Science.gov

Sample records for nucleolar protein recognized

  1. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera

    SciTech Connect

    Aris, J.P.; Blobel, G. )

    1991-02-01

    The authors have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is {approximately}1,300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain {approximately}75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.

  2. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress.

    PubMed

    Nicolas, Emilien; Parisot, Pascaline; Pinto-Monteiro, Celina; de Walque, Roxane; De Vleeschouwer, Christophe; Lafontaine, Denis L J

    2016-01-01

    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology. PMID:27265389

  3. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress

    PubMed Central

    Nicolas, Emilien; Parisot, Pascaline; Pinto-Monteiro, Celina; de Walque, Roxane; De Vleeschouwer, Christophe; Lafontaine, Denis L. J.

    2016-01-01

    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology. PMID:27265389

  4. Topogenesis of a nucleolar protein: determination of molecular segments directing nucleolar association.

    PubMed

    Zirwes, R F; Kouzmenko, A P; Peters, J M; Franke, W W; Schmidt-Zachmann, M S

    1997-02-01

    To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein NO38 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large alpha-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleo-plasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein NO38 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino- and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein NO38 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture. PMID:9190204

  5. Effects of altered gravity on a distribution of rDNA and nucleolar proteins and the expression of nucleolar proteins in plants

    NASA Astrophysics Data System (ADS)

    Sobol, Margaryta; Kordyum, Elizabeth; Medina, Francisco Javier

    predominantly in FCs in the form of condensed chromatin inclusions and internal non condensed fibrils, redistributing from the DFC and the transition zone between FCs and the DFC, recognized as the site of rDNA transcription. Regarding nucleolar proteins, a general decrease in the levels of fibrillarin and the nucleolin homologues, evaluated by estimating the density of immunogold labeling on the nucleolus, was recorded firstly in clinorotated samples, compared to controls. Furthermore, the intranucleolar location of the investigated proteins was also observed to change in response to the growth in altered gravity conditions. In particular, a decrease in the quantity of these proteins in the transition zone FCs-DFC as well as in the bulk of the DFC was observed in the experimental samples, compared to controls, whereas the content of the proteins was much higher in the inner space of FCs. Concerning the two-dimensional nuclear proteome, we revealed a decrease in the isoelectric point (pI) range of soluble proteins, which are known to be actively engaged in RNA (including rRNA) metabolism, and a shortening in the molecular weight range of them under clinorotation. Besides, minor and major protein spots in clinorotated samples showed decreased optical densities in comparison to control ones. Moreover, we showed the shortening of both the pI and the molecular weight ranges of the spots corresponding to the major nucleolin homologue NhL90 (detected by cross-reaction with anti-onion NopA100) in the fraction of soluble proteins in altered gravity. Based on these data, an effect of altered gravity in lowering the level of rDNA transcription as well as rRNA processing, that could be the evidence of a decrease in the level of nucleolar functional activity, is suggested.

  6. Nucleolar protein B23 has molecular chaperone activities.

    PubMed Central

    Szebeni, A.; Olson, M. O.

    1999-01-01

    Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. PMID:10211837

  7. Nuclear and nucleolar targeting of human ribosomal protein S6.

    PubMed Central

    Schmidt, C; Lipsius, E; Kruppa, J

    1995-01-01

    Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Images PMID:8590812

  8. Nucleolar protein GLTSCR2 stabilizes p53 in response to ribosomal stresses

    PubMed Central

    Lee, S; Kim, J-Y; Kim, Y-J; Seok, K-O; Kim, J-H; Chang, Y-J; Kang, H-Y; Park, J-H

    2012-01-01

    p53 is a key regulator of cell growth and death by controlling cell cycle progression and apoptosis under conditions of stress such as DNA damage or oncogenic stimulation. As these processes are critical for cell function and inhibition of tumor development, p53 regulatory pathways are strictly monitored in cells. Recently, it was recognized that nucleolar proteins, including nucleophosmin/B23, ribosomal protein L11, and alternate reading frame (ARF), form the nucleolus-ARF-murine double minute 2 (MDM2) axis in p53 regulatory pathways, which increases p53 stability by suppressing the activity of MDM2. In this work, we show that nucleolar protein glioma tumor-suppressor candidate region gene 2 (GLTSCR2) translocates to the nucleoplasm under ribosomal stress, where it interacts with and stabilizes p53 and inhibits cell cycle progression without the involvement of the major upstream p53 regulator, ARF. Furthermore, ectopic expression of GLTSCR2 significantly suppressed growth of cancer cells in a xenograft animal model via p53-dependent pathway. Our data identify GLTSCR2 as a new member of the nucleolus–nucleoplasmic axis for p53 regulation. ARF-independent direct regulation of p53 by GLTSCR2 may be a key mechanism and therapeutic target for cell death or growth inhibition when nucleolus-ARF-p53 pathways are inactivated by genetic or epigenetic modifications of ARF, which are the second most common types of genetic change observed in human cancers. PMID:22522597

  9. Melanoma antigen-D2: A nucleolar protein undergoing delocalization during cell cycle and after cellular stress.

    PubMed

    Pirlot, Céline; Thiry, Marc; Trussart, Charlotte; Di Valentin, Emmanuel; Piette, Jacques; Habraken, Yvette

    2016-04-01

    Melanoma antigen D2 (MAGE-D2) is recognized as a cancer diagnostic marker; however, it has poorly characterized functions. Here, we established its intracellular localization and shuttling during cell cycle progression and in response to cellular stress. In normal conditions, MAGE-D2 is present in the cytoplasm, nucleoplasm, and nucleoli. Within the latter, MAGE-D2 is mostly found in the granular and the dense fibrillar components, and it interacts with nucleolin. Transfection of MAGE-D2 deletion mutants demonstrated that Δ203-254 leads to confinement of MAGE-D2 to the cytoplasm, while Δ248-254 prevents its accumulation in nucleoli but still allows its presence in the nucleoplasm. Consequently, this short sequence belongs to a nucleolar localization signal. MAGE-D2 deletion does not alter the nucleolar organization or rRNA levels. However, its intracellular localization varies with the cell cycle in a different kinetic than nucleolin. After genotoxic and nucleolar stresses, MAGE-D2 is excluded from nucleoli and concentrates in the nucleoplasm. We demonstrated that its camptothecin-related delocalization results from two distinct events: a rapid nucleolar release and a slower phospho-ERK-dependent cytoplasm to nucleoplasm translocation, which results from an increased flux from the cytoplasm to nucleoplasm. In conclusion, MAGE-D2 is a dynamic protein whose shuttling properties could suggest a role in cell cycle regulation. PMID:26705694

  10. The protein arginine methyltransferase PRMT6 inhibits HIV-1 Tat nucleolar retention.

    PubMed

    Fulcher, Alex J; Sivakumaran, Haran; Jin, Hongping; Rawle, Daniel J; Harrich, David; Jans, David A

    2016-02-01

    The human immunodeficiency virus (HIV)-1 transactivator protein Tat is known to play a key role in HIV infection, integrally related to its role in the host cell nucleus/nucleolus. Here we show for the first time that Tat localisation can be modulated by specific methylation, whereby overexpression of active but not catalytically inactive PRMT6 methyltransferase specifically leads to exclusion of Tat from the nucleolus. An R52/53A mutated Tat derivative does not show this redistribution, implying that R52/53, within Tat's nuclear/nucleolar localisation signal, are the targets of PRMT6 activity. Analysis using fluorescence recovery after photobleaching indicate that Tat nucleolar accumulation is largely through binding to nucleolar components, with methylation of Tat by PRMT6 preventing this. To our knowledge, this is the first report of specific protein methylation inhibiting nucleolar retention. PMID:26611710

  11. Tau protein binds to pericentromeric DNA: a putative role for nuclear tau in nucleolar organization.

    PubMed

    Sjöberg, Marcela K; Shestakova, Elena; Mansuroglu, Zeyni; Maccioni, Ricardo B; Bonnefoy, Eliette

    2006-05-15

    The microtubule-associated tau protein participates in the organization and integrity of the neuronal cytoskeleton. A nuclear form of tau has been described in neuronal and non-neuronal cells, which displays a nucleolar localization during interphase but is associated with nucleolar-organizing regions in mitotic cells. In the present study, based on immunofluorescence, immuno-FISH and confocal microscopy, we show that nuclear tau is mainly present at the internal periphery of nucleoli, partially colocalizing with the nucleolar protein nucleolin and human AT-rich alpha-satellite DNA sequences organized as constitutive heterochromatin. By using gel retardation, we demonstrate that tau not only colocalizes with, but also specifically binds to, AT-rich satellite DNA sequences apparently through the recognition of AT-rich DNA stretches. Here we propose a functional role for nuclear tau in relation to the nucleolar organization and/or heterochromatinization of a portion of RNA genes. Since nuclear tau has also been found in neurons from patients with Alzheimer's disease (AD), aberrant nuclear tau could affect the nucleolar organization during the course of AD. We discuss nucleolar tau associated with AT-rich alpha-satellite DNA sequences as a potential molecular link between trisomy 21 and AD. PMID:16638814

  12. The nucleolar structure and nucleolar proteins in proliferating cells of Arabidopsis seeds germinated in the International Space Station

    NASA Astrophysics Data System (ADS)

    Matía, I.; González-Camacho, F.; Marco, R.; Kiss, J. Z.; Gasset, G.; Medina, F. J.

    Seeds of Arabidopsis thaliana were sent to the ISS in the ``Cervantes Mission'' (Spanish Soyuz Mission) within MAMBA Biocontainers (Dutch Space B.V.). These Biocontainers are capable of supplying liquids to the biosample by means of a motorized mechanism based on the ``Berlingot-Ampoule'' concept. Seed germination was activated by supplying culture medium to them, and the process progressed for 4 days at 22°C. Then, growth was stopped by the addition of paraformaldehyde (PFA) fixative. Once back on the ground, samples were immediately processed for microscopical observation. A parallel ground control experiment was simultaneously replicated, following the same schedule and conditions. Seed germination occurred at a high rate in the Space. No differences in the germination rate were observed with respect to the ground control, although Space-grown seedlings were substantially longer (affecting the roots and also the hypocotyl) than the parallel samples grown at 1 g. The mitotic index and the cellular morphometric parameters (length, width, nuclear size) were measured and compared in both the experimental and control conditions. Bidimensional protein electrophoresis was performed on samples in which PFA fixation was reverted by prolonged (two weeks) storage in PBS buffer. The total proteomic profile of seedlings showed differences between the Space sample and the ground control, affecting to nearly one third of the spots. Remarkably, a set of spots around 35 kDa and pI 8.0 are conspicuous in the Space sample and do not appear in the ground control. A more specialized proteomic analysis, with functional significance, was carried out using the AgNOR staining method on Western blots, a technique revealing nucleolar proteins associated with cell proliferation. Immunocytochemical experiments showed the in situ distribution of nucleolin, a nucleolar multifunctional protein regulated by kinases related with cell cycle and proliferation control mechanisms. Finally, the

  13. C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

    PubMed

    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-09-11

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. PMID:26231209

  14. Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus

    PubMed Central

    Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.

    2012-01-01

    The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

  15. Evaluation of Nucleolar Proliferating Protein 1 as a Candidate Gene for Beef Carcass Characteristics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNP) identified in four regions of nucleolar proliferating protein 1 (Nol1: A, B, C and D) were evaluated for associations with carcass traits. Genotyping was performed in a group of 42 bulls (from which breed and age) were assigned to three supplementation treatment...

  16. Nucleolar Enrichment of Brain Proteins with Critical Roles in Human Neurodevelopment.

    PubMed

    Slomnicki, Lukasz P; Malinowska, Agata; Kistowski, Michal; Palusinski, Antoni; Zheng, Jing-Juan; Sepp, Mari; Timmusk, Tonis; Dadlez, Michal; Hetman, Michal

    2016-06-01

    To study nucleolar involvement in brain development, the nuclear and nucleolar proteomes from the rat cerebral cortex at postnatal day 7 were analyzed using LC-MS/iTRAQ methodology. Data of the analysis are available via ProteomeXchange with identifier PXD002188. Among 504 candidate nucleolar proteins, the overrepresented gene ontology terms included such cellular compartmentcategories as "nucleolus", "ribosome" and "chromatin". Consistent with such classification, the most overrepresented functional gene ontology terms were related to RNA metabolism/ribosomal biogenesis, translation, and chromatin organization. Sixteen putative nucleolar proteins were associated with neurodevelopmental phenotypes in humans. Microcephaly and/or cognitive impairment were the most common phenotypic manifestations. Although several such proteins have links to ribosomal biogenesis and/or genomic stability/chromatin structure (e.g. EMG1, RPL10, DKC1, EIF4A3, FLNA, SMC1, ATRX, MCM4, NSD1, LMNA, or CUL4B), others including ADAR, LARP7, GTF2I, or TCF4 have no such connections known. Although neither the Alazami syndrome-associated LARP7nor the Pitt-Hopkins syndrome-associated TCF4 were reported in nucleoli of non-neural cells, in neurons, their nucleolar localization was confirmed by immunostaining. In cultured rat hippocampal neurons, knockdown of LARP7 reduced both perikaryal ribosome content and general protein synthesis. Similar anti-ribosomal/anti-translation effects were observed after knockdown of the ribosomal biogenesis factor EMG1 whose deficiency underlies Bowen-Conradi syndrome. Finally, moderate reduction of ribosome content and general protein synthesis followed overexpression of two Pitt-Hopkins syndrome mutant variants of TCF4. Therefore, dysregulation of ribosomal biogenesis and/or other functions of the nucleolus may disrupt neurodevelopment resulting in such phenotypes as microcephaly and/or cognitive impairment. PMID:27053602

  17. Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig.

    PubMed

    Maddox-Hyttel, P; Svarcova, O; Laurincik, J

    2007-09-01

    The nucleolus is the site of ribosomal RNA (rRNA) and ribosome production. In the bovine primordial follicle oocyte, this organelle is inactive, but in the secondary follicle an active fibrillo-granular nucleolus develops and proteins involved in rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) localize to it. At the end of the oocyte growth phase, the nucleolus is inactivated again and transforms into a solid remnant. The nucleolar remnant is dissolved when meiosis is resumed. Upon fertilization, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities are engaged in the re-establishment of fibrillo-granular nucleoli at the major activation of the embryonic genome. This nucleolar formation can be classified into two different modes: one where nucleolus development occurs inside NPBs (internal; e.g. cattle) and the other where it occurs on the surface of NPBs (external; e.g. pig). Oocyte derived proteins engaged in late rRNA processing (nucleolin and nucleophosmin) may to some degree be re-used for nucleolar formation in the embryo, while the other nucleolar proteins require de novo embryonic transcription in order to be allocated to the developing nucleoli. Moreover, unprocessed rRNA inherited from the oocyte targets to the developing embryonic nucleoli. In conclusion, the nucleolus is important for the development of oocytes and embryos and may serve as a marker for the completion of oocyte growth and the normality of activation of the embryonic genome. PMID:17466364

  18. Heat shock protein 70 inhibits hydrogen peroxide-induced nucleolar fragmentation via suppressing cleavage and down-regulation of nucleolin.

