:26764228

  19. [Structure and function of the eukaryotic ribosome].

    PubMed

    Bakowska-Zywicka, Kamilla; Twardowski, Tomasz

    2008-01-01

    The protein biosynthesis is a complicated process and not fully understood yet. According to smaller size and less complicated structure, understanding of prokaryotic 70S ribosomes is much more advanced. Eucaryotic 80S ribosomes are more complex and generate more difficulties in research. The morphology of 80S ribosome has been pretty well resolved and we know a lot about mechanism of functioning. Determination of the interactions between the ribosomes and the factors taking part in protein biosynthesis is still a great challenge. Dynamic changes of these interactions during particular steps of elongation cycle are quite difficult to understand. Conformational changes of the ribosome are of great functional and regulatory importance during protein biosynthesis. They are essential for the whole gene expression process. Only further research of the structure and function of the ribosome will lead us to knowledge about specificity of the mechanism of their action. In this article we present current opinions concerning structure and function of the eukaryotic ribosomes.

  20. Histone Deacetylase Inhibitors Enhance the Apoptotic Activity of Insulin-Like Growth Factor Binding Protein-3 by Blocking PKC-Induced IGFBP-3 Degradation

    PubMed Central

    Oh, Seung Hyun; Whang, Young Mi; Min, Hye-Young; Han, Seung Ho; Kang, Ju-Hee; Song, Ki-Hoon; Glisson, Bonnie S.; Kim, Yeul Hong; Lee, Ho-Young

    2012-01-01

    Overexpression of insulin-like growth factor binding protein (IGFBP)-3 induces apoptosis of cancer cells. However, preexisting resistance to IGFBP-3 could limit its antitumor activities. This study characterizes the efficacy and mechanism of the combination of recombinant IGFBP-3 (rIGFBP-3) and HDAC inhibitors to overcome IGFBP-3 resistance in a subset of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cells. The effects of the combination of rIGFBP-3 and a number of HDAC inhibitors on cell proliferation and apoptosis were assessed in vitro and in vivo by using the MTT assay, a flow cytometry-based TUNEL assay, western blot analyses, and the NSCLC xenograft tumor model. Combined treatment with HDAC inhibitors and rIGFBP-3 had synergistic antiproliferative effects accompanied by increased apoptosis rates in a subset of NSCLC and HNSCC cell lines in vitro. Moreover, combined treatment with depsipeptide and rIGFBP-3 completely suppressed tumor growth and increased the apoptosis rate in vivo in H1299 NSCLC xenografts. Evidence suggests that HDAC inhibitors increased the half-life of rIGFBP-3 protein by blocking protein kinase C (PKC)-mediated phosphorylation and degradation of rIGFBP-3. In addition, combined treatment of IGFBP-3 with an HDAC inhibitor facilitates apoptosis through up-regulation of rIGFBP-3 stability and Akt signaling inhibition. The ability of HDAC inhibitors to decrease PKC activation may enhance apoptotic activities of rIGFBP-3 in NSCLC cells in vitro and in vivo. These results indicated that combined treatment with HDAC inhibitor and rIGFBP-3 could be an effective treatment strategy for NSCLC and HNSCC with highly activated PKC. PMID:22362554

  1. Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

    SciTech Connect

    Ho, Meng-Chiao; Sturm, Matthew B.; Almo, Steven C.; Schramm, Vern L.

    2010-01-12

    Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple {pi}-stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the {pi}-stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H{sub 2}O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.

  2. Intestinal transmission of macromolecules (BSA and FITC-dextran) in the neonatal pig: enhancing effect of colostrum, proteins and proteinase inhibitors.

    PubMed

    Weström, B R; Ohlsson, B G; Svendsen, J; Tagesson, C; Karlsson, B W

    1985-01-01

    The effects of colostrum and constituents/factors in colostrum which may influence intestinal macromolecular transmission in the newborn preclosure pig were investigated. Unsuckled piglets were given, by use of a stomach tube, bovine serum albumin (BSA) and fluorescein-isothiocyanate (FITC)-labelled dextran 70,000 (FITC-D) as markers together with colostrum or the factors under study. The serum levels of BSA and FITC-D 4 h after feeding were then determined as a measure of the transfer. It was found that the two colostrums tested, bovine and especially porcine, markedly enhanced the transmission of both BSA and FITC-D. Furthermore, increasing amounts of the model proteins, BSA and bovine IgG (50-200 mg/ml), significantly increased the transfer of FITC-D, whereas unlabelled dextran 70,000 given in similar amounts did not. Proteinase inhibitors obtained from sow colostrum or soy bean also enhanced the transmission of both BSA and FITC-D while the inactive inhibitors, given as trypsin-inhibitor complexes, had no effect. On the other hand, addition of a proteinase, porcine trypsin, significantly decreased the transmission of FITC-D. These findings indicate that the intestinal transmission of macromolecules in the preclosure piglet is governed by the amount of protein available in the intestine. Therefore, feeding colostrum with a high protein content and proteinase inhibitors is likely to favour efficient intestinal transmission, although other colostrum factors may also be of importance.

  3. Interrelationships between Yeast Ribosomal Protein Assembly Events and Transient Ribosome Biogenesis Factors Interactions in Early Pre-Ribosomes

    PubMed Central

    Jakob, Steffen; Ohmayer, Uli; Neueder, Andreas; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Hochmuth, Eduard; Deutzmann, Rainer; Griesenbeck, Joachim; Tschochner, Herbert; Milkereit, Philipp

    2012-01-01

    Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains. PMID:22431976

  4. Enhanced release of plasminogen activator inhibitor(s) but not of plasminogen activators by cultured rat glial cells treated with interleukin-1.

    PubMed

    Rogister, B; Leprince, P; Delree, P; Van Damme, J; Billiau, A; Moonen, G

    1990-01-01

    Astroglial cells are known to proliferate during development of the nervous system, as well as during post-traumatic gliosis. We have previously shown that the proliferation of cultured astrocytes can be stimulated by the urokinase-type (uPA) of plasminogen activator (PA) and that astrocytes are able to release such uPA upon stimulation with basic fibroblast growth factor, which is known to act as a mitogen for these cells. Here we report studies on the effects of human interleukin-1 (IL-1) on the release of PA activity by cultured newborn rat astroglial cells. Whereas there is controversy in the literature as to whether IL-1 stimulates multiplication of astroglial cells, we failed to observe such an effect in our system. We did observe, however, a dose-dependent decrease in PA activity in the supernatant of the IL-1 treated cultures. Further analysis revealed that this apparent decrease in PA release was in fact due to an increased release of plasminogen activator inhibitor (PAI). A similar IL-1 induced increase in PAI release was also found to occur in cultures of transformed astrocytes (human glioma LN18) and in cultured Schwann cells, but not in cultures of neurons or neuronal tumour cells. Since protease inhibitors are known to possess neuritogenic properties, our results suggest that IL-1, by its capacity to induce PAI, may promote neuritogenesis.

  5. S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

    PubMed Central

    Liu, Tsang-Pai; Lo, Hsiang-Ling; Wei, Li-Shan; Hao-yun Hsiao, Heidi

    2015-01-01

    The enhancer of zeste homolog 2 (EZH2) has emerged as a novel anticancer target. Various EZH2 inhibitors have been developed in recent years. Among these, 3-deazaneplanocin A (DZNep) is known to deplete EZH2 protein expression through an indirect pathway. In contrast, GSK343 directly inhibits enzyme activity through an S-adenosyl-l-methionine-competitive pathway. Therefore, we proposed that DZNep and GSK343 may exert differential effects against cancer cells. In this study, we found that GSK343 but not DZNep induced autophagic cell death of cancer cells. Inhibition of EZH2 expression was not required for GSK343-induced autophagy. In addition, GSK343 enhanced the anticancer activity of a multikinase inhibitor, sorafenib, in human hepatocellular carcinoma cells. Our results show that GSK343 is a more potent anticancer agent than DZNep, and for the first time, we show that it acts as an autophagy inducer. PMID:25203626

  6. S-Adenosyl-L-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells.

    PubMed

    Liu, Tsang-Pai; Lo, Hsiang-Ling; Wei, Li-Shan; Hsiao, Heidi Hao-yun; Yang, Pei-Ming

    2015-02-01

    The enhancer of zeste homolog 2 (EZH2) has emerged as a novel anticancer target. Various EZH2 inhibitors have been developed in recent years. Among these, 3-deazaneplanocin A (DZNep) is known to deplete EZH2 protein expression through an indirect pathway. In contrast, GSK343 directly inhibits enzyme activity through an S-adenosyl-L-methionine-competitive pathway. Therefore, we proposed that DZNep and GSK343 may exert differential effects against cancer cells. In this study, we found that GSK343 but not DZNep induced autophagic cell death of cancer cells. Inhibition of EZH2 expression was not required for GSK343-induced autophagy. In addition, GSK343 enhanced the anticancer activity of a multikinase inhibitor, sorafenib, in human hepatocellular carcinoma cells. Our results show that GSK343 is a more potent anticancer agent than DZNep, and for the first time, we show that it acts as an autophagy inducer.

  7. Phylogenomics of Prokaryotic Ribosomal Proteins

    PubMed Central

    Yutin, Natalya; Puigbò, Pere; Koonin, Eugene V.; Wolf, Yuri I.

    2012-01-01

    Archaeal and bacterial ribosomes contain more than 50 proteins, including 34 that are universally conserved in the three domains of cellular life (bacteria, archaea, and eukaryotes). Despite the high sequence conservation, annotation of ribosomal (r-) protein genes is often difficult because of their short lengths and biased sequence composition. We developed an automated computational pipeline for identification of r-protein genes and applied it to 995 completely sequenced bacterial and 87 archaeal genomes available in the RefSeq database. The pipeline employs curated seed alignments of r-proteins to run position-specific scoring matrix (PSSM)-based BLAST searches against six-frame genome translations, mitigating possible gene annotation errors. As a result of this analysis, we performed a census of prokaryotic r-protein complements, enumerated missing and paralogous r-proteins, and analyzed the distributions of ribosomal protein genes among chromosomal partitions. Phyletic patterns of bacterial and archaeal r-protein genes were mapped to phylogenetic trees reconstructed from concatenated alignments of r-proteins to reveal the history of likely multiple independent gains and losses. These alignments, available for download, can be used as search profiles to improve genome annotation of r-proteins and for further comparative genomics studies. PMID:22615861

  8. 3-aminobenzamide, a poly (ADP ribose) polymerase inhibitor, enhances wound healing in whole body gamma irradiated model.

    PubMed

    El-Hamoly, Tarek; El-Denshary, Ezzeddin S; Saad, Shokry Mohamed; El-Ghazaly, Mona A

    2015-09-01

    The custom use of radiotherapy was found to participate in the development of chronic unhealed wounds. In general, exposure to gamma radiation stimulates the production of reactive oxygen species (ROS) that eventually leads to damaging effect. Conversely, overexpression of a nuclear poly (ADP-ribose) polymerase enzyme (PARP) after oxidative insult extremely brings about cellular injury due to excessive consumption of NAD and ATP. Here, we dedicated our study to investigate the role of 3-aminobenzamide (3-AB), a PARP inhibitor, on pregamma irradiated wounds. Two full-thickness (6 mm diameter) wounds were created on the dorsum of Swiss albino mouse. The progression of wound contraction was monitored by capturing daily photo images. Exposure to gamma radiation (6Gy) exacerbated the normal healing of excisional wounds. Remarkably, topical application of 3-AB cream (50 µM) revealed a marked acceleration in the rate of wound contraction. Likewise, PARP inhibition ameliorated the unbalanced oxidative/nitrosative status of granulated skin tissues. Such effect was significantly revealed by the correction of the reduced antioxidant capacity and the enhanced lipid peroxidation, hydrogen peroxide, and myeloperoxidase contents. Moreover, application of 3-AB modified the cutaneous nitrite content throughout healing process. Conversely, the expressions of pro-inflammatory cytokines were down-regulated by PARP inhibition. The mitochondrial ATP content showed a lower consumption rate on 3-AB-treated wound bed as well. In parallel, the mRNA expressions of Sirt-1 and acyl-COA oxidase-2 (ACOX-2) were up-regulated; whom functions control the mitochondrial ATP synthesis and lipid metabolism. The current data suggested that inhibition of PARP-1 enzyme may accelerate the delayed wound healing in whole body gamma irradiated mice by early modifying the oxidative stress as well as the inflammatory response. PMID:26080614

  9. In Vitro and In Vivo Enhancement of Chemoradiation Using the Oral PARP Inhibitor ABT-888 in Colorectal Cancer Cells

    SciTech Connect

    Shelton, Joseph W.; Waxweiler, Timothy V.; Landry, Jerome; Gao, Huiying; Xu, Yanbo; Wang, Lanfang; El-Rayes, Bassel; Shu, Hui-Kuo G.

    2013-07-01

    Purpose: Poly(ADP-ribose) polymerase plays a critical role in the recognition and repair of DNA single-strand breaks and double-strand breaks (DSBs). ABT-888 is an orally available inhibitor of this enzyme. This study seeks to evaluate the use of ABT-888 combined with chemotherapy and radiation therapy (RT) in colorectal carcinoma models. Methods and Materials: RT clonogenic assays were performed on HCT116 and HT29 cells treated with 5-fluorouracil, irinotecan, or oxaliplatin with or without ABT. The surviving fraction at 2 Gy and dose-modifying factor at 10% survival were analyzed. Synergism was assessed by isobologram analysis for combination therapies. γH2AX and neutral comet assays were performed to assess the effect of therapy on DSB formation/repair. In vivo assessments were made by use of HCT116 cells in a xenograft mouse model. Tumor growth delay was measured at a volume of 500 mm{sup 3}. Results: Both lines were radiosensitized by ABT alone, and ABT further increased chemotherapy dose-modifying factors to the 1.6 to 1.8 range. All combinations were synergistic (combination indices <0.9). ABT treatment significantly increased DSB after RT (γH2AX, 69% vs 43%; P=.017) and delayed repair. We found tumor growth delays of 7.22 days for RT; 11.90 days for RT and ABT; 13.5 days for oxaliplatin, RT, and ABT; 14.17 days for 5-fluorouracil, RT, and ABT; and 23.81 days for irinotecan, RT, and ABT. Conclusion: ABT-888 radiosensitizes at similar or higher levels compared with classic chemotherapies and acts synergistically with these chemotherapies to enhance RT effects. In vivo confirmation of these results indicates a potential role for combining its use with existing chemoradiation regimens.

  10. Neutron scattering in the ribosome structure

    NASA Astrophysics Data System (ADS)

    Serdyuk, Igor N.

    1997-02-01

    Thermal neutron scattering has become a powerful instrument for studying the ribosome and its components. The application of neutron scattering allowed to establish some principal features of the ribosome structure: non-homogeneous distribution of the RNA and protein within ribosomal particles, the RNA role as a framework in the arrangement and maintenance of the structure of ribosomal particles, and the globular character of ribosomal proteins. The use of selective deuteration of separate ribosomal proteins in combination with the triangulation method revealed mutual spatial arrangement (the 3D-map) of all the ribosomal proteins within the small particle and in the most part of the large ribosomal particle. An essential impact has been made in the structural studies of ribosomes with the development of novel experimental approaches: triple isotopic substitution and spin contrast variation. These approaches with direct interpretation of spherical harmonics provide new possibilities for constructing models of ribosomal particles, opening principally new perspectives for joint use of X-ray synchrotron diffraction in crystals and small-angle neutron scattering in solution.

  11. Ribosome engineering to promote new crystal forms

    SciTech Connect

    Selmer, Maria; Gao, Yong-Gui; Weixlbaumer, Albert; Ramakrishnan, V.

    2012-05-01

    Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

  12. Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

    SciTech Connect

    Ahmed, Salahuddin; Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V.; Bhansali, Pravin; Tillekeratne, L.M. Viranga

    2013-07-15

    In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

  13. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32

  14. Inhibition of JNK-mediated autophagy enhances NSCLC cell sensitivity to mTORC1/2 inhibitors

    PubMed Central

    Jin, Hyeon-Ok; Hong, Sung-Eun; Park, Jin-Ah; Chang, Yoon Hwan; Hong, Young Jun; Park, In-Chul; Lee, Jin Kyung

    2016-01-01

    As the activation of autophagy contributes to the efficacy of many anticancer therapies, deciphering the precise role of autophagy in cancer therapy is critical. Here, we report that the dual mTORC1/2 inhibitors PP242 and OSI-027 decreased cell viability but did not induce apoptosis in the non-small cell lung cancer (NSCLC) cell lines H460 and A549. PP242 induced autophagy in NSCLC cells as demonstrated by the formation of massive vacuoles and acidic vesicular organelles and the accumulation of LC3-II. JNK was activated by PP242, and PP242-induced autophagy was blocked by inhibiting JNK pathway with SP600125 or JNK siRNA, suggesting that JNK activation is required for the mTORC1/2 inhibitor-mediated induction of autophagy in NSCLC cells. Inhibiting JNK or autophagy increased the sensitivity of H460 cells to mTORC1/2 inhibitors, indicating that JNK or autophagy promoted survival in NSCLC cells treated with mTORC1/2 inhibitors. Together, these data suggest that combining mTORC1/2 inhibitors with inhibitors of JNK or autophagy might be an effective approach for improving therapeutic outcomes in NSCLC. PMID:27358039

  15. Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts.

    PubMed

    Ahmed, Salahuddin; Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V; Bhansali, Pravin; Tillekeratne, L M Viranga

    2013-07-15

    In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1-5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5-5 μM) inhibited the constitutive expression of HDAC1 (0-30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ~220% with a concomitant decrease in HDAC5 [30-58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α+LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA.

  16. RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis

    PubMed Central

    Goto, Simon; Kato, Shingo; Kimura, Takatsugu; Muto, Akira; Himeno, Hyouta

    2011-01-01

    RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level. PMID:21102555

  17. Ribosome-associated protein quality control

    PubMed Central

    Brandman, Onn; Hegde, Ramanujan S

    2016-01-01

    Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation. PMID:26733220

  18. Seeing is Believing in Ribosome Assembly.

    PubMed

    Warner, Jonathan R

    2016-07-14

    Many proteins have been implicated genetically and biochemically in the assembly of eukaryotic ribosomes. Now, Kornprobst et al. show us how they are put together with a cryoEM structure of the 90S processome that initiates ribosome assembly, revealing the arrangement of U3 RNA and the several UTP complexes that form a chaperone-like structure around and within the developing 40S ribosomal subunit. PMID:27419867

  19. Enhanced anti-tumor activity induced by adoptive T cell transfer and the adjunctive use of the HDAC Inhibitor LAQ824

    PubMed Central

    Vo, Dan D.; Prins, Robert M.; Begley, Jonathan L.; Donahue, Timothy R.; Morris, Lilah F.; Bruhn, Kevin W.; de la Rocha, Pilar; Yang, Meng-Yin; Mok, Stephen; Garban, Hermes J.; Craft, Noah; Economou, James S.; Marincola, Francesco M.; Wang, Ena; Ribas, Antoni

    2009-01-01

    Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a pro-apoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 together with the adoptive transfer (AT) of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: 1) increase in MHC and tumor-associated antigen (TAA) expression by tumor cells; 2) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and 3) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein-2 (TRP2), which also demonstrated a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes. PMID:19861533

  20. The nucleolus and transcription of ribosomal genes.

    PubMed

    Raska, Ivan; Koberna, Karel; Malínský, Jan; Fidlerová, Helena; Masata, Martin

    2004-10-01

    Ribosome biogenesis is a highly dynamic, steady-state nucleolar process that involves synthesis and maturation of rRNA, its transient interactions with non-ribosomal proteins and RNPs and assembly with ribosomal proteins. In the few years of the 21st century, an exciting progress in the molecular understanding of rRNA and ribosome biogenesis has taken place. In this review, we discuss the recent results on the regulation of rRNA synthesis in relation to the functional organization of the nucleolus, and put an emphasis on the situation encountered in mammalian somatic cells.

  1. Scattering studies on ribosomes in solution

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, V.

    1986-02-01

    Ribosomes are organelles that play a central role in protein synthesis. They are complexes of protein and nucleic acid, and can be analysed as two-component systems by neutron scattering. Moreover, ribosomes can be biochemically prepared that have specific proteins deuterated. Both these properties have been exploited to study the structure of the ribosome by neutron scattering. This article reviews the studies carried out on the small ribosomal subunit, and describes a recent study that has resolved a conflict between the results of two classes of experiments.

  2. Ribosome biogenesis in the yeast Saccharomyces cerevisiae.

    PubMed

    Woolford, John L; Baserga, Susan J

    2013-11-01

    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

  3. Enhanced histone acetylation in somatic cells induced by a histone deacetylase inhibitor improved inter-generic cloned leopard cat blastocysts.

    PubMed

    Lee, Hyo-Sang; Yu, Xian-Feng; Bang, Jae-Il; Cho, Su-Jin; Deb, Gautam Kumar; Kim, Byeong-Woo; Kong, Il-Keun

    2010-11-01

    The objective was to determine whether alterations of histone acetylation status in donor cells affected inter-generic SCNT (igSCNT)-cloned embryo development. Leopard cat cells were treated with trichostatin A (TSA; a histone deacetylase inhibitor) for 48 h, and then donor cells were transferred into enucleated oocytes from domestic cats. Compared to non-treated cells, the acetylated histone 3 at lysine 9 (AcH3K9) and histone 4 at lysine 5 (AcH4K5) in the TSA group increased for up to 48 h (P < 0.05). The AcH3K9 signal ratios of igSCNT group was higher than control group 3 h after activation (P < 0.05). Treatment with TSA significantly increased total cell number of blastocysts (109.1 ± 6.9 vs. 71.8 ± 2.9, mean ± SEM), with no significant effects on rates of cleavage or blastocyst development (71.1 ± 2.8 vs. 67.6 ± 2.9 and 12.2 ± 2.6 vs. 11.0 ± 2.6, respectively). When igSCNT cloned embryos were transferred into a domestic cat oviduct and recovered after 8 d, blastocyst development rates and total cell numbers were greater in the TSA-igSCNT group (20.7 ± 3.0% and 2847.6 ± 37.2) than in the control igSCNT group (5.7 ± 2.2% and 652.1 ± 17.6, P < 0.05). Average total cell numbers of blastocysts were approximately 4.4-fold higher in the TSA-igSCNT group (2847.6 ± 37.2, n = 10) than in the control group (652.1 ± 17.6, n = 8; P < 0.05), but were ∼2.9-fold lower than in vivo cat blastocysts produced by intrauterine insemination (8203.8 ± 29.6, n = 5; P < 0.001). Enhanced histone acetylation levels of donor cells improved in vivo developmental competence and quality of inter-generic cloned embryos; however, fewer cells in blastocysts derived from igSCNT than blastocysts produced by insemination may reduce development potential following intergeneric cloning (none of the cloned embryos were maintained to term).

  4. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. PMID:25910649

  5. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress.

  6. Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

    PubMed Central

    Cacan, Ercan; Spring, Alexander M.; Kumari, Anita; Greer, Susanna F.; Garnett-Benson, Charlie

    2015-01-01

    Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity. PMID:26703577

  7. Satratoxin G Interaction with 40S and 60S Ribosomal Subunits Precedes Apoptosis in the Macrophage

    PubMed Central

    Bae, Hee Kyong; Shinozuka, Junko; Islam, Zahidul; Pestka, James J.

    2011-01-01

    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 resultant 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. PMID:19306889

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

    SciTech Connect

    Bae, Hee Kyong; Shinozuka, Junko; Islam, Zahidul; Pestka, James J.

    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 resultant 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.

  9. Modulation of decoding fidelity by ribosomal proteins S4 and S5.

    PubMed

    Agarwal, Deepali; Kamath, Divya; Gregory, Steven T; O'Connor, Michael

    2015-03-01

    Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.

  10. AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells

    SciTech Connect

    Raghavan, Pavithra; Tumati, Vasu; Yu Lan; Chan, Norman; Tomimatsu, Nozomi; Burma, Sandeep; Bristow, Robert G.; Saha, Debabrata

    2012-11-15

    Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. Methods and Materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used. Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G{sub 2}-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.

  11. Opioid-sparing effect of selective cyclooxygenase-2 inhibitors on surgical outcomes after open colorectal surgery within an enhanced recovery after surgery protocol

    PubMed Central

    Lohsiriwat, Varut

    2016-01-01

    AIM: To evaluate the opioid-sparing effect of selective cyclooxygenase-2 (COX-2) inhibitors on short-term surgical outcomes after open colorectal surgery. METHODS: Patients undergoing open colorectal resection within an enhanced recovery after surgery protocol from 2011 to 2015 were reviewed. Patients with combined general anesthesia and epidural anesthesia, and those with acute colonic obstruction or perforation were excluded. Patients receiving selective COX-2 inhibitor were compared with well-matched individuals without such a drug. Outcome measures included numeric pain score and morphine milligram equivalent (MME) consumption on postoperative day (POD) 1-3, gastrointestinal recovery (time to tolerate solid diet and time to defecate), complications and length of postoperative stay. RESULTS: There were 75 patients in each group. Pain score on POD 1-3 was not significantly different between two groups. However, MME consumption and MME consumption per kilogram body weight on POD 1-3 was significantly less in patients receiving a selective COX-2 inhibitor (P < 0.001). Median MME consumption per kilogram body weight on POD 1-3 was 0.09, 0.06 and nil, respectively in patients receiving a selective COX-2 inhibitor and 0.22, 0.25 and 0.07, respectively in the comparative group (P < 0.001), representing at least 59% opioid reduction. Patients prescribing a selective COX-2 inhibitor had a shorter median time to resumption of solid diet [1 (IQR 1-2) d vs 2 (IQR 2-3) d; P < 0.001] and time to first defecation [2 (IQR 2-3) d vs 3 (IQR 3-4) d; P < 0.001]. There was no significant difference in overall postoperative complications between two groups. However, median postoperative stay was significantly 1-d shorter in patients prescribing a selective COX-2 inhibitor [4 (IQR 3-5) d vs 5 (IQR 4-6) d; P < 0.001]. CONCLUSION: Perioperative administration of oral selective COX-2 inhibitors significantly decreased intravenous opioid consumption, shortened time to gastrointestinal

  12. Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer

    PubMed Central

    Howe, Grant A.; Xiao, Bin; Zhao, Huijun; Al-Zahrani, Khalid N.; Hasim, Mohamed S.; Villeneuve, James; Sekhon, Harmanjatinder S.; Goss, Glenwood D.; Sabourin, Luc A.; Dimitroulakos, Jim; Addison, Christina L.

    2016-01-01

    Blockade of epidermal growth factor receptor (EGFR) activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC). As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs), there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK) activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975) were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271) both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be particularly

  13. High-fat diet enhances and plasminogen activator inhibitor-1 deficiency attenuates bone loss in mice with Lewis Lung carcinoma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study determined the effects of a high-fat diet and plasminogen activator inhibitor-1 deficiency (PAI-1-/-) on bone structure in mice bearing Lewis lung carcinoma (LLC) in lungs. Reduction in bone volume fraction (BV/TV) by 22% and 21%, trabecular number (Tb.N) by 8% and 4% and bone mineral de...

  14. Evolution of the ribosome at atomic resolution

    PubMed Central

    Petrov, Anton S.; Bernier, Chad R.; Hsiao, Chiaolong; Norris, Ashlyn M.; Kovacs, Nicholas A.; Waterbury, Chris C.; Stepanov, Victor G.; Harvey, Stephen C.; Fox, George E.; Wartell, Roger M.; Hud, Nicholas V.; Williams, Loren Dean

    2014-01-01

    The origins and evolution of the ribosome, 3–4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be “observed” by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call “insertion fingerprints.” Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel. PMID:24982194

  15. Ribosomes: lifting the nuclear export ban.

    PubMed

    Johnson, Arlen W

    2014-02-01

    A recent study shows that nuclear export of the large ribosomal subunit is regulated by a GTPase that blocks recruitment of the nuclear export factor Nmd3 until remodeling of the pre-ribosome by the AAA-ATPase Rea1 (Midasin).

  16. Suppression of PRKAR1A expression enhances anti-proliferative and apoptotic effects of protein kinase inhibitors and chemotherapeutic drugs on cholangiocarcinoma cells.

    PubMed

    Loilome, Watcharin; Juntana, Sirinun; Pinitsoontorn, Chadamas; Namwat, Nisana; Tassaneeyakul, Wichittra; Yongvanit, Puangrat

    2012-01-01

    Suppression of protein kinase A regulatory subunit 1 alpha (PRKAR1A) has been proven to inhibit cholangiocarcinoma (CCA) cell growth and enhance apoptosis. In the present study, we aimed to determine synergistic and/or additive effects of chemotherapeutic agents, including protein kinase inhibitors (i.e. sorafenib, sunitinib, gefitinib, Met inhibitor) and conventional chemotherapeutic drugs (i.e. 5-fluorouracil, doxorubicin, paclitaxel, gemcitabine), in PRKARIA knockdown CCA cell lines. The results revealed that PRKAR1A suppressed CCA cell lines demonstrated enhanced sensitivity to some chemotherapeutic drugs when compared to control cells. Moreover, PRKAR1A knockdown in combination with either sorafenib or 5-fluorouracil increased apoptotic effects on CCA cell lines. Therefore, selective inhibition of PRKAR1A appears to enhance the growth inhibitory effects of chemotherapeutic drugs as well as induce apoptotic cell death. Our findings suggest that additional suppression of PRKAR1A expression may increase the efficacy of conventional CCA chemotherapeutic treatment. Clinical studies in CCA patients now need to be conducted. PMID:23480756

  17. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functions via epigenetic modulation of Sca-1 and Pw1 promoters.

    PubMed

    Smeriglio, Piera; Alonso-Martin, Sonia; Masciarelli, Silvia; Madaro, Luca; Iosue, Ilaria; Marrocco, Valeria; Relaix, Frédéric; Fazi, Francesco; Marazzi, Giovanna; Sassoon, David A; Bouché, Marina

    2016-04-01

    Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fatein vitro In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regenerationin vivofollowing transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potentialin vivoand replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, includingSca-1andPw1 Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functionsviaepigenetic modulation of Sca-1 and Pw1 promoters.

  18. Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination

    PubMed Central

    2010-01-01

    Background Activating Transcription Factor (ATF) 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC) inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor. Results The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) as well as chromatin immunoprecipitation (ChIP) assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. Conclusion This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity. PMID:20828393

  19. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  20. Differential Stoichiometry among Core Ribosomal Proteins.

    PubMed

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-11-01

    Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  1. Ribosome defects in disorders of erythropoiesis.

    PubMed

    Narla, Anupama; Hurst, Slater N; Ebert, Benjamin L

    2011-02-01

    Over the past decade, genetic lesions that cause ribosome dysfunction have been identified in both congenital and acquired human disorders. These discoveries have established a new category of disorders, known as ribosomopathies, in which the primary pathophysiology is related to impaired ribosome function. The protoptypical disorders are Diamond-Blackfan anemia, a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome. In both of these disorders, impaired ribosome function causes a severe macrocytic anemia. In this review, we will discuss the evidence that defects in ribosomal biogenesis cause the hematologic phenotype of Diamond-Blackfan anemia and the 5q- syndrome. We will also explore the potential mechanisms by which a ribosomal defect, which would be expected to have widespread consequences, may lead to specific defects in erythropoiesis. PMID:21279816

  2. Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth

    PubMed Central

    Ghalei, Homa; Schaub, Franz X.; Doherty, Joanne R.; Noguchi, Yoshihiko; Roush, William R.; Cleveland, John L.; Stroupe, M. Elizabeth

    2015-01-01

    Casein kinase 1δ/ε (CK1δ/ε) and their yeast homologue Hrr25 are essential for cell growth. Further, CK1δ is overexpressed in several malignancies, and CK1δ inhibitors have shown promise in several preclinical animal studies. However, the substrates of Hrr25 and CK1δ/ε that are necessary for cell growth and survival are unknown. We show that Hrr25 is essential for ribosome assembly, where it phosphorylates the assembly factor Ltv1, which causes its release from nascent 40S subunits and allows subunit maturation. Hrr25 inactivation or expression of a nonphosphorylatable Ltv1 variant blocked Ltv1 release in vitro and in vivo, and prevented entry into the translation-like quality control cycle. Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion. Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly. These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics. PMID:25778921

  3. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-01

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  4. The BET bromodomain inhibitor, JQ1, facilitates c-FLIP degradation and enhances TRAIL-induced apoptosis independent of BRD4 and c-Myc inhibition.

    PubMed

    Yao, Weilong; Yue, Ping; Khuri, Fadlo R; Sun, Shi-Yong

    2015-10-27

    Inhibition of BET bromodomains (BRDs) has emerged as a promising cancer therapeutic strategy. Accordingly, inhibitors of BRDs such as JQ1 have been actively developed and some have reached clinical testing. However, the mechanisms by which this group of inhibitors exerts their anticancer activity, including induction of apoptosis, have not been fully elucidated. This report reveals a previously uncovered activity of JQ1 in inducing c-FLIP degradation and enhancing TRAIL-induced apoptosis. JQ1 potently decreased c-FLIP (both long and short forms) levels in multiple cancer cell lines without apparently increasing the expression of DR5 and DR4. Consequently, JQ1, when combined with TRAIL, synergistically induced apoptosis; this enhanced apoptosis-inducing activity could be abolished by enforced expression of ectopic FLIPL or FLIPS. Hence it appears that JQ1 decreases c-FLIP levels, resulting in enhancement of TRAIL-induced apoptosis. Inhibition of proteasome with MG132 prevented JQ1-induced c-FLIP reduction. Moreover, JQ1 decreased c-FLIP stability. Therefore, JQ1 apparently decreases c-FLIP levels through facilitating its proteasomal degradation. Genetic inhibition of either BRD4 or c-Myc by knocking down their expression failed to mimic JQ1 in decreasing c-FLIP and enhancing TRAIL-induced apoptosis, suggesting that JQ1 induces c-FLIP degradation and enhances TRAIL-induced apoptosis independent of BRD4 or c-Myc inhibition. In summary, our findings in this study highlights a novel biological function of JQ1 in modulating apoptosis and warrant further study of the potential treatment of cancer with the JQ1 and TRAIL combination. PMID:26415225

  5. JAK2 inhibitor combined with DC-activated AFP-specific T-cells enhances antitumor function in a Fas/FasL signal-independent pathway

    PubMed Central

    Liu, Yang; Wang, Yue-ru; Ding, Guang-hui; Yang, Ting-song; Yao, Le; Hua, Jie; He, Zhi-gang; Qian, Ming-ping

    2016-01-01

    Objective Combination therapy for cancer is more effective than using only standard chemo- or radiotherapy. Our previous results showed that dendritic cell-activated α-fetoprotein (AFP)-specific T-cells inhibit tumor in vitro and in vivo. In this study, we focused on antitumor function of CD8+ T-cells combined with or without JAK2 inhibitor. Methods Proliferation and cell cycle were analyzed by CCK-8 and flow cytometry. Western blot was used to analyze the expression level of related protein and signaling pathway. Results We demonstrated reduced viability and induction of apoptosis of tumor cells with combination treatment. Intriguingly, cell cycle was blocked at the G1 phase by using AFP-specific CD8+ T-cells combined with JAK2 inhibitor (AG490). Furthermore, an enhanced expression of BAX but no influence on Fas/FasL was detected from the tumor cells. Conclusion These results indicate a Fas/FasL-independent pathway for cellular apoptosis in cancer therapies with the treatment of AFP-specific CD8+ T-cells combined with JAK2 inhibitor. PMID:27499636

  6. The novel mTORC1/2 dual inhibitor INK128 enhances radiosensitivity of breast cancer cell line MCF-7.

    PubMed

    Liu, Zhi-Gang; Tang, Jiao; Chen, Zhenghu; Zhang, Huiyuan; Wang, Hui; Yang, Jianhua; Zhang, Hong

    2016-09-01

    mTOR, a member of the PIKK family, is crucial for cell growth, survival, motility, proliferation, protein synthesis and DNA transcription. Many studies have demonstrated that mTOR inhibitor could enhance radiosensitivity. However, the effect of the novel mTORC1/2 dual inhibitor, INK128, on the radiosensitivity of breast cancer and the underlying mechanisms are still vague. In the present study, the cell viability was estimated using CCK-8 assay, and the dose-survival relationship was analyzed using a clonogenic survival assay. Cell cycle was evaluated by flow cytometry. The staining of γH2AX foci was assessed by immunofluorescence. In addition, we used western blots to verify the downregulating signal protein and to detect the potential related pathway. We found that the exposure of MCF-7 cells to INK128 decreased the cell viability. Exposure of MCF-7 cells to INK128 and combined ionizing radiation greatly reduced the survival rate. INK128 combined radiotherapy significantly induced G2/M arrest, double strand breaks and inhibited its repair. Furthermore, INK128 plus radiation downregulated p-Chk2, p21 and upregulated cleaved PARP, LC3B expression. These findings suggest that mTOR inhibitor could be used as a novel radiosensitizing target for breast cancer patients. PMID:27574017

  7. Ccr4-Not Regulates RNA Polymerase I Transcription and Couples Nutrient Signaling to the Control of Ribosomal RNA Biogenesis

    PubMed Central

    Laribee, R. Nicholas; Hosni-Ahmed, Amira; Workman, Jason J.; Chen, Hongfeng

    2015-01-01

    Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis. PMID:25815716

  8. Antiestrogen fulvestrant enhances the antiproliferative effects of epidermal growth factor receptor inhibitors in human non-small cell lung cancer

    PubMed Central

    Garon, Edward B.; Pietras, Richard J.; Finn, Richard S.; Kamranpour, Naeimeh; Pitts, Sharon; Márquez-Garbán, Diana C.; Desai, Amrita J.; Dering, Judy; Hosmer, Wylie; von Euw, Erika M.; Dubinett, Steven M.; Slamon, Dennis J.

    2012-01-01

    Introduction Estrogen receptor (ER) signaling and its interaction with epidermal growth factor receptor (EGFR) is a potential therapeutic target in non-small cell lung cancer (NSCLC). To explore cross-communication between ER and EGFR, we have correlated ER pathway gene and protein expression profiles and examined effects of antiestrogens with or without EGFR inhibitors in preclinical models of human NSCLC. Methods We evaluated 54 NSCLC cell lines for growth inhibition with EGFR inhibitors, antiestrogen treatment or the combination. Each line was evaluated for baseline ER pathway protein expression. The majority were also evaluated for baseline ER pathway gene expression. Human NSCLC xenografts were evaluated for effects of inhibition of each pathway either individually or in combination. Results The specific antiestrogen fulvestrant has modest single agent activity in vitro, but in many lines fulvestrant adds to effects of EGFR inhibitors, including synergy in the EGFR mutant, erlotinib-resistant H1975 line. ERα, ERβ, progesterone receptor (PR)-A, PR-B and aromatase proteins are expressed in all lines to varying degrees, with trends towards lower aromatase in more sensitive cell lines. Sensitivity to fulvestrant correlates with greater baseline ERα gene expression. Tumor stability is achieved in human tumor xenografts with either fulvestrant or EGFR inhibitors, but tumors regress significantly when both pathways are inhibited. Conclusions These data provide a rationale for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-EGFR agents in the clinic. Future work should also evaluate dual ER and EGFR inhibition in the setting of secondary resistance to EGFR inhibition. PMID:23399957

  9. Diverse effects of residues 74-78 in ribosomal protein S12 on decoding and antibiotic sensitivity.

    PubMed

    Agarwal, Deepali; O'Connor, Michael

    2014-03-01

    Ribosomal protein S12 plays key roles in the ribosome's response to the error-promoting antibiotic streptomycin and in modulating the accuracy of translation. The discovery that substitutions at His76 in S12, distant from the streptomycin binding site, conferred streptomycin resistance in the thermophilic bacterium Thermus thermophilus prompted us to make similar alterations in the S12 protein of Escherichia coli. While, none of the E. coli S12 mutations confers streptomycin resistance, they all have distinct effects on the accuracy of translation. In addition, a subset of the S12 alterations renders the cells hypersensitive to fusidic acid, an inhibitor of the translocation step of translation. These results indicate that the His 76 region of ribosomal protein S12 plays key roles in tRNA selection and translocation steps of protein synthesis, consistent with its interaction with elongation factors EF-Tu and EF-G, as deduced from structural studies of ribosomal complexes. PMID:24530394

  10. Mechanisms for Ribotoxin-induced Ribosomal RNA Cleavage

    PubMed Central

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-01-01

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10 ng/ml) and ribosome-inactivating protein ricin (≥300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspase 8, 9 and 3 concurrently with apoptosis further suggested rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors cathepsin L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. PMID:23022514

  11. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    PubMed

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, José A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

  12. GSK356278, a potent, selective, brain-penetrant phosphodiesterase 4 inhibitor that demonstrates anxiolytic and cognition-enhancing effects without inducing side effects in preclinical species.

    PubMed

    Rutter, A Richard; Poffe, Alessandro; Cavallini, Palmina; Davis, T Gregg; Schneck, Jessica; Negri, Michele; Vicentini, Elena; Montanari, Dino; Arban, Roberto; Gray, Frank A; Davies, Ceri H; Wren, Paul B

    2014-07-01

    Small molecule phosphodiesterase (PDE) 4 inhibitors have long been known to show therapeutic benefit in various preclinical models of psychiatric and neurologic diseases because of their ability to elevate cAMP in various cell types of the central nervous system. Despite the registration of the first PDE4 inhibitor, roflumilast, for the treatment of chronic obstructive pulmonary disease, the therapeutic potential of PDE4 inhibitors in neurologic diseases has never been fulfilled in the clinic due to severe dose-limiting side effects such as nausea and vomiting. In this study, we describe the detailed pharmacological characterization of GSK356278 [5-(5-((2,4-dimethylthiazol-5-yl)methyl)-1,3,4-oxadiazol-2-yl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine], a potent, selective, and brain-penetrant PDE4 inhibitor that shows a superior therapeutic index to both rolipram and roflumilast in various preclinical species and has potential for further development in the clinic for the treatment of psychiatric and neurologic diseases. GSK356278 inhibited PDE4B enzyme activity with a pIC50 of 8.8 and bound to the high-affinity rolipram binding site with a pIC50 of 8.6. In preclinical models, the therapeutic index as defined in a rodent lung inflammation model versus rat pica feeding was >150 compared with 0.5 and 6.4 for rolipram and roflumilast, respectively. In a model of anxiety in common marmosets, the therapeutic index for GSK356278 was >10 versus <1 for rolipram. We also demonstrate that GSK356278 enhances performance in a model of executive function in cynomolgus macaques with no adverse effects, a therapeutic profile that supports further evaluation of GSK356278 in a clinical setting.

  13. Kaempferia parviflora, a plant used in traditional medicine to enhance sexual performance contains large amounts of low affinity PDE5 inhibitors

    PubMed Central

    Temkitthawon, Prapapan; Hinds, Thomas R.; Beavo, Joseph A.; Viyoch, Jarupa; Suwanborirux, Khanit; Pongamornkul, Wittaya; Sawasdee, Pattara; Ingkaninan, Kornkanok

    2014-01-01

    Aim of the study A number of medicinal plants are used in traditional medicine to treat erectile dysfunction. Since cyclic nucleotide PDEs inhibitors underlie several current treatments for this condition, we sought to show whether these plants might contain substantial amounts of PDE5 inhibitors. Materials and methods Forty one plant extracts and eight 7-methoxyflavones from Kaempferia parviflora Wall. ex Baker were screened for PDE5 and PDE6 inhibitory activities using the two-step radioactive assay. The PDE5 and PDE6 were prepared from mice lung and chicken retinas, respectively. All plant extracts were tested at 50 μg/ml whereas the pure compounds were tested at 10 μM. Results From forty one plant extracts tested, four showed the PDE5 inhibitory effect. The chemical constituents isolated from rhizomes of Kaempferia parviflora were further investigated on inhibitory activity against PDE5 and PDE6. The results showed that 7-methoxyflavones from this plant showed inhibition toward both enzymes. The most potent PDE5 inhibitor was 5,7-dimethoxyflavone (IC50 = 10.64 ± 2.09 μM, selectivity on PDE5 over PDE6 = 3.71). Structure activity relationship showed that the methoxyl group at C-5 position of 7-methoxyflavones was necessary for PDE5 inhibition. Conclusions Kaempferia parviflora rhizome extract and its 7-methoxyflavone constituents had moderate inhibitory activity against PDE5. This finding provides an explanation for enhancing sexual performance in the traditional use of Kaempferia parviflora. Moreover, 5,7-dimethoxyflavones should make a useful lead compound to further develop clinically efficacious PDE5 inhibitors. PMID:21884777

  14. Ribosomes and Ribosomal Protein from Neurospora crassa I. Physical, Chemical, and Immunochemical Properties1

    PubMed Central

    Alberghina, F. A. M.; Suskind, S. R.

    1967-01-01

    Ribosomes from Neurospora crassa, initially characterized by ultracentrifugal and immunochemical analyses, have been used to prepare ribosomal protein for physical, chemical, and immunochemical study. The acrylamide gel disc electrophoretic profiles of Neurospora ribosomal protein exhibit a degree of heterogeneity comparable to what has been observed in other systems. Only by chemical modification or by aggregation of the protein do alterations in the profile become apparent. Disulfide-bond formation appears to play a role in the aggregation of ribosomal protein to complexes of S20,w = 200. The aggregation can be prevented by alkylation of −SH groups, and protein treated in this fashion has a subunit molecular weight of about 20,000 as determined by equilibrium centrifugation. Finger-printing of tryptic peptides indicates that more than one unique sequence of amino acids must be present in ribosomal protein, although gross primary structural heterogeneity is questioned. Antigenic heterogeneity is much less apparent; only a few precipitin bands are resolved by immunodiffusion tests, although complete reactivity of total ribosomal protein is suggested by quantitative precipitin analysis. The antigenically active ribosomal protein components appear to reside in at least two fractions; one is removed readily from the ribosome by CsC1 treatment. Ribosomal protein of N. crassa possesses antigenic determinants present in E. coli ribosomal protein as judged by spur formation in immunodiffusion tests. Images PMID:4962303

  15. Second generation bisheteroarylpiperazine (BHAP) HIV-1 reverse transcriptasae inhibitors: Enhancement of antiviral activity and aqueous solubility via 5- and 6-substitution of the indole ring

    SciTech Connect

    Poel, T.; Thomas, R.C.; Romero, D.L.; Hosley, M.J.; Morge, R.A.; Biles, C.; Reusser, F.; Althaus, I.W.; Schinzer, W.C.; Platzer, D.J.