    PubMed

    Wang, Kangkai; Deng, Gonghua; Chen, Guangwen; Liu, Meidong; Yi, Yuxin; Yang, Tubao; McMillan, Daniel R; Xiao, Xiangzhong

    2012-01-01

    It has been reported that nucleolar fragmentation is a part of the overall apoptotic morphology, however, it is currently obscure whether and how nucleolar fragmentation can be induced by hydrogen peroxide (H(2)O(2)) and heat shock protein 70 (Hsp70) can prevent nucleolar fragmentation. To dissect these two questions, C(2)C(12) myogenic cells and immortalized mouse embryonic fibroblasts (MEFs) with heat shock transcriptional factor 1 (HSF1) null mutation were treated with heat shock response (HS) (42.5 ± 0.5°C for 1 h and recovery at 37°C for 24 h) and then were insulted with 0.5 mmol/L H(2)O(2). Morphological changes of nucleoli were observed under contrast microscope or electronic microscope. It was found that (1) stimulation with H(2)O(2)-induced nucleolar fragmentation by mediating cleavage and down-regulation of nucleolar protein, nucleolin in C(2)C(12) myocytes and MEFs; (2) HS suppressed nucleolar fragmentation by inducing the expression of Hsp70 in an HSF1-dependent manner as indicated by assays of transfection with Hsp70 antisense oligonucleotides (AS-ONs) or recombinant plasmids of full-length Hsp70 cDNA; (3) protection of Hsp70 against nucleolar fragmentation was related to its accumulation in nucleolus mediated by nuclear localization sequence and its inhibition against cleavage and down-regulation of nucleolin. These results suggested that H(2)O(2)-induced nucleolar fragmentation and HS or Hsp70 inhibit H(2)O(2)-induced nucleolar fragmentation through the translocation of Hsp70 into nucleolar and its protection against impairment of nucleolin. PMID:21960124

  19. Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition*

    PubMed Central

    Matafora, Vittoria; D'Amato, Alfonsina; Mori, Silvia; Blasi, Francesco; Bachi, Angela

    2009-01-01

    Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity. PMID:19596686

  20. Influence of heart failure on nucleolar organization and protein expression in human hearts

    SciTech Connect

    Rosello-Lleti, Esther; Rivera, Miguel; Cortes, Raquel; Azorin, Inmaculada; Sirera, Rafael; Martinez-Dolz, Luis; Hove, Leif; Cinca, Juan; Lago, Francisca; Gonzalez-Juanatey, Jose R.; Salvador, Antonio; Portoles, Manuel

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Heart failure alters nucleolar morphology and organization. Black-Right-Pointing-Pointer Nucleolin expression is significant increased in ischemic and dilated cardiomyopathy. Black-Right-Pointing-Pointer Ventricular function of heart failure patients was related with nucleolin levels. -- Abstract: We investigate for the first time the influence of heart failure (HF) on nucleolar organization and proteins in patients with ischemic (ICM) or dilated cardiomyopathy (DCM). A total of 71 human hearts from ICM (n = 38) and DCM (n = 27) patients, undergoing heart transplantation and control donors (n = 6), were analysed by western-blotting, RT-PCR and cell biology methods. When we compared protein levels according to HF etiology, nucleolin was increased in both ICM (117%, p < 0.05) and DCM (141%, p < 0.01). Moreover, mRNA expression were also upregulated in ICM (1.46-fold, p < 0.05) and DCM (1.70-fold, p < 0.05. Immunofluorescence studies showed that the highest intensity of nucleolin was into nucleolus (p < 0.0001), and it was increased in pathological hearts (p < 0.0001). Ultrastructure analysis by electron microscopy showed an increase in the nucleus and nucleolus size in ICM (17%, p < 0.05 and 131%, p < 0.001) and DCM (56%, p < 0.01 and 69%, p < 0.01). Nucleolar organization was influenced by HF irrespective of etiology, increasing fibrillar centers (p < 0.001), perinucleolar chromatin (p < 0.01) and dense fibrillar components (p < 0.01). Finally, left ventricular function parameters were related with nucleolin levels in ischemic hearts (p < 0.0001). The present study demonstrates that HF influences on morphology and organization of nucleolar components, revealing changes in the expression and in the levels of nucleolin protein.

  1. Determinants of Mammalian Nucleolar Architecture

    PubMed Central

    Farley, Katherine I.; Surovtseva, Yulia; Merkel, Janie; Baserga, Susan J.

    2015-01-01

    The nucleolus is responsible for the production of ribosomes, essential machines which synthesize all proteins needed by the cell. The structure of human nucleoli is highly dynamic and is directly related to its functions in ribosome biogenesis. Despite the importance of this organelle, the intricate relationship between nucleolar structure and function remains largely unexplored. How do cells control nucleolar formation and function? What are the minimal requirements for making a functional nucleolus? Here we review what is currently known regarding mammalian nucleolar formation at nucleolar organizer regions (NORs), which can be studied by observing the dissolution and reformation of the nucleolus during each cell division. Additionally, the nucleolus can be examined by analyzing how alterations in nucleolar function manifest in differences in nucleolar architecture. Furthermore, changes in nucleolar structure and function are correlated with cancer, highlighting the importance of studying the determinants of nucleolar formation. PMID:25670395

  2. The Subcellular Localization and Functional Analysis of Fibrillarin2, a Nucleolar Protein in Nicotiana benthamiana

    PubMed Central

    Zheng, Luping; Yao, Jinai; Gao, Fangluan; Chen, Lin; Zhang, Chao; Lian, Lingli; Xie, Liyan; Wu, Zujian; Xie, Lianhui

    2016-01-01

    Nucleolar proteins play important roles in plant cytology, growth, and development. Fibrillarin2 is a nucleolar protein of Nicotiana benthamiana (N. benthamiana). Its cDNA was amplified by RT-PCR and inserted into expression vector pEarley101 labeled with yellow fluorescent protein (YFP). The fusion protein was localized in the nucleolus and Cajal body of leaf epidermal cells of N. benthamiana. The N. benthamiana fibrillarin2 (NbFib2) protein has three functional domains (i.e., glycine and arginine rich domain, RNA-binding domain, and α-helical domain) and a nuclear localization signal (NLS) in C-terminal. The protein 3D structure analysis predicted that NbFib2 is an α/β protein. In addition, the virus induced gene silencing (VIGS) approach was used to determine the function of NbFib2. Our results showed that symptoms including growth retardation, organ deformation, chlorosis, and necrosis appeared in NbFib2-silenced N. benthamiana. PMID:26885505

  3. The Wnt Target Protein Peter Pan Defines a Novel p53-independent Nucleolar Stress-Response Pathway.

    PubMed

    Pfister, Astrid S; Keil, Marina; Kühl, Michael

    2015-04-24

    Proper ribosome formation is a prerequisite for cell growth and proliferation. Failure of this process results in nucleolar stress and p53-mediated apoptosis. The Wnt target Peter Pan (PPAN) is required for 45 S rRNA maturation. So far, the role of PPAN in nucleolar stress response has remained elusive. We demonstrate that PPAN localizes to mitochondria in addition to its nucleolar localization and inhibits the mitochondrial apoptosis pathway in a p53-independent manner. Loss of PPAN induces BAX stabilization, depolarization of mitochondria, and release of cytochrome c, demonstrating its important role as an anti-apoptotic factor. Staurosporine-induced nucleolar stress and apoptosis disrupt nucleolar PPAN localization and induce its accumulation in the cytoplasm. This is accompanied by phosphorylation and subsequent cleavage of PPAN by caspases. Moreover, we show that PPAN is a novel interaction partner of the anti-apoptotic protein nucleophosmin (NPM). PPAN depletion induces NPM and upstream-binding factor (UBF) degradation, which is independent of caspases. In summary, we provide evidence for a novel nucleolar stress-response pathway involving PPAN, NPM, and BAX to guarantee cell survival in a p53-independent manner. PMID:25759387

  4. Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress.

    PubMed

    Klibanov, S A; O'Hagan, H M; Ljungman, M

    2001-05-01

    The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes. PMID:11329373

  5. Influence of heart failure on nucleolar organization and protein expression in human hearts.

    PubMed

    Roselló-Lletí, Esther; Rivera, Miguel; Cortés, Raquel; Azorín, Inmaculada; Sirera, Rafael; Martínez-Dolz, Luis; Hove, Leif; Cinca, Juan; Lago, Francisca; González-Juanatey, José R; Salvador, Antonio; Portolés, Manuel

    2012-02-10

    We investigate for the first time the influence of heart failure (HF) on nucleolar organization and proteins in patients with ischemic (ICM) or dilated cardiomyopathy (DCM). A total of 71 human hearts from ICM (n=38) and DCM (n=27) patients, undergoing heart transplantation and control donors (n=6), were analysed by western-blotting, RT-PCR and cell biology methods. When we compared protein levels according to HF etiology, nucleolin was increased in both ICM (117%, p<0.05) and DCM (141%, p<0.01). Moreover, mRNA expression were also upregulated in ICM (1.46-fold, p<0.05) and DCM (1.70-fold, p<0.05. Immunofluorescence studies showed that the highest intensity of nucleolin was into nucleolus (p<0.0001), and it was increased in pathological hearts (p<0.0001). Ultrastructure analysis by electron microscopy showed an increase in the nucleus and nucleolus size in ICM (17%, p<0.05 and 131%, p<0.001) and DCM (56%, p<0.01 and 69%, p<0.01). Nucleolar organization was influenced by HF irrespective of etiology, increasing fibrillar centers (p<0.001), perinucleolar chromatin (p<0.01) and dense fibrillar components (p<0.01). Finally, left ventricular function parameters were related with nucleolin levels in ischemic hearts (p<0.0001). The present study demonstrates that HF influences on morphology and organization of nucleolar components, revealing changes in the expression and in the levels of nucleolin protein. PMID:22244875

  6. Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription.

    PubMed

    Deng, Wensheng; Lopez-Camacho, Cesar; Tang, Jen-Yang; Mendoza-Villanueva, Daniel; Maya-Mendoza, Apolinar; Jackson, Dean A; Shore, Paul

    2012-01-31

    Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease. PMID:22307607

  7. Aluminum can induce alterations in the cellular localization and expression of three major nucleolar proteins in root tip cells of Allium cepa var. agrogarum L.

    PubMed

    Qin, Rong; Jiang, Wusheng; Liu, Donghua

    2013-01-01

    A 50 μM aluminum (Al) could induce nucleolar materials containing the argyrophilic proteins scattered in the nuclei and extruded from the nuclei into the cytoplasm in the root tip cells of Allium cepa. Unfortunately, what kinds of nucleolar proteins are affected has not been reported till now. In order to go deeper into the understanding of the cytological effects of Al on nucleolus and nucleolar proteins, alterations in the cellular localization and expression of three major nucleolar proteins: nucleophosmin, nucleolin, and fibrillarin were further examined under the treatment with Al in the root tip cells of A. cepa in the present study. Cytological effects of Al on nucleolus were observed by silver-staining method and three major nucleolar proteins: nucleophosmin, nucleolin, and fibrillarin were examined by western blotting. The results indicated that in the presence of 50 μM Al for 48 h the nucleolar proteins were translocated from nucleolus to nucleoplasm and cytoplasm. Western blotting data demonstrated the relatively higher expression of the three major nucleolar proteins when compared with control. Evidence from the present investigation indicated that Al had toxic effects on Ag-NOR proteins, nucleophosmin and nucleolin, and other kinds of nucleolar proteins, fibrillarin. PMID:23111171

  8. Nucleolar localization of cirhin, the protein mutated in North American Indian childhood cirrhosis

    SciTech Connect

    Yu, Bin; Mitchell, Grant A.; Richter, Andrea . E-mail: andrea.richter@umontreal.ca

    2005-12-10

    Cirhin (NP{sub 1}16219), the product of the CIRH1A gene is mutated in North American Indian childhood cirrhosis (NAIC/CIRH1A, OMIM 604901), a severe autosomal recessive intrahepatic cholestasis. It is a 686-amino-acid WD40-repeat containing protein of unknown function that is predicted to contain multiple targeting signals, including an N-terminal mitochondrial targeting signal, a C-terminal monopartite nuclear localization signal (NLS) and a bipartite nuclear localization signal (BNLS). We performed the direct determination of subcellular localization of cirhin as a crucial first step in unraveling its biological function. Using EGFP and His-tagged cirhin fusion proteins expressed in HeLa and HepG2, cells we show that cirhin is a nucleolar protein and that the R565W mutation, for which all NAIC patients are homozygous, has no effect on subcellular localization. Cirhin has an active C-terminal monopartite nuclear localization signal (NLS) and a unique nucleolar localization signal (NrLS) between residues 315 and 432. The nucleolus is not known to be important specifically for intrahepatic cholestasis. These observations provide a new dimension in the study of hereditary cholestasis.

  9. A constitutive nucleolar protein identified as a member of the nucleoplasmin family.

    PubMed Central

    Schmidt-Zachmann, M S; Hügle-Dörr, B; Franke, W W

    1987-01-01

    Using monoclonal antibodies we have localized a polypeptide, appearing on gel electrophoresis with a Mr of approximately 38,000 and a pI of approximately 5.6, to the granular component of the nucleoli of Xenopus laevis oocytes and a broad range of cells from various species. The protein (NO38) also occurs in certain distinct nucleoplasmic particles but is not detected in ribosomes and other cytoplasmic components. During mitosis NO38-containing material dissociates from the nucleolar organizer region and distributes over the chromosomal surfaces and the perichromosomal cytoplasm; in telophase it re-populates the forming nucleoli. With these antibodies we have isolated from a X. laevis ovary lambda gt11 expression library a cDNA clone encoding a polypeptide which, on one- and two-dimensional gel electrophoresis, co-migrates with authentic NO38. The amino acid sequence deduced from this clone defines a polypeptide of 299 amino acids of mol. wt 33,531 which is characterized by the presence of two domains exceptionally rich in aspartic and glutamic acid, one of them flanked by two putative karyophilic signal heptapeptides. Comparison with other protein sequences shows that NO38 is closely related to the histone-binding, karyophilic protein nucleoplasmin: the first 124 amino acids have 58 amino acid positions in common. Protein NO38 also shows striking homologies to the phosphopeptide region of rat nucleolar protein B23 and the carboxyterminal region of human B23. We propose that protein NO38, which forms distinct homo-oligomers of approximately 7S and Mr of approximately 230,000, is a member of a family of karyophilic proteins, the 'nucleoplasmin family'. It is characterized by its specific association with the nucleolus and might be involved in nuclear accumulation, nucleolar storage and pre-rRNA assembly of ribosomal proteins in a manner similar to that discussed for the role of nucleoplasmin in histone storage and chromatin assembly. Images Fig. 1. Fig. 2. Fig. 3

  10. Mesenchyme-specific overexpression of nucleolar protein 66 in mice inhibits skeletal growth and bone formation

    PubMed Central

    Chen, Qin; Zhang, Liping; de Crombrugghe, Benoit; Krahe, Ralf

    2015-01-01

    Previous studies showed that nucleolar protein 66 (NO66), the Jumonji C-domain-containing histone demethylase for methylated histone H3K4 and H3K36 (H3K36me), negatively regulates osteoblast differentiation in vitro by inhibiting the activity of transcription factor osterix (Osx). However, whether NO66 affects mammalian skeletogenesis in vivo is not yet known. Here, we generated transgenic (TG) mice overexpressing a flag-tagged NO66 transgene driven by the Prx1 (paired related homeobox 1) promoter. We found that NO66 overexpression in Prx1-expressing mesenchymal cells inhibited skeletal growth and bone formation. The inhibitory phenotype was associated with >50% decreases in chondrocyte/osteoblast proliferation and differentiation. Moreover, we found that in bones of NO66-TG mice, expression of Igf1, Igf1 receptor (Igf1r), runt-related transcription factor 2, and Osx was significantly down-regulated (P < 0.05). Consistent with these results, we observed >50% reduction in levels of phosphorylated protein kinase B (Akt) and H3K36me3 in bones of NO66-TG mice, suggesting an inverse correlation between NO66 histone demethylase and the activity of IGF1R/Akt signaling. This correlation was further confirmed by in vitro assays of C2C12 cells with NO66 overexpression. We propose that the decrease in the IGF1R/Akt signaling pathway in mice with mesenchymal overexpression of NO66 may contribute in part to the inhibition of skeletal growth and bone formation.—Chen, Q., Zhang, L., de Crombrugghe, B., Krahe, R. Mesenchyme-specific overexpression of nucleolar protein 66 in mice inhibits skeletal growth and bone formation. PMID:25746793

  11. Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos.

    PubMed

    Borah, Supriya; Barrodia, Praveen; Swain, Rajeeb K

    2016-01-01

    The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut. PMID:26934290

  12. Saccharomyces cerevisiae nucleolar protein Nop7p is necessary for biogenesis of 60S ribosomal subunits.

    PubMed Central

    Adams, Cynthia C; Jakovljevic, Jelena; Roman, Judibelle; Harnpicharnchai, Piyanun; Woolford, John L

    2002-01-01

    To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A. PMID:11911362

  13. Tracking the interactions of rRNA processing proteins during nucleolar assembly in living cells.

    PubMed

    Angelier, Nicole; Tramier, Marc; Louvet, Emilie; Coppey-Moisan, Maïté; Savino, Tula M; De Mey, Jan R; Hernandez-Verdun, Danièle

    2005-06-01

    Reorganization of the nuclear machinery after mitosis is a fundamental but poorly understood process. Here, we investigate the recruitment of the nucleolar processing proteins in the nucleolus of living cells at the time of nucleus formation. We question the role of the prenucleolar bodies (PNBs), during migration of the processing proteins from the chromosome periphery to sites of rDNA transcription. Surprisingly, early and late processing proteins pass through the same PNBs as demonstrated by rapid two-color four-dimensional imaging and quantification, whereas a different order of processing protein recruitment into nucleoli is supported by differential sorting. Protein interactions along the recruitment pathway were investigated using a promising time-lapse analysis of fluorescence resonance energy transfer. For the first time, it was possible to detect in living cells the interactions between proteins of the same rRNA processing machinery in nucleoli. Interestingly interactions between such proteins also occur in PNBs but not at the chromosome periphery. The dynamics of these interactions suggests that PNBs are preassembly platforms for rRNA processing complexes. PMID:15814843

  14. Identification of a small, very acidic constitutive nucleolar protein (NO29) as a member of the nucleoplasmin family

    PubMed Central

    Zirwes, Rudolf F.; Schmidt-Zachmann, Marion S.; Franke, Werner W.

    1997-01-01

    We report the discovery and molecular characterization of a small and very acidic nucleolar protein of an SDS/PAGE mobility corresponding to Mr 29,000 (NO29). The cDNA-deduced sequence of the Xenopus laevis protein defines a polypeptide of a calculated molecular mass of 20,121 and a pI of 3.75, with an extended acidic region near its C terminus, and is related to the major nucleolar protein, NO38, and the histone-binding protein, nucleoplasmin. This member of the nucleoplasmin family of proteins was immunolocalized to nucleoli in Xenopus oocytes and diverse somatic cells. Protein NO29 is associated with nuclear particles from Xenopus oocytes, partly complexed with protein NO38, and occurs in preribosomes but not in mature ribosomes. The location and the enormously high content of negatively charged amino acids lead to the hypothesis that NO29 might be involved in the nuclear and nucleolar accumulation of ribosomal proteins and the coordinated assembly of pre-ribosomal particles. PMID:9326619

  15. The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera

    PubMed Central

    Martins, Juliana Ramos; Bitondi, Márcia Maria Gentile

    2016-01-01

    Hexamerins are insect storage proteins abundantly secreted by the larval fat body into the haemolymph. The canonical role of hexamerins consists of serving as an amino acid reserve for development toward the adult stage. However, in Apis mellifera, immunofluorescence assays coupled to confocal laser-scanning microscopy, and high-throughput sequencing, have recently shown the presence of hexamerins in other organs than the fat body. These findings have led us to study these proteins with the expectation of uncovering additional functions in insect development. We show here that a honeybee hexamerin, HEX 110, localizes in the cytoplasm and nucleus of ovarian cells. In the nucleus of somatic and germline cells, HEX 110 colocalized with a nucleolar protein, fibrillarin, suggesting a structural or even regulatory function in the nucleolus. RNase A provoked the loss of HEX 110 signals in the ovarioles, indicating that the subcellular localization depends on RNA. This was reinforced by incubating ovaries with pyronin Y, a RNA-specific dye. Together, the colocalization with fibrillarin and pyronin Y, and the sensitivity to RNase, highlight unprecedented roles for HEX110 in the nucleolus, the nuclear structure harbouring the gene cluster involved in ribosomal RNA production. However, the similar patterns of HEX 110 foci distribution in the active and inactive ovaries of queens and workers preclude its association with the functional status of these organs. PMID:26954256

  16. The TORMOZ Gene Encodes a Nucleolar Protein Required for Regulated Division Planes and Embryo Development in Arabidopsis[W

    PubMed Central

    Griffith, Megan E.; Mayer, Ulrike; Capron, Arnaud; Ngo, Quy A.; Surendrarao, Anandkumar; McClinton, Regina; Jürgens, Gerd; Sundaresan, Venkatesan

    2007-01-01

    Embryogenesis in Arabidopsis thaliana is marked by a predictable sequence of oriented cell divisions, which precede cell fate determination. We show that mutation of the TORMOZ (TOZ) gene yields embryos with aberrant cell division planes and arrested embryos that appear not to have established normal patterning. The defects in toz mutants differ from previously described mutations that affect embryonic cell division patterns. Longitudinal division planes of the proembryo are frequently replaced by transverse divisions and less frequently by oblique divisions, while divisions of the suspensor cells, which divide only transversely, appear generally unaffected. Expression patterns of selected embryo patterning genes are altered in the mutant embryos, implying that the positional cues required for their proper expression are perturbed by the misoriented divisions. The TOZ gene encodes a nucleolar protein containing WD repeats. Putative TOZ orthologs exist in other eukaryotes including Saccharomyces cerevisiae, where the protein is predicted to function in 18S rRNA biogenesis. We find that disruption of the Sp TOZ gene results in cell division defects in Schizosaccharomyces pombe. Previous studies in yeast and animal cells have identified nucleolar proteins that regulate the exit from M phase and cytokinesis, including factors involved in pre-rRNA processing. Our study suggests that in plant cells, nucleolar functions might interact with the processes of regulated cell divisions and influence the selection of longitudinal division planes during embryogenesis. PMID:17616738

  17. Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

    PubMed Central

    Peeples, M E; Wang, C; Gupta, K C; Coleman, N

    1992-01-01

    A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization. Images PMID:1560547

  18. Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

    SciTech Connect

    Amin, Mohammed Abdullahel; Matsunaga, Sachihiro; Ma, Nan; Takata, Hideaki; Yokoyama, Masami; Uchiyama, Susumu; Fukui, Kiichi . E-mail: kfukui@bio.eng.osaka-u.ac.jp

    2007-08-24

    Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.

  19. NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

    SciTech Connect

    Shen, Qi; Zheng, Xingzheng; McNutt, Michael A.; Guang, Lizhao; Sun, Ying; Wang, Jiaochen; Gong, Yilei; Hou, Lin; Zhang, Bo

    2009-06-10

    The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated {alpha}-tubulin, suggesting that it plays a role in maintaining or enhancing stability of {alpha}-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated {alpha}-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.

  20. Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency

    PubMed Central

    Deschamps-Francoeur, Gabrielle; Garneau, Daniel; Dupuis-Sandoval, Fabien; Roy, Audrey; Frappier, Marie; Catala, Mathieu; Couture, Sonia; Barbe-Marcoux, Mélissa; Abou-Elela, Sherif; Scott, Michelle S.

    2014-01-01

    Small nucleolar RNAs (snoRNAs) are among the first discovered and most extensively studied group of small non-coding RNA. However, most studies focused on a small subset of snoRNAs that guide the modification of ribosomal RNA. In this study, we annotated the expression pattern of all box C/D snoRNAs in normal and cancer cell lines independent of their functions. The results indicate that C/D snoRNAs are expressed as two distinct forms differing in their ends with respect to boxes C and D and in their terminal stem length. Both forms are overexpressed in cancer cell lines but display a conserved end distribution. Surprisingly, the long forms are more dependent than the short forms on the expression of the core snoRNP protein NOP58, thought to be essential for C/D snoRNA production. In contrast, a subset of short forms are dependent on the splicing factor RBFOX2. Analysis of the potential secondary structure of both forms indicates that the k-turn motif required for binding of NOP58 is less stable in short forms which are thus less likely to mature into a canonical snoRNP. Taken together the data suggest that C/D snoRNAs are divided into at least two groups with distinct maturation and functional preferences. PMID:25074380

  1. NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

    PubMed Central

    de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

    2013-01-01

    NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species. PMID:23516598

  2. Lrrc34, a novel nucleolar protein, interacts with npm1 and ncl and has an impact on pluripotent stem cells.

    PubMed

    Lührig, Sandra; Siamishi, Iliana; Tesmer-Wolf, Marieke; Zechner, Ulrich; Engel, Wolfgang; Nolte, Jessica

    2014-12-01

    The gene Lrrc34 (leucine rich repeat containing 34) is highly expressed in pluripotent stem cells and its expression is strongly downregulated upon differentiation. These results let us to suggest a role for Lrrc34 in the regulation and maintenance of pluripotency. Expression analyses revealed that Lrrc34 is predominantly expressed in pluripotent stem cells and has an impact on the expression of known pluripotency genes, such as Oct4. Methylation studies of the Lrrc34 promoter showed a hypomethylation in undifferentiated stem cells and chromatin immunoprecipitation-quantitative polymerase chain reaction analyses of histone modifications revealed an enrichment of activating histone modifications on the Lrrc34 promoter region. Further, we could verify the nucleolus-the place of ribosome biogenesis-as the major subcellular localization of the LRRC34 protein. We have verified the interaction of LRRC34 with two major nucleolar proteins, Nucleophosmin and Nucleolin, by two independent methods, suggesting a role for Lrrc34 in ribosome biogenesis of pluripotent stem cells. In conclusion, LRRC34 is a novel nucleolar protein that is predominantly expressed in pluripotent stem cells. Its altered expression has an impact on pluripotency-regulating genes and it interacts with proteins known to be involved in ribosome biogenesis. Therefore we suggest a role for Lrrc34 in ribosome biogenesis of pluripotent stem cells. PMID:24991885

  3. Nucleolar targeting of proteins by the tandem array of basic amino acid stretches identified in the RNA polymerase I-associated factor PAF49

    SciTech Connect

    Ushijima, Ryujiro; Matsuyama, Toshifumi; Nagata, Izumi; Yamamoto, Kazuo

    2008-05-16

    There is accumulating evidence to indicate that the regulation of subnuclear compartmentalization plays important roles in cellular processes. The RNA polymerase I-associated factor PAF49 has been shown to accumulate in the nucleolus in growing cells, but disperse into the nucleoplasm in growth-arrested cells. Serial deletion analysis revealed that amino acids 199-338 were necessary for the nucleolar localization of PAF49. Combinatorial point mutation analysis indicated that the individual basic amino acid stretches (BS) within the central (BS1-4) and the C-terminal (BS5 and 6) regions may cooperatively confer the nucleolar localization of PAF49. Addition of the basic stretches in tandem to a heterologous protein, such as the interferon regulatory factor-3, translocated the tagged protein into the nucleolus, even in the presence of an intrinsic nuclear export sequence. Thus, tandem array of the basic amino acid stretches identified here functions as a dominant nucleolar targeting sequence.

  4. [Identification of the protein partners of the human nucleolar protein SURF6 in HeLa cells by GST pull-down assay].

    PubMed

    Kordiukova, M Iu; Polzikov, M A; Shishova, K V; Zatsepina, O V

    2014-01-01

    The eukaryotic proteins comprising the SURF6 protein family are evolutionary conservative and housekeeping proteins however, functional roles of human SURF6 have not been studied so far. To shed light to this question in the present work we applied GST pull-down assay and used two proteins fused with GST, namely human GST-SURF6 and the conservative C-terminal domain of mouse Surf6 that has 85% homology with the C-terminus of the human SURF6 conservative domain (GST-Surf6-dom), to identify SURF6-interacting proteins in human HeLa cells. The results obtained showed that GST-SURF6 interacts with several key nucleolar RNA processing factors (B23/nucleophosmin, nucleolin, EBP2), and also with the specific cofactor of RNA polymerase I, protein UBE These results are the first experimental evidences in favor of participation of the human SURF6 protein in ribosome biogenesis, including transcription of rDNA and processing of rRNAs. The same results were obtained, when GST-Surf6-dom was used to pull-down proteins in HeLa cells. In addition, the panel of the GST-Surf6-dom protein partners, which were identified by mass-spectrometry, points to putative interactions of human SURF6 with a number of nuclear and nucleolar, proteins of other functional groups, i.e. to the protein plurifunctionality. PMID:25898752

  5. Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

    PubMed

    Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

    2014-09-01

    Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations. PMID:24870286

  6. gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

    PubMed Central

    Gulli, M P; Girard, J P; Zabetakis, D; Lapeyre, B; Melese, T; Caizergues-Ferrer, M

    1995-01-01

    Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA. Images PMID:7596817

  7. Identification and fine mapping of nuclear and nucleolar localization signals within the human ribosomal protein S17.

    PubMed

    Kenney, Scott P; Meng, Xiang-Jin

    2015-01-01

    Human ribosomal protein S17 (RPS17) is mutated in Diamond-Blackfan Anemia (DBA), a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV) from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7). RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP), we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs), one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes. PMID:25853866

  8. Overexpression and Nucleolar Localization of γ-Tubulin Small Complex Proteins GCP2 and GCP3 in Glioblastoma.

    PubMed

    Dráberová, Eduarda; D'Agostino, Luca; Caracciolo, Valentina; Sládková, Vladimíra; Sulimenko, Tetyana; Sulimenko, Vadym; Sobol, Margaryta; Maounis, Nicoletta F; Tzelepis, Elias; Mahera, Eleni; Křen, Leoš; Legido, Agustin; Giordano, Antonio; Mörk, Sverre; Hozák, Pavel; Dráber, Pavel; Katsetos, Christos D

    2015-07-01

    The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating γ-tubulin small complex (γTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma cells versus normal human astrocytes; these were associated with higher amounts of both γTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with γ-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that γTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype. PMID:26079448

  9. Evidence for nucleolar subcompartments in Dictyostelium

    SciTech Connect

    Catalano, Andrew; O’Day, Danton H.

    2015-01-24

    Highlights: • Two nucleolar subcompartments (NoSC1, NoSC2) were found in Dictyostelium. • Specific nucleolar proteins localize to different nucleolar subcompartments. • Specific proteins exit NoSC1 and NoSC2 differently upon Actinomycin D treatment. • KRKR appears to function as an NoSC2 nucleolar subcompartment localization signal. - Abstract: The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during

  10. Rad53 homologue forkhead-associated kinase A (FhkA) and Ca2+-binding protein 4a (CBP4a) are nucleolar proteins that differentially redistribute during mitosis in Dictyostelium

    PubMed Central

    2013-01-01

    Background During mitosis most nucleolar proteins redistribute to other locales providing an opportunity to study the relationship between nucleolar protein localization and function. Dictyostelium is a model organism for the study of several fundamental biological processes and human diseases but only two nucleolar proteins have been studied during mitosis: NumA1 and Snf12. Both of them are linked to the cell cycle. To acquire a better understanding of nucleolar protein localization and dynamics in Dictyostelium we studied the nucleolar localization of two additional proteins during mitosis: Snf12-linked forkhead-associated kinase A (FhkA), which is involved in the cell cycle, and Ca2+-binding protein 4a (CBP4a), which is a binding partner of NumA1. Methods Polyclonal antibodies were produced in-house. Cells were fixed and probed with either anti-FhkA or anti-CBP4a in order to determine cellular localization during interphase and throughout the stages of mitosis. Colocalization with DAPI nuclear stain allowed us to determine the location of the nucleus and nucleolus while colocalization with anti-α-tubulin allowed us to determine the cell cycle stage. Results Here we verify two novel nucleolar proteins, Rad53 homologue FhkA which localized around the edge of the nucleolus and CBP4a which was detected throughout the entire nucleolus. Treatment with the Ca2+ chelator BAPTA (5 mM) showed that the nucleolar localization of CBP4a is Ca2+-dependent. In response to actinomycin D (0.05 mg/mL) CBP4a disappeared from the nucleolus while FhkA protruded from the nucleus, eventually pinching off as cytoplasmic circles. FhkA and CBP4a redistributed differently during mitosis. FhkA redistributed throughout the entire cell and at the nuclear envelope region from prometaphase through telophase. In contrast, during prometaphase CBP4a relocated to many large, discrete “CBP4a islands” throughout the nucleoplasm. Two larger “CBP4a islands” were also detected specifically

  11. Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics.