    1993-12-31

    U-87201E, a potent HIV-1 reverse transcriptase inhibitor (RTI) discovered at Upjohn, is currently in Phase II clinical trials. Additional structure-activity studies have identified second-generation BHAPs with enhanced antiviral activity and improved pharmaceutical properties, notably increased aqueous solubility. Capitalizing on initial SAR studies which demonstrated a tolerance for substitution in the indole ring, a series of BHAPs bearing 5- and 6-substituted indoles was evaluated. Substituents such as ethers, sulfonamides, ureas, and sulfamides containing water-solubilizing groups such as polyethers or basic amines provided highly potent BHAPs with greatly enhanced solubility, such as U-93923. The synthesis, antiviral evaluation and solubility properties of these potent HIV-1 RTIs will be detailed.

  16. Ribosomopathies: human disorders of ribosome dysfunction.

    PubMed

    Narla, Anupama; Ebert, Benjamin L

    2010-04-22

    Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases. PMID:20194897

  17. Inhibition of soluble epoxide hydrolase enhances the anti-inflammatory effects of aspirin and 5-lipoxygenase activation protein inhibitor in a murine model.

    PubMed

    Liu, Jun-Yan; Yang, Jun; Inceoglu, Bora; Qiu, Hong; Ulu, Arzu; Hwang, Sung-Hee; Chiamvimonvat, Nipavan; Hammock, Bruce D

    2010-03-15

    Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects. PMID:19896470

  18. Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity.

    PubMed

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V; Schuetz, John D

    2013-09-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV-associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  19. Human Immunodeficiency Virus Protease Inhibitors Interact with ATP Binding Cassette Transporter 4/Multidrug Resistance Protein 4: A Basis for Unanticipated Enhanced Cytotoxicity

    PubMed Central

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B.; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V.

    2013-01-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  20. The clinically active PARP inhibitor AG014699 ameliorates cardiotoxicity but does not enhance the efficacy of doxorubicin, despite improving tumor perfusion and radiation response in mice.

    PubMed

    Ali, Majid; Kamjoo, Marzieh; Thomas, Huw D; Kyle, Suzanne; Pavlovska, Ivanda; Babur, Muhammed; Telfer, Brian A; Curtin, Nicola J; Williams, Kaye J

    2011-12-01

    AG014699 was the first inhibitor of the DNA repair enzyme PARP-1 to enter clinical trial in cancer patients. In addition to enhancing the cytotoxic effect of DNA-damaging chemotherapies, we have previously shown that AG014699 is vasoactive, thereby having the potential to improve drug biodistribution. The effectiveness of the clinical agent doxorubicin is confounded both by poor tumor penetration and cardiotoxicity elicited via PARP hyperactivation. In this study, we analyzed the impact of AG014699 on doxorubicin tolerance and response in breast (MDA-MB-231) and colorectal (SW620, LoVo) tumor models in vitro and in vivo. As anticipated, AG014699 did not potentiate the response to doxorubicin in vitro. In vivo, AG014699 did not influence the pharmacokinetics of doxorubicin; however, it did ameliorate cardiotoxicity. Both toxicity and extent of amelioration were more pronounced in male than in female mice. AG014699 improved vessel perfusion in both MDA-MB-231 and SW620 tumors; however, this neither led to improved tumor-accumulation of doxorubicin nor enhanced therapeutic response. In contrast, when combined with radiotherapy, AG014699 significantly enhanced response both in vitro and in vivo. Real-time assessment of tumor vessel function and companion histologic studies indicate that doxorubicin causes a profound antivascular effect that counters the positive effect of AG014699 on perfusion. These data indicate that although AG014699 can enhance response to some chemotherapeutic drugs via improved delivery, this does not apply to doxorubicin. PARP inhibitors may still be of use to counter doxorubicin toxicity, and if the gender effect translates from rodents to humans, this would have greater effect in males. PMID:21926192

  1. Origin and evolution of the ribosome.

    PubMed

    Fox, George E

    2010-09-01

    The modern ribosome was largely formed at the time of the last common ancestor, LUCA. Hence its earliest origins likely lie in the RNA world. Central to its development were RNAs that spawned the modern tRNAs and a symmetrical region deep within the large ribosomal RNA, (rRNA), where the peptidyl transferase reaction occurs. To understand pre-LUCA developments, it is argued that events that are coupled in time are especially useful if one can infer a likely order in which they occurred. Using such timing events, the relative age of various proteins and individual regions within the large rRNA are inferred. An examination of the properties of modern ribosomes strongly suggests that the initial peptides made by the primitive ribosomes were likely enriched for l-amino acids, but did not completely exclude d-amino acids. This has implications for the nature of peptides made by the first ribosomes. From the perspective of ribosome origins, the immediate question regarding coding is when did it arise rather than how did the assignments evolve. The modern ribosome is very dynamic with tRNAs moving in and out and the mRNA moving relative to the ribosome. These movements may have become possible as a result of the addition of a template to hold the tRNAs. That template would subsequently become the mRNA, thereby allowing the evolution of the code and making an RNA genome useful. Finally, a highly speculative timeline of major events in ribosome history is presented and possible future directions discussed. PMID:20534711

  2. Enhanced magnetic resonance imaging of experimental pancreatic tumor in vivo by block copolymer-coated magnetite nanoparticles with TGF-beta inhibitor.

    PubMed

    Kumagai, Michiaki; Kano, Mitsunobu R; Morishita, Yasuyuki; Ota, Motomi; Imai, Yutaka; Nishiyama, Nobuhiro; Sekino, Masaki; Ueno, Shoogo; Miyazono, Kohei; Kataoka, Kazunori

    2009-12-16

    Early detection of solid tumors, particularly pancreatic cancer, is of substantial importance in clinics. Enhanced magnetic resonance imaging (MRI) with iron oxide nanoparticles is an available way to detect the cancer. The effective and selective accumulation of these nanoparticles in the tumor tissue is needed for improved imaging, and in this regard, their longevity in the blood circulation time is crucial. We developed here block copolymer-coated magnetite nanoparticles for pancreatic cancer imaging, by means of a chelation between the carboxylic acid groups in poly(ethylene glycol)-poly(aspartic acid) block copolymer (PEG-PAsp) and Fe on the surface of the iron oxide nanoparticles. These nanoparticles had considerably narrow distribution, even upon increased ionic strength or in the presence of fetal bovine serum. The PEG-PAsp-coated nanoparticles were further shown to be potent as a contrast agent for enhanced MRI for an experimental pancreatic cancer, xenografts of the human-derived BxPC3 cell line in BALB/c nude mice, with combined administration of TGF-beta inhibitor. Iron staining of tumor tissue confirmed the accumulation of the nanoparticles in tumor tissue. Use of the PEG-PAsp-coated magnetite nanoparticles, combined with the TGF-beta inhibitor, is of promising clinical importance for the detection of intractable solid cancers, including pancreatic cancer.

  3. Adhesion of ZAP-70+ chronic lymphocytic leukemia cells to stromal cells is enhanced by cytokines and blocked by inhibitors of the PI3-kinase pathway.

    PubMed

    Lafarge, Sandrine T; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2014-01-01

    CLL cell survival and proliferation is enhanced through direct contact with supporting cells present in lymphoid tissues. PI3Ks are critical signal transduction enzymes controlling B cell survival and activation. PI3K inhibitors have entered clinical trials and show promising therapeutic activity; however, it is unclear whether PI3K inhibitor drugs differentially affect ZAP-70 positive versus negative CLL cells or target specific microenvironmental interactions. Here we provide evidence that CD40L+IL-4, IL-8 or IL-6 enhance adhesion to stromal cells, with IL-6 showing a selective effect on ZAP-70 positive cells. Stimulatory effects of IL-8 or IL-6 are fully reversed by PI3K inhibition, while the effects of CD40L+IL-4 are partially reversed. While CD40L+IL-4 is the only stimulation increasing CLL cell survival for all patient groups, IL-6 protects ZAP-70 positive cells from cell death induced by PI3K inhibition. Altogether, our results indicate that targeting the PI3K pathway can reverse protective CLL-microenvironment interactions in both ZAP-70 positive and negative CLL despite their differences in cytokine responsiveness.

  4. Intersubunit Bridges of the Bacterial Ribosome.

    PubMed

    Liu, Qi; Fredrick, Kurt

    2016-05-22

    The ribosome is a large two-subunit ribonucleoprotein machine that translates the genetic code in all cells, synthesizing proteins according to the sequence of the mRNA template. During translation, the primary substrates, transfer RNAs, pass through binding sites formed between the two subunits. Multiple interactions between the ribosomal subunits, termed intersubunit bridges, keep the ribosome intact and at the same time govern dynamics that facilitate the various steps of translation such as transfer RNA-mRNA movement. Here, we review the molecular nature of these intersubunit bridges, how they change conformation during translation, and their functional roles in the process.

  5. A new system for naming ribosomal proteins

    PubMed Central

    Ban, Nenad; Beckmann, Roland; Cate, Jamie HD; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis LJ; Lindahl, Lasse; Liljas, Anders; Lipton, Jeffrey M; McAlear, Michael A; Moore, Peter B; Noller, Harry F; Ortega, Joaquin; Panse, Vikram Govind; Ramakrishnan, V; Spahn, Christian MT; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

    2015-01-01

    A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as ‘new system’ names. PMID:24524803

  6. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  7. Dipeptidyl peptidase-4 inhibitor MK-626 restores insulin secretion through enhancing autophagy in high fat diet-induced mice.

    PubMed

    Liu, Limei; Liu, Jian; Yu, Xiaoxing

    2016-02-12

    Autophagy is cellular machinery for maintenance of β-cell function and mass. The current study aimed to investigate the regulatory effects of MK-626, a dipeptidyl peptidase-4 inhibitor, on insulin secretion through the activation of autophagy in high fat diet-induced obese mice. C57BL/6 mice were fed with a rodent diet containing 45 kcal% fat for 16 weeks to induce obesity and then were received either vehicle or MK-626 (3 mg/kg/day) orally during the final 4 weeks. Mouse islets were isolated. Phosphorylation of serine/threonine-protein kinase mTOR and levels of light chain 3B I (LC3B I), LC3B II, sequestosome-1 (SQSTM1/p62) and autophagy-related protein-7 (Atg7) were examined by Western blotting. Glucagon like-peptide-1 (GLP-1) level and insulin secretion were measured by ELISA. GLP-1 level in plasma was decreased in obese mice, which was elevated by dipeptidyl peptidase-4 inhibitor MK-626. In the islets of obese mice, phosphorylation of mTOR, ratio of LC3B I and LC3B II, and level of p62 were elevated and the expression of Atg7 and insulin secretion were reduced compared to those of C57BL/6 mice. However, such effects were reversed by MK-626. Autophagy activator rapamycin stimulated insulin secretion in obese mice but autophagy inhibitor chloroquine treatment inhibited insulin secretion in obese mice administrated by MK-626. Furthermore, the beneficial effects of MK-626 were inhibited by GLP-1 receptor antagonist exendin 9-39. The present study reveals the activation of autophagy to mediate the anti-diabetic effect of GLP-1.

  8. Enhancement of ovalbumin-induced pulmonary eosinophilia by intranasal administration of alpha1-proteinase inhibitor type 2 antisense oligonucleotides.

    PubMed

    Sung, Ha-Na; Jeon, Chang-Hwan; Gill, Byoung-Chul; Kim, Hye-Rin; Cheong, Sun-Woo; Park, Joo-Hung

    2009-01-29

    To identify asthma-susceptibility genes, we did proteome analyses of the lung from control and ovalbumin-sensitized BALB/c mice. Among the 6 up-regulated proteins is alpha(1)-protease inhibitor (alpha(1)-PI) type 2, which is a member of the serine protease inhibitor superfamily of protease inhibitors that participate in a variety of physiological functions, including extracellular matrix remodeling and inflammation. The up-regulated expression of alpha(1)-PI type 2 was confirmed by real-time PCR. Then we examined mRNA expression of five members of the alpha(1)-PI family genes (alpha(1)-PI types 1-5) in several organs of BALB/c mice and found that in addition to the liver, all the organs tested also expressed different isoforms of alpha(1)-PI in a tissue-specific manner, albeit to a lesser extent compared with the liver. When a similar study was performed with C57BL/6 mice, which have been shown to be more susceptible to ovalbumin-induced asthma than BALB/c mice, a pair of remarkable differences between the mouse strains were revealed: (1) the magnitude of alpha(1)-PI type 2 mRNA in all the organs was much higher in BALB/c than in C57BL/6 mice and (2) alpha(1)-PI type 2 is the only isoform expressed in the lung of BALB/c, but not of C57BL/c mice. Using the antisense oligonucleotide technology to specifically down-regulate expression of alpha(1)-PI type 2, we demonstrated that pulmonary infiltration of eosinophils was significantly increased by intranasal administration of alpha(1)-PI type 2 antisense oligonucleotides in OVA-sensitized mice, suggesting that alpha(1)-PI type 2 may suppress the progress of asthma, probably by acting on neutrophil elastase, which can produce many of the pathological features of asthma.

  9. Maize reas1 Mutant Stimulates Ribosome Use Efficiency and Triggers Distinct Transcriptional and Translational Responses1[OPEN

    PubMed Central

    Qi, Weiwei; Zhu, Jie; Wu, Qiao; Wang, Qun; Li, Xia; Yao, Dongsheng; Jin, Ying; Wang, Gang; Wang, Guifeng

    2016-01-01

    Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects; however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic initiation factor 2α and the increased elongation factor 1α level indicate an enhancement of general translational efficiency in dek* cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs. Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis. PMID:26645456

  10. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    PubMed Central

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  11. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation.

    PubMed

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu Hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  12. Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli.

    PubMed

    Tsai, Yi-Chun; Du, Dijun; Domínguez-Malfavón, Lilianha; Dimastrogiovanni, Daniela; Cross, Jonathan; Callaghan, Anastasia J; García-Mena, Jaime; Luisi, Ben F

    2012-11-01

    The RNA degradosome is a multi-enzyme assembly that contributes to key processes of RNA metabolism, and it engages numerous partners in serving its varied functional roles. Small domains within the assembly recognize collectively a diverse range of macromolecules, including the core protein components, the cytoplasmic lipid membrane, mRNAs, non-coding regulatory RNAs and precursors of structured RNAs. We present evidence that the degradosome can form a stable complex with the 70S ribosome and polysomes, and we demonstrate the proximity in vivo of ribosomal proteins and the scaffold of the degradosome, RNase E. The principal interactions are mapped to two, independent, RNA-binding domains from RNase E. RhlB, the RNA helicase component of the degradosome, also contributes to ribosome binding, and this is favoured through an activating interaction with RNase E. The catalytic activity of RNase E for processing 9S RNA (the ribosomal 5S RNA precursor) is repressed in the presence of the ribosome, whereas there is little affect on the cleavage of single-stranded substrates mediated by non-coding RNA, suggestings that the enzyme retains capacity to cleave unstructured substrates when associated with the ribosome. We propose that polysomes may act as antennae that enhance the rates of capture of the limited number of degradosomes, so that they become recruited to sites of active translation to act on mRNAs as they become exposed or tagged for degradation.

  13. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis

    NASA Astrophysics Data System (ADS)

    Finley, Daniel; Bartel, Bonnie; Varshavsky, Alexander

    1989-03-01

    Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.

  14. Enhanced Dopamine Release by Dopamine Transport Inhibitors Described by a Restricted Diffusion Model and Fast-Scan Cyclic Voltammetry.

    PubMed

    Hoffman, Alexander F; Spivak, Charles E; Lupica, Carl R

    2016-06-15

    Fast-scan cyclic voltammetry (FSCV) using carbon fiber electrodes is widely used to rapidly monitor changes in dopamine (DA) levels in vitro and in vivo. Current analytical approaches utilize parameters such as peak oxidation current amplitude and decay times to estimate release and uptake processes, respectively. However, peak amplitude changes are often observed with uptake inhibitors, thereby confounding the interpretation of these parameters. To overcome this limitation, we demonstrate that a simple five-parameter, two-compartment model mathematically describes DA signals as a balance of release (r/ke) and uptake (ku), summed with adsorption (kads and kdes) of DA to the carbon electrode surface. Using nonlinear regression, we demonstrate that our model precisely describes measured DA signals obtained in brain slice recordings. The parameters extracted from these curves were then validated using pharmacological manipulations that selectively alter vesicular release or DA transporter (DAT)-mediated uptake. Manipulation of DA release through altering the Ca(2+)/Mg(2+) ratio or adding tetrodotoxin reduced the release parameter with no effect on the uptake parameter. DAT inhibitors methylenedioxypyrovalerone, cocaine, and nomifensine significantly reduced uptake and increased vesicular DA release. In contrast, a low concentration of amphetamine reduced uptake but had no effect on DA release. Finally, the kappa opioid receptor agonist U50,488 significantly reduced vesicular DA release but had no effect on uptake. Together, these data demonstrate a novel analytical approach to distinguish the effects of manipulations on DA release or uptake that can be used to interpret FSCV data. PMID:27018734

  15. Enhanced xylose fermentation and hydrolysate inhibitor tolerance of Scheffersomyces shehatae for efficient ethanol production from non-detoxified lignocellulosic hydrolysate.

    PubMed

    Senatham, Srisuda; Chamduang, Thada; Kaewchingduang, Yotin; Thammasittirong, Anon; Srisodsuk, Malee; Elliston, Adam; Roberts, Ian N; Waldron, Keith W; Thammasittirong, Sutticha Na-Ranong

    2016-01-01

    Effective conversion of xylose into ethanol is important for lignocellulosic ethanol production. In the present study, UV-C mutagenesis was used to improve the efficiency of xylose fermentation. The mutated Scheffersomyces shehatae strain TTC79 fermented glucose as efficiently and xylose more efficiently, producing a higher ethanol concentration than the wild-type. A maximum ethanol concentration of 29.04 g/L was produced from 71.31 g/L xylose, which was 58.95 % higher than that of the wild-type. This mutant also displayed significantly improved hydrolysate inhibitors tolerance and increased ethanol production from non-detoxified lignocellulosic hydrolysates. The ethanol yield, productivity and theoretical yield by TTC79 from sugarcane bagasse hydrolysate were 0.46 g/g, 0.20 g/L/h and 90.61 %, respectively, while the corresponding values for the wild-type were 0.20 g/g, 0.04 g/L/h and 39.20 %, respectively. These results demonstrate that S. shehatae TTC79 is a useful non-recombinant strain, combining efficient xylose consumption and high inhibitor tolerance, with potential for application in ethanol production from lignocellulose hydrolysates. PMID:27462488

  16. Combination delivery of TGF-β inhibitor and IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy

    NASA Astrophysics Data System (ADS)

    Park, Jason; Wrzesinski, Stephen H.; Stern, Eric; Look, Michael; Criscione, Jason; Ragheb, Ragy; Jay, Steven M.; Demento, Stacey L.; Agawu, Atu; Licona Limon, Paula; Ferrandino, Anthony F.; Gonzalez, David; Habermann, Ann; Flavell, Richard A.; Fahmy, Tarek M.

    2012-10-01

    The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-β (TGF-β), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-β inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8+ T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.

  17. The immunological properties of Brucella ribosomal preparations.

    PubMed

    Corbel, M J

    1976-01-01

    Ribosomes were isolated from Brucella abortus strains 19 and 45/20 by disruption of the cells followed by differential ultracentrifugation. The ribosome preparations contained 2-3 components reacting in immunodiffusion tests but were free of detectable lipopolysaccharide-protein agglutinogen. They crossreacted with antisera to Br. abortus, Br. melitensis, Br. suis and Br. ovis and elicited intradermal delayed hypersensitivity reactions in animals infected with Br. abortus, Br. melitensis or Br. suis. The ribosomes were antigenic in rabbits, guinea pigs and mice. Those from Br. abortus S19 induced agglutinins reaction with smooth brucella strains whereas those from Br. abortus 45/20 induced agglutinins reacting with rough brucella strains. Cattle vaccinated with S19 or 45/20 vaccines or infected with Br. abortus developed pricipitins to ribosomal components at an early stage in the immune response. PMID:816681

  18. The tmRNA ribosome rescue system

    PubMed Central

    Janssen, Brian D.; Hayes, Christopher S.

    2012-01-01

    The bacterial tmRNA quality control system monitors protein synthesis and recycles stalled translation complexes in a process termed “ribosome rescue”. During rescue, tmRNA acts first as a transfer RNA to bind stalled ribosomes, then as a messenger RNA to add the ssrA peptide tag to the C-terminus of the nascent polypeptide chain. The ssrA peptide targets tagged peptides for proteolysis, ensuring rapid degradation of potentially deleterious truncated polypeptides. Ribosome rescue also facilitates turnover of the damaged messages responsible for translational arrest. Thus, tmRNA increases the fidelity of gene expression by promoting the synthesis of full-length proteins. In addition to serving as a global quality control system, tmRNA also plays important roles in bacterial development, pathogenesis and environmental stress responses. This review focuses on the mechanism of tmRNA-mediated ribosome rescue and the role of tmRNA in bacterial physiology. PMID:22243584

  19. Potential extra-ribosomal functions of ribosomal proteins in Saccharomyces cerevisiae.

    PubMed

    Lu, Hui; Zhu, Yi-Fei; Xiong, Juan; Wang, Rong; Jia, Zhengping

    2015-08-01

    Ribosomal proteins (RPs), are essential components of the ribosomes, the molecular machines that turn mRNA blueprints into proteins, as they serve to stabilize the structure of the rRNA, thus improving protein biosynthesis. In addition, growing evidence suggests that RPs can function in other cellular roles. In the present review, we summarize several potential extra-ribosomal functions of RPs in ribosomal biogenesis, transcription activity, translation process, DNA repair, replicative life span, adhesive growth, and morphological transformation in Saccharomyces cerevisiae. However, the future in-depth studies are needed to identify these novel secondary functions of RPs in S. cerevisiae.

  20. Ribosome Inactivating Proteins from Rosaceae.

    PubMed

    Shang, Chenjing; Rougé, Pierre; Van Damme, Els J M

    2016-01-01

    Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins. PMID:27556443

  1. Ribosome Inactivating Proteins from Rosaceae.

    PubMed

    Shang, Chenjing; Rougé, Pierre; Van Damme, Els J M

    2016-01-01

    Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins.

  2. Combinatorial action of transcription factors orchestrates cell cycle-dependent expression of the ribosomal protein genes and ribosome biogenesis.