    PubMed

    Marciniak, K; Bilecka, A

    1986-01-01

    Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication. PMID:3733472

  12. Proteomic, cellular, and network analyses reveal new DUSP3 interactions with nucleolar proteins in HeLa cells.

    PubMed

    Panico, Karine; Forti, Fabio Luis

    2013-12-01

    DUSP3 (or Vaccinia virus phosphatase VH1-related; VHR) is a small dual-specificity phosphatase known to dephosphorylate c-Jun N-terminal kinases and extracellular signal-regulated kinases. In human cervical cancer cells, DUSP3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. Other DUSP3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates DNA damage response or repair processes. In this study, we sought to identify new interactions between DUSP3 and proteins directly or indirectly involved in or correlated with its biological roles in HeLa cells exposed to gamma or UV radiation. By using GST-DUSP as bait, we pulled down interacting proteins and identified them by LC-MS/MS. Of the 46 proteins obtained, six hits were extensively validated by immune techniques; the proteins Nucleophosmin, HnRNP C1/C2, and Nucleolin were the most promising targets found to directly interact with DUSP3. We then analyzed the DUSP3 interactomes using physical protein-protein interaction networks using our hits as the seed list. The validated hits as well as unvalidated hits fluctuated on the DUSP3 interactomes of HeLa cells, independent of the time post radiation, which confirmed our proteomic and experimental data and clearly showed the proximity of DUSP3 to proteins involved in processes intimately related to DNA repair and senescence, such as Ku70 and Tert, via interactions with nucleolar proteins, which were identified in this study, that regulate DNA/RNA structure and functions. PMID:24245651

  13. Pontin is localized in nucleolar fibrillar centers

    PubMed Central

    2008-01-01

    Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I. Nucleolar localization of Pontin was confirmed by its detection in nucleolar extracts and by electron microscopy, which revealed Pontin accumulation specifically in the nucleolar fibrillar centers. Pontin localization in the nucleolus was dynamic and Pontin accumulated in large nucleolar dots mainly during S-phase. Pontin concentration in the large nucleolar dots correlated with reduced transcriptional activity of nucleoli. In addition, Pontin was found to associate with RNA polymerase I and to interact in a complex with c-Myc with rDNA sequences indicating that Pontin is involved in the c-Myc-dependent regulation of rRNA synthesis. PMID:18548265

  14. Recognizing the fold of a protein structure.

    PubMed

    Harrison, Andrew; Pearl, Frances; Sillitoe, Ian; Slidel, Tim; Mott, Richard; Thornton, Janet; Orengo, Christine

    2003-09-22

    This paper reports a graph-theoretic program, GRATH, that rapidly, and accurately, matches a novel structure against a library of domain structures to find the most similar ones. GRATH generates distributions of scores by comparing the novel domain against the different types of folds that have been classified previously in the CATH database of structural domains. GRATH uses a measure of similarity that details the geometric information, number of secondary structures and number of residues within secondary structures, that any two protein structures share. Although GRATH builds on well established approaches for secondary structure comparison, a novel scoring scheme has been introduced to allow ranking of any matches identified by the algorithm. More importantly, we have benchmarked the algorithm using a large dataset of 1702 non-redundant structures from the CATH database which have already been classified into fold groups, with manual validation. This has facilitated introduction of further constraints, optimization of parameters and identification of reliable thresholds for fold identification. Following these benchmarking trials, the correct fold can be identified with the top score with a frequency of 90%. It is identified within the ten most likely assignments with a frequency of 98%. GRATH has been implemented to use via a server (http://www.biochem.ucl.ac.uk/cgi-bin/cath/Grath.pl). GRATH's speed and accuracy means that it can be used as a reliable front-end filter for the more accurate, but computationally expensive, residue based structure comparison algorithm SSAP, currently used to classify domain structures in the CATH database. With an increasing number of structures being solved by the structural genomics initiatives, the GRATH server also provides an essential resource for determining whether newly determined structures are related to any known structures from which functional properties may be inferred. PMID:14512345

  15. Human H/ACA Small Nucleolar RNPs and Telomerase Share Evolutionarily Conserved Proteins NHP2 and NOP10

    PubMed Central

    Pogacic, Vanda; Dragon, François; Filipowicz, Witold

    2000-01-01

    The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. In the yeast Saccharomyces cerevisiae, four common proteins are associated with H/ACA snoRNAs: Gar1p, Cbf5p, Nhp2p, and Nop10p. In vitro reconstitution studies showed that four proteins also specifically interact with H/ACA snoRNAs in mammalian cell extracts. Two mammalian proteins, NAP57/dyskerin (the ortholog of Cbf5p) and hGAR1, have been characterized. In this work we describe properties of hNOP10 and hNHP2, human orthologs of yeast Nop10p and Nhp2p, respectively, and further characterize hGAR1. hNOP10 and hNHP2 complement yeast cells depleted of Nhp2p and Nop10p, respectively. Immunoprecipitation experiments with extracts from transfected HeLa cells indicated that epitope-tagged hNOP10 and hNHP2 specifically associate with hGAR1 and H/ACA RNAs; they also interact with the RNA subunit of telomerase, which contains an H/ACA-like domain in its 3′ moiety. Immunofluorescence microscopy experiments showed that hGAR1, hNOP10, and hNHP2 are localized in the dense fibrillar component of the nucleolus and in Cajal (coiled) bodies. Deletion analysis of hGAR1 indicated that its evolutionarily conserved core domain contains all the signals required for localization, but progressive deletions from either the N or the C terminus of the core domain abolish localization in the nucleolus and/or the Cajal bodies. PMID:11074001

  16. Multifunctional roles for the N-terminal basic motif of Alfalfa mosaic virus coat protein: nucleolar/cytoplasmic shuttling, modulation of RNA-binding activity, and virion formation.

    PubMed

    Herranz, Mari Carmen; Pallas, Vicente; Aparicio, Frederic

    2012-08-01

    In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation. PMID:22746826

  17. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

    SciTech Connect

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

    2008-08-15

    Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.

  18. Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA.

    PubMed

    Shishova, Kseniya V; Lavrentyeva, Elena A; Dobrucki, Jurek W; Zatsepina, Olga V

    2015-01-15

    It is well known that fully-grown mammalian oocytes, rather than typical nucleoli, contain prominent but structurally homogenous bodies called "nucleolus-like bodies" (NLBs). NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain. To clarify the composition of the NLB material in mouse GV oocytes, we devised an assay to detect internal oocyte proteins with fluorescein-5-isothiocyanate (FITC) and applied the fluorescent RNA-binding dye acridine orange to examine whether NLBs contain RNA. Our results unequivocally show that, similarly to typical nucleoli, proteins and RNA are major constituents of transcriptionally active (or non-surrounded) NLBs as well as of transcriptionally silent (or surrounded) NLBs. We also show, by exposing fixed oocytes to a mild proteinase K treatment, that the NLB mass in oocytes of both types contains nucleolar proteins that are involved in all major steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), and late rRNA processing (NPM1/nucleophosmin/B23, nucleolin/C23), but none of the nuclear proteins tested, including SC35, NOBOX, topoisomerase II beta, HP1α, and H3. The ribosomal RPL26 protein was detected within the NLBs of NSN-type oocytes but is virtually absent from NLBs of SN-type oocytes. Taking into account that the major class of nucleolar RNA is ribosomal RNA (rRNA), we applied fluorescence in situ hybridization with oligonucleotide probes targeting 18S and 28S rRNAs. The results show that, in contrast to active nucleoli, NLBs of fully-grown oocytes are impoverished for the rRNAs, which is consistent with the absence of transcribed ribosomal genes in the NLB mass. Overall, the results of this study suggest that NLBs of fully-grown mammalian oocytes serve for storing major nucleolar proteins but not rRNA. PMID:25481757

  19. Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals.

    PubMed

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Estraño, Carlos E

    2014-01-01

    Giardia lamblia mitosomes are believed to be vestigial mitochondria which lack a genome. Similar to higher eukaryotes, mitosomal proteins possess either N-terminal or internal mitosomal targeting sequences. To date, some components of the higher eukaryote archetypal mitochondrial protein import apparatus have been identified and characterized in Giardia mitosomes; therefore, it is expected that mitochondrial signals will be recognized by the mitosomal protein import system. To further determine the level of conservation of the Giardia mitosome protein import apparatus, we expressed mitochondrial proteins from higher eukaryotes in Giardia. These recombinant proteins include Tom20 and Tom22; two components of the mitochondrial protein import machinery. Our results indicate that N-terminal mitochondrial targeting sequence is recognized by the mitosomal protein import machinery; however, interestingly the internal mitochondrial targeting sequences of higher eukaryotes are not recognized by the mitosome. Our results indicate that Giardia mitosome protein transport machinery shows differential recognition of higher eukaryotic mitochondria transfer signals, suggesting a divergence of the transport system in G. lamblia. Therefore, our data support the hypothesis that the protein import machinery in Giardia lamblia mitosome is an incomplete vestigial derivative of mitochondria components. PMID:25159305

  20. A transducible nuclear/nucleolar protein, mLLP, regulates neuronal morphogenesis and synaptic transmission

    PubMed Central

    Yu, Nam-Kyung; Kim, Hyoung F.; Shim, Jaehoon; Kim, Somi; Kim, Dae Won; Kwak, Chuljung; Sim, Su-Eon; Choi, Jun-Hyeok; Ahn, Seohee; Yoo, Juyoun; Choi, Sun-Lim; Jang, Deok-Jin; Lim, Chae-Seok; Lee, Yong-Seok; Kang, Chulhun; Choi, Soo Young; Kaang, Bong-Kiun

    2016-01-01

    Cell-permeable proteins are emerging as unconventional regulators of signal transduction and providing a potential for therapeutic applications. However, only a few of them are identified and studied in detail. We identify a novel cell-permeable protein, mouse LLP homolog (mLLP), and uncover its roles in regulating neural development. We found that mLLP is strongly expressed in developing nervous system and that mLLP knockdown or overexpression during maturation of cultured neurons affected the neuronal growth and synaptic transmission. Interestingly, extracellular addition of mLLP protein enhanced dendritic arborization, demonstrating the non-cell-autonomous effect of mLLP. Moreover, mLLP interacts with CCCTC-binding factor (CTCF) as well as transcriptional machineries and modulates gene expression involved in neuronal growth. Together, these results illustrate the characteristics and roles of previously unknown cell-permeable protein mLLP in modulating neural development. PMID:26961175

  1. A transducible nuclear/nucleolar protein, mLLP, regulates neuronal morphogenesis and synaptic transmission.

    PubMed

    Yu, Nam-Kyung; Kim, Hyoung F; Shim, Jaehoon; Kim, Somi; Kim, Dae Won; Kwak, Chuljung; Sim, Su-Eon; Choi, Jun-Hyeok; Ahn, Seohee; Yoo, Juyoun; Choi, Sun-Lim; Jang, Deok-Jin; Lim, Chae-Seok; Lee, Yong-Seok; Kang, Chulhun; Choi, Soo Young; Kaang, Bong-Kiun

    2016-01-01

    Cell-permeable proteins are emerging as unconventional regulators of signal transduction and providing a potential for therapeutic applications. However, only a few of them are identified and studied in detail. We identify a novel cell-permeable protein, mouse LLP homolog (mLLP), and uncover its roles in regulating neural development. We found that mLLP is strongly expressed in developing nervous system and that mLLP knockdown or overexpression during maturation of cultured neurons affected the neuronal growth and synaptic transmission. Interestingly, extracellular addition of mLLP protein enhanced dendritic arborization, demonstrating the non-cell-autonomous effect of mLLP. Moreover, mLLP interacts with CCCTC-binding factor (CTCF) as well as transcriptional machineries and modulates gene expression involved in neuronal growth. Together, these results illustrate the characteristics and roles of previously unknown cell-permeable protein mLLP in modulating neural development. PMID:26961175

  2. Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins.

    PubMed

    Shiohama, Aiko; Sasaki, Takashi; Noda, Setsuko; Minoshima, Shinsei; Shimizu, Nobuyoshi

    2007-12-10

    We identified 11 proteins that are associated with DGCR8 by immunoprecipitation assay and mass spectrometry. These proteins included Nucleolin, ILF3 and others, most of which appeared to be involved in the RNA processing or RNA transportation. We detected at least four kinds of protein complex, such as DROSHA/DGCR8, DGCR8/Nucleolin, DGCR8/ILF3 and ILF3/XPO5, by co-immunoprecipitation. The complex formation of DGCR8 with Nucleolin was dependent on RNA. Subcellular localization analysis by the immunofluorescent microscopy and immunoelectron microscopy indicated that DGCR8 locates at the nucleolus and small foci adjacent to splicing speckles in the nucleoplasm. Furthermore, the localization of DGCR8 at the nucleolus was changed by the inhibition of RNA transcription. Thus, our studies provided additional new evidence for the involvement of various protein complexes in the molecular mechanisms of apparently complex innate RNA interference machinery. PMID:17765891

  3. Nucleolar localization of Small G protein RhoA is associated with active RNA synthesis in human carcinoma HEp-2 cells

    PubMed Central

    LI, YUEYING; HU, YONG; CHE, LILONG; JIA, JUNHAI; CHEN, MIN

    2016-01-01

    Previous studies have demonstrated that the nuclear localization of ras homolog family member A (RhoA), with prominent concentration in the nucleolus, is a common feature in human cancer tissues and cancer cell lines. Although a previous study has demonstrated that the nuclear translocation of RhoA occurs via active transport, a process that occurs through importin α in a nuclear factor-κB-dependent manner, the mechanism, biological function and pathological meaning of the nucleolar residency of RhoA remain to be elucidated. As the cell nucleolus is the site of ribosome biosynthesis, the aim of the present study was to investigate the association between RNA synthesis and the nucleolar localization of RhoA, as well as the molecular mechanisms underlying the residency of RhoA in the nucleolus of HEp-2 (human larynx epithelial carcinoma) cells. Indirect immunofluorescence microscopy was used to evaluate the subcellular distribution of nuclear RhoA, and immunoblotting analysis was used to determine the total cellular protein level of RhoA. Consistent with the results of previous studies, untreated HEp-2 cells exhibited bright nucleolar staining, indicating an increased concentration of RhoA in the nucleoli. Treatment with actinomycin D for the inhibition of RNA synthesis caused a redistribution of RhoA from the nucleoli to the nucleoplasm with a speckled staining pattern. Immunoblotting revealed that neither the total cellular amount of RhoA nor the integrity of RhoA was affected by treatment with actinomycin D. In cells that were treated at a decreased concentration (0.05 mg/l) of actinomycin D, the redistribution of RhoA was reversible following the removal of the drug from the culture medium. However, this reversal was not observed at an increased drug concentration (1 mg/l). Overall, to the best of our knowledge, the results of the present study provide the first in situ evidence that the inhibition of RNA synthesis induces a redistribution of nucleolar RhoA to

  4. SAP-like domain in nucleolar spindle associated protein mediates mitotic chromosome loading as well as interphase chromatin interaction

    SciTech Connect

    Verbakel, Werner; Carmeliet, Geert; Engelborghs, Yves

    2011-08-12

    Highlights: {yields} The SAP-like domain in NuSAP is a functional DNA-binding domain with preference for dsDNA. {yields} This SAP-like domain is essential for chromosome loading during early mitosis. {yields} NuSAP is highly dynamic on mitotic chromatin, as evident from photobleaching experiments. {yields} The SAP-like domain also mediates NuSAP-chromatin interaction in interphase nucleoplasm. -- Abstract: Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy. The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase Nu

  5. PAK1IP1, a ribosomal stress-induced nucleolar protein, regulates cell proliferation via the p53–MDM2 loop

    PubMed Central

    Yu, Weishi; Qiu, Zhongwei; Gao, Na; Wang, Liren; Cui, Hengxiang; Qian, Yu; Jiang, Li; Luo, Jian; Yi, Zhengfang; Lu, Hua; Li, Dali; Liu, Mingyao

    2011-01-01

    Cell growth and proliferation are tightly controlled via the regulation of the p53–MDM2 feedback loop in response to various cellular stresses. In this study, we identified a nucleolar protein called PAK1IP1 as another regulator of this loop. PAK1IP1 was induced when cells were treated with chemicals that disturb ribosome biogenesis. Overexpression of PAK1IP1 inhibited cell proliferation by inducing p53-dependent G1 cell-cycle arrest. PAK1IP1 bound to MDM2 and inhibited its ability to ubiquitinate and to degrade p53, consequently leading to the accumulation of p53 levels. Interestingly, knockdown of PAK1IP1 in cells also inhibited cell proliferation and induced p53-dependent G1 arrest. Deficiency of PAK1IP1 increased free ribosomal protein L5 and L11 which were required for PAK1IP1 depletion-induced p53 activation. Taken together, our results reveal that PAK1IP1 is a new nucleolar protein that is crucial for rRNA processing and plays a regulatory role in cell proliferation via the p53–MDM2 loop. PMID:21097889

  6. Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

    PubMed Central

    Earley, Lauriel F.; Kawano, Yasuhiro; Adachi, Kei; Sun, Xiao-Xin; Dai, Mu-Shui

    2014-01-01

    ABSTRACT Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2144–184). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase–AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2144–184 has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly. IMPORTANCE Adeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of

  7. Nucleolar protein PES1 is a marker of neuroblastoma outcome and is associated with neuroblastoma differentiation

    PubMed Central

    Nakaguro, Masato; Kiyonari, Shinichi; Kishida, Satoshi; Cao, Dongliang; Murakami-Tonami, Yuko; Ichikawa, Hitoshi; Takeuchi, Ichiro; Nakamura, Shigeo; Kadomatsu, Kenji

    2015-01-01

    Neuroblastoma (NB) is a childhood malignant tumor that arises from precursor cells of the sympathetic nervous system. Spontaneous regression is a phenomenon unique to NBs and is caused by differentiation of tumor cells. PES1 is a multifunctional protein with roles in both neural development and ribosome biogenesis. Various kinds of models have revealed the significance of PES1 in neurodevelopment. However, the roles of PES1 in NB tumorigenesis and differentiation have remained unknown. Here we show that NB cases with MYCN amplification and clinically unfavorable stage (INSS stage 4) express higher levels of PES1. High PES1 expression was associated with worse overall and relapse-free survival. In NB cell lines, PES1 knockdown suppressed tumor cell growth and induced apoptosis. This growth inhibition was associated with the expression of NB differentiation markers. However, when the differentiation of NB cell lines was induced by the use of all-trans retinoic acid, there was a corresponding decrease in PES1 expression. Pes1 expression of tumorspheres originated from MYCN transgenic mice also diminished after the induction of differentiation with growth factors. We also reanalyzed the distribution of PES1 in the nucleolus. PES1 was localized in the dense fibrillar component, but not in the granular component of nucleoli. After treatment with the DNA-damaging agent camptothecin, this distribution was dramatically changed to diffuse nucleoplasmic. These data suggest that PES1 is a marker of NB outcome, that it regulates NB cell proliferation, and is associated with NB differentiation. PMID:25557119

  8. Nucleolar localization of SmMAK16 protein from Schistosoma mansoni is regulated by three distinct signals that function independent of pH or phosphorylation status.