    PubMed

    Nosrati, Nagisa; Kapoor, Neetu R; Kumar, Vijay

    2014-05-01

    Nucleolar assembly begins at the early G1 phase of the cell cycle and is a hub of ribosomal DNA transcription and rRNA biosynthesis. The newly-formed rRNAs together with ribosomal proteins (RPs) constitute the building block of the ribosomal machinery. Although RPs play a major role in protein biosynthesis, their own regulation and expression is rather poorly understood. In the present study, we investigated the regulation of RP genes RPS27a, RPS24, RPS6, RPL9 and RPL4 in synchronized mammalian cell culture. Quantitative RT-PCR analysis indicated their expression during the mid to late G1 phase, whereas the rRNA genes were expressed during the early G1 phase of the cell cycle. The promoter reporter analysis of the RPS27a gene revealed that it could be synergistically stimulated by the transcription factors specificity protein 1 (Sp1) and cAMP response element-binding protein (CREB). However, E2F transcription factor 1 (E2F1) appeared to negatively regulate gene expression. Chromatin immunoprecipitation studies confirmed the promoter occupancy of Sp1, CREB and E2F1. Although Sp1 and CREB binding enhanced the promoter occupancy of histone acetyltransferases PCAF, p300 and CREB binding protein, E2F1 facilitated the recruitment of histone deacetylases. Both acetylation (histone H4 pan-acetyl, histone H3 acetyl Lys 14) and methylation (histone H3 trimethyl Lys 9) marks were observed in the RPS27a promoter region, suggesting their important regulatory role in gene expression. Because the promoter regions of most RP genes are well conserved, we propose that their orchestrated regulation and synthesis during the cell cycle facilitates ribosome biogenesis. PMID:24646001

  3. Frozen spin targets in ribosomal structure research.

    PubMed

    Stuhrmann, H B

    1991-01-01

    Polarized neutron scattering strongly depends on nuclear spin polarisation, particularly on proton spin polarisation. A single proton in a deuterated environment then is as efficient as 10 electrons in X-ray anomalous diffraction. Neutron scattering from the nuclear spin label is controlled by the polarisation of neutron spins and nuclear spins. Pure deuteron spin labels and proton spin labels are created by NMR saturation. We report on results obtained from the large subunit of E. coli ribosomes which have been obtained at the research reactor of GKSS using the polarized target facility developed by CERN. The nuclear spins were oriented with respect to an external field by dynamic nuclear polarisation. Proton spin polarisations of more than 80% were obtained in ribosomes at temperatures below 0.5 K. At T = 130 mK the relaxation time of the polarized target is one month (frozen spin target). Polarized small-angle neutron scattering of the in situ structure of rRNA and the total ribosomal protein (TP) has been determined from the frozen spin targets of the large ribosomal subunit, which has been deuterated in the TP and rRNA respectively. The results agree with those from neutron scattering in H2O/D2O mixtures obtained at room temperature. This is a necessary prerequisite for the planned determination of the in situ structure of individual ribosomal proteins and especially of that of ribosome bound mRNA and tRNAs. PMID:1720669

  4. Dissociation of ribosomes into large and small subunits.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-04-01

    Structural and functional studies of ribosomal subunits require the dissociation of intact ribosomes into individual small and large ribosomal subunits. The dissociation of the prokaryotic 70S ribosomes into the 50S and 30S subunits is achieved by dialysis against a buffer containing a lower Mg(2+) concentration. Eukaryotic 80S ribosomes are dissociated into 60S and 40S subunits by incubation in a buffer containing puromycin and higher KCl and Mg(2+) concentrations.

  5. Development of Selective Inhibitors for Human Aldehyde Dehydrogenase 3A1 (ALDH3A1) for the Enhancement of Cyclophosphamide Cytotoxicity

    PubMed Central

    Parajuli, Bibek; Georgiadis, Taxiarchis M.; Fishel, Melissa L.; Hurley, Thomas D.

    2014-01-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including cancer chemo-resistance by metabolizing activated forms of oxazaphosphorine drugs such as cyclophosphamide (CP) and its analogues such as mafosfamide (MF), ifosfamide (IFM), 4-hydroperoxycyclophosphamide (4-HPCP). Compounds that can selectively target ALDH3A1 may permit delineation of its roles in these processes and could restore chemosensitivity in cancer cells that express this isoenzyme. Here we report the detailed kinetic and structural characterization of an ALDH3A1 selective inhibitor, CB29, previously identified in a high throughput screen. Kinetic and crystallographic studies demonstrate that CB29 binds within the aldehyde substrate-binding site of ALDH3A1. Cellular proliferation of ALDH3A1-expressing lung adenocarcinoma (A549) and glioblastoma (SF767) cell lines, as well as the ALDH3A1 non-expressing lung fibroblast cells, CCD-13Lu, is unaffected by treatment with CB29 and its analogues alone. However, the sensitivity toward the anti-proliferative effects of mafosfamide is enhanced by treatment with CB29 and its analogue in the tumour cells. In contrast, the sensitivity of CCD-13Lu cells toward mafosfamide was unaffected by the addition of these same compounds. CB29 is chemically distinct from the previously reported small molecule inhibitors of ALDH isoenzymes and does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1 or ALDH2 isoenzymes at concentrations up to 250 μM. Thus, CB29 is a novel small molecule inhibitor of ALDH3A1, which may be useful as a chemical tool to delineate the role of ALDH3A1 in numerous metabolic pathways, including sensitizing ALDH3A1-positive cancer cells to oxazaphosphorines. PMID:24677340

  6. PI3K inhibitors prime neuroblastoma cells for chemotherapy by shifting the balance towards pro-apoptotic Bcl-2 proteins and enhanced mitochondrial apoptosis.

    PubMed

    Bender, A; Opel, D; Naumann, I; Kappler, R; Friedman, L; von Schweinitz, D; Debatin, K-M; Fulda, S

    2011-01-27

    We recently identified activation of phosphatidylinositol 3'-kinase (PI3K)/Akt as a novel predictor of poor outcome in neuroblastoma. Here, we investigated the effect of small-molecule PI3K inhibitors on chemosensitivity. We provide first evidence that PI3K inhibitors, for example PI103, synergize with various chemotherapeutics (Doxorubicin, Etoposide, Topotecan, Cisplatin, Vincristine and Taxol) to trigger apoptosis in neuroblastoma cells (combination index: high synergy). Mechanistic studies reveal that PI103 cooperates with Doxorubicin to reduce Mcl-1 expression and Bim(EL) phosphorylation and to upregulate Noxa and Bim(EL) levels. This shifted ratio of pro- and antiapoptotic Bcl-2 proteins results in increased Bax/Bak conformational change, loss of mitochondrial membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Although Mcl-1 knockdown enhances Doxorubicin- and PI103-induced apoptosis, silencing of Noxa, Bax/Bak or p53 reduces apoptosis, underscoring the functional relevance of the Doxorubicin- and PI103-mediated modulation of these proteins for chemosensitization. Bcl-2 overexpression inhibits Bax activation, mitochondrial perturbations, cleavage of caspases and Bid, and apoptosis, confirming the central role of the mitochondrial pathway for chemosensitization. Interestingly, the broad-range caspase inhibitor zVAD.fmk does not interfere with Bax activation or mitochondrial outer membrane permeabilization, whereas it blocks caspase activation and apoptosis, thus placing mitochondrial events upstream of caspase activation. Importantly, PI103 and Doxorubicin cooperate to induce apoptosis and to suppress tumor growth in patients' derived primary neuroblastoma cells and in an in vivo neuroblastoma model, underlining the clinical relevance of the results. Thus, targeting PI3K presents a novel and promising strategy to sensitize neuroblastoma cells for chemotherapy-induced apoptosis, which has important implications for the

  7. Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP.

    PubMed

    Kuai, L; Wu, C; Qiu, Q; Zhang, J; Zhou, A; Wang, S; Zhang, H; Song, Q; Liao, S; Han, Y; Liu, J; Ma, Z

    2000-08-01

    Erythropoietin (EPO) mimetic peptide (EMP) encoding sequence was inserted into the gene of plasminogen activator inhibitor-1 (PAI-1) between Ala348 and Pro349 (P2'-P3'), generating a novel gene, PAI-1/EMP (PMP). This was cloned into pET32a expression vector, fused with TrxA peptide in the vector, and a 63-kDa protein was expressed in inclusion bodies with an expression level >50%. The TrxA/PMP protein was purified by Ni-NTA-agarose metal-ligand affinity chromatography to a purity >90%, showing a single, silver-stained band on SDS-PAGE. Using a reticulocyte counting assay, the EPO activity of PMP was determined to be 5,000 IU/mg, 2,500-fold that of EMP.

  8. Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides.

    PubMed

    da Cunha Morales Álvares, Alice; Schwartz, Elisabeth Ferroni; Amaral, Nathalia Oda; Trindade, Neidiane Rosa; Pedrino, Gustavo Rodrigues; Silva, Luciano Paulino; de Freitas, Sonia Maria

    2014-10-30

    The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

  9. Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides.

    PubMed

    da Cunha Morales Álvares, Alice; Schwartz, Elisabeth Ferroni; Amaral, Nathalia Oda; Trindade, Neidiane Rosa; Pedrino, Gustavo Rodrigues; Silva, Luciano Paulino; de Freitas, Sonia Maria

    2014-01-01

    The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ. PMID:25361421

  10. Enhancing yield of infectious Bursal disease virus structural proteins in baculovirus expression systems: focus on media, protease inhibitors, and dissolved oxygen.

    PubMed

    Hu, Y C; Bentley, W E

    1999-01-01

    Structural proteins of the poultry pathogen, infectious bursal disease virus (IBDV), were expressed in the baculovirus/insect cell expression system. To date, several reports have indicated that animal virus structural proteins are expressed only at low yield in this system. In this article, several factors were examined to enhance yield. These include medium, dissolved oxygen level, and the addition (in vivo and in vitro) of protease inhibitors. Specifically, two media were compared, and SF-900 II was superior to Ex-Cell 401 for cell growth and IBDV protein expression. A cocktail of protease inhibitors including phenylmethyl sulfonyl fluoride (PMSF), leupeptin, and ethylenediamine tetraacetic acid (EDTA) minimized proteolysis in vitro. Also, aprotinin and pepstatin A deterred product degradation in vivo and increased the product yield nearly 2-fold. Finally, in 3 L bioreactors, a dissolved oxygen tension of 50% DO (air saturation) was optimal. Results demonstrated that several relatively simple adjustments to the baculovirus system significantly improved the yield of IBD virus structural proteins. PMID:10585191

  11. Enhancing yield of infectious Bursal disease virus structural proteins in baculovirus expression systems: focus on media, protease inhibitors, and dissolved oxygen.

    PubMed

    Hu, Y C; Bentley, W E

    1999-01-01

    Structural proteins of the poultry pathogen, infectious bursal disease virus (IBDV), were expressed in the baculovirus/insect cell expression system. To date, several reports have indicated that animal virus structural proteins are expressed only at low yield in this system. In this article, several factors were examined to enhance yield. These include medium, dissolved oxygen level, and the addition (in vivo and in vitro) of protease inhibitors. Specifically, two media were compared, and SF-900 II was superior to Ex-Cell 401 for cell growth and IBDV protein expression. A cocktail of protease inhibitors including phenylmethyl sulfonyl fluoride (PMSF), leupeptin, and ethylenediamine tetraacetic acid (EDTA) minimized proteolysis in vitro. Also, aprotinin and pepstatin A deterred product degradation in vivo and increased the product yield nearly 2-fold. Finally, in 3 L bioreactors, a dissolved oxygen tension of 50% DO (air saturation) was optimal. Results demonstrated that several relatively simple adjustments to the baculovirus system significantly improved the yield of IBD virus structural proteins.

  12. Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

    PubMed Central

    Terras, FRG.; Schoofs, HME.; Thevissen, K.; Osborn, R. W.; Vanderleyden, J.; Cammue, BPA.; Broekaert, W. F.

    1993-01-01

    Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes. PMID:12232024

  13. Inhibition of CHK1 enhances cell death induced by the Bcl-2-selective inhibitor ABT-199 in acute myeloid leukemia cells

    PubMed Central

    Zhao, Jianyun; Niu, Xiaojia; Li, Xinyu; Edwards, Holly; Wang, Guan; Wang, Yue; Taub, Jeffrey W; Lin, Hai; Ge, Yubin

    2016-01-01

    Resistance to standard chemotherapy agents remains a major obstacle for improving treatment outcomes for acute myeloid leukemia (AML). The Bcl-2-selective inhibitor ABT-199 has demonstrated encouraging preclinical results, drug resistance remains a concern. Mcl-1 has been demonstrated to contribute to ABT-199 resistance, thus combining with therapies that target Mcl-1 could overcome such resistance. In this study, we utilized a CHK1 inhibitor, LY2603618, to decrease Mcl-1 and enhance ABT-199 efficacy. We found that LY2603618 treatment resulted in abolishment of the G2/M cell cycle checkpoint and increased DNA damage, which was partially dependent on CDK activity. LY2603618 treatment resulted in decrease of Mcl-1, which coincided with the initiation of apoptosis. Overexpression of Mcl-1 in AML cells significantly attenuated apoptosis induced by LY2603618, confirming the critical role of Mcl-1 in apoptosis induced by the agent. Simultaneous treatment with LY2603618 and ABT-199 resulted in synergistic induction of apoptosis in both AML cell lines and primary patient samples. Our findings provide new insights into overcoming a mechanism of intrinsic ABT-199 resistance in AML cells and support the clinical development of combined ABT-199 and CHK1 inhibition. PMID:27166183

  14. Enhancement of Radiation Sensitivity in Lung Cancer Cells by a Novel Small Molecule Inhibitor That Targets the β-Catenin/Tcf4 Interaction

    PubMed Central

    Luo, Guifen; Han, Xiaofeng; Bao, Wenjing; Cheng, Yanyan; Tian, Wang; Yan, Maocai; Yang, Guanlin; An, Jing

    2016-01-01

    Radiation therapy is an important treatment choice for unresectable advanced human lung cancers, and a critical adjuvant treatment for surgery. However, radiation as a lung cancer treatment remains far from satisfactory due to problems associated with radiation resistance in cancer cells and severe cytotoxicity to non-cancer cells, which arise at doses typically administered to patients. We have recently identified a promising novel inhibitor of β-catenin/Tcf4 interaction, named BC-23 (C21H14ClN3O4S), which acts as a potent cell death enhancer when used in combination with radiation. Sequential exposure of human p53-null non-small cell lung cancer (NSCLC) H1299 cells to low doses of x-ray radiation, followed 1 hour later by administration of minimally cytotoxic concentrations of BC-23, resulted in a highly synergistic induction of clonogenic cell death (combination index <1.0). Co-treatment with BC-23 at low concentrations effectively inhibits Wnt/β-catenin signaling and down-regulates c-Myc and cyclin D1 expression. S phase arrest and ROS generation are also involved in the enhancement of radiation effectiveness mediated by BC-23. BC-23 therefore represents a promising new class of radiation enhancer. PMID:27014877

  15. Enhancement of Radiation Sensitivity in Lung Cancer Cells by a Novel Small Molecule Inhibitor That Targets the β-Catenin/Tcf4 Interaction.

    PubMed

    Zhang, Qinghao; Gao, Mei; Luo, Guifen; Han, Xiaofeng; Bao, Wenjing; Cheng, Yanyan; Tian, Wang; Yan, Maocai; Yang, Guanlin; An, Jing

    2016-01-01

    Radiation therapy is an important treatment choice for unresectable advanced human lung cancers, and a critical adjuvant treatment for surgery. However, radiation as a lung cancer treatment remains far from satisfactory due to problems associated with radiation resistance in cancer cells and severe cytotoxicity to non-cancer cells, which arise at doses typically administered to patients. We have recently identified a promising novel inhibitor of β-catenin/Tcf4 interaction, named BC-23 (C21H14ClN3O4S), which acts as a potent cell death enhancer when used in combination with radiation. Sequential exposure of human p53-null non-small cell lung cancer (NSCLC) H1299 cells to low doses of x-ray radiation, followed 1 hour later by administration of minimally cytotoxic concentrations of BC-23, resulted in a highly synergistic induction of clonogenic cell death (combination index <1.0). Co-treatment with BC-23 at low concentrations effectively inhibits Wnt/β-catenin signaling and down-regulates c-Myc and cyclin D1 expression. S phase arrest and ROS generation are also involved in the enhancement of radiation effectiveness mediated by BC-23. BC-23 therefore represents a promising new class of radiation enhancer.

  16. Enhancement of Radiation Sensitivity in Lung Cancer Cells by a Novel Small Molecule Inhibitor That Targets the β-Catenin/Tcf4 Interaction.

    PubMed

    Zhang, Qinghao; Gao, Mei; Luo, Guifen; Han, Xiaofeng; Bao, Wenjing; Cheng, Yanyan; Tian, Wang; Yan, Maocai; Yang, Guanlin; An, Jing

    2016-01-01

    Radiation therapy is an important treatment choice for unresectable advanced human lung cancers, and a critical adjuvant treatment for surgery. However, radiation as a lung cancer treatment remains far from satisfactory due to problems associated with radiation resistance in cancer cells and severe cytotoxicity to non-cancer cells, which arise at doses typically administered to patients. We have recently identified a promising novel inhibitor of β-catenin/Tcf4 interaction, named BC-23 (C21H14ClN3O4S), which acts as a potent cell death enhancer when used in combination with radiation. Sequential exposure of human p53-null non-small cell lung cancer (NSCLC) H1299 cells to low doses of x-ray radiation, followed 1 hour later by administration of minimally cytotoxic concentrations of BC-23, resulted in a highly synergistic induction of clonogenic cell death (combination index <1.0). Co-treatment with BC-23 at low concentrations effectively inhibits Wnt/β-catenin signaling and down-regulates c-Myc and cyclin D1 expression. S phase arrest and ROS generation are also involved in the enhancement of radiation effectiveness mediated by BC-23. BC-23 therefore represents a promising new class of radiation enhancer. PMID:27014877

  17. Enhancement of antibiotic activity against multidrug-resistant bacteria by the efflux pump inhibitor 3,4-dibromopyrrole-2,5-dione isolated from a Pseudoalteromonas sp.

    PubMed

    Whalen, Kristen E; Poulson-Ellestad, Kelsey L; Deering, Robert W; Rowley, David C; Mincer, Tracy J

    2015-03-27

    Members of the resistance nodulation cell division (RND) of efflux pumps play essential roles in multidrug resistance (MDR) in Gram-negative bacteria. Here, we describe the search for new small molecules from marine microbial extracts to block efflux and thus restore antibiotic susceptibility in MDR bacterial strains. We report the isolation of 3,4-dibromopyrrole-2,5-dione (1), an inhibitor of RND transporters, from Enterobacteriaceae and Pseudomonas aeruginosa, from the marine bacterium Pseudoalteromonas piscicida. 3,4-Dibromopyrrole-2,5-dione decreased the minimum inhibitory concentrations (MICs) of two fluoroquinolones, an aminoglycoside, a macrolide, a beta-lactam, tetracycline, and chloramphenicol between 2- and 16-fold in strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM). 3,4-Dibromopyrrole-2,5-dione also increased the intracellular accumulation of Hoechst 33342 in wild-type but not in transporter-deficient strains and prevented H33342 efflux (IC50 = 0.79 μg/mL or 3 μM), a hallmark of efflux pump inhibitor (EPI) functionality. A metabolomic survey of 36 Pseudoalteromonas isolates mapped the presence of primarily brominated metabolites only within the P. piscicida phylogenetic clade, where a majority of antibiotic activity was also observed, suggesting a link between halogenation and enhanced secondary metabolite biosynthetic potential. In sum, 3,4-dibromopyrrole-2,5-dione is a potent EPI and deserves further attention as an adjuvant to enhance the effectiveness of existing antibiotics. PMID:25646964

  18. Distribution of dwell times of a ribosome: effects of infidelity, kinetic proofreading and ribosome crowding

    NASA Astrophysics Data System (ADS)

    Sharma, Ajeet K.; Chowdhury, Debashish

    2011-04-01

    Ribosome is a molecular machine that polymerizes a protein where the sequence of the amino acid residues, the monomers of the protein, is dictated by the sequence of codons (triplets of nucleotides) on a messenger RNA (mRNA) that serves as the template. The ribosome is a molecular motor that utilizes the template mRNA strand also as the track. Thus, in each step the ribosome moves forward by one codon and, simultaneously, elongates the protein by one amino acid. We present a theoretical model that captures most of the main steps in the mechanochemical cycle of a ribosome. The stochastic movement of the ribosome consists of an alternating sequence of pause and translocation; the sum of the durations of a pause and the following translocation is the time of dwell of the ribosome at the corresponding codon. We derive the analytical expression for the distribution of the dwell times of a ribosome in our model. Wherever experimental data are available, our theoretical predictions are consistent with those results. We suggest appropriate experiments to test the new predictions of our model, particularly the effects of the quality control mechanism of the ribosome and that of their crowding on the mRNA track.

  19. Structure of the mammalian antimicrobial peptide Bac7(1-16) bound within the exit tunnel of a bacterial ribosome.

    PubMed

    Seefeldt, A Carolin; Graf, Michael; Pérébaskine, Natacha; Nguyen, Fabian; Arenz, Stefan; Mardirossian, Mario; Scocchi, Marco; Wilson, Daniel N; Innis, C Axel

    2016-03-18

    Proline-rich antimicrobial peptides (PrAMPs) produced as part of the innate immune response of animals, insects and plants represent a vast, untapped resource for the treatment of multidrug-resistant bacterial infections. PrAMPs such as oncocin or bactenecin-7 (Bac7) interact with the bacterial ribosome to inhibit translation, but their supposed specificity as inhibitors of bacterial rather than mammalian protein synthesis remains unclear, despite being key to developing drugs with low toxicity. Here, we present crystal structures of the Thermus thermophilus 70S ribosome in complex with the first 16 residues of mammalian Bac7, as well as the insect-derived PrAMPs metalnikowin I and pyrrhocoricin. The structures reveal that the mammalian Bac7 interacts with a similar region of the ribosome as insect-derived PrAMPs. Consistently, Bac7 and the oncocin derivative Onc112 compete effectively with antibiotics, such as erythromycin, which target the ribosomal exit tunnel. Moreover, we demonstrate that Bac7 allows initiation complex formation but prevents entry into the elongation phase of translation, and show that it inhibits translation on both mammalian and bacterial ribosomes, explaining why this peptide needs to be stored as an inactive pro-peptide. These findings highlight the need to consider the specificity of PrAMP derivatives for the bacterial ribosome in future drug development efforts. PMID:26792896

  20. RatA (YfjG), An Escherichia Coli Toxin, Inhibits 70S Ribosome Association to Block Translation Initiation

    PubMed Central

    Zhang, Yonglong; Inouye, Masayori

    2011-01-01

    Summary RatA (YfjG) is a toxin encoded by the ratA-ratB (yfjG-yfjF) operon on the Escherichia coli genome. Induction of RatA led to the inhibition of protein synthesis, while DNA and RNA synthesis was not affected. The stability of mRNAs was also unchanged as judged by in vivo primer extension experiments and by Northern blotting analysis. The ribosome profile of the cells overexpressing RatA showed that 70S ribosomes as well as polysomes significantly decreased with concomitant increase of 50S and 30S subunits. The addition of purified RatA to a cell-free system inhibited the formation of 70S ribosomes even in the presence of 6 mM Mg2+. RatA was specifically associated with 50S subunits, indicating that it binds to 50S subunits to block its association with 30S subunits leading to the inhibition of formation of 70S ribosomes. However, RatA did not cause dissociation of 70S ribosomes and its anti-association activity was blocked by paromomycin, an inhibitor for IF3, an essential initiation factor, having 21% sequence homology with RatA. Here we demonstrate that RatA is a new E. coli toxin, which effectively blocks the translation initiation step. We propose that this toxin of previously unknown function be renamed as RatA (Ribosome association toxin A). PMID:21323758

  1. In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

    PubMed Central

    Yates, J L; Arfsten, A E; Nomura, M

    1980-01-01

    Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins. Images PMID:6445562

  2. HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.

    PubMed

    Zhang, Yanqing; Mandava, Chandra Sekhar; Cao, Wei; Li, Xiaojing; Zhang, Dejiu; Li, Ningning; Zhang, Yixiao; Zhang, Xiaoxiao; Qin, Yan; Mi, Kaixia; Lei, Jianlin; Sanyal, Suparna; Gao, Ning

    2015-11-01

    Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions. PMID:26458047

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

    SciTech Connect

    Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen; Bashan, Anat; Belousoff, Matthew; Wekselman, Itai; Zimmerman, Ella; Xiong, Liqun; Klepacki, Dorota; Arakawa, Kenji; Kinashi, Haruyasu; Mankin, Alexander S.; Yonath, Ada

    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, the 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.