    PubMed

    Hoellerich, Elisa; Dunagan, Christie; Maring, Daniel; Wong, Yun-Lan C; Shouldice, Daniel; Stripe, Jennifer; Kline, Tayah; Albert, Thomas J; Milhon, Jon L

    2014-01-01

    SmMAK16 from the trematode Schistosoma mansoni is a protein that is known to localize in the nucleolus. Recent findings show that SmMAK16 is involved in 60S ribosomal subunit synthesis. Although the SmMAK16 protein contains putative nuclear localization signals (NLS), little is known about their precise function, redundancy or regulation. The goal of the current study was to identify and characterize the presence and functional regulation of the localization signals in SmMAK16. The SmMAK16 coding sequence and specific fragments were individually cloned in-frame into the pEGFP-C2 expression vector to encode Green Fluorescent Protein (GFP) fusion proteins. Constructs were individually transfected into COS-7 cells and fluorescent microscopy used to determine the cellular location and thus the presence of signals regulating nuclear and nucleolar localization. SmMAK16 was found to contain two NLSs and one nucleolar localization signal (NoLS). One of the signals contains a sequence identical to an established nucleolar detention signal that reportedly functions only under acidic cellular conditions. The localization of the SmMAK16-GFP constructs was analyzed under acidic conditions; however, altering pH did not influence the localization of SmMAK16. It has been previously reported that casein kinase 2 (CK2) can phosphorylate SmMAK16 at serines adjacent to one of the NLSs. One of these CK2 sites and the adjacent NLS are conserved with that of the SV40 Large T Antigen (LTA) and phosphorylation of this site in the SV40 LTA regulates the kinetics of the NLS. To discover if kinetic regulation also occurs in SmMAK16, mutant and wild type SmMAK16-GFP proteins were purified and injected into individual COS-7 cells. No difference in the rate of transport was found between wt and mutant SmMAK16 proteins. Therefore, SmMAK16 localizes to the nucleolus using three separate signals, two NLSs and one NoLS, however, these signals appear to function independently of pH and

  9. Nucleolar stress in Diamond Blackfan anemia pathophysiology.

    PubMed

    Ellis, Steven R

    2014-06-01

    Diamond Blackfan anemia is a red cell hypoplasia that typically presents within the first year of life. Most cases of Diamond Blackfan anemia are caused by ribosome assembly defects linked to haploinsufficiency for structural proteins of either ribosomal subunit. Nucleolar stress associated with abortive ribosome assembly leads to p53 activation via the interaction of free ribosomal proteins with HDM2, a negative regulator of p53. Significant challenges remain in linking this nucleolar stress signaling pathway to the clinical features of Diamond Blackfan anemia. Defining aspects of disease presentation may relate to developmental and physiological triggers that work in conjunction with nucleolar stress signaling to heighten the p53 response in the developing erythron after birth. The growing number of ribosomopathies provides additional challenges for linking molecular mechanisms with clinical phenotypes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease. PMID:24412987

  10. A polybasic motif in ErbB3-binding protein 1 (EBP1) has key functions in nucleolar localization and polyphosphoinositide interaction.

    PubMed

    Karlsson, Thomas; Altankhuyag, Altanchimeg; Dobrovolska, Olena; Turcu, Diana C; Lewis, Aurélia E

    2016-07-15

    Polyphosphoinositides (PPIns) are present in the nucleus where they participate in crucial nuclear processes, such as chromatin remodelling, transcription and mRNA processing. In a previous interactomics study, aimed to gain further insight into nuclear PPIns functions, we identified ErbB3 binding protein 1 (EBP1) as a potential nuclear PPIn-binding protein in a lipid pull-down screen. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In the present study, we show that EBP1 binds directly to several PPIns via two distinct PPIn-binding sites consisting of clusters of lysine residues and positioned at the N- and C-termini of the protein. Using interaction mutants, we show that the C-terminal PPIn-binding motif contributes the most to the localization of EBP1 in the nucleolus. Importantly, a K372N point mutation, located within the C-terminal motif and found in endometrial tumours, is sufficient to alter the nucleolar targeting of EBP1. Our study reveals also the presence of the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110β and its product PtdIns(3,4,5)P3 together with EBP1 in the nucleolus. Using NMR, we further demonstrate an association between EBP1 and PtdIns(3,4,5)P3 via both electrostatic and hydrophobic interactions. Taken together, these results show that EBP1 interacts directly with PPIns and associate with PtdIns(3,4,5)P3 in the nucleolus. The presence of p110β and PtdIns(3,4,5)P3 in the nucleolus indicates their potential role in regulating nucleolar processes, at least via EBP1. PMID:27118868

  11. A polybasic motif in ErbB3-binding protein 1 (EBP1) has key functions in nucleolar localization and polyphosphoinositide interaction

    PubMed Central

    Karlsson, Thomas; Altankhuyag, Altanchimeg; Dobrovolska, Olena; Turcu, Diana C.; Lewis, Aurélia E.

    2016-01-01

    Polyphosphoinositides (PPIns) are present in the nucleus where they participate in crucial nuclear processes, such as chromatin remodelling, transcription and mRNA processing. In a previous interactomics study, aimed to gain further insight into nuclear PPIns functions, we identified ErbB3 binding protein 1 (EBP1) as a potential nuclear PPIn-binding protein in a lipid pull-down screen. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In the present study, we show that EBP1 binds directly to several PPIns via two distinct PPIn-binding sites consisting of clusters of lysine residues and positioned at the N- and C-termini of the protein. Using interaction mutants, we show that the C-terminal PPIn-binding motif contributes the most to the localization of EBP1 in the nucleolus. Importantly, a K372N point mutation, located within the C-terminal motif and found in endometrial tumours, is sufficient to alter the nucleolar targeting of EBP1. Our study reveals also the presence of the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110β and its product PtdIns(3,4,5)P3 together with EBP1 in the nucleolus. Using NMR, we further demonstrate an association between EBP1 and PtdIns(3,4,5)P3 via both electrostatic and hydrophobic interactions. Taken together, these results show that EBP1 interacts directly with PPIns and associate with PtdIns(3,4,5)P3 in the nucleolus. The presence of p110β and PtdIns(3,4,5)P3 in the nucleolus indicates their potential role in regulating nucleolar processes, at least via EBP1. PMID:27118868

  12. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging.

    PubMed

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K; Osorio, Fernando A; Hiscox, Julian A

    2008-08-15

    Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus. PMID:18550142

  13. Isolation and characterization of a carrot nucleolar protein with structural and sequence similarity to the vertebrate PESCADILLO protein.

    PubMed

    Ueda, Kenji; Xu, Zheng-Jun; Miyagi, Nobuaki; Ono, Michiyuki; Wabiko, Hiroetsu; Masuda, Kiyoshi; Inoue, Masayasu

    2013-07-01

    The nuclear matrix is involved in many nuclear events, but its protein architecture in plants is still not fully understood. A cDNA clone was isolated by immunoscreening with a monoclonal antibody raised against nuclear matrix proteins of Daucus carota L. Its deduced amino acid sequence showed about 40% identity with the PESCADILLO protein of zebrafish and humans. Primary structure analysis of the protein revealed a Pescadillo N-terminus domain, a single breast cancer C-terminal domain, two nuclear localization signals, and a potential coiled-coil region as also found in animal PESCADILLO proteins. Therefore, we designated this gene DcPES1. Although DcPES1 mRNA was detected in all tissues examined, its levels were highest in tissues with proliferating cells. Immunofluorescence using specific antiserum against the recombinant protein revealed that DcPES1 localized exclusively in the nucleolus. Examination of fusion proteins with green fluorescent protein revealed that the N-terminal portion was important for localization to the nucleoli of tobacco and onion cells. Moreover, when the nuclear matrix of carrot cells was immunostained with an anti-DcPES1 serum, the signal was detected in the nucleolus. Therefore, the DcPES1 protein appears to be a component of or tightly bound to components of the nuclear matrix. PMID:23683933

  14. The localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence.

    PubMed

    Rowland, R R; Kervin, R; Kuckleburg, C; Sperlich, A; Benfield, D A

    1999-10-01

    The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) possesses two regions in the N-terminal half of the protein that are enriched in basic amino acids. Presumably, these basic regions are important for packaging the RNA genome within the nucleocapsid of the virus. The PSORT computer program identified the same regions as nuclear localization signal (NLS) sequence motifs. N protein localization to the nucleus of infected MARC-145 and porcine pulmonary macrophages was observed following staining with SDOW-17 and SR-30 anti-N monoclonal antibodies. Furthermore, the co-localization of SR-30 antibody with human ANA-N autoimmune serum identified the nucleolus as the primary site for N protein localization within the nucleus. The localization of the N protein in the absence of infection was studied by following fluorescence in MARC-145 cells transfected with a plasmid, which expressed the nucleocapsid protein fused to an enhanced green fluorescent protein (N-EGFP). Similar to infected cells, N-EGFP localized to the cytoplasm and the nucleolus. Results following the transfection of cells with pEGFP fused to truncated portions of the N gene identified a region containing the second basic stretch of amino acids as the nucleolar localization signal (NoLS) sequence. Another outcome following transfection was the rapid disappearance of cells that expressed high levels of N-EGFP. However, cell death did not correlate with localization of N-EGFP to the nucleolus. PMID:10500278

  15. Determination of the functional domains involved in nucleolar targeting of nucleolin.

    PubMed Central

    Créancier, L; Prats, H; Zanibellato, C; Amalric, F; Bugler, B

    1993-01-01

    Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus. Images PMID:8167407

  16. The yeast NOP4 gene product is an essential nucleolar protein required for pre-rRNA processing and accumulation of 60S ribosomal subunits.

    PubMed Central

    Sun, C; Woolford, J L

    1994-01-01

    The Saccharomyces cerevisiae NOP4 gene was isolated by screening a lambda gt11 yeast genomic DNA library with a monoclonal antibody against a yeast nucleolar protein. NOP4 encodes a 78 kDa protein that contains two prototypical RNA recognition motifs (RRMs) flanking an imperfect RRM lacking characteristic RNP1 and RNP2 motifs. In addition, there is a fourth incomplete RRM. NOP4 is a single copy essential gene present on chromosome XVI, between RAD1 and PEP4. To examine the function of Nop4p, we constructed a conditional null allele of NOP4 by placing this gene under the control of the glucose-repressible GAL1 promoter. When cells are shifted from galactose-containing medium to glucose-containing medium, NOP4 transcription is terminated, Nop4 protein is depleted and cell growth is impaired. Nop4 protein depletion results in diminished accumulation of 60S ribosomal subunits, assignable to a defect in ribosome biogenesis arising from a lack of production of mature 25S rRNA from 27S precursor rRNA. Images PMID:8039505

  17. The leukemogenic t(8;21) fusion protein AML1-ETO controls ribosomal RNA genes and associates with nucleolar organizing regions at mitotic chromosomes

    PubMed Central

    Bakshi, Rachit; Zaidi, Sayyed K.; Pande, Sandhya; Hassan, Mohammad Q.; Young, Daniel W.; Lian, Jane B.; van Wijnen, Andre J.; Stein, Janet L.; Stein, Gary S.

    2010-01-01

    SUMMARY RUNX1/AML1 is required for definitive hematopoiesis and is frequently targeted by chromosomal translocation in acute myeloid leukemias (AML). The t(8;21) related AML1-ETO fusion protein blocks differentiation of myeloid progenitors. Here, we show by immunofluorescence microscopy that during interphase, endogenous AML1-ETO localizes to nuclear microenvironments distinct from those containing native RUNX1/AML1 protein. At mitosis, we clearly detect binding of AML1-ETO to nucleolar organizing regions (NORs) in AML derived Kasumi-1 cells and binding of RUNX1/AML1 to NORs in Jurkat cells. Both RUNX1/AML1 and AML1-ETO occupy ribosomal DNA repeats during interphase, as well as interact with the endogenous RNA Pol I transcription factor UBF-1. Promoter cytosine methylation analysis indicates that RUNX1/AML1 binds to rDNA repeats that are more highly CpG methylated than those bound by AML1-ETO. Down-regulation by RNA interference reveals that RUNX1/AML1 negatively regulates rDNA transcription, while AML1-ETO is a positive regulator in Kasumi-1 cells. Taken together, our findings identify a novel role for the leukemia-related AML1-ETO protein in epigenetic control of cell growth through upregulation of RNA Pol I-mediated ribosomal gene transcription, consistent with the hyper-proliferative phenotype of myeloid cells in AML patients. PMID:19001502

  18. The Relationship Between Human Nucleolar Organizer Regions and Nucleoli, Probed by 3D-ImmunoFISH.

    PubMed

    van Sluis, Marjolein; van Vuuren, Chelly; McStay, Brian

    2016-01-01

    3D-immunoFISH is a valuable technique to compare the localization of DNA sequences and proteins in cells where three-dimensional structure has been preserved. As nucleoli contain a multitude of protein factors dedicated to ribosome biogenesis and form around specific chromosomal loci, 3D-immunoFISH is a particularly relevant technique for their study. In human cells, nucleoli form around transcriptionally active ribosomal gene (rDNA) arrays termed nucleolar organizer regions (NORs) positioned on the p-arms of each of the acrocentric chromosomes. Here, we provide a protocol for fixing and permeabilizing human cells grown on microscope slides such that nucleolar proteins can be visualized using antibodies and NORs visualized by DNA FISH. Antibodies against UBF recognize transcriptionally active rDNA/NORs and NOP52 antibodies provide a convenient way of visualizing the nucleolar volume. We describe a probe designed to visualize rDNA and introduce a probe comprised of NOR distal sequences, which can be used to identify or count individual NORs. PMID:27576706

  19. Downregulation of nucleolar and spindle-associated protein 1 expression suppresses cell migration, proliferation and invasion in renal cell carcinoma.

    PubMed

    Fang, Lu; Zhang, Meng; Chen, Lei; Xiong, Hu; Ge, Yukun; Lu, Wei; Wu, Xun; Heng, Baoli; Yu, Dexin; Wu, Song

    2016-09-01

    Nucleolar and spindle-associated protein 1 (NUSAP1) is a microtubule-binding protein that plays an essential role in mitosis and cancer. Previous studies have demonstrated that NUSAP1 expression is relatively elevated in several malignancies. However, the biological roles of NUSAP1 in renal cell carcinoma (RCC) remain unknown. In the present study, we firstly performed reverse transcription‑polymerase chain reaction (RT-PCR) and western blot analysis to reveal that the expression of NUSAP1 was relatively elevated in clear cell RCC (ccRCC) tissue specimens and RCC cell lines. Immunohistochemical analysis showed that upregulation of NUSAP1 was significantly correlated with Fuhrman grade (P<0.001), tumor size (P=0.016), clinical stage (P<0.001) and distant metastasis (P=0.023). Additionally, high expression of NUSAP1 was closely associated with a shorter overall survival time of the ccRCC patients (P=0.006). Furthermore, we investigated the biological behaviors of RCC cells in vitro, and we identified that NUSAP1 depletion inhibited RCC cell migration, proliferation and invasion, and apoptosis was induced and the cell cycle was arrested. On the basis of our studies, NUSAP1 was identified as a potential prognostic indicator and a novel therapeutic target for RCC patients. PMID:27461786

  20. The nucleolar protein NIFK promotes cancer progression via CK1α/β-catenin in metastasis and Ki-67-dependent cell proliferation.

    PubMed

    Lin, Tsung-Chieh; Su, Chia-Yi; Wu, Pei-Yu; Lai, Tsung-Ching; Pan, Wen-An; Jan, Yi-Hua; Chang, Yu-Chang; Yeh, Chi-Tai; Chen, Chi-Long; Ger, Luo-Ping; Chang, Hong-Tai; Yang, Chih-Jen; Huang, Ming-Shyan; Liu, Yu-Peng; Lin, Yuan-Feng; Shyy, John Y-J; Tsai, Ming-Daw; Hsiao, Michael

    2016-01-01

    Nucleolar protein interacting with the FHA domain of pKi-67 (NIFK) is a Ki-67-interacting protein. However, its precise function in cancer remains largely uninvestigated. Here we show the clinical significance and metastatic mechanism of NIFK in lung cancer. NIFK expression is clinically associated with poor prognosis and metastasis. Furthermore, NIFK enhances Ki-67-dependent proliferation, and promotes migration, invasion in vitro and metastasis in vivo via downregulation of casein kinase 1α (CK1α), a suppressor of pro-metastatic TCF4/β-catenin signaling. Inversely, CK1α is upregulated upon NIFK knockdown. The silencing of CK1α expression in NIFK-silenced cells restores TCF4/β-catenin transcriptional activity, cell migration, and metastasis. Furthermore, RUNX1 is identified as a transcription factor of CSNK1A1 (CK1α) that is negatively regulated by NIFK. Our results demonstrate the prognostic value of NIFK, and suggest that NIFK is required for lung cancer progression via the RUNX1-dependent CK1α repression, which activates TCF4/β-catenin signaling in metastasis and the Ki-67-dependent regulation in cell proliferation. PMID:26984280

  1. The nucleolar protein NIFK promotes cancer progression via CK1α/β-catenin in metastasis and Ki-67-dependent cell proliferation

    PubMed Central

    Lin, Tsung-Chieh; Su, Chia-Yi; Wu, Pei-Yu; Lai, Tsung-Ching; Pan, Wen-An; Jan, Yi-Hua; Chang, Yu-Chang; Yeh, Chi-Tai; Chen, Chi-Long; Ger, Luo-Ping; Chang, Hong-Tai; Yang, Chih-Jen; Huang, Ming-Shyan; Liu, Yu-Peng; Lin, Yuan-Feng; Shyy, John Y-J; Tsai, Ming-Daw; Hsiao, Michael

    2016-01-01

    Nucleolar protein interacting with the FHA domain of pKi-67 (NIFK) is a Ki-67-interacting protein. However, its precise function in cancer remains largely uninvestigated. Here we show the clinical significance and metastatic mechanism of NIFK in lung cancer. NIFK expression is clinically associated with poor prognosis and metastasis. Furthermore, NIFK enhances Ki-67-dependent proliferation, and promotes migration, invasion in vitro and metastasis in vivo via downregulation of casein kinase 1α (CK1α), a suppressor of pro-metastatic TCF4/β-catenin signaling. Inversely, CK1α is upregulated upon NIFK knockdown. The silencing of CK1α expression in NIFK-silenced cells restores TCF4/β-catenin transcriptional activity, cell migration, and metastasis. Furthermore, RUNX1 is identified as a transcription factor of CSNK1A1 (CK1α) that is negatively regulated by NIFK. Our results demonstrate the prognostic value of NIFK, and suggest that NIFK is required for lung cancer progression via the RUNX1-dependent CK1α repression, which activates TCF4/β-catenin signaling in metastasis and the Ki-67-dependent regulation in cell proliferation. DOI: http://dx.doi.org/10.7554/eLife.11288.001 PMID:26984280

  2. [Prognistic value of a study of the expression of argyrophilic nucleolar organizer region associated proteins in case of papillary thyroid cancer].

    PubMed

    Raĭkhlin, N T; Bukaeva, I A; Smirnova, E A; Pavlovskaia, A I; Brzhezovskiĭ, V Zh; Bogatyrev, V N; Ponomareva, M V

    2010-01-01

    The prognosis in papillary thyroid cancer (PTC) is usually good. Ten-year survival can be seen in 90-98% of patients. Immunohistochemical study (antigen K-67) ascertained that a female patient with PTC had a low number of proliferating cells, which is usually seen in the favorable course of the disease. However, in the presented case, PTC was highly aggressive and showed a significant invasive growth, provided regional and distant metastases, rapidly progressed and, despite the performed surgical treatment, the patient died due to disease progression 3 months after surgery. This discrepancy between the number of proliferating cells and the aggressive course of PTC should be explained by the high expression of argyrophilic nucleolar organizer region associated proteins nucleofozmin and nucleolin, detected by immunohistochemical study, which is known to cause an increase in the rate of a mitotic cycle rate and to promote intercellular adhesion and enhancement of invasive growth and metastatic spread. Various factors involved in the regulation of proliferation of cells and their capacity for invasion and metastasis should be studied to make the most objective estimation of the degree of malignancy of a tumor and its prognosis. PMID:21086640

  3. Structure and Function of the N-terminal Nucleolin Binding Domain of Nuclear Valosin-containing Protein-like 2 (NVL2) Harboring a Nucleolar Localization Signal*

    PubMed Central

    Fujiwara, Yoshie; Fujiwara, Ken-ichiro; Goda, Natsuko; Iwaya, Naoko; Tenno, Takeshi; Shirakawa, Masahiro; Hiroaki, Hidekazu

    2011-01-01

    The N-terminal regions of AAA-ATPases (ATPase associated with various cellular activities) often contain a domain that defines the distinct functions of the enzymes, such as substrate specificity and subcellular localization. As described herein, we have determined the solution structure of an N-terminal unique domain isolated from nuclear valosin-containing protein (VCP)-like protein 2 (NVL2UD). NVL2UD contains three α helices with an organization resembling that of a winged helix motif, whereas a pair of β-strands is missing. The structure is unique and distinct from those of other known type II AAA-ATPases, such as VCP. Consequently, we identified nucleolin from a HeLa cell extract as a binding partner of this domain. Nucleolin contains a long (∼300 amino acids) intrinsically unstructured region, followed by the four tandem RNA recognition motifs and the C-terminal glycine/arginine-rich domain. Binding analyses revealed that NVL2UD potentially binds to any of the combinations of two successive RNA binding domains in the presence of RNA. Furthermore, NVL2UD has a characteristic loop, in which the key basic residues RRKR are exposed to the solvent at the edge of the molecule. The mutation study showed that these residues are necessary and sufficient for nucleolin-RNA complex binding as well as nucleolar localization. Based on the observations presented above, we propose that NVL2 serves as an unfoldase for the nucleolin-RNA complex. As inferred from its RNA dependence and its ATPase activity, NVL2 might facilitate the dissociation and recycling of nucleolin, thereby promoting efficient ribosome biogenesis. PMID:21474449

  4. Are there any effects of chronic carbon monoxide exposure on argyrophilic nucleolar-organizing region-associated protein synthesis in rat myocardium?

    PubMed

    Saritas, A; Gunes, H; Colakoglu, S; Eroz, R; Akoz, A; Oktay, M; Buyukkaya, A; Kandis, H; Ozkan, A

    2016-09-01

    The aims of the study are to detect whether there are any possible effects of chronic carbon monoxide (CO) exposure on the argyrophilic nucleolar-organizing region (AgNOR)-associated protein synthesis and evaluate any possible relationship between the amount of AgNOR protein and the level of myocardial injury also and between AgNOR and histopathological evaluation methods. Adult male albino Wistar rats (n = 18) were randomly divided into three groups (groups A, B, and C). Group A served as control, while groups B and C were rats exposed to CO gas chronically (1000 and 3000 ppm CO concentration with a flow rate of 4 L/min for 30 min/day for 7 days, respectively). Total AgNOR area/nuclear area (TAA/NA) and the mean AgNOR numbers for each myocyte nucleus were determined. There were significant differences among all groups for TAA/NA ratio. These differences were not significant for mean AgNOR numbers. According to the histopathological evaluation scores, there were significant differences between the groups. The differences were significant among the groups for loss of sarcomere pattern. A strong positive correlation between histopathological injury scores and TAA/NA ratio was found (Rsq = 0.48; p = 0.002), however, the correlation was not significant for mean AgNOR numbers (Rsq = 0.08; p = 0.25). In conclusion, TAA/NA ratio can be used as an indicator for obtaining information about the level of myocardial damage instead of histopathological evaluation scores. PMID:26462711

  5. The dynamics of the alternatively spliced NOL7 gene products and role in nucleolar architecture

    PubMed Central

    Kinor, Noa

    2011-01-01

    Three alternatively spliced forms of the human NOL7 gene coding for relatively small proteins were identified. The two shorter forms were generated by intron retention events, and each isoform was differently localized within the cell. The NOL7-SP1 long form (29 kD) localized to the nucleolus, SP2 was nucleoplasmic, while SP3 was distributed throughout the whole cell. NOL7-SP1 was confined to the nucleolar granular component, and during cell division disassociated from the nucleolus. Knockdown of NOL7-SP1 levels abrogated nucleolar architecture, in particular the internal regions, and reduced cell proliferation. Analysis of the nucleolar dynamics of the SP1 protein during interphase showed nucleolar high binding affinity. Dissection of protein domains showed that nucleolar targeting was mediated by a unique C-terminal nucleolar localization sequence (NoLS). However, this sequence was not sufficient for conferring high binding affinity, which required additional regions of the protein. Our analysis shows that NOL7 is important for maintaining internal nucleolar structure and cell growth rates, and that while specific protein localization can be obtained by specific short localization motifs, nucleolar residency through binding must be mediated by a synergistic combination of protein modules. PMID:21818416

  6. 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

  7. [Parkinson's disease and nucleolar stress].

    PubMed

    Zhou, Qingqing; Chen, Yongping; Wei, Qianqian; Shang, Huifang

    2016-06-01

    Parkinson's disease (PD) is a common age-related neurodegenerative disorder characterized mainly by motor dysfunction resulting in bradykinesia, rigidity, tremor, gait impairment, and postural instability. The classic pathogenic feature of PD is preferential loss of dopaminergic neurons in the substantia nigra. Downregulation of rRNA transcription is one of major mechanisms to maintain cellular homeostasis under stress conditions. Nucleolar stress has emerged as a component of the degenerative process caused by impaired rRNA transcription and altered nucleolar integrity. Recent study has indicated that the response to stress conditions and quality control mechanisms are impaired in PD, and that metabolic stress may be a trigger mechanism for PD. This review aims to present evidence for a role of nucleolar stress in PD and has summarized mechanisms by which nucleolar stress may play a role in the progression of PD. PMID:27264829

  8. The Fragile X Mental Retardation Protein, FMRP, Recognizes G-Quartets

    ERIC Educational Resources Information Center

    Darnell, Jennifer C.; Warren, Stephen T.; Darnell, Robert B.

    2004-01-01

    Fragile X mental retardation is a disease caused by the loss of function of a single RNA-binding protein, FMRP. Identifying the RNA targets recognized by FMRP is likely to reveal much about its functions in controlling some aspects of memory and behavior. Recent evidence suggests that one of the predominant RNA motifs recognized by the FMRP…

  9. The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae.

    PubMed

    Paci, Alexandr; Liu, Peter X H; Zhang, Lingjie; Zhao, Rongmin

    2016-05-27

    Pih1 is a scaffold protein of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) protein complex, which is conserved in fungi and animals. The chaperone-like activity of the R2TP complex has been implicated in the assembly of multiple protein complexes, such as the small nucleolar RNA protein complex. However, the mechanism of the R2TP complex activity in vivo and the assembly of the complex itself are still largely unknown. Pih1 is an unstable protein and tends to aggregate when expressed alone. The C-terminal fragment of Pih1 contains multiple destabilization factors and acts as a degron when fused to other proteins. In this study, we investigated Pih1 interactors and identified a specific interaction between Pih1 and the proteasome subunit Rpn8 in yeast Saccharomyces cerevisiae when HSP90 co-chaperone Tah1 is depleted. By analyzing truncation mutants, we identified that the C-terminal 30 amino acids of Rpn8 are sufficient for the binding to Pih1 C terminus. With in vitro and in vivo degradation assays, we showed that the Pih1 C-terminal fragment Pih1(282-344) is able to induce a ubiquitin-independent degradation of GFP. Additionally, we demonstrated that truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits the degradation of the GFP-Pih1(282-344) fusion protein in vivo and Pih1 in vitro We propose that Pih1 is a ubiquitin-independent proteasome substrate, and the direct interaction with Rpn8 C terminus mediates its proteasomal degradation. PMID:27053109

  10. Proteomic characterization of the nucleolar linker histone H1 interaction network

    PubMed Central

    Szerlong, Heather J.; Herman, Jacob A.; Krause, Christine M.; DeLuca, Jennifer G.; Skoultchi, Arthur; Winger, Quinton A.; Prenni, Jessica E.; Hansen, Jeffrey C.

    2015-01-01

    To investigate the relationship between linker histone H1 and protein-protein interactions in the nucleolus, biochemical and proteomics approaches were used to characterize nucleoli purified from cultured human and mouse cells. Mass spectrometry identified 175 proteins in human T-cell nucleolar extracts that bound to sepharose-immobilized H1 in vitro. Gene ontology analysis found significant enrichment for H1 binding proteins with functions related to nucleolar chromatin structure and RNA polymerase I transcription regulation, rRNA processing, and mRNA splicing. Consistent with the affinity binding results, H1 existed in large (400 to >650 kDa) macromolecular complexes in human T cell nucleolar extracts. To complement the biochemical experiments, the effects of in vivo H1 depletion on protein content and structural integrity of the nucleolus were investigated using the H1 triple isoform knock out (H1ΔTKO) mouse embryonic stem cell (mESC) model system. Proteomic profiling of purified wild type mESC nucleoli identified a total of 613 proteins, only ~60% of which were detected in the H1 mutant nucleoli. Within the affected group, spectral counting analysis quantitated 135 specific nucleolar proteins whose levels were significantly altered in H1ΔTKO mESC. Importantly, the functions of the affected proteins in mESC closely overlapped with those of the human T cell nucleolar H1 binding proteins. Immunofluorescence microscopy of intact H1ΔTKO mESC demonstrated both a loss of nucleolar RNA content and altered nucleolar morphology resulting from in vivo H1 depletion. We conclude that H1 organizes and maintains an extensive protein-protein interaction network in the nucleolus required for nucleolar structure and integrity. PMID:25584861

  11. Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement, Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana1[OPEN

    PubMed Central

    Lukhovitskaya, Nina I.; Cowan, Graham H.; Vetukuri, Ramesh R.; Tilsner, Jens; Torrance, Lesley

    2015-01-01

    Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement. PMID:25576325

  12. Primary Central Nervous System (CNS) Lymphoma B Cell Receptors Recognize CNS Proteins.

    PubMed

    Montesinos-Rongen, Manuel; Purschke, Frauke G; Brunn, Anna; May, Caroline; Nordhoff, Eckhard; Marcus, Katrin; Deckert, Martina

    2015-08-01

    Primary lymphoma of the CNS (PCNSL) is a diffuse large B cell lymphoma confined to the CNS. To elucidate its peculiar organ tropism, we generated recombinant Abs (recAbs) identical to the BCR of 23 PCNSLs from immunocompetent patients. Although none of the recAbs showed self-reactivity upon testing with common autoantigens, they recognized 1547 proteins present on a large-scale protein microarray, indicating polyreactivity. Interestingly, proteins (GRINL1A, centaurin-α, BAIAP2) recognized by the recAbs are physiologically expressed by CNS neurons. Furthermore, 87% (20/23) of the recAbs, including all Abs derived from IGHV4-34 using PCNSL, recognized galectin-3, which was upregulated on microglia/macrophages, astrocytes, and cerebral endothelial cells upon CNS invasion by PCNSL. Thus, PCNSL Ig may recognize CNS proteins as self-Ags. Their interaction may contribute to BCR signaling with sustained NF-κB activation and, ultimately, may foster tumor cell proliferation and survival. These data may also explain, at least in part, the affinity of PCNSL cells for the CNS. PMID:26116512

  13. Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

    PubMed

    Gonzalez-Ceron, L; Rodriguez, M H; Wirtz, R A; Sina, B J; Palomeque, O L; Nettel, J A; Tsutsumi, V

    1998-11-01

    The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur. PMID:9806864

  14. Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy.