  4. HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.

    PubMed

    Zhang, Yanqing; Mandava, Chandra Sekhar; Cao, Wei; Li, Xiaojing; Zhang, Dejiu; Li, Ningning; Zhang, Yixiao; Zhang, Xiaoxiao; Qin, Yan; Mi, Kaixia; Lei, Jianlin; Sanyal, Suparna; Gao, Ning

    2015-11-01

    Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.

  5. Exposure to histone deacetylase inhibitors during Pavlovian conditioning enhances subsequent cue-induced reinstatement of operant behavior.

    PubMed

    Ploense, Kyle L; Kerstetter, Kerry A; Wade, Matthew A; Woodward, Nicholas C; Maliniak, Dan; Reyes, Michael; Uchizono, Russell S; Bredy, Timothy W; Kippin, Tod E

    2013-06-01

    Histone deacetylase inhibitors (HDACIs) strengthen memory following fear conditioning and cocaine-induced conditioned place preference. Here, we examined the effects of two nonspecific HDACIs, valproic acid (VPA) and sodium butyrate (NaB), on appetitive learning measured by conditioned stimulus (CS)-induced reinstatement of operant responding. Rats were trained to lever press for food reinforcement and then injected with VPA (50-200 mg/kg, i.p.), NaB (250-1000 mg/kg, i.p.), or saline vehicle (1.0 ml/kg), 2 h before receiving pairings of noncontingent presentation of food pellets preceded by a tone+light cue CS. Rats next underwent extinction of operant responding followed by response-contingent re-exposure to the CS. Rats receiving VPA (100 mg/kg) or NaB (1000 mg/kg) before conditioning displayed significantly higher cue-induced reinstatement than did saline controls. Rats that received either vehicle or VPA (100 mg/kg) before a conditioning session with a randomized relation between presentation of food pellets and the CS failed to show subsequent cue-induced reinstatement with no difference between the two groups. These findings indicate that, under certain contexts, HDACIs strengthen memory formation by specifically increasing the associative strength of the CS, not through an increasing motivation to seek reinforcement.

  6. Mammary-Derived Growth Inhibitor Targeting Peptide-Modified PEG-PLA Nanoparticles for Enhanced Targeted Glioblastoma Therapy.

    PubMed

    Feng, Xingye; Gao, Xiaoling; Kang, Ting; Jiang, Di; Yao, Jianhui; Jing, Yixian; Song, Qingxiang; Jiang, Xinguo; Liang, Jianying; Chen, Jun

    2015-08-19

    Targeting delivery of chemotherapeutics to neovasculature represents a promising means for tumor therapy since angiogenesis has been a featured hallmark of glioblastma. However, anti-angiogenic therapy would induce the occurrence of metastatic tumor and even neoplasm recurrence. Simultaneous targeting of tumor cells and neovasculature perfectly overcome such defects and has been proven to be an efficacious strategy for suppressing tumor growth. In the present study, a tumor homing peptide CooP selective binding to mammary-derived growth inhibitor that overexpressed in glioma cells and blood vessel endothelial cells was decorated on the surface of paclitaxel-loading PEG-PLA nanoparticles (NP-PTX) to obtain the dual targeting nanovector CooP-NP-PTX. In vitro antiproliferation study showed that HUVEC cells and U87MG cells were much more sensitive to CooP-NP-PTX than NP-PTX. In vivo imaging demonstrated that CooP-NP accumulated more selectively and penetrated deeper into the tumor site. In addition, the glioma-bearing mice treated with CooP-NP-PTX achieved the longest survival time compared to NP-PTX and Taxol. The findings observed above indicated that CooP peptide-functionalized anti-neoplastic agent-loaded nanoparticles might possess promising potential for glioblastoma therapy. PMID:26222392

  7. Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo.

    PubMed

    Czuczman, Natalie M; Barth, Matthew J; Gu, Juan; Neppalli, Vishala; Mavis, Cory; Frys, Sarah E; Hu, Qiang; Liu, Song; Klener, Pavel; Vockova, Petra; Czuczman, Myron S; Hernandez-Ilizaliturri, Francisco J

    2016-03-01

    Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 μM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL. PMID:26675347

  8. Enhanced expression of early mitotic inhibitor-1 predicts a poor prognosis in esophageal squamous cell carcinoma patients

    PubMed Central

    GUAN, CHENGQI; ZHANG, JIANFENG; ZHANG, JIANGUO; SHI, HUI; NI, RUNZHOU

    2016-01-01

    Early mitotic inhibitor-1 (Emi1), as a key cell cycle regulatory gene, induces S phase and mitotic entry by controlling anaphase-promoting complex substrates. Emi1 overexpression may be a prognostic factor for patients with invasive breast cancer. However, its expression and clinical significance in esophageal squamous cell carcinoma (ESCC) remain unknown. In the present study, Emi1 was overexpressed in ESCC samples, contrarily to their neighboring normal tissues. The expression of Emi1 was correlated with histological differentiation (P=0.032), lymphatic metastasis (P=0.006) and Ki-67 expression (P=0.028). Multivariate analysis indicated that the presence of lymphatic metastasis and the protein expression levels of Emi1 and Ki-67 were all independent prognostic factors for ESCC patients (P=0.042, 0.018 and 0.001, respectively). In vitro, however, the expression of Emi1 was upregulated in the ECA109 cell line following release from serum starvation. In addition, depletion of endogenous Emi1 by small interfering RNA could effectively reduce cell proliferation. Thus, the present data indicated that Emi1 expression was upregulated in ESCC tissues and correlated with poor survival in ESCC patients, and suggested that Emi1 may be an independent prognostic factor for ESCC patients. PMID:27347110

  9. The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines

    PubMed Central

    Cosenza, Maria; Civallero, Monica; Fiorcari, Stefania; Pozzi, Samantha; Marcheselli, Luigi; Bari, Alessia; Ferri, Paola; Sacchi, Stefano

    2016-01-01

    ABSTRACT We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas-299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou–Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, −9, −3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies. PMID:27657380

  10. Intracellular colocalization of HAP1/STBs with steroid hormone receptors and its enhancement by a proteasome inhibitor

    SciTech Connect

    Fujinaga, Ryutaro; Takeshita, Yukio; Yoshioka, Kazuhiro; Nakamura, Hiroyuki; Shinoda, Shuhei; Islam, Md. Nabiul; Jahan, Mir Rubayet; Yanai, Akie; Kokubu, Keiji; Shinoda, Koh

    2011-07-15

    The stigmoid body (STB) is a cytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), and HAP1/STB formation is induced by transfection of the HAP1 gene into cultured cells. In the present study, we examined the intracellular colocalization of HAP1/STBs with steroid hormone receptors (SHRs), including the androgen receptor (AR), estrogen receptor, glucocorticoid receptor (GR), and mineralocorticoid receptor, in COS-7 cells cotransfected with HAP1 and each receptor. We found that C-terminal ligand-binding domains of all SHRs had potential for colocalization with HAP1/STBs, whereas only AR and GR were clearly colocalized with HAP1/STBs when each full-length SHR was coexpressed with HAP1. In addition, it appeared that HAP1/STBs did not disrupt GR and AR functions because the receptors on HAP1/STBs maintained nuclear translocation activity in response to their specific ligands. When the cells were treated with a proteasome inhibitor, GR and AR localized outside HAP1/STBs translocated into the nucleus, whereas the receptors colocalized with HAP1/STBs persisted in their colocalization even after treatment with their ligands. Therefore, HAP1/STBs may be involved in cytoplasmic modifications of the nuclear translocation of GR and AR in a ubiquitin-proteasome system.

  11. The novel tankyrase inhibitor (AZ1366) enhances irinotecan activity in tumors that exhibit elevated tankyrase and irinotecan resistance

    PubMed Central

    Quackenbush, Kevin S.; Bagby, Stacey; Tai, Wai Meng; Messersmith, Wells A.; Schreiber, Anna; Greene, Justin; Kim, Jihye; Wang, Guoliang; Purkey, Alicia; Pitts, Todd M.; Nguyen, Anna; Gao, Dexiang; Blatchford, Patrick; Capasso, Anna; Schuller, Alwin G.; Eckhardt, S. Gail; Arcaroli, John J.

    2016-01-01

    Background Dysregulation of the canonical Wnt signaling pathway has been implicated in colorectal cancer (CRC) development as well as incipient stages of malignant transformation. In this study, we investigated the antitumor effects of AZ1366 (a novel tankyrase inhibitor) as a single agent and in combination with irinotecan in our patient derived CRC explant xenograft models. Results Six out of 18 CRC explants displayed a significant growth reduction to AZ1366. There was one CRC explant (CRC040) that reached the threshold of sensitivity (TGII ≤ 20%) in this study. In addition, the combination of AZ1366 + irinotecan demonstrated efficacy in 4 out of 18 CRC explants. Treatment effects on the WNT pathway revealed that tankyrase inhibition was ineffective at reducing WNT dependent signaling. However, the anti-tumor effects observed in this study were likely a result of alternative tankyrase effects whereby tankyrase inhibition reduced NuMA levels. Materials and Methods Eighteen CRC explants were treated with AZ1366 single agent or in combination for 28 days and treatment responses were assessed. Pharmacokinetic (AZ1366 drug concentrations) and pharmacodynamic effects (Axin2 levels) were investigated over 48 hours. Immunohistochemistry of nuclear β-catenin levels as well as western blot was employed to examine the treatment effects on the WNT pathway as well as NuMA. Conclusions Combination AZ1366 and irinotecan achieved greater anti-tumor effects compared to monotherapy. Activity was limited to CRC explants that displayed irinotecan resistance and increased protein levels of tankyrase and NuMA. PMID:27070088

  12. High-resolution structure of the Escherichia coli ribosome

    PubMed Central

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; Altman, Roger B.; Blanchard, Scott C.; Cate, Jamie H. D.

    2015-01-01

    Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development. PMID:25775265

  13. [Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method].

    PubMed

    Iusupov, M M; Spirin, A S

    1986-11-01

    A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure. PMID:3542056

  14. Ribosomes in the squid giant axon.

    PubMed

    Bleher, R; Martin, R

    2001-01-01

    Ribosome clusters, referred to as endoaxoplasmic plaques, were documented and quantitatively analyzed in the squid giant axon at the light and electron microscopic levels. The methods included nonspecific high affinity fluorescence staining of RNA by YOYO-1, specific immunofluorescence labeling of ribosomal RNA, electron energy loss spectroscopic mapping of ribosomal phosphorus, and conventional transmission electron microscopy. The endoaxoplasmic plaques were sharply defined, oval in shape, and less than 2 microm in diameter. While they were very numerous in the postsynaptic axonal area of the giant synapse, the frequency of occurrence was much lower in the peripheral giant axon, with a density of about 1 plaque/1000 microm3. Their distribution was random within axoplasm, with no preferential localization near the membrane. The several thousand ribosomes in a plaque usually were not membrane bound, but vesicular structures were observed in or near plaques; plaques were often surrounded by mitochondria. We conclude that ribosomes, a requisite machinery for protein synthesis, are present in the squid giant axon in discrete configurations.

  15. c-Met inhibitor SU11274 enhances the response of the prostate cancer cell line DU145 to ionizing radiation

    SciTech Connect

    Yu, Hongliang; Li, Xiaoying; Sun, Shaoqian; Gao, Xianshu; Zhou, Demin

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer c-Met inhibition could significantly enhance the radiosensitivity of DU145 cells. Black-Right-Pointing-Pointer The mechanisms of the radiosensitization effect of c-Met inhibition on DU145 cells were also presented in this paper. Black-Right-Pointing-Pointer This is the first study demonstrating the effectiveness of c-Met inhibition on treating HRPC cells with radiotherapy. -- Abstract: Hormone-refractory prostate cancer shows substantial resistance to most conventional therapies including radiotherapy, constitutes a key impediment to curing patients with the disease. c-Met overexpression plays a key role in prostate cancer tumorigenesis and disease progression. Here, we demonstrate that c-Met inhibition by SU11274 could significantly suppress cell survival and proliferation as well as enhance the radiosensitivity of DU145 cells. The underlying mechanisms of the effects of SU11274 on DU145 cells may include the inhibition of c-Met signaling, depolarization of the mitochondrial membrane potential, impairment of DNA repair function, abrogation of cell cycle arrest, and enhancement of cell death. Our study is the first to show the effectiveness of combining c-Met inhibition with ionizing radiation to cure hormone-refractory prostate cancer.

  16. Bile enhances glucose uptake, reduces permeability, and modulates effects of lectins, trypsin inhibitors and saponins on intestinal tissue.

    PubMed

    Bakke, Anne Marie; Chikwati, Elvis M; Venold, Fredrik F; Sahlmann, Christian; Holm, Halvor; Penn, Michael H; Oropeza-Moe, Marianne; Krogdahl, Åshild

    2014-02-01

    Antinutritional factors (ANFs) can disrupt digestive and other intestinal functions. ANFs in soybean meal (SBM) are implicated in proliferative and inflammatory responses in the intestine of various (functionally) monogastric animals, including Atlantic salmon (Salmo salar L.). The goal of the current study was to investigate the effect of ex vivo exposure of mid and distal intestinal tissue of salmon to soybean saponins (SAP), lectin (LEC) and Kunitz' trypsin inhibitor (KTI), singly and in combination, on epithelial function, as assessed by measuring in vitro glucose uptake pathways along a glucose concentration gradient. As solubilization of SAP in the calcium-containing Ringer's solution was problematic but resolved with the addition of a physiological concentration of bile collected from the gall bladder of salmon, an evaluation of bile effects became an added element. Results indicated that bile increased baseline glucose absorption and possibly transport, and also had a protective effect on the epithelial barrier, at least partially due to taurocholate. Compared to controls, tissues exposed to LEC+bile, KTI+bile and LEC+KTI+bile exhibited increased glucose uptake at the higher glucose concentrations, apparently due to markedly increased tissue permeability. Addition of SAP, however, attenuated the response, possibly by binding bile components. SAP+bile, also in combination with LEC and/or KTI, as well as LEC, KTI and LEC+KTI without bile often reduced transcellular glucose uptake pathways, while maintaining low tissue permeability. SAP+LEC+KTI+bile, LEC and KTI caused the most marked reductions. The distal intestine was more affected, reflecting the restriction of in vivo SBM-induced inflammatory changes to this region. PMID:24291392

  17. Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations.

    PubMed

    Cong, Xiaojing; Cremer, Christian; Nachreiner, Thomas; Barth, Stefan; Carloni, Paolo

    2016-08-01

    Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 µs replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/G86R (GGRRmut ), Q117G (QGmut ), and G85R/G86R/Q117G (GGRR/QGmut ). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut -based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut -based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut -based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity. PMID:27110669

  18. Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations.

    PubMed

    Cong, Xiaojing; Cremer, Christian; Nachreiner, Thomas; Barth, Stefan; Carloni, Paolo

    2016-08-01

    Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 µs replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/G86R (GGRRmut ), Q117G (QGmut ), and G85R/G86R/Q117G (GGRR/QGmut ). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut -based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut -based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut -based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.

  19. Histone deacetylase inhibitor sodium butyrate enhances cellular radiosensitivity by inhibiting both DNA nonhomologous end joining and homologous recombination.

    PubMed

    Koprinarova, Miglena; Botev, Peter; Russev, George

    2011-09-01

    HDAC inhibitors have been proposed as radiosensitizers in cancer therapy. Their application would permit the use of lower radiation doses and would reduce the adverse effects of the treatment. However, the molecular mechanisms of their action remain unclear. In the present article, we have studied the radiosensitizing effect of sodium butyrate on HeLa cells. FACS analysis showed that it did not abrogate the γ-radiation imposed G2 cell cycle arrest. The dynamics of γ-H2AX foci disappearance in the presence and in the absence of butyrate, however, demonstrated that butyrate inhibited DSB repair. In an attempt to clarify which one of the two major DSBs repair pathways was affected, we synchronized HeLa cells in G1 phase and after γ-irradiation followed the repair of the DSBs by agarose gel electrophoresis. Since HR is not operational during G1 phase, by this approach we determined the rates of NHEJ only. The results showed that NHEJ decreased in the presence of butyrate. In another set of experiments, we followed the dynamics of disappearance of RAD51 foci in the presence and in the absence of butyrate after γ-radiation of HeLa cells. Since RAD51 takes part in HR only, this experiment allows the effect of butyrate on DSB repair by homologous recombination to be assessed. It showed that HR was also obstructed by butyrate. These results were confirmed by host cell reactivation assays in which the repair of plasmids containing a single DSB by NHEJ or HR was monitored. We suggest that after a DSB is formed, HDACs deacetylated core histones in the vicinity of the breaks in order to compact the chromatin structure and prevent the broken DNA ends from moving apart from each other, thus ensuring effective repair.

  20. Molecular interactions of ribosomal components. IV: Cooperative interactions during assembly in vitro.

    PubMed

    Green, M; Kurland, C G

    1973-08-01

    Cooperative interactions between different 30S ribosomal proteins during assembly in vitro are described. The site specific binding of S7 to 16S RNA is enhanced by S20; that of S16 requires S4 and S20; and S7 is required for the maximum binding of S9, S13 and S19. Some of these interactions are reflected in the protein neighborhoods of the functional ribosome, but this may not be a general rule. Finally, we suggest that the assembly cooperativety observed may not be a consequence of direct-protein interactions.

  1. Genome Mining for Ribosomally Synthesized Natural Products

    PubMed Central

    Velásquez, Juan E.; van der Donk, Wilfred

    2011-01-01

    In recent years, the number of known peptide natural products that are synthesized via the ribosomal pathway has rapidly grown. Taking advantage of sequence homology among genes encoding precursor peptides or biosynthetic proteins, in silico mining of genomes combined with molecular biology approaches has guided the discovery of a large number of new ribosomal natural products, including lantipeptides, cyanobactins, linear thiazole/oxazole-containing peptides, microviridins, lasso peptides, amatoxins, cyclotides, and conopeptides. In this review, we describe the strategies used for the identification of these ribosomally-synthesized and posttranslationally modified peptides (RiPPs) and the structures of newly identified compounds. The increasing number of chemical entities and their remarkable structural and functional diversity may lead to novel pharmaceutical applications. PMID:21095156

  2. Structural snapshots of actively translating human ribosomes.

    PubMed

    Behrmann, Elmar; Loerke, Justus; Budkevich, Tatyana V; Yamamoto, Kaori; Schmidt, Andrea; Penczek, Pawel A; Vos, Matthijn R; Bürger, Jörg; Mielke, Thorsten; Scheerer, Patrick; Spahn, Christian M T

    2015-05-01

    Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

  3. Single-molecule observations of ribosome function.

    PubMed

    Blanchard, Scott C

    2009-02-01

    Single-molecule investigations promise to greatly advance our understanding of basic and regulated ribosome functions during the process of translation. Here, recent progress towards directly imaging the elemental translation elongation steps using fluorescence resonance energy transfer (FRET)-based imaging methods is discussed, which provide striking evidence of the highly dynamic nature of the ribosome. In this view, global rates and fidelities of protein synthesis reactions may be regulated by interactions of the ribosome with mRNA, tRNA, translation factors and potentially many other cellular ligands that modify intrinsic conformational equilibria in the translating particle. Future investigations probing this model must aim to visualize translation processes from multiple structural and kinetic perspectives simultaneously, to provide direct correlations between factor binding and conformational events.

  4. p38 mitogen-activated protein kinase inhibitor LY2228820 enhances bortezomib-induced cytotoxicity and inhibits osteoclastogenesis in multiple myeloma; therapeutic implications.

    PubMed

    Ishitsuka, Kenji; Hideshima, Teru; Neri, Paola; Vallet, Sonia; Shiraishi, Norihiko; Okawa, Yutaka; Shen, Zhenxin; Raje, Noopur; Kiziltepe, Tanyel; Ocio, Enrique M; Chauhan, Dharminder; Tassone, Pierfrancesco; Munshi, Nikhil; Campbell, Robert M; Dios, Alfonso De; Shih, Chuan; Starling, James J; Tamura, Kazuo; Anderson, Kenneth C

    2008-05-01

    The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment induces proliferation and survival of MM cells, as well as osteoclastogenesis. This study investigated the therapeutic potential of novel p38 mitogen-activated protein kinase (p38MAPK) inhibitor LY2228820 (LY) in MM. Although cytotoxicity against MM cell lines was modest, LY significantly enhanced the toxicity of bortezomib by down-regulating bortezomib-induced heat shock protein 27 phosphorylation. LY inhibited interleukin-6 secretion from long term cultured-BM stromal cells and BM mononuclear cells (BMMNCs) derived from MM patients in remission. LY also inhibited macrophage inflammatory protein-1alpha secretion from patient MM cells and BMMNCs as well as normal CD14 positive osteoclast precursor cells. Moreover, LY significantly inhibited in vitro osteoclastogenesis from CD14 positive cells induced by macrophage-colony stimulating factor and soluble receptor activator of nuclear factor-kappaB ligand. Finally, LY also inhibited in vivo osteoclatogenesis in a severe combined immunodeficiency mouse model of human MM. These results suggest that LY represents a promising novel targeted approach to improve MM patient outcome both by enhancing the effect of bortezomib and by reducing osteoskeletal events.

  5. Structure and dynamics of the mammalian ribosomal pre-translocation complex

    PubMed Central

    Budkevich, Tatyana; Giesebrecht, Jan; Altman, Roger B.; Munro, James B.; Mielke, Thorsten; Nierhaus, Knud H.; Blanchard, Scott C.; Spahn, Christian M.T.