    PubMed

    Park, Hweon; Han, Sung-Sik; Sako, Yasushi; Pack, Chan-Gi

    2015-03-01

    Organization and functions of the nucleolus is maintained by mobilities and interactions of nucleolar factors. Because the nucleolus is a densely packed structure, molecular crowding effects determined by the molecular concentrations and mobilities in the nucleolus should also be important for regulating nucleolar organization and functions. However, such molecular property of nucleolar organization is not fully understood. To understand the biophysical property of nucleolar organization, the diffusional behaviors of inert green fluorescent protein (GFP) oligomers with or without nuclear localization signals (NLSs) were analyzed under various conditions by fluorescence correlation spectroscopy. Our result demonstrates that the mobility of GFPs inside the nucleolus and the nucleoplasm can be represented by single free diffusion under normal conditions, even though the mobility in the nucleolus is considerably slower than that in the chromatin region. Moreover, the free diffusion of GFPs is found to be significantly size- and NLS-dependent only in the nucleolus. Interestingly, the mobility in the nucleolus is highly sensitive to ATP depletion, as well as actinomycin D (ActD) treatment. In contrast, the ultra-structure of the nucleolus was not significantly changed by ATP depletion but was changed by ActD treatment. These results suggest that the nucleolus behaves similarly to an open aqueous-phase medium with an increased molecular crowding effect that depends on both energy and transcription. PMID:25404711

  15. CAG expansion induces nucleolar stress in polyglutamine diseases.

    PubMed

    Tsoi, Ho; Lau, Terrence Chi-Kong; Tsang, Suk-Ying; Lau, Kwok-Fai; Chan, Ho Yin Edwin

    2012-08-14

    The cell nucleus is a major site for polyglutamine (polyQ) toxicity, but the underlying mechanisms involved have yet been fully elucidated. Here, we report that mutant RNAs that carry an expanded CAG repeat (expanded CAG RNAs) induce apoptosis by activating the nucleolar stress pathway in both polyQ patients and transgenic animal disease models. We showed that expanded CAG RNAs interacted directly with nucleolin (NCL), a protein that regulates rRNA transcription. Such RNA-protein interaction deprived NCL of binding to upstream control element (UCE) of the rRNA promoter, which resulted in UCE DNA hypermethylation and subsequently perturbation of rRNA transcription. The down-regulation of rRNA transcription induced nucleolar stress and provoked apoptosis by promoting physical interaction between ribosomal proteins and MDM2. Consequently, p53 protein was found to be stabilized in cells and became concentrated in the mitochondria. Finally, we showed that mitochondrial p53 disrupted the interaction between the antiapoptotic protein, Bcl-xL, and the proapoptotic protein, Bak, which then caused cytochrome c release and caspase activation. Our work provides in vivo evidence that expanded CAG RNAs trigger nucleolar stress and induce apoptosis via p53 and describes a polyQ pathogenic mechanism that involves the nucleolus. PMID:22847428

  16. Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies.

    PubMed

    Cheng, Hsien-Jen; Lin, Chiou-Feng; Lei, Huan-Yao; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Luo, Yueh-Hsia; Lin, Yee-Shin

    2009-01-01

    We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope. PMID:18997103

  17. Coexisting Liquid Phases Underlie Nucleolar Subcompartments.

    PubMed

    Feric, Marina; Vaidya, Nilesh; Harmon, Tyler S; Mitrea, Diana M; Zhu, Lian; Richardson, Tiffany M; Kriwacki, Richard W; Pappu, Rohit V; Brangwynne, Clifford P

    2016-06-16

    The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP. PMID:27212236

  18. Nucleolar organizer regions: genomic 'dark matter' requiring illumination.

    PubMed

    McStay, Brian

    2016-07-15

    Nucleoli form around tandem arrays of a ribosomal gene repeat, termed nucleolar organizer regions (NORs). During metaphase, active NORs adopt a characteristic undercondensed morphology. Recent evidence indicates that the HMG-box-containing DNA-binding protein UBF (upstream binding factor) is directly responsible for this morphology and provides a mitotic bookmark to ensure rapid nucleolar formation beginning in telophase in human cells. This is likely to be a widely employed strategy, as UBF is present throughout metazoans. In higher eukaryotes, NORs are typically located within regions of chromosomes that form perinucleolar heterochromatin during interphase. Typically, the genomic architecture of NORs and the chromosomal regions within which they lie is very poorly described, yet recent evidence points to a role for context in their function. In Arabidopsis, NOR silencing appears to be controlled by sequences outside the rDNA (ribosomal DNA) array. Translocations reveal a role for context in the expression of the NOR on the X chromosome in Drosophila Recent work has begun on characterizing the genomic architecture of human NORs. A role for distal sequences located in perinucleolar heterochromatin has been inferred, as they exhibit a complex transcriptionally active chromatin structure. Links between rDNA genomic stability and aging in Saccharomyces cerevisiae are now well established, and indications are emerging that this is important in aging and replicative senescence in higher eukaryotes. This, combined with the fact that rDNA arrays are recombinational hot spots in cancer cells, has focused attention on DNA damage responses in NORs. The introduction of DNA double-strand breaks into rDNA arrays leads to a dramatic reorganization of nucleolar structure. Damaged rDNA repeats move from the nucleolar interior to form caps at the nucleolar periphery, presumably to facilitate repair, suggesting that the chromosomal context of human NORs contributes to their genomic

  19. The epitope recognized by a monoclonal antibody in the myelin-associated protein CNP.

    PubMed

    Stricker, R; Kalbacher, H; Reiser, G

    1997-08-18

    The epitope recognized by a monoclonal antibody (MAb-46-1) directed against the myelin-associated protein CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase; EC 3.1.4.37) from several species was characterized. MAb-46-1 can be employed for immunoprecipitation, immunostaining in Western blots and in immunohistochemistry. Short peptides derived from the human CNP1 peptide sequence were synthesized and used in enzyme linked immunosorbent assays to test the reactivity of MAb-46-1. Coarse screening experiments enabled us to localize the epitope recognized by MAb-46-1 to the amino acid residues 9 to 19 close to the N-terminus. Further investigations using shorter peptides comprising this part of the protein allowed us to identify a 9 amino acid residue long peptide (amino acids 11 to 19: ELQFPFLQD) which represents the minimal epitope recognized by MAb-46-1, probably through a 3-dimensional structure and less likely a straight linear peptide. The epitope seems to be stabilized also by the attached amino acids 7 to 10 (KDKP). The peptide sequence 9-19 is conserved in all CNP sequences described so far. Thus, MAb-46-1 might be of general usefulness for further studies of the not yet identified function of the myelin-associated protein CNP. PMID:9268698

  20. A nucleolar localizing Rev binding element inhibits HIV replication

    PubMed Central

    Michienzi, Alessandro; De Angelis, Fernanda G; Bozzoni, Irene; Rossi, John J

    2006-01-01

    The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment. PMID:16712721

  1. PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin.

    PubMed

    Kozakai, Yuuki; Kamada, Rui; Furuta, Junya; Kiyota, Yuhei; Chuman, Yoshiro; Sakaguchi, Kazuyasu

    2016-01-01

    An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1. PMID:27619510

  2. Proteomic Analysis of Trichinella spiralis Muscle Larval Excretory-Secretory Proteins Recognized by Early Infection Sera

    PubMed Central

    Wang, Li; Wang, Zhong Quan; Hu, Dan Dan; Cui, Jing

    2013-01-01

    Although the excretory-secretory (ES) proteins of Trichinella spiralis muscle larvae are the most commonly used diagnostic antigens for trichinellosis, their main disadvantage is the false negative results during the early stage of infection and cross-reaction of their main components (43, 45, 49, and 53 kDa) with sera of patients with other helminthiasis. The aim of this study was to identify early specific diagnostic antigens in T. spiralis ES proteins with 30–40 kDa. The ES proteins were analyzed by two-dimensional electrophoresis (2-DE), and a total of approximately 150 proteins spots were detected with isoelectric point (pI) varying from 4 to 7 and molecular weight from 14 to 66 kDa. When probed with sera from infected mice at 18 days postinfection, ten protein spots with molecular weight of 30–40 kDa were recognized and identified by MALDI-TOF/TOF-MS. All of ten spots were successfully identified and characterized to correlate with five different proteins, including two potential serine proteases, one antigen targeted by protective antibodies, one deoxyribonuclease (DNase) II, and one conserved hypothetical protein. These proteins might be the early specific diagnostic antigens for trichinellosis. PMID:23844355

  3. Nucleolin: a multifunctional major nucleolar phosphoprotein.

    PubMed

    Tuteja, R; Tuteja, N

    1998-01-01

    Nucleolin is a major protein of exponentially growing eukaryotic cells where it is present in abundance at the heart of the nucleolus. It is highly conserved during evolution. Nucleolin contains a specific bipartite nuclear localization signal sequence and possesses a number of unusual structural features. It has unique tripartite structure and each domain performs a specific function by interacting with DNA or RNA or proteins. Nucleolin exhibits intrinsic self-cleaving, DNA helicase, RNA helicase and DNA-dependent ATPase activities. Nucleolin also acts as a sequence-specific RNA binding protein, an autoantigen, and as the component of a B cell specific transcription factor. Its phosphorylation by cdc2, CK2, and PKC-zeta modulate some of its activities. This multifunctional protein has been implicated to be involved directly or indirectly in many metabolic processes such as ribosome biogenesis (which includes rDNA transcription, pre-rRNA synthesis, rRNA processing, ribosomal assembly and maturation), cytokinesis, nucleogenesis, cell proliferation and growth, cytoplasmic-nucleolar transport of ribosomal components, transcriptional repression, replication, signal transduction, inducing chromatin decondensation and many more (see text). In plants it is developmentally, cell-cycle, and light regulated. The regulation of all these functions of a single protein seems to be a challenging puzzle. PMID:9918513

  4. Staphylococcus aureus proteins differentially recognized by the ovine immune response in mastitis or nasal carriage.

    PubMed

    Seyffert, Nubia; Le Maréchal, Caroline; Jardin, Julien; McCulloch, John A; Rosado, Fabio R; Miyoshi, Anderson; Even, Sergine; Jan, Gwenaël; Berkova, Nadia; Vautor, Eric; Thiéry, Richard; Azevedo, Vasco; Le Loir, Yves

    2012-06-15

    Staphylococcus aureus is an opportunistic pathogen in dairy ruminants where it is found in healthy carriage and can be a major cause of mastitis. A better knowledge of the host-pathogen interactions is needed to tackle this serious animal health problem. This study aimed at identifying S. aureus proteins differentially expressed by S. aureus in nasal colonization versus mastitis. Serological proteome analysis (SERPA) was used to examine protein samples prepared from culture supernatants of S. aureus strains originally isolated from gangrenous mastitis and nasal carriage (O11) or subclinical mastitis (O46) and to compare patterns of immune-reactive proteins. These staphylococcal proteins were revealed by sera obtained from ewes suffering from S. aureus mastitis and by sera obtained from healthy nulliparous ewes (i.e. no lactation and no mastitis or other symptoms) that were nasally colonized by S. aureus. Altogether 49 staphylococcal immune-reactive proteins were identified in this study. Patterns of proteins revealed by sera from infected- or healthy carrier- animals were comparable and analysis singled out one immune-reactive protein, N-acetylmuramyl-L-alanine amidase, which was recognized by each of the 6 sera from infected animals, when tested individually, and not by the sera of healthy carriers. This is the first study that compares the S. aureus seroproteome in colonization versus mastitis context in ruminants. These results open avenues for studies aiming at a better understanding of the balance between infection and commensal lifestyle in this opportunistic pathogen and at new prevention strategies. PMID:22342493

  5. Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.

    PubMed

    Kirigiti, P; Yang, Y F; Li, X; Li, B; Midson, C N; Machida, C A

    2000-03-01

    will bind to both the beta 1-AR GS-1 promoter fragment and commercially available AP-2 consensus element control probes. Interestingly, using antibody supershift and immunoblotting experiments, no supershifts were observed and the major 117-kDa protein was not immunoreactive to antibodies recognizing either AP-2 alpha or AP-2 beta. These results support our contention that this beta 1-AR regulatory region contains AP-2 consensus elements that recognize novel transactivator proteins. PMID:10860867

  6. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

    PubMed

    Buffalo, Cosmo Z; Bahn-Suh, Adrian J; Hirakis, Sophia P; Biswas, Tapan; Amaro, Rommie E; Nizet, Victor; Ghosh, Partho

    2016-01-01

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ∼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design. PMID:27595425

  7. Protein folds recognized by an intelligent predictor based-on evolutionary and structural information.

    PubMed

    Cheung, Ngaam J; Ding, Xue-Ming; Shen, Hong-Bin

    2016-02-01

    Protein fold recognition is an important and essential step in determining tertiary structure of a protein in biological science. In this study, a model termed NiRecor is developed for recognizing protein folds based on artificial neural networks incorporated in an adaptive heterogeneous particle swarm optimizer. The main contribution of NiRecor is that it is a data-driven and highly-performing predictor without manually tuning control parameters for different data sets. In biological science, since evolutionary- and structure-based information of amino acid sequences is greatly important in determination of tertiary structure of a protein, accordingly, in NiRecor we employ two different feature sets, which involve position specific scoring matrix and secondary structure prediction matrix, to predict the structural classes of protein folds. The experimental results demonstrate the proposed method is powerful in predicting protein folds with higher precisions by improvements of 1.1 ∼7.8 percentages on three benchmark datasets by comparing with several existing predictors. PMID:26502837

  8. FRAN and RBF-PSO as two components of a hyper framework to recognize protein folds.

    PubMed

    Abbasi, Elham; Ghatee, Mehdi; Shiri, M E

    2013-09-01

    In this paper, an intelligent hyper framework is proposed to recognize protein folds from its amino acid sequence which is a fundamental problem in bioinformatics. This framework includes some statistical and intelligent algorithms for proteins classification. The main components of the proposed framework are the Fuzzy Resource-Allocating Network (FRAN) and the Radial Bases Function based on Particle Swarm Optimization (RBF-PSO). FRAN applies a dynamic method to tune up the RBF network parameters. Due to the patterns complexity captured in protein dataset, FRAN classifies the proteins under fuzzy conditions. Also, RBF-PSO applies PSO to tune up the RBF classifier. Experimental results demonstrate that FRAN improves prediction accuracy up to 51% and achieves acceptable multi-class results for protein fold prediction. Although RBF-PSO provides reasonable results for protein fold recognition up to 48%, it is weaker than FRAN in some cases. However the proposed hyper framework provides an opportunity to use a great range of intelligent methods and can learn from previous experiences. Thus it can avoid the weakness of some intelligent methods in terms of memory, computational time and static structure. Furthermore, the performance of this system can be enhanced throughout the system life-cycle. PMID:23930812

  9. Amino Acid Signature Enables Proteins to Recognize Modified tRNA

    PubMed Central

    2015-01-01

    Human tRNALys3UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNALys3UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNALys3UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N6-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNALys3UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA’s fully modified anticodon stem and loop domain, hASLLys3UUU. Peptides of this sequence specifically recognized and bound modified htRNALys3UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions. PMID:24483944

  10. Nucleolar Separation from Chromosomes during Aspergillus nidulans Mitosis Can Occur Without Spindle Forces

    PubMed Central

    Ukil, Leena; De Souza, Colin P.; Liu, Hui-Lin

    2009-01-01

    How the nucleolus is segregated during mitosis is poorly understood and occurs by very different mechanisms during closed and open mitosis. Here we report a new mechanism of nucleolar segregation involving removal of the nucleolar-organizing regions (NORs) from nucleoli during Aspergillus nidulans mitosis. This involves a double nuclear envelope (NE) restriction which generates three NE-associated structures, two daughter nuclei (containing the NORs), and the nucleolus. Therefore, a remnant nucleolar structure can exist in the cytoplasm without NORs. In G1, this parental cytoplasmic nucleolus undergoes sequential disassembly releasing nucleolar proteins to the cytoplasm as nucleoli concomitantly reform in daughter nuclei. By depolymerizing microtubules and mutating spindle assembly checkpoint function, we demonstrate that a cycle of nucleolar “segregation” can occur without a spindle in a process termed spindle-independent mitosis (SIM). During SIM physical separation of the NOR from the nucleolus occurs, and NE modifications promote expulsion of the nucleolus to the cytoplasm. Subsequently, the cytoplasmic nucleolus is disassembled and rebuilt at a new site around the nuclear NOR. The data demonstrate the existence of a mitotic machinery for nucleolar segregation that is normally integrated with mitotic spindle formation but that can function without it. PMID:19211837

  11. Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans.

    PubMed

    Guerreiro, H; Croda, J; Flannery, B; Mazel, M; Matsunaga, J; Galvão Reis, M; Levett, P N; Ko, A I; Haake, D A

    2001-08-01

    Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine

  12. Leptospiral Proteins Recognized during the Humoral Immune Response to Leptospirosis in Humans

    PubMed Central

    Guerreiro, Hygia; Croda, Júlio; Flannery, Brendan; Mazel, Mary; Matsunaga, James; Reis, Mitermayer Galvão; Levett, Paul N.; Ko, Albert I.; Haake, David A.

    2001-01-01

    Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine

  13. Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants.