    2011-01-01

    Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pre-translocation complex reveal spontaneous, large-scale conformational changes including an inter-subunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favoured within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor, cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action. PMID:22017870

  6. Eudistomin C, an Antitumor and Antiviral Natural Product, Targets 40S Ribosome and Inhibits Protein Translation.

    PubMed

    Ota, Yu; Chinen, Takumi; Yoshida, Keisuke; Kudo, Shun; Nagumo, Yoko; Shiwa, Yuh; Yamada, Ryosuke; Umihara, Hirotatsu; Iwasaki, Kotaro; Masumoto, Hiroshi; Yokoshima, Satoshi; Yoshikawa, Hirofumi; Fukuyama, Tohru; Kobayashi, Junichi; Usui, Takeo

    2016-09-01

    Eudistomin C (EudiC), a natural product, shows potent antitumor and antiviral activities, but the target molecule and the mechanism of action remain to be revealed. Here, we show that the 40S ribosome is the target in EudiC cytotoxicity. We isolated EudiC-resistant mutants from a multidrug-sensitive yeast strain, and a genetic analysis classified these YER (yeast EudiC resistance) mutants into three complementation groups. A genome-wide study revealed that the YER1-6 mutation is in the uS11 gene (RPS14A). Biotinylated EudiC pulled down Rps14p-containing complexes from 40S and 80S ribosomes, but not from the 60S ribosome. EudiC strongly inhibited translation of the wild-type strain but not of YER1-6 in cells and in vitro. These results indicate that EudiC is a protein synthesis inhibitor targeting the uS11-containing ribosomal subunit, and shows cytotoxicity by inhibiting protein translation. PMID:27304596

  7. Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center

    PubMed Central

    Marks, James; Kannan, Krishna; Roncase, Emily J.; Klepacki, Dorota; Kefi, Amira; Orelle, Cédric; Vázquez-Laslop, Nora; Mankin, Alexander S.

    2016-01-01

    The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center. PMID:27791002

  8. Ribosome-dependent activation of stringent control.

    PubMed

    Brown, Alan; Fernández, Israel S; Gordiyenko, Yuliya; Ramakrishnan, V

    2016-06-01

    In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics. PMID:27279228

  9. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    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.

  10. Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis.

    PubMed

    Ren, Jinqi; Wang, Yaqing; Liang, Yuheng; Zhang, Yongqing; Bao, Shilai; Xu, Zhiheng

    2010-04-23

    Modulation of ribosomal assembly is a fine tuning mechanism for cell number and organ size control. Many ribosomal proteins undergo post-translational modification, but their exact roles remain elusive. Here, we report that ribosomal protein s10 (RPS10) is a novel substrate of an oncoprotein, protein-arginine methyltransferase 5 (PRMT5). We show that PRMT5 interacts with RPS10 and catalyzes its methylation at the Arg(158) and Arg(160) residues. The methylation of RPS10 at Arg(158) and Arg(160) plays a role in the proper assembly of ribosomes, protein synthesis, and optimal cell proliferation. The RPS10-R158K/R160K mutant is not efficiently assembled into ribosomes and is unstable and prone to degradation by the proteasomal pathway. In nucleoli, RPS10 interacts with nucleophosmin/B23 and is predominantly concentrated in the granular component region, which is required for ribosome assembly. The RPS10 methylation mutant interacts weakly with nucleophosmin/B23 and fails to concentrate in the granular component region. Our results suggest that PRMT5 is likely to regulate cell proliferation through the methylation of ribosome proteins, and thus reveal a novel mechanism for PRMT5 in tumorigenesis.

  11. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    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

  12. 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

  13. Ribosomal RNA pseudouridines and pseudouridine synthases.

    PubMed

    Ofengand, James

    2002-03-01

    Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.

  14. Interplay between the ribosomal tunnel, nascent chain, and macrolides influences drug inhibition.

    PubMed

    Starosta, Agata L; Karpenko, Viktoriya V; Shishkina, Anna V; Mikolajka, Aleksandra; Sumbatyan, Natalia V; Schluenzen, Frank; Korshunova, Galina A; Bogdanov, Alexey A; Wilson, Daniel N

    2010-05-28

    Accumulating evidence suggests that, during translation, nascent chains can form specific interactions with ribosomal exit tunnel to regulate translation and promote initial folding events. The clinically important macrolide antibiotics bind within the exit tunnel and inhibit translation by preventing progression of the nascent chain and inducing peptidyl-tRNA drop-off. Here, we have synthesized amino acid- and peptide-containing macrolides, which are used to demonstrate that distinct amino acids and peptides can establish interaction with components of the ribosomal tunnel and enhance the ribosome-binding and inhibitory properties of the macrolide drugs, consistent with the concept that the exit tunnel is not simply a Teflon-like channel. Surprisingly, we find that macrolide antibiotics do not inhibit translation of all nascent chains similarly, but rather exhibit polypeptide-specific inhibitory effects, providing a change to our general mechanistic understanding of macrolide inhibition.

  15. Transcriptional effects of polyamines on ribosomal proteins and on polyamine-synthesizing enzymes in Escherichia coli.

    PubMed

    Huang, S C; Panagiotidis, C A; Canellakis, E S

    1990-05-01

    We find that the transcription of various ribosomal proteins can be differentially affected by polyamines and by changes in growth rates. Using strain MG1655 of Escherichia coli K-12 (F-, lambda-), we have determined the effects of polyamines and changes in growth rate on the transcription of several ribosomal genes and the polyamine-synthesizing enzymes ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17) and arginine decarboxylase (L-arginine carboxylyase; EC 4.1.1.19). Ribosomal proteins S20 and L34 can be differentiated from the other ribosomal proteins studied; the transcription of S20 and L34 is especially sensitive to polyamines and less sensitive to changes in growth rates. In contrast, the transcription of S10, S15, S19, L2, L4, L20, L22, and L23 is insensitive to polyamines although it is particularly sensitive to changes in growth rates. Like S20 and L34, the transcription of ornithine decarboxylase and arginine decarboxylase is especially sensitive to polyamines. Polyamines specifically enhance the transcription of ribosomal proteins S20 and L34, and decrease that of ornithine decarboxylase and arginine decarboxylase. It is evident that polyamines can exert both positive and negative regulation of gene expression in E. coli that can be differentiated from the effects caused by changes in growth rates.

  16. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    SciTech Connect

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via

  17. Enhanced skin permeation of 5α-reductase inhibitors entrapped into surface-modified liquid crystalline nanoparticles.

    PubMed

    Madheswaran, Thiagarajan; Baskaran, Rengarajan; Sundaramoorthy, Pasupathi; Yoo, Bong Kyu

    2015-04-01

    The objective of this study is to enhance skin permeation of finasteride and dutasteride for the treatment of androgenetic alopecia using surface-modified liquid crystalline nanoparticle (sm-LCN) dispersion. LCN entrapped with the drugs was prepared by using monoolein as a liquid crystal former, and surface modification was performed by treatment of the LCN dispersion with same volume of 1 % v/v acetic acid solution containing chitosan. Physicochemical properties of the LCN's were studied with regard to particle size, polydispersity index, zeta potential, and release of the drugs. Skin permeation of drugs entrapped into the LCN and sm-LCN was investigated with porcine abdominal skin using Franz diffusion cell. Cytotoxicity of the LCN's was also studied using human skin keratinocytes. The particle size and zeta potential of the LCN were 197.9 ± 2.5 nm and -20.2 ± 1.9 mV, respectively, and sm-LCN showed slightly bigger size and positive zeta potential due to the presence of thin coating on the surface of the nanoparticles. Compared to LCN, sm-LCN resulted in significantly enhanced skin permeation of the drugs whereas in vitro release was significantly reduced. Cell viability as a measure of cytotoxicity was above 80 % up to 20 μg/ml concentration of both LCN and sm-LCN. In conclusion, sm-LCN may provide a strategy to maximize therapeutic efficacy minimizing unwanted systemic side effects associated with the use of the drugs for the treatment of androgenetic alopecia. PMID:25085659

  18. Enzastaurin, an inhibitor of PKCβ, Enhances Antiangiogenic Effects and Cytotoxicity of Radiation against Endothelial Cells1,2

    PubMed Central

    Spalding, Aaron C; Zeitlin, Benjamin D; Wilder-Romans, Kari; Davis, Mary E; Nor, Jacques E; Lawrence, Theodore S; Ben-Josef, Edgar

    2008-01-01

    PURPOSE: Angiogenesis plays an important role in pancreas cancer pathobiology. Pancreatic tumor cells secrete vascular endothelial growth factor (VEGF), activating endothelial cell protein kinase C beta (PKCβ) that phosphorylates GSK3β to suppress apoptosis and promote endothelial cell proliferation and microvessel formation. We used Enzastaurin (Enz) to test the hypothesis that inhibition of PKCβ results in radiosensitization of endothelial cells in culture and in vivo. MATERIALS/METHODS: We measured PKCβ phosphorylation, VEGF pathway signaling, colony formation, and capillary sprout formation in primary human dermal microvessel endothelial cells (HDMECs) after Enz or radiation (RT) treatment. Microvessel density and tumor volume of human pancreatic cancer xenografts in nude mice were measured after treatment with Enz, RT, or both. RESULTS: Enz inhibited PKCβ and radiosensitized HDMEC with an enhancement ratio of 1.31 ± 0.05. Enz combined with RT reduced HDMEC capillary sprouting to a greater extent than either agent alone. Enz prevented radiation-induced GSK3β phosphorylation of serine 9 while having no direct effect on VEGFR phosphorylation. Treatment of xenografts with Enz and radiation produced greater reductions in microvessel density than either treatment alone. The reduction in microvessel density corresponded with increased tumor growth delay. CONCLUSIONS: Enz-induced PKCβ inhibition radiosensitizes human endothelial cells and enhances the antiangiogenic effects of RT. The combination of Enz and RT reduced microvessel density and resulted in increased growth delay in pancreatic cancer xenografts, without increase in toxicity. These results provide the rationale for combining PKCβ inhibition with radiation and further investigating such regimens in pancreatic cancer. PMID:19043530

  19. The Arabidopsis Cytosolic Ribosomal Proteome: From form to Function

    PubMed Central

    Carroll, Adam J.

    2013-01-01

    The cytosolic ribosomal proteome of Arabidopsis thaliana has been studied intensively by a range of proteomics approaches and is now one of the most well characterized eukaryotic ribosomal proteomes. Plant cytosolic ribosomes are distinguished from other eukaryotic ribosomes by unique proteins, unique post-translational modifications and an abundance of ribosomal proteins for which multiple divergent paralogs are expressed and incorporated. Study of the A. thaliana ribosome has now progressed well beyond a simple cataloging of protein parts and is focused strongly on elucidating the functions of specific ribosomal proteins, their paralogous isoforms and covalent modifications. This review summarises current knowledge concerning the Arabidopsis cytosolic ribosomal proteome and highlights potentially fruitful areas of future research in this fast moving and important area. PMID:23459595

  20. Dom34 rescues ribosomes in 3' untranslated regions.

    PubMed

    Guydosh, Nicholas R; Green, Rachel

    2014-02-27

    Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here, we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3' UTRs. These ribosomes appear to gain access to the 3' UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them.

  1. Protein synthesis inhibitor from potato tuber

    SciTech Connect

    Romaen, R. )

    1989-04-01

    A protein fraction capable of inhibit in vitro protein synthesis was found in potato tubers in fresh and wounded tissue. Inhibitor activity from fresh tissue decays with wounding. Inhibition activity was detected absorbed to ribsomal fraction and cytosol of potato tuber tissue by a partially reconstituted in vitro system from potato tuber and wheat germ. Adsorbed ribosomal fraction was more suitable of purification. This fraction was washed from ribosomes with 0.3M KCl, concentrated with ammonium sulfate precipitation and purified through sephadex G100 and sephadex G-75 columns chromatography. After 61 fold purification adsorbed protein fraction can inhibit germination of maize, wheat and sesame seeds, as well as {sup 3}H-leucine incorporation into protein by imbibed maize embryos. Inhibition activity was lost by temperature, alkali and protease-K hydrolysis. Preliminar analysis could not show presence of reductor sugars. Physiological role of this inhibitor in relation to rest and active tissue remains to be studied.

  2. Enhanced activity of carbosilane dendrimers against HIV when combined with reverse transcriptase inhibitor drugs: searching for more potent microbicides

    PubMed Central

    Vacas-Córdoba, Enrique; Galán, Marta; de la Mata, Francisco J; Gómez, Rafael; Pion, Marjorie; Muñoz-Fernández, M Ángeles

    2014-01-01

    Self-administered topical microbicides or oral preexposure prophylaxis could be very helpful tools for all risk groups to decrease the human immunodeficiency virus (HIV)-1 infection rates. Up until now, antiretrovirals (ARVs) have been the most advanced microbicide candidates. Nevertheless, the majority of clinical trials has failed in HIV-1 patients. Nanotechnology offers suitable approaches to develop novel antiviral agents. Thereby, new nanosystems, such as carbosilane dendrimers, have been shown to be safe and effective compounds against HIV with great potential as topical microbicides. In addition, because most of the attempts to develop effective topical microbicides were unsuccessful, combinatorial strategies could be a valid approach when designing new microbicides. We evaluated various combinations of anionic carbosilane dendrimers with sulfated (G3-S16) and naphthyl sulfonated (G2-NF16) ended groups with different ARVs against HIV-1 infection. The G3-S16 and G2-NF16 dendrimers showed a synergistic or additive activity profile with zidovudine, efavirenz, and tenofovir in the majority of the combinations tested against the X4 and R5 tropic HIV-1 in cell lines, as well as in human primary cells. Therefore, the combination of ARVs and polyanionic carbosilane dendrimers enhances the antiviral potency of the individual compounds, and our findings support further clinical research on combinational approaches as potential microbicides to block the sexual transmission of HIV-1. PMID:25114528

  3. Enhancement of CD8(+) T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor.

    PubMed

    Ren, Hongwei; Ferguson, Brian J; Maluquer de Motes, Carlos; Sumner, Rebecca P; Harman, Laura E R; Smith, Geoffrey L

    2015-05-01

    Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8(+) T cells and this correlates with its inhibition of nuclear factor-κB (NF-κB) activation. Infection with VACVs that either have the N1L gene deleted (vΔN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-κB resulted in increased central and memory CD8(+) T-cell populations, increased CD8(+) T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8(+) memory T-cell function was increased following infection with vN1.I6E, with more interferon-γ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8(+) T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-κB activation within infected cells for long-term CD8(+) T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8(+) T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety.

  4. Enhancement of lipase catalyzed-fatty acid methyl esters production from waste activated bleaching earth by nullification of lipase inhibitors.

    PubMed

    Dwiarti, Lies; Ali, Ehsan; Park, Enoch Y

    2010-01-01

    This study sought to identify inhibitory factors of lipase catalyzed-fatty acid methyl esters (FAME) production from waste activated bleaching earth (wABE). During the vegetable oil refinery process, activated bleaching earth (ABE) is used for removing the impure compounds, but adsorbs vegetable oil up to 35-40% as on a weight basis, and then the wABE is discarded as waste material. The impurities were extracted from the wABE with methanol and evaluated by infra-red (IR) spectroscopy, which revealed that some were chlorophyll-plant pigments. The chlorophylls inhibited the lipase during FAME conversion from wABE. The inhibition by a mixture of chlorophyll a and b was found to be competitive. The inhibition of the enzymatic hydrolysis of waste vegetable oil contained in wABE by chlorophyll a alone was competitive, while the inhibition by chlorophyll b alone was non-competitive. Furthermore, the addition of a small amount of alkali nullified this inhibitory effect and accelerated the FAME production rate. When 0.9% KOH (w/w wABE) was added to the transesterification reaction with only 0.05% lipase (w/w wABE), the maximum FAME production rate improved 120-fold, as compared to that without the addition of KOH. The alkali-combined lipase significantly enhanced the FAME production rate from wABE, in spite of the presence of the plant pigments, and even when a lower amount of lipase was used as a catalyst.

  5. The Synthesis of Ribosomes in E. coli

    PubMed Central

    McCarthy, B. J.; Britten, R. J.; Roberts, R. B.

    1962-01-01

    Techniques of chromatography on columns of DEAE1 cellulose and sedimentation analysis through a sucrose gradient have been used to study the flow of C14-uracil label through precursors to completed ribosomes. Analysis by chromatography shows the existence of two sequential precursors constituting together some 10 per cent of the total ribosomal RNA. The chromatographic separation into three fractions is ascribed to the lower protein/RNA ratios of the precursor. By sedimentation the primary precursor (eosome) is identified as a component of average sedimentation coefficient 14S. The second precursor stage (neosome) is divided among at least two particles, one of 43S and the other of about 30S. Detailed kinetic analysis shows that all the radioactivity passes through the eosome on its way to finished 50S and 30S ribosomes. The delay in the entry of radioactivity to ribosomes is that expected from the quantity of eosome precursor. The obvious conclusion that there exists a precursor-product relationship is discussed together with possible interpretations. PMID:19431315

  6. Evaluation of a ribosomal vaccine against pertussis.

    PubMed Central

    Field, L H; Parker, C D; Manclark, C R; Berry, L J

    1979-01-01

    A crude ribosomal vaccine derived from Bordetella pertussis administered to ICR and N:NIH (SW) strains of mice protected them effectively against a standardized intracranial challenge. The dose of vaccine that protected half the mice was less for N:NIH (SW) than for ICR mice and compared favorably with a killed reference vaccine. Ribosomes prepared from bacteria ground with washed sea sand were more immunogenic than those obtained by rupture with alumina or with a Braun homogenizer. The protective effect of the crude ribosomes was not an innate part of the organelle but was due to a substance or substances that could be removed from them by a 1 M NH4Cl wash. The material in the wash was highly immunogenic and retained both the histamine-sensitizing and leukocytosis-promoting properties. It lost much of the dermonecrotic activity and was poorly pyrogenic in rabbits. The most potent pyrogen was present in the washed ribosomes, which apparently, retained the endotoxic components of the cell wall. The best vaccines permitted acceptable weight gain in the immunized mice. PMID:222684

  7. Peptide Bond Formation Mechanism Catalyzed by Ribosome

    PubMed Central

    Świderek, Katarzyna; Marti, Sergio; Tuñón, Iñaki; Moliner, Vicent; Bertran, Juan

    2015-01-01

    In this paper we present a study of the peptide bond formation reaction catalyzed by ribosome. Different mechanistic proposals have been explored by means of Free Energy Perturbation methods within hybrid QM/MM potentials, where the chemical system has been described by the M06-2X functional and the environment by means of the AMBER force field. According to our results, the most favourable mechanism in the ribosome would proceed through an eight-membered ring transition state, involving a proton shuttle mechanism through the hydroxyl group of the sugar and a water molecule. This transition state is similar to that described for the reaction in solution (J. Am. Chem. Soc. 2013, 135, 8708–8719) but the reaction mechanisms are noticeable different. Our simulations reproduce the experimentally determined catalytic effect of ribosome that can be explained by the different behaviour of the two environments. While the solvent reorganizes during the chemical process involving an entropic penalty, the ribosome is preorganized in the formation of the Michaelis complex and does not suffer important changes along the reaction, dampening the charge redistribution of the chemical system. PMID:26325003

  8. Ribosome crystals in the oocyte of Gerris najas (Heteroptera).

    PubMed

    Choi, W C; Nagl, W

    1977-01-01

    Oocytes of the pond skater, Gerris najas, display ribosome tetramers that are arranged in the form of sheets in the vicinity of the nucleus. This is the first finding of ribosome crystals in an insect and suggests that ribosome crystallization may be a common phenomenon of cells that are inactive in protein synthesis.

  9. The HSP70 and autophagy inhibitor pifithrin-μ enhances the antitumor effects of TRAIL on human pancreatic cancer.

    PubMed

    Monma, Hiroyuki; Harashima, Nanae; Inao, Touko; Okano, Shinji; Tajima, Yoshitsugu; Harada, Mamoru

    2013-04-01

    TRAIL and agonistic death receptor-specific antibodies can induce apoptosis in cancer cells with little cytotoxicity to normal cells. To improve TRAIL-induced antitumor effects, we tested its effectiveness in combination with pifithrin (PFT)-μ, which has the potential to inhibit HSP70 function and autophagy, both of which participate in TRAIL resistance in cancer cells. Among the four human pancreatic cancer cell lines tested, MiaPaca-2, Panc-1, and BxPC-3 cells showed varying sensitivities to TRAIL. In MiaPaca-2 and Panc-1 cells, knockdown of HSP70 or beclin-1, the latter an autophagy-related molecule, by RNA interference augmented TRAIL-induced antitumor effects, decreasing cell viability, and increasing apoptosis. On the basis of these findings, we next determined whether the TRAIL-induced antitumor effects could be augmented by its combination with PFT-μ. The combination of TRAIL plus PFT-μ significantly decreased the viability and colony-forming ability of MiaPaca-2 and Panc-1 cells compared with cells treated with either agent alone. When applied alone, PFT-μ increased Annexin V(+) cells in both caspase-dependent and -independent manners. It also promoted TRAIL-induced apoptosis and arrested cancer cell growth. Furthermore, PFT-μ antagonized TRAIL-associated NF-κB activation in cancer cells. In a xenograft mouse model, combination therapy significantly inhibited MiaPaca-2 tumor growth compared with treatment with either agent alone. The results of this study suggest protective roles for HSP70 and autophagy in TRAIL resistance in pancreatic cancer cells and suggest that PFT-μ is a promising agent for use in therapies intended to enhance the antitumor effects of TRAIL.

  10. Protection of rat liver 80 S ribosomes against ricin A chain inactivation by proteins extracted from rat liver and wheat germ ribosomal subunits with ammonium chloride/magnesium chloride.

    PubMed

    Chang, M S; Houston, L L

    1981-09-28

    Proteins extracted from wheat germ 60 S ribosomal subunits and rat liver 60 S and 40 S ribosomal subunits with 3 M NH4Cl/75 mM MgCl2 were able to prevent the ricin A chain-mediated inactivation of untreated 80 S rat liver ribosomes. The protection of polyphenylalanine synthetic capability of 80 S ribosomes was saturable and reached 100% protection in the presence of about 20 micrograms of extracted protein using a uniform set of assay conditions. No protection was observed using proteins extracted from wheat germ 40 S subunits or the core fraction of rat liver 60 S subunits or protein extracted from Escherichia coli ribosomes or ribosomal subunits. The conclusion that the protective effect of extracted 60 S subunit proteins was specific, was further strengthened by showing that unrelated proteins such as alpha-lactalbumin, bovine serum albumin and lysozyme, and polypeptides such as polylysine and poly(aspartic acid), also showed no protection. If 80 S ribosomes were first treated with ricin A chain and then incubated with proteins extracted from rat liver 60 S subunits, no protection was observed. Proteins extracted with NH4Cl/MgCl2 from 60 S rat liver subunits were applied to carboxymethylcellulose column equilibrated with 6 M urea. Stepwise elution with increasing concentrations of LiCl resulted in seven fractions. One fraction (D) contained most of the protective factor; one fraction (E) contained a lesser amount of the protective factor. Two-dimensional polyacrylamide gel electrophoresis of fraction D showed the presence of ten proteins. These data are consistent with the idea that the enzymatic target of ricin A chain is protein is nature and that fraction D contains one or more proteins that appear to act as a inhibitor against ricin A chain.

  11. Mescaline-induced changes of brain-cortex ribosomes. Effect of mescaline on the stability of brain-cortex ribosomes.

    PubMed

    Datta, R K; Ghosh, J J

    1970-05-01

    1. During the action of mescaline sulphate on goat brain-cortex slices the ribosomal particles become susceptible to breakdown, releasing protein, RNA, acidsoluble nucleotides and ninhydrin-positive materials, resulting in loss of ribosomal enzyme activities. 2. Ribosomes of the mescaline-treated cortex slices undergo rapid degradation in the presence of trypsin and ribonuclease. 3. Mescaline does not alter the chemical and nucleotide compositions or the u.v.-absorption characteristics of ribosomal particles, however.

  12. Molecular Simulations of Cotranslational Protein Folding: Fragment Stabilities, Folding Cooperativity, and Trapping in the Ribosome

    PubMed Central

    Elcock, Adrian H

    2006-01-01

    Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these

  13. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  14. Radiation Enhancement of Head and Neck Squamous Cell Carcinoma by the Dual PI3K/mTOR Inhibitor PF-05212384

    PubMed Central

    Leiker, Andrew J.; DeGraff, William; Choudhuri, Rajani; Sowers, Anastasia L.; Thetford, Angela; Cook, John A.; Van Waes, Carter; Mitchell, James B.

    2015-01-01

    Purpose Radiation remains a mainstay for the treatment of non-metastatic head and neck squamous cell carcinoma (HNSCC), a malignancy characterized by a high rate of PI3K/mTOR signaling axis activation. We investigated the ATP-competitive dual PI3K/mTOR inhibitor, PF-05212384, as a radiosensitizer in pre-clinical HNSCC models. Experimental Design Extent of radiation enhancement of two HNSCC cell lines (UMSCC1-wtP53, UMSCC46-mtP53) and normal human fibroblast (1522) was assessed by in vitro clonogenic assay with appropriate target inhibition verified by immunoblotting. Radiation induced DNA damage repair was evaluated by γH2AX western blots with mechanism of DNA-DSB repair abrogation investigated by cell cycle analysis, immunoblotting, and RT-PCR. PF-05212384 efficacy in vivo was assessed by UMSCC1 xenograft tumor regrowth delay, xenograft lysate immunoblotting, and tissue section immunohistochemistry. Results PF-05212384 effectively inhibited PI3K and mTOR resulting in significant radiosensitization of exponentially growing and plateau-phase cells with 24 hr treatment following irradiation, and variable radiation enhancement with 24 hr treatment prior to irradiation. Tumor cells radiosensitized to a greater extent than normal human fibroblasts. Post-irradiation PF-05212384 treatment delays γ-H2AX foci resolution. PF-05212384 24 hr exposure resulted in an evident G1/S phase block in p53 competent cells. Fractionated radiation plus IV PF-05212384 synergistically delayed nude-mice bearing UMSCC1 xenograft regrowth, with potential drug efficacy biomarkers identified, including pS6, pAkt, p4EBP1, and Ki67. Conclusions Taken together, our results of significant radiosensitization both in vitro and in vivo validates the PI3K/mTOR axis as a radiation modification target and PF-05212384 as a potential clinical radiation modifier of non-metastatic HNSCC. PMID:25724523

  15. Sildenafil, a phosphodiesterase type 5 inhibitor, enhances the activity of two atypical antidepressant drugs, mianserin and tianeptine, in the forced swim test in mice.

    PubMed

    Socała, Katarzyna; Nieoczym, Dorota; Wyska, Elżbieta; Poleszak, Ewa; Wlaź, Piotr

    2012-08-01

    Sildenafil, a selective phosphodiesterase type 5 inhibitor, has recently been reported to abolish anti-immobility action of antidepressant drugs, i.e., bupropion, venlafaxine and S-citalopram, in the forced swim test in mice. The present study was designed to investigate the influence of sildenafil on the potential of two atypical antidepressants, namely mianserin and tianeptine. Swim sessions were conducted by placing mice in glass cylinders filled with water for 6 min and the duration of the behavioral immobility during the last 4 min of the test was evaluated. Locomotor activity was measured with photoresistor actimeters. To evaluate the potential pharmocokinetic interaction, total brain concentrations of the studied antidepressants were determined by HPLC method. Sildenafil at a dose of 2.5 mg/kg did not affect the activity of mianserin (20 mg/kg) in the forced swim test. Interestingly, at higher doses (5 and 10 mg/kg), sildenafil significantly enhanced the anti-immobility action of mianserin. Likewise, sildenafil (5, 10 and 20 mg/kg) robustly augmented the antidepressant activity of tianeptine (30 mg/kg). Mianserin alone, as well as in a combination with sildenafil at the highest dose, caused a potent reduction in locomotor activity. However, the changes in motor activity did not interfere with the data obtained in the forced swim test. Sildenafil significantly increased the total brain tianeptine concentration. No alteration in mianserin level in the brain after sildenafil co-administration was observed. The present study suggests that sildenafil enhances the activity of mianserin and tianeptine in the forced swim test in mice. The changes in the antidepressant activity of mianserin evoked by sildenafil co-administration were related to pharmacodynamic interaction while the interaction between tianeptine and sildenafil was, at least in part, pharmacokinetic in nature.

  16. Sildenafil, a phosphodiesterase type 5 inhibitor, enhances the activity of two atypical antidepressant drugs, mianserin and tianeptine, in the forced swim test in mice.

    PubMed

    Socała, Katarzyna; Nieoczym, Dorota; Wyska, Elżbieta; Poleszak, Ewa; Wlaź, Piotr

    2012-08-01

    Sildenafil, a selective phosphodiesterase type 5 inhibitor, has recently been reported to abolish anti-immobility action of antidepressant drugs, i.e., bupropion, venlafaxine and S-citalopram, in the forced swim test in mice. The present study was designed to investigate the influence of sildenafil on the potential of two atypical antidepressants, namely mianserin and tianeptine. Swim sessions were conducted by placing mice in glass cylinders filled with water for 6 min and the duration of the behavioral immobility during the last 4 min of the test was evaluated. Locomotor activity was measured with photoresistor actimeters. To evaluate the potential pharmocokinetic interaction, total brain concentrations of the studied antidepressants were determined by HPLC method. Sildenafil at a dose of 2.5 mg/kg did not affect the activity of mianserin (20 mg/kg) in the forced swim test. Interestingly, at higher doses (5 and 10 mg/kg), sildenafil significantly enhanced the anti-immobility action of mianserin. Likewise, sildenafil (5, 10 and 20 mg/kg) robustly augmented the antidepressant activity of tianeptine (30 mg/kg). Mianserin alone, as well as in a combination with sildenafil at the highest dose, caused a potent reduction in locomotor activity. However, the changes in motor activity did not interfere with the data obtained in the forced swim test. Sildenafil significantly increased the total brain tianeptine concentration. No alteration in mianserin level in the brain after sildenafil co-administration was observed. The present study suggests that sildenafil enhances the activity of mianserin and tianeptine in the forced swim test in mice. The changes in the antidepressant activity of mianserin evoked by sildenafil co-administration were related to pharmacodynamic interaction while the interaction between tianeptine and sildenafil was, at least in part, pharmacokinetic in nature. PMID:22406168

  17. Sildenafil, a phosphodiesterase type 5 inhibitor, enhances the antidepressant activity of amitriptyline but not desipramine, in the forced swim test in mice.

    PubMed

    Socała, Katarzyna; Nieoczym, Dorota; Wyska, Elżbieta; Poleszak, Ewa; Wlaź, Piotr

    2012-06-01

    The cholinergic theory of depression highlights the involvement of muscarinic acetylcholine receptors in the neurobiology of mood disorders. The present study was designed to investigate the effect of sildenafil, a phosphodiesterase type 5 inhibitor which exhibits cholinomimetic properties, alone and in combination with scopolamine in the forced swim test in mice. Moreover, we assessed the ability of sildenafil to modify the antidepressant activity of two tricyclic antidepressants with distinct cholinolytic activity, amitriptyline and desipramine. Swim sessions were conducted by placing mice in glass cylinders filled with water for 6 min and the duration of behavioral immobility during the last 4 min of the test was evaluated. Locomotor activity was measured with photoresistor actimeters. To evaluate the potential pharmacokinetic interaction between amitriptyline and sildenafil, brain and serum concentrations of amitriptyline were determined by HPLC. Sildenafil (1.25-20 mg/kg) as well as scopolamine (0.5 mg/kg) and its combination with sildenafil (1.25 mg/kg) did not affect the total immobility time duration. However, joint administration of scopolamine with sildenafil at doses of 2.5 and 5 mg/kg significantly reduced immobility time as compared to control group. Moreover, co-administration of scopolamine with sildenafil at the highest dose (5 mg/kg) significantly decreased immobility time as compared to scopolamine-treated group. Sildenafil (1.25, 2.5 and 5 mg/kg) significantly enhanced the antidepressant activity of amitriptyline (5 mg/kg). No changes in anti-immobility action of desipramine (20 mg/kg) in combination with sildenafil (5, 10 and 20 mg/kg) were observed. Sildenafil did not affect amitriptyline level in both brain and serum. In conclusion, the present study suggests that sildenafil may enhance the activity of antidepressant drugs which exhibit cholinolytic activity.

  18. Mutually enhancing anti-inflammatory activities of dimethyl fumarate and NF-κB inhibitors--implications for dose-sparing combination therapies.

    PubMed

    Hund, Anna-Carina; Lockmann, Anike; Schön, Michael P

    2016-02-01

    Fumaric acid esters, dimethyl fumarate (DMF) in particular, have been established for the therapy of psoriasis and, more recently, multiple sclerosis. In the light of therapy-limiting dose-dependent side effects, such as gastrointestinal irritation, reducing the effective doses of FAE is a worthwhile goal. In search of strategies to maintain the anti-inflammatory activity of DMF at reduced concentrations, we found that NF-κB inhibition augmented key anti-inflammatory effects of DMF in two complementary experimental settings in vitro. At non-toxic concentrations, both proteasome inhibition with bortezomib as well as blocking NF-κB activation through KINK-1, a small molecule inhibitor of IKKβ-profoundly enhanced DMF-dependent inhibition of nuclear NF-κB translocation in TNFα-stimulated human endothelial cells. This resulted in significant and selective co-operative down-regulation of endothelial adhesion molecules crucial for leucocyte extravasation, namely E-selectin (CD62E), VCAM-1 (CD106) and ICAM-1 (CD54), on both mRNA and protein levels. Functionally, these molecular changes led to synergistically decreased rolling and firm adhesion of human lymphocytes on TNF-activated endothelial cells, as demonstrated in a dynamic flow chamber system. If our in vitro findings can be translated into clinical settings, it is conceivable that anti-inflammatory effects of DMF can be achieved with lower doses than currently used, thus potentially reducing unwanted side effects. PMID:26513635

  19. NBM-T-L-BMX-OS01, Semisynthesized from Osthole, Is a Novel Inhibitor of Histone Deacetylase and Enhances Learning and Memory in Rats.

    PubMed

    Yang, Ying-Chen; Chen, Chia-Nan; Wu, Carol-Imei; Huang, Wei-Jan; Kuo, Tsun-Yung; Kuan, Ming-Chung; Tsai, Tung-Hu; Huang, Jing-Shi; Huang, Chung-Yang

    2013-01-01

    NBM-T-L-BMX-OS01 (BMX) was derived from the semisynthesis of osthole, isolated from Cnidium monnieri (L.) Cuss., and was identified to be a potent inhibitor of HDAC8. This study shows that HDAC8 is highly expressed in the pancreas and the brain. The function of HDAC8 in the brain has not been adequately studied. Because BMX enhances neurite outgrowth and cAMP response element-binding protein (CREB) activation, the effect of BMX on neural plasticity such as learning and memory is examined. To examine declarative and nondeclarative memory, a water maze, a passive one-way avoidance task, and a novel object recognition task were performed. Results from the water maze revealed that BMX and suberoylanilide-hydroxamic-acid-(SAHA-) treated rats showed shorter escape latency in finding the hidden platform. The BMX-treated animals spent more time in the target quadrant in the probe trial performance. An analysis of the passive one-way avoidance results showed that the BMX-treated animals stayed longer in the illuminated chamber by 1 day and 7 days after footshock. The novel object recognition task revealed that the BMX-treated animals showed a marked increase in the time spent exploring novel objects. Furthermore, BMX ameliorates scopolamine-(Sco-) induced learning and memory impairment in animals, indicating a novel role of BMX in learning and memory. PMID:23606881

  20. HIF-1α is required for hematopoietic stem cell mobilization and 4-prolyl hydroxylase inhibitors enhance mobilization by stabilizing HIF-1α.

    PubMed

    Forristal, C E; Nowlan, B; Jacobsen, R N; Barbier, V; Walkinshaw, G; Walkley, C R; Winkler, I G; Levesque, J P

    2015-06-01

    Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic.

  1. HIF-1α is required for hematopoietic stem cell mobilization and 4-prolyl hydroxylase inhibitors enhance mobilization by stabilizing HIF-1α

    PubMed Central

    Forristal, C E; Nowlan, B; Jacobsen, R N; Barbier, V; Walkinshaw, G; Walkley, C R; Winkler, I G; Levesque, J P

    2015-01-01

    Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic. PMID:25578474

  2. NBM-T-L-BMX-OS01, Semisynthesized from Osthole, Is a Novel Inhibitor of Histone Deacetylase and Enhances Learning and Memory in Rats

    PubMed Central

    Yang, Ying-Chen; Chen, Chia-Nan; Wu, Carol-Imei; Huang, Wei-Jan; Kuo, Tsun-Yung; Kuan, Ming-Chung; Tsai, Tung-Hu; Huang, Jing-Shi; Huang, Chung-Yang

    2013-01-01

    NBM-T-L-BMX-OS01 (BMX) was derived from the semisynthesis of osthole, isolated from Cnidium monnieri (L.) Cuss., and was identified to be a potent inhibitor of HDAC8. This study shows that HDAC8 is highly expressed in the pancreas and the brain. The function of HDAC8 in the brain has not been adequately studied. Because BMX enhances neurite outgrowth and cAMP response element-binding protein (CREB) activation, the effect of BMX on neural plasticity such as learning and memory is examined. To examine declarative and nondeclarative memory, a water maze, a passive one-way avoidance task, and a novel object recognition task were performed. Results from the water maze revealed that BMX and suberoylanilide-hydroxamic-acid-(SAHA-) treated rats showed shorter escape latency in finding the hidden platform. The BMX-treated animals spent more time in the target quadrant in the probe trial performance. An analysis of the passive one-way avoidance results showed that the BMX-treated animals stayed longer in the illuminated chamber by 1 day and 7 days after footshock. The novel object recognition task revealed that the BMX-treated animals showed a marked increase in the time spent exploring novel objects. Furthermore, BMX ameliorates scopolamine-(Sco-) induced learning and memory impairment in animals, indicating a novel role of BMX in learning and memory. PMID:23606881

  3. Rho kinase inhibitor Y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

    PubMed

    van den Bogaard, Ellen H; Rodijk-Olthuis, Diana; Jansen, Patrick A M; van Vlijmen-Willems, Ivonne M J J; van Erp, Piet E; Joosten, Irma; Zeeuwen, Patrick L J M; Schalkwijk, Joost

    2012-09-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes. PMID:22519508

  4. Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman-Birk protease inhibitor in seeds.

    PubMed

    Kim, Won-Seok; Chronis, Demosthenis; Juergens, Matthew; Schroeder, Amy C; Hyun, Seung Won; Jez, Joseph M; Krishnan, Hari B

    2012-01-01

    Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58-74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22-32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman-Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.

  5. Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.

    PubMed

    Sun, Linxiao; Yu, Yonghua; Hu, Weiqin; Min, Qiming; Kang, Huiling; Li, Yilu; Hong, Yue; Wang, Xuemin; Hong, Yueyun

    2016-07-01

    Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice. PMID:27102613

  6. Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.

    PubMed

    Sun, Linxiao; Yu, Yonghua; Hu, Weiqin; Min, Qiming; Kang, Huiling; Li, Yilu; Hong, Yue; Wang, Xuemin; Hong, Yueyun

    2016-07-01

    Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice.

  7. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

    Antithrombotic medications have become standard of care for management of acute coronary syndrome. Platelet adhesion, activation, and aggregation are essential components of platelet function; platelet-inhibiting medications interfere with these components and reduce incidence of thrombosis. Active bleeding is a contraindication for administration of platelet inhibitors. There is currently no reversal agent for platelet inhibitors, although platelet transfusion may be used to correct active bleeding after administration of platelet inhibitors. PMID:26897422

  8. pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

    PubMed Central

    Muth, G W; Chen, L; Kosek, A B; Strobel, S A

    2001-01-01

    A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known. PMID:11680845

  9. Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis

    PubMed Central

    Juntawong, Piyada; Girke, Thomas; Bazin, Jérémie; Bailey-Serres, Julia

    2014-01-01

    Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia–up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions. PMID:24367078

  10. Reduction of Ribosome Level Triggers Flocculation of Fission Yeast Cells

    PubMed Central

    Li, Rongpeng; Li, Xuesong; Sun, Lei; Chen, Feifei; Liu, Zhenxing; Gu, Yuyu; Gong, Xiaoyan; Liu, Zhonghua; Wei, Hua; Huang, Ying

    2013-01-01

    Deletion of ribosomal protein L32 genes resulted in a nonsexual flocculation of fission yeast. Nonsexual flocculation also occurred when two other ribosomal protein genes, rpl21-2 and rpl9-2, were deleted. However, deletion of two nonribosomal protein genes, mpg and fbp, did not cause flocculation. Overall transcript levels of rpl32 in rpl32-1Δ and rpl32-2Δ cells were reduced by 35.9% and 46.9%, respectively, and overall ribosome levels in rpl32-1Δ and rpl32-2Δ cells dropped 31.1% and 27.8%, respectively, compared to wild-type cells. Interestingly, ribosome protein expression levels and ribosome levels were also reduced greatly in sexually flocculating diploid YHL6381/WT (h+/h−) cells compared to a mixture of YHL6381 (h+) and WT (h−) nonflocculating haploid cells. Transcriptome analysis indicated that the reduction of ribosomal levels in sexual flocculating cells was caused by more-extensive suppression of ribosomal biosynthesis gene expression, while the reduction of ribosomal levels caused by deleting ribosomal protein genes in nonsexual flocculating cells was due to an imbalance between ribosomal proteins. We propose that once the reduction of ribosomal levels is below a certain threshold value, flocculation is triggered. PMID:23355005

  11. History of the ribosome and the origin of translation

    PubMed Central

    Petrov, Anton S.; Gulen, Burak; Norris, Ashlyn M.; Kovacs, Nicholas A.; Lanier, Kathryn A.; Fox, George E.; Harvey, Stephen C.; Wartell, Roger M.; Hud, Nicholas V.; Williams, Loren Dean

    2015-01-01

    We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA. PMID:26621738

  12. Tertiary interactions within the ribosomal exit tunnel.

    PubMed

    Kosolapov, Andrey; Deutsch, Carol

    2009-04-01

    Although tertiary folding of whole protein domains is prohibited by the cramped dimensions of the ribosomal tunnel, dynamic tertiary interactions may permit folding of small elementary units within the tunnel. To probe this possibility, we used a beta-hairpin and an alpha-helical hairpin from the cytosolic N terminus of a voltage-gated potassium channel and determined a probability of folding for each at defined locations inside and outside the tunnel. Minimalist tertiary structures can form near the exit port of the tunnel, a region that provides an entropic window for initial exploration of local peptide conformations. Tertiary subdomains of the nascent peptide fold sequentially, but not independently, during translation. These studies offer an approach for diagnosing the molecular basis for folding defects that lead to protein malfunction and provide insight into the role of the ribosome during early potassium channel biogenesis.

  13. Tertiary Interactions within the Ribosomal Exit Tunnel

    PubMed Central

    Kosolapov, Andrey; Deutsch, Carol

    2009-01-01

    Although tertiary folding of whole protein domains is prohibited by the cramped dimensions of the ribosomal tunnel, dynamic tertiary interactions may permit folding of small elementary units within the tunnel. To probe this possibility, we used a β-hairpin as well as an α-helical hairpin from the cytosolic N-terminus of a voltage-gated potassium channel and determined a probability of folding for each at defined locations inside and outside the tunnel. Minimalist tertiary structures can form near the exit port of the tunnel, a region that provides an entropic window for initial exploration of local peptide conformations. Tertiary subdomains of the nascent peptide fold sequentially, but not independently, during translation. These studies offer an approach for diagnosing the molecular basis for folding defects that lead to protein malfunction and provide insight into the role of the ribosome during early potassium channel biogenesis. PMID:19270700

  14. Linker scanner mutagenesis of the Xenopus laevis ribosomal gene promoter.

    PubMed Central

    Reeder, R H; Pennock, D; McStay, B; Roan, J; Tolentino, E; Walker, P

    1987-01-01

    We have assayed a series of linker scanner mutants which cover the Xenopus laevis ribosomal gene promoter at approximately ten base pair intervals. All of these mutations adversely affect promoter activity with the exception of one mutation which stimulates activity. Thus, none are neutral. We show that most of the mutations can be partially rescued by ligating a block of enhancer elements upstream of the promoter. In addition, we have made extracts from liver nuclei which produce DNaseI protection footprints over the promoter. Analysis of both strands reveals a prominent footprinting domain from about -5 to -30. However, lesser changes in the digestion pattern are detected over most of the promoter. Previously published analyses have suggested that this promoter might be composed of three functional domains. The experiments presented here suggest that either 1) the three putative domains are so closely arranged that the boundaries are difficult to discern, or 2) the situation is more complex. Images PMID:3658698

  15. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

  16. Structure and Function of the Mitochondrial Ribosome.

    PubMed

    Greber, Basil J; Ban, Nenad

    2016-06-01

    Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate production in eukaryotic cells. Throughout evolution, mitoribosomes have become functionally specialized for synthesizing mitochondrial membrane proteins, and this has been accompanied by large changes to their structure and composition. We review recent high-resolution structural data that have provided unprecedented insight into the structure and function of mitoribosomes in mammals and fungi. PMID:27023846

  17. Structural snapshots of actively translating human ribosomes

    PubMed Central

    Behrmann, Elmar; Loerke, Justus; Budkevich, Tatyana V.; Yamamoto, Kaori; Schmidt, Andrea; Penczek, Pawel A.; Vos, Matthijn R.; Bürger, Jörg; Mielke, Thorsten; Scheerer, Patrick; Spahn, Christian M.T.

    2015-01-01

    Summary Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. While their mode of action is often compared to that of mechanical machines, a crucial difference is that at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and three-dimensional variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements. PMID:25957688

  18. Quantitative profiling of initiating ribosomes in vivo.

    PubMed

    Gao, Xiangwei; Wan, Ji; Liu, Botao; Ma, Ming; Shen, Ben; Qian, Shu-Bing

    2015-02-01

    Cells have evolved exquisite mechanisms to fine-tune the rate of protein synthesis in response to stress. Systemic mapping of start-codon positions and precise measurement of the corresponding initiation rate would transform our understanding of translational control. Here we present quantitative translation initiation sequencing (QTI-seq), with which the initiating ribosomes can be profiled in real time at single-nucleotide resolution. Resultant initiation maps not only delineated variations of start-codon selection but also highlighted a dynamic range of initiation rates in response to nutrient starvation. The integrated data set provided unique insights into principles of alternative translation and mechanisms controlling different aspects of translation initiation. With RiboTag mice, QTI-seq permitted tissue-specific profiling of initiating ribosomes in vivo. Liver cell-specific ribosome profiling uncovered a robust translational reprogramming of the proteasome system in fasted mice. Our findings illuminated the prevalence and dynamic nature of translational regulation pivotal to physiological adaptation in vivo.

  19. Pseudomonas ribosomal vaccines: preparation, properties, and immunogenicity.

    PubMed Central

    Lieberman, M M

    1978-01-01

    The preparation, properties, and immunogenicity of ribosomal vaccines from Pseudomonas aeruginosa are described. These preparations, containing protein and RNA, were tested for immunogenicity by active immunization of mice and subsequent challenge with homologous, live bacteria. The results demonstrated that vaccines prepared from a majority of serotypes used were immunogenic, i.e., afforded 60 to 100% mouse protection against a challenge inoculum containing 8 to 50 50% lethal doses. In some cases vaccine doses as low as 1 microgram of RNA provided 100% mouse protection. Molecular sieve chromatography of a highly immunogenic ribosomal preparation on Sepharose 4B demonstrated the presence of two molecular weight fractions: (i) peak A, an excluded peak (thus having a molecular weight of at least 2 times 10(7)), and (ii) peak B, considerably retarded, with an elution position corresponding to a molecular weight of about 2.2 X 10(6), approximating that of typical 70S ribosomes. Both peaks A and B were immunogenic; however, the immunogenicity of peak A was greater (i.e., a smaller immunizing dose was required) than that of peak B. Peak A was shown to contain components of lipopolysaccharide in addition to protein and RNA (which comprised 80% of the dry weight of peak A). On the other hand, peak B was shown to be free of lipopolysaccharide, and 100% of its dry weight consisted of protein and RNA. PMID:101464

  20. The ribosome challenge to the RNA world.

    PubMed

    Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean

    2015-04-01

    An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system. PMID:25739364

  1. Mitochondrial ribosome assembly in health and disease

    PubMed Central

    De Silva, Dasmanthie; Tu, Ya-Ting; Amunts, Alexey; Fontanesi, Flavia; Barrientos, Antoni

    2015-01-01

    The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health. PMID:26030272

  2. The ribosome challenge to the RNA world.

    PubMed

    Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean

    2015-04-01

    An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

  3. Mitochondrial ribosome assembly in health and disease.

    PubMed

    De Silva, Dasmanthie; Tu, Ya-Ting; Amunts, Alexey; Fontanesi, Flavia; Barrientos, Antoni

    2015-01-01

    The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

  4. Corrosion inhibitor

    SciTech Connect

    Wisotsky, M.J.; Metro, S.J.

    1989-10-31

    A corrosion inhibitor for use in synthetic ester lubricating oils is disclosed. It comprises an effective amount of: at least one aromatic amide; and at least one hydroxy substituted aromatic compound. The corrosion inhibitor thus formed is particularly useful in synthetic ester turbo lubricating oils.

  5. Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly

    PubMed Central

    Davis, Joseph H.; Williamson, James R.; Britton, Robert A.

    2014-01-01

    RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins. PMID:25330043

  6. Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

    PubMed Central

    Fuchs, Gabriele; Petrov, Alexey N.; Marceau, Caleb D.; Popov, Lauren M.; Chen, Jin; O’Leary, Seán E.; Wang, Richard; Carette, Jan E.; Sarnow, Peter; Puglisi, Joseph D.

    2015-01-01

    Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors. PMID:25516984

  7. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

    Orelle, Cédric; Carlson, Skylar; Kaushal, Bindiya; Almutairi, Mashal M.; Liu, Haipeng; Ochabowicz, Anna; Quan, Selwyn; Pham, Van Cuong; Squires, Catherine L.; Murphy, Brian T.

    2013-01-01

    Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol. PMID:24041905

  8. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  9. Ribosomal history reveals origins of modern protein synthesis.

    PubMed

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  10. Enhancement of TPA-induced growth inhibition and apoptosis in myeloid leukemia cells by BAY 11-7082, an NF-kappaB inhibitor.

    PubMed

    Hansson, Annette; Marín, Yarí E; Suh, Junghan; Rabson, Arnold B; Chen, Suzie; Huberman, Eliezer; Chang, Richard L; Conney, Allan H; Zheng, Xi

    2005-10-01

    The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation and apoptosis in myeloid leukemia cells. In the present study, we investigated the role of the transcription factor NF-kappaB in TPA-induced growth inhibition and apoptosis in the myeloid leukemia HL-60 cell line and its TPA-resistant cell variant HL-525. Unlike the parental cell line, HL-525 cells are protein kinase C (PKC)-beta deficient and resistant to TPA-induced differentiation and apoptosis. We found that treatment of HL-60 cells with TPA resulted in a concentration-dependent growth inhibition and an increase in apoptotic cells. TPA only had a small effect on growth and apoptosis in HL-525 cells. Treatment of HL-60 cells with TPA (0.64-3.2 nM) caused a rapid activation of NF-kappaB as determined by electrophoresis mobility shift assay (EMSA) and immunocytochemistry. Although the basal level of NF-kappaB activity was low in HL-60 cells, TPA-resistant HL-525 cells had a high basal level of NF-kappaB activity. Treatment of HL-525 cells with higher concentrations of TPA (16-80 nM) resulted in a further increase in NF-kappaB activity. (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), which inhibits IkappaB alpha phosphorylation and thus decreases NF-kappaB activation, was found to decrease TPA-induced nuclear translocation of NF-kappaB. Furthermore, BAY enhanced TPA-induced growth inhibition and apoptosis in both HL-60 and HL-525 cells. Results from the present study indicate that inhibition of NF-kappaB by BAY was associated with enhanced TPA-induced growth inhibition and apoptosis in human myeloid leukemia cells. TPA in combination with pharmacological inhibitors of NF-kappaB may improve the therapeutic efficacy of TPA and overcome the resistance to TPA in some myeloid leukemia patients.

  11. The structure and function of the eukaryotic ribosome.

    PubMed

    Wilson, Daniel N; Doudna Cate, Jamie H

    2012-05-01

    Structures of the bacterial ribosome have provided a framework for understanding universal mechanisms of protein synthesis. However, the eukaryotic ribosome is much larger than it is in bacteria, and its activity is fundamentally different in many key ways. Recent cryo-electron microscopy reconstructions and X-ray crystal structures of eukaryotic ribosomes and ribosomal subunits now provide an unprecedented opportunity to explore mechanisms of eukaryotic translation and its regulation in atomic detail. This review describes the X-ray crystal structures of the Tetrahymena thermophila 40S and 60S subunits and the Saccharomyces cerevisiae 80S ribosome, as well as cryo-electron microscopy reconstructions of translating yeast and plant 80S ribosomes. Mechanistic questions about translation in eukaryotes that will require additional structural insights to be resolved are also presented.

  12. The properties of ribosomal proteins from a moderate halophile.

    PubMed

    Falkenberg, P; Matheson, A T; Rollin, C F

    1976-06-15

    The ribosomes from the extreme halophile Halobacterium cutirubrum are unusual in that their ribosomal proteins are acidic rather than basic as is the case with almost all bacterial ribosomes (Bayley, S.T. (1966) J. Mol. Biol. 15, 420-427). To determine whether the ribosomes of a moderate halophile show similar properties the ribosomal proteins from an unidentified moderate halophile, which grows over a wide range of NaCl concentrations (0.04-4.3 M), were compared to those of Escherichia coli and H. cutirubrum. The proteins are slightly more acidic than those of E. coli but much less acidic than those from the extreme halophile as judged by their mobility on polyacrylamide gels and their amino acid composition. The electrophoretic profile on polyacrylamide gels of the ribosomal proteins from the moderate halophile is similar whether the cells are grown in 0.5 M or 4.25 M NaCl.

  13. Structure of the mammalian antimicrobial peptide Bac7(1–16) bound within the exit tunnel of a bacterial ribosome

    PubMed Central

    Seefeldt, A. Carolin; Graf, Michael; Pérébaskine, Natacha; Nguyen, Fabian; Arenz, Stefan; Mardirossian, Mario; Scocchi, Marco; Wilson, Daniel N.; Innis, C. Axel

    2016-01-01

    Proline-rich antimicrobial peptides (PrAMPs) produced as part of the innate immune response of animals, insects and plants represent a vast, untapped resource for the treatment of multidrug-resistant bacterial infections. PrAMPs such as oncocin or bactenecin-7 (Bac7) interact with the bacterial ribosome to inhibit translation, but their supposed specificity as inhibitors of bacterial rather than mammalian protein synthesis remains unclear, despite being key to developing drugs with low toxicity. Here, we present crystal structures of the Thermus thermophilus 70S ribosome in complex with the first 16 residues of mammalian Bac7, as well as the insect-derived PrAMPs metalnikowin I and pyrrhocoricin. The structures reveal that the mammalian Bac7 interacts with a similar region of the ribosome as insect-derived PrAMPs. Consistently, Bac7 and the oncocin derivative Onc112 compete effectively with antibiotics, such as erythromycin, which target the ribosomal exit tunnel. Moreover, we demonstrate that Bac7 allows initiation complex formation but prevents entry into the elongation phase of translation, and show that it inhibits translation on both mammalian and bacterial ribosomes, explaining why this peptide needs to be stored as an inactive pro-peptide. These findings highlight the need to consider the specificity of PrAMP derivatives for the bacterial ribosome in future drug development efforts. PMID:26792896

  14. The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells.

    PubMed

    Martin, F L; Cole, K J; Orme, M H; Grover, P L; Phillips, D H; Venitt, S

    1999-09-15

    We have found previously that the metabolically-competent human MCL-5 cell line did not appear to be usefully sensitive to the DNA-damaging effects of several carcinogens, as measured by the alkaline single-cell gel electrophoresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resynthesis during nucleotide excision repair. The following compounds were tested, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4, 8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-9H-pyrido[2,3-b]indole (A[alpha]C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA[alpha]C), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MCA), 7, 12-dimethylbenz[a]anthracene (DMBA), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), aniline, o-toluidine, benzene, lindane, bleomycin, cisplatin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium chromate, chromic chloride, and diethylstilboestrol (DES). We made the following observations. The background level of comet formation was reasonably constant over several months and was increased only slightly, but significantly, in the presence of the DNA-repair inhibitors. All compounds that induced comet formation did so without appreciable cytotoxicity as assessed by trypan blue exclusion. Of the compounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbons (with the exceptions of PhIP and B[a]P) failed to induce convincing levels of comet formation in the absence of repair inhibitors. In their presence the heterocyclic amines tested induced comet formation (with the exception of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marked comet formation even in the presence of HU

  15. Extrachromosomal circular ribosomal DNA in the yeast Saccharomyces carlsbergensis.

    PubMed Central

    Meyerink, J H; Klootwijk, J; Planta, R J; van der Ende, A; van Bruggen, E F

    1979-01-01

    Purified ribosomal DNA from Saccharomyces carlsbergensis contains a small proportion of circular DNA molecules with a contour length of 3 micron or integral multiples thereof. Hybridization of yeast ribosomal DNA with 26 S rRNA, using the R-loop technique, reveals that these circular molecules contain sequences complementary to yeast ribosomal RNA. We suggest that these extrachromosomal rRNA genes may be intermediates in the amplification of rRNA genes in yeast. Images PMID:493145

  16. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  17. Virological effects of ISIS 14803, an antisense oligonucleotide inhibitor of hepatitis C virus (HCV) internal ribosome entry site (IRES), on HCV IRES in chronic hepatitis C patients and examination of the potential role of primary and secondary HCV resistance in the outcome of treatment.

    PubMed

    Soler, Muriel; McHutchison, John G; Kwoh, T Jesse; Dorr, F Andrew; Pawlotsky, Jean-Michel

    2004-12-01

    Antisense oligonucleotides represent a promising class of antiviral agents. ISIS 14803 is a 20-unit phosphorothioate oligodeoxynucleotide that inhibited hepatitis C virus (HCV) replication and protein expression in cell culture and mouse models. A Phase I dose-escalation clinical study of ISIS 14803 was performed in 24 patients with HCV genotype 1 chronic hepatitis C. The patients received 0.5, 1.0, 2.0 or 3.0 mg/kg of ISIS 14803 for 4 weeks. Two of them receiving 2.0 mg/kg, experienced a significant (>1.0 log10) viral load reduction and nine other patients experienced minor (<1.0 log10) viral load reductions that were difficult to definitively distinguish from assay or patient variations. The aims of this study were to examine the effect of ISIS 14803 on its target site and neighbouring region quasispecies evolution, and to determine whether primary and secondary HCV resistance contributed to the observed virological response rate. The HCV internal ribosome entry site (IRES), including the ISIS 14803 target site in virus specimens collected from patients at baseline and end-of-treatment, was sequenced. An extensive IRES quasispecies analysis was performed in 10 of the patients at various time points before, during and after ISIS 14803 treatment. A significant IRES genetic evolution was found in three out of 10 patients through quasispecies analysis suggesting that treatment with ISIS 14803, a drug designed to bind to HCV RNA, exerted a selective pressure on HCV IRES. However, no mutations in the ISIS 14803 target site, which would inhibit binding of the oligonucleotide to HCV RNA, were detected before (primary resistance) or after treatment (secondary resistance) with the oligonucleotide. Furthermore, no obvious nucleotide changes in the surrounding IRES region that might possibly affect oligonucleotide binding were detected.

  18. -1 Programmed Ribosomal Frameshifting as a Force-Dependent Process.

    PubMed

    Visscher, Koen

    2016-01-01

    -1 Programmed ribosomal frameshifting is a translational recoding event in which ribosomes slip backward along messenger RNA presumably due to increased tension disrupting the codon-anticodon interaction at the ribosome's coding site. Single-molecule physical methods and recent experiments characterizing the physical properties of mRNA's slippery sequence as well as the mechanical stability of downstream mRNA structure motifs that give rise to frameshifting are discussed. Progress in technology, experimental assays, and data analysis methods hold promise for accurate physical modeling and quantitative understanding of -1 programmed ribosomal frameshifting. PMID:26970190

  19. Ribosome hibernation factor promotes Staphylococcal survival and differentially represses translation

    PubMed Central

    Basu, Arnab; Yap, Mee-Ngan F.

    2016-01-01

    In opportunistic Gram-positive Staphylococcus aureus, a small protein called hibernation-promoting factor (HPFSa) is sufficient to dimerize 2.5-MDa 70S ribosomes into a translationally inactive 100S complex. Although the 100S dimer is observed in only the stationary phase in Gram-negative gammaproteobacteria, it is ubiquitous throughout all growth phases in S. aureus. The biological significance of the 100S ribosome is poorly understood. Here, we reveal an important role of HPFSa in preserving ribosome integrity and poising cells for translational restart, a process that has significant clinical implications for relapsed staphylococcal infections. We found that the hpf null strain is severely impaired in long-term viability concomitant with a dramatic loss of intact ribosomes. Genome-wide ribosome profiling shows that eliminating HPFSa drastically increased ribosome occupancy at the 5′ end of specific mRNAs under nutrient-limited conditions, suggesting that HPFSa may suppress translation initiation. The protective function of HPFSa on ribosomes resides at the N-terminal conserved basic residues and the extended C-terminal segment, which are critical for dimerization and ribosome binding, respectively. These data provide significant insight into the functional consequences of 100S ribosome loss for protein synthesis and stress adaptation. PMID:27001516

  20. Dynamic Behavior of Trigger Factor on the Ribosome.

    PubMed

    Deeng, J; Chan, K Y; van der Sluis, E O; Berninghausen, O; Han, W; Gumbart, J; Schulten, K; Beatrix, B; Beckmann, R

    2016-09-11

    Trigger factor (TF) is the only ribosome-associated chaperone in bacteria. It interacts with hydrophobic segments in nascent chain (NCs) as they emerge from the ribosome. TF binds via its N-terminal ribosome-binding domain (RBD) mainly to ribosomal protein uL23 at the tunnel exit on the large ribosomal subunit. Whereas earlier structural data suggested that TF binds as a rigid molecule to the ribosome, recent comparisons of structural data on substrate-bound, ribosome-bound, and TF in solution from different species suggest that this chaperone is a rather flexible molecule. Here, we present two cryo-electron microscopy structures of TF bound to ribosomes translating an mRNA coding for a known TF substrate from Escherichia coli of a different length. The structures reveal distinct degrees of flexibility for the different TF domains, a conformational rearrangement of the RBD upon ribosome binding, and an increase in rigidity within TF when the NC is extended. Molecular dynamics simulations agree with these data and offer a molecular basis for these observations. PMID:27320387

  1. Ribosome hibernation factor promotes Staphylococcal survival and differentially represses translation.

    PubMed

    Basu, Arnab; Yap, Mee-Ngan F

    2016-06-01

    In opportunistic Gram-positive Staphylococcus aureus, a small protein called hibernation-promoting factor (HPFSa) is sufficient to dimerize 2.5-MDa 70S ribosomes into a translationally inactive 100S complex. Although the 100S dimer is observed in only the stationary phase in Gram-negative gammaproteobacteria, it is ubiquitous throughout all growth phases in S. aureus The biological significance of the 100S ribosome is poorly understood. Here, we reveal an important role of HPFSa in preserving ribosome integrity and poising cells for translational restart, a process that has significant clinical implications for relapsed staphylococcal infections. We found that the hpf null strain is severely impaired in long-term viability concomitant with a dramatic loss of intact ribosomes. Genome-wide ribosome profiling shows that eliminating HPFSa drastically increased ribosome occupancy at the 5' end of specific mRNAs under nutrient-limited conditions, suggesting that HPFSa may suppress translation initiation. The protective function of HPFSa on ribosomes resides at the N-terminal conserved basic residues and the extended C-terminal segment, which are critical for dimerization and ribosome binding, respectively. These data provide significant insight into the functional consequences of 100S ribosome loss for protein synthesis and stress adaptation. PMID:27001516

  2. Dissociability of free and peptidyl-tRNA bound ribosomes.

    PubMed

    Surguchov, A P; Fominykch, E S; Lyzlova, L V

    1978-06-16

    The influence of peptidyl-tRNA on the dissociation of yeast 80 S ribosomes into subunits was studied. For this purpose temperature-sensitive (ts) suppressor strain of yeast Saccharomyces cervisiae carrying a defect in peptide chain termination was used. It was found that peptidyl-tRNA did not influence the dissociation of ribosomes either at high salt concentration or in the presence of dissociation factor (DF) from yeast. After dissociation of yeast ribosomes in 0.5 M KCl, peptidyl-tRNA remains bound to the 60 S subunit. Some characteristics of the termination process and release of nascent polypeptides from yeast ribosomes are discussed. PMID:355860

  3. Can Structures Lead to Better Drugs? Lessons from Ribosome Research

    NASA Astrophysics Data System (ADS)

    Yonath, Ada

    Ribosome research has undergone astonishing progress in recent years. Crystal structures have shed light on the functional properties of the translation machinery and revealed how the ribosome's striking architecture is ingeniously designed as the framework for its unique capabilities: precise decoding, substrate mediated peptide-bond formation and efficient poly-merase activity. New findings include the two concerted elements of tRNA translocation: sideways shift and a ribosomal-navigated rotatory motion; the dynamics of the nascent chain exit tunnel and the shelter formed by the ribosome-bound trigger-factor, which acts as a chaperone to prevent nascent chain aggregation and misfolding.

  4. Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

    PubMed Central

    Body, Barbara A.; Brownstein, Bernard H.

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

  5. Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

    PubMed

    Body, A; Brownstein, B H

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

  6. Homoiterons and expansion in ribosomal RNAs.

    PubMed

    Parker, Michael S; Sallee, Floyd R; Park, Edwards A; Parker, Steven L

    2015-01-01

    Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks. PMID:26636029

  7. Homoiterons and expansion in ribosomal RNAs

    PubMed Central

    Parker, Michael S.; Sallee, Floyd R.; Park, Edwards A.; Parker, Steven L.

    2015-01-01

    Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks. PMID:26636029

  8. Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells

    PubMed Central

    Wittenberg, Avigail Dreazen; Azar, Shahar; Klochendler, Agnes; Stolovich-Rain, Miri; Avraham, Shlomit; Birnbaum, Lea; Binder Gallimidi, Adi; Katz, Maximiliano; Dor, Yuval; Meyuhas, Oded

    2016-01-01

    Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation. PMID:26919188

  9. Feedback regulation of ribosomal protein gene expression in Escherichia coli: structural homology of ribosomal RNA and ribosomal protein MRNA.

    PubMed Central

    Nomura, M; Yates, J L; Dean, D; Post, L E

    1980-01-01

    Certain ribosomal proteins (r proteins) in Escherichia coli, such as S4 and S7, function as feedback repressors in the regulation of r-protein synthesis. These proteins inhibit the translation of their own mRNA. The repressor r proteins so far identified are also known to bind specifically to rRNA at an initial stage in ribosome assembly. We have found structural homology between the S7 binding region on 16S rRNA and a region of the mRNA where S7 acts as a translational repressor. Similarly, there is structural homology between one of the reported S4 binding regions on 16S rRNA and the mRNA target site for S4. The observed homology supports the concept that regulation by repressor r proteins is based on competition between rRNA and mRNA for these proteins and that the same structural features and of the r proteins are used in their interactions with both rRNA and mRNA. PMID:7012833

  10. Feedback regulation of ribosomal protein gene expression in Escherichia coli: structural homology of ribosomal RNA and ribosomal protein MRNA.

    PubMed

    Nomura, M; Yates, J L; Dean, D; Post, L E

    1980-12-01

    Certain ribosomal proteins (r proteins) in Escherichia coli, such as S4 and S7, function as feedback repressors in the regulation of r-protein synthesis. These proteins inhibit the translation of their own mRNA. The repressor r proteins so far identified are also known to bind specifically to rRNA at an initial stage in ribosome assembly. We have found structural homology between the S7 binding region on 16S rRNA and a region of the mRNA where S7 acts as a translational repressor. Similarly, there is structural homology between one of the reported S4 binding regions on 16S rRNA and the mRNA target site for S4. The observed homology supports the concept that regulation by repressor r proteins is based on competition between rRNA and mRNA for these proteins and that the same structural features and of the r proteins are used in their interactions with both rRNA and mRNA.

  11. Ribosomal RNA: a key to phylogeny

    NASA Technical Reports Server (NTRS)

    Olsen, G. J.; Woese, C. R.

    1993-01-01

    As molecular phylogeny increasingly shapes our understanding of organismal relationships, no molecule has been applied to more questions than have ribosomal RNAs. We review this role of the rRNAs and some of the insights that have been gained from them. We also offer some of the practical considerations in extracting the phylogenetic information from the sequences. Finally, we stress the importance of comparing results from multiple molecules, both as a method for testing the overall reliability of the organismal phylogeny and as a method for more broadly exploring the history of the genome.

  12. Molecular morphology of ribosomes. Iodination of Escherichia coli ribosomal proteins with solid-state lactoperoxidase.

    PubMed

    Michalski, C J; Sells, B H

    1975-03-17

    Using either soluble or solid-state lactoperoxidase, a comparison was made between the enzymic iodination of ribosomal proteins iodinated as 30-S and 50-S subunits or as 70-S monosomes. Proteins S7, S11 and S12 of the 30-S subunit and proteins L2, L11, L26 and L28 of the 50-S subunit were labelled to a greater extent in isolated particles than in the 70-S ribosome. In contrast, proteins S4, S19 and S20 were labelled to a lesser extent in the isolated subunit. No significant differences were observed in the iodination patterns of ribosomes iodinated in the presence of soluble lactoperoxidase and those iodinated in the presence of lactoperoxidase bound to Sepharose 4B. It is suggested that the 30-S subunit undergoes a conformational cha