    PubMed

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-03-11

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  14. Nuclear/Nucleolar GTPase 2 Proteins as a Subfamily of YlqF/YawG GTPases Function in Pre-60S Ribosomal Subunit Maturation of Mono- and Dicotyledonous Plants*

    PubMed Central

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I. Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-01-01

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with kcat values 0.12 ± 0.007 min−1 (n = 6) and 0.087 ± 0.002 min−1 (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  15. Mechanism by which a LINE protein recognizes its 3′ tail RNA

    PubMed Central

    Hayashi, Yoshinori; Kajikawa, Masaki; Matsumoto, Takuma; Okada, Norihiro

    2014-01-01

    LINEs mobilize their own copies via retrotransposition. LINEs can be divided into two types. One is a stringent type, which constitutes a majority of LINEs. The other is a relaxed type. To elucidate the molecular mechanism of retrotransposition, we used here two different zebrafish LINEs belonging to the stringent type. By using retrotransposition assays, we demonstrated that proteins (ORF2) encoded by an individual LINE recognize the cognate 3′ tail sequence of the LINE RNA strictly. By conducting in vitro binding assays with a variety of ORF2 proteins, we demonstrated that the region between the endonuclease and reverse transcriptase domains in ORF2 is the site at which the proteins bind the stem-loop structure of the 3′ tail RNA, showing that the strict recognition of the stem-loop structure by the cognate ORF2 protein is an important step in retrotransposition. This recognition can be bipartite, involving the general recognition of the stem by cTBR (conserved tail-binding region) of ORF2 and the specific recognition of the loop by vTBR (variable tail-binding region). This is the first report that clearly characterized the RNA-binding region in ORF2, providing the generality for the recognition mechanism of the RNA tail by the ORF2 protein encoded by LINEs. PMID:25143533

  16. Exploration of Gated Ligand Binding Recognizes an Allosteric Site for Blocking FABP4-Protein Interaction

    PubMed Central

    Li, Yan; Li, Xiang; Dong, Zigang

    2015-01-01

    Fatty acid binding protein 4 (FABP4), reversibly binding to fatty acids and other lipids with high affinities, is a potential target for treatment of cancers. The binding site of FABP4 is buried in an interior cavity and thereby ligand binding/unbinding is coupled with opening/closing of FABP4. It is a difficult task both experimentally and computationally to illuminate the entry or exit pathway, especially with the conformational gating. In this report we combine extensive computer simulations, clustering analysis, and Markov state model to investigate the binding mechanism of FABP4 and troglitazone. Our simulations capture spontaneous binding and unbinding events as well as the conformational transition of FABP4 between the open and closed states. An allosteric binding site on the protein surface is recognized for development of novel FABP4 inhibitors. The binding affinity is calculated and compared with the experimental value. The kinetic analysis suggests that ligand residence on the protein surface may delay the binding process. Overall, our results provide a comprehensive picture of ligand diffusion on the protein surface, ligand migration into the buried cavity, and the conformational change of FABP4 at an atomic level. PMID:26580122

  17. Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV-1

    PubMed Central

    Borthwick, Nicola; Ahmed, Tina; Ondondo, Beatrice; Hayes, Peter; Rose, Annie; Ebrahimsa, Umar; Hayton, Emma-Jo; Black, Antony; Bridgeman, Anne; Rosario, Maximillian; Hill, Adrian VS; Berrie, Eleanor; Moyle, Sarah; Frahm, Nicole; Cox, Josephine; Colloca, Stefano; Nicosia, Alfredo; Gilmour, Jill; McMichael, Andrew J; Dorrell, Lucy; Hanke, Tomáš

    2014-01-01

    Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4+ cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8+ T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro. PMID:24166483

  18. Phosphorylation of DNA damage-recognizing proteins at heavy-ion track

    NASA Astrophysics Data System (ADS)

    Ohnishi, T.; Takahashi, A.; Nojima, K.; Furusawa, Y.; Ohnishi, K.

    To identify the repair dynamics for high LET-radiation-induced DNA damage we analyzed the focus formation after exposure to iron-ion beams 500 MeV u 200 KeV um using immunocytochemical methods Since the focus formation of phospho-H2AX gamma-H2AX which is well understood to be activated at radiation-induced double strand breaks DSBs we performed the visualization of the tracks spatial distribution of lesions from an aspect of dose dependency The number of this track induced by iron-ion beams was well corresponded with the value of a calculation well In addition we demonstrate that DNA damage-recognizing proteins such as phospho-serine 1981 of ATM phospho-threonine 2609 of DNA-PKcs phospho-serine 343 of NBS1 and phospho-threonine 68 of Chk2 co-localized with gamma-H2AX at high LET-radiation-induced portion These findings suggest that iron-ion beams were quite effective for detection of DNA damages of DSBs recognized with DNA repair enzymes used here after phosphorylation of them because iron-ion beams can be used to generate extremely localized at DNA damages within restricted regions of the nuclei

  19. NTTMUNSW BioC modules for recognizing and normalizing species and gene/protein mentions

    PubMed Central

    Dai, Hong-Jie; Singh, Onkar; Jonnagaddala, Jitendra; Su, Emily Chia-Yu

    2016-01-01

    In recent years, the number of published biomedical articles has increased as researchers have focused on biological domains to investigate the functions of biological objects, such as genes and proteins. However, the ambiguous nature of genes and their products have rendered the literature more complex for readers and curators of molecular interaction databases. To address this challenge, a normalization technique that can link variants of biological objects to a single, standardized form was applied. In this work, we developed a species normalization module, which recognizes species names and normalizes them to NCBI Taxonomy IDs. Unlike most previous work, which ignored the prefix of a gene name that represents an abbreviation of the species name to which the gene belongs, the recognition results of our module include the prefixed species. The developed species normalization module achieved an overall F-score of 0.954 on an instance-level species normalization corpus. For gene normalization, two separate modules were respectively employed to recognize gene mentions and normalize those mentions to their Entrez Gene IDs by utilizing a multistage normalization algorithm developed for processing full-text articles. All of the developed modules are BioC-compatible .NET framework libraries and are publicly available from the NuGet gallery. Database URL: https://sites.google.com/site/hjdairesearch/Projects/isn-corpus PMID:27465130

  20. NTTMUNSW BioC modules for recognizing and normalizing species and gene/protein mentions.

    PubMed

    Dai, Hong-Jie; Singh, Onkar; Jonnagaddala, Jitendra; Su, Emily Chia-Yu

    2016-01-01

    In recent years, the number of published biomedical articles has increased as researchers have focused on biological domains to investigate the functions of biological objects, such as genes and proteins. However, the ambiguous nature of genes and their products have rendered the literature more complex for readers and curators of molecular interaction databases. To address this challenge, a normalization technique that can link variants of biological objects to a single, standardized form was applied. In this work, we developed a species normalization module, which recognizes species names and normalizes them to NCBI Taxonomy IDs. Unlike most previous work, which ignored the prefix of a gene name that represents an abbreviation of the species name to which the gene belongs, the recognition results of our module include the prefixed species. The developed species normalization module achieved an overall F-score of 0.954 on an instance-level species normalization corpus. For gene normalization, two separate modules were respectively employed to recognize gene mentions and normalize those mentions to their Entrez Gene IDs by utilizing a multistage normalization algorithm developed for processing full-text articles. All of the developed modules are BioC-compatible .NET framework libraries and are publicly available from the NuGet gallery.Database URL: https://sites.google.com/site/hjdairesearch/Projects/isn-corpus. PMID:27465130

  1. fastSCOP: a fast web server for recognizing protein structural domains and SCOP superfamilies.

    PubMed

    Tung, Chi-Hua; Yang, Jinn-Moon

    2007-07-01

    The fastSCOP is a web server that rapidly identifies the structural domains and determines the evolutionary superfamilies of a query protein structure. This server uses 3D-BLAST to scan quickly a large structural classification database (SCOP1.71 with <95% identity with each other) and the top 10 hit domains, which have different superfamily classifications, are obtained from the hit lists. MAMMOTH, a detailed structural alignment tool, is adopted to align these top 10 structures to refine domain boundaries and to identify evolutionary superfamilies. Our previous works demonstrated that 3D-BLAST is as fast as BLAST, and has the characteristics of BLAST (e.g. a robust statistical basis, effective search and reliable database search capabilities) in large structural database searches based on a structural alphabet database and a structural alphabet substitution matrix. The classification accuracy of this server is approximately 98% for 586 query structures and the average execution time is approximately 5. This server was also evaluated on 8700 structures, which have no annotations in the SCOP; the server can automatically assign 7311 (84%) proteins (9420 domains) to the SCOP superfamilies in 9.6 h. These results suggest that the fastSCOP is robust and can be a useful server for recognizing the evolutionary classifications and the protein functions of novel structures. The server is accessible at http://fastSCOP.life.nctu.edu.tw. PMID:17485476

  2. Cohesion promotes nucleolar structure and function.

    PubMed

    Harris, Bethany; Bose, Tania; Lee, Kenneth K; Wang, Fei; Lu, Shuai; Ross, Rhonda Trimble; Zhang, Ying; French, Sarah L; Beyer, Ann L; Slaughter, Brian D; Unruh, Jay R; Gerton, Jennifer L

    2014-02-01

    The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus. PMID:24307683

  3. Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

    PubMed Central

    Wang, Chen; Meier, U Thomas

    2004-01-01

    Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57–NOP10–NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein–RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis. PMID:15044956

  4. Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins.

    PubMed

    Brändle, Simone M; Obermeier, Birgit; Senel, Makbule; Bruder, Jessica; Mentele, Reinhard; Khademi, Mohsen; Olsson, Tomas; Tumani, Hayrettin; Kristoferitsch, Wolfgang; Lottspeich, Friedrich; Wekerle, Hartmut; Hohlfeld, Reinhard; Dornmair, Klaus

    2016-07-12

    Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases. PMID:27325759

  5. Nucleolar stress with and without p53.

    PubMed

    James, Allison; Wang, Yubo; Raje, Himanshu; Rosby, Raphyel; DiMario, Patrick

    2014-01-01

    A veritable explosion of primary research papers within the past 10 years focuses on nucleolar and ribosomal stress, and for good reason: with ribosome biosynthesis consuming ~80% of a cell's energy, nearly all metabolic and signaling pathways lead ultimately to or from the nucleolus. We begin by describing p53 activation upon nucleolar stress resulting in cell cycle arrest or apoptosis. The significance of this mechanism cannot be understated, as oncologists are now inducing nucleolar stress strategically in cancer cells as a potential anti-cancer therapy. We also summarize the human ribosomopathies, syndromes in which ribosome biogenesis or function are impaired leading to birth defects or bone narrow failures; the perplexing problem in the ribosomopathies is why only certain cells are affected despite the fact that the causative mutation is systemic. We then describe p53-independent nucleolar stress, first in yeast which lacks p53, and then in other model metazoans that lack MDM2, the critical E3 ubiquitin ligase that normally inactivates p53. Do these presumably ancient p53-independent nucleolar stress pathways remain latent in human cells? If they still exist, can we use them to target >50% of known human cancers that lack functional p53? PMID:25482194

  6. Nucleolar stress with and without p53

    PubMed Central

    James, Allison; Wang, Yubo; Raje, Himanshu; Rosby, Raphyel; DiMario, Patrick

    2014-01-01

    A veritable explosion of primary research papers within the past 10 years focuses on nucleolar and ribosomal stress, and for good reason: with ribosome biosynthesis consuming ~80% of a cell’s energy, nearly all metabolic and signaling pathways lead ultimately to or from the nucleolus. We begin by describing p53 activation upon nucleolar stress resulting in cell cycle arrest or apoptosis. The significance of this mechanism cannot be understated, as oncologists are now inducing nucleolar stress strategically in cancer cells as a potential anti-cancer therapy. We also summarize the human ribosomopathies, syndromes in which ribosome biogenesis or function are impaired leading to birth defects or bone narrow failures; the perplexing problem in the ribosomopathies is why only certain cells are affected despite the fact that the causative mutation is systemic. We then describe p53-independent nucleolar stress, first in yeast which lacks p53, and then in other model metazoans that lack MDM2, the critical E3 ubiquitin ligase that normally inactivates p53. Do these presumably ancient p53-independent nucleolar stress pathways remain latent in human cells? If they still exist, can we use them to target >50% of known human cancers that lack functional p53? PMID:25482194

  7. Comprehensive Mapping of Common Immunodominant Epitopes in the Eastern Equine Encephalitis Virus E2 Protein Recognized by Avian Antibody Responses

    PubMed Central

    Sun, EnCheng; Zhao, Jing; Sun, Liang; Xu, QingYuan; Yang, Tao; Qin, YongLi; Wang, WenShi; Wei, Peng; Sun, Jing; Wu, DongLai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211–226 and 331–352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11–26, 30–45 and 151–166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein. PMID:23922704

  8. Comprehensive mapping of common immunodominant epitopes in the eastern equine encephalitis virus E2 protein recognized by avian antibody responses.

    PubMed

    Sun, Encheng; Zhao, Jing; Sun, Liang; Xu, Qingyuan; Yang, Tao; Qin, Yongli; Wang, Wenshi; Wei, Peng; Sun, Jing; Wu, Donglai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211-226 and 331-352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11-26, 30-45 and 151-166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein. PMID:23922704

  9. Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication

    PubMed Central

    Ugrinova, Iva; Monier, Karine; Ivaldi, Corinne; Thiry, Marc; Storck, Sébastien; Mongelard, Fabien; Bouvet, Philippe

    2007-01-01

    Background Nucleolin is a major component of the nucleolus, but is also found in other cell compartments. This protein is involved in various aspects of ribosome biogenesis from transcription regulation to the assembly of pre-ribosomal particles; however, many reports suggest that it could also play an important role in non nucleolar functions. To explore nucleolin function in cell proliferation and cell cycle regulation we used siRNA to down regulate the expression of nucleolin. Results We found that, in addition to the expected effects on pre-ribosomal RNA accumulation and nucleolar structure, the absence of nucleolin results in a cell growth arrest, accumulation in G2, and an increase of apoptosis. Numerous nuclear alterations, including the presence of micronuclei, multiple nuclei or large nuclei are also observed. In addition, a large number of mitotic cells showed a defect in the control of centrosome duplication, as indicated by the presence of more than 2 centrosomes per cell associated with a multipolar spindle structure in the absence of nucleolin. This phenotype is very similar to that obtained with the inactivation of another nucleolar protein, B23. Conclusion Our findings uncovered a new role for nucleolin in cell division, and highlight the importance of nucleolar proteins for centrosome duplication. PMID:17692122

  10. Critical Epitopes in the Nucleocapsid Protein of SFTS Virus Recognized by a Panel of SFTS Patients Derived Human Monoclonal Antibodies

    PubMed Central

    Yu, Li; Zhang, Li; Sun, Lina; Lu, Jing; Wu, Wei; Li, Chuan; Zhang, Quanfu; Zhang, Fushun; Jin, Cong; Wang, Xianjun; Bi, Zhenqiang; Li, Dexin; Liang, Mifang

    2012-01-01

    Background SFTS virus (SFTSV) is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS) in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. Methodology/Principal Findings We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs) recognized the nucleocapsid (N) protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. Conclusions/Significance The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection. PMID:22719874

  11. Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation.

    PubMed

    Cheng, Hsien-Jen; Lei, Huan-Yao; Lin, Chiou-Feng; Luo, Yueh-Hsia; Wan, Shu-Wen; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Lin, Yee-Shin

    2009-12-01

    Hemorrhagic syndrome is a hallmark of severe dengue diseases. We previously suggested a mechanism of molecular mimicry in which antibodies against dengue virus (DV) nonstructural protein 1 (NS1) cross-react with platelets. In the present study, we demonstrate that protein disulfide isomerase (PDI) on the platelet surface is recognized by anti-DV NS1 antibodies. Anti-DV NS1 obtained from hyperimmunized mouse sera inhibited PDI activity and platelet aggregation, and both inhibitory effects were prevented when anti-DV NS1 antibodies were preabsorbed with PDI. Anti-PDI antibodies bound to a peptide consisting of amino acid residues 311-330 (P311-330) of NS1. This peptide was a predicted epitope analyzed by homologous sequence alignments between DV NS1 and PDI. The platelet binding activities of anti-PDI and anti-DV NS1 antibodies were both reduced by P311-330 preabsorption. Similar to the findings using anti-DV NS1, antibodies against P311-330 bound to PDI and platelets, followed by inhibition of PDI activity and platelet aggregation. Furthermore, the cross-reactivity of dengue hemorrhagic fever patient sera with platelets was reduced when patient sera were preabsorbed with PDI or P311-330. Dengue hemorrhagic fever patient sera also inhibited platelet aggregation, while PDI or P311-330 reduced this inhibitory effect. In conclusion, anti-DV NS1 antibodies cross-react with PDI on platelet surface causing inhibition of platelet aggregation, which may provide implications in dengue disease pathogenesis. PMID:19822367

  12. Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA

    PubMed Central

    Michienzi, Alessandro; Cagnon, Laurence; Bahner, Ingrid; Rossi, John J.

    2000-01-01

    The HIV regulatory proteins Tat and Rev have a nucleolar localization property in human cells. However, no functional role has been attributed to this localization. Recently it has been demonstrated that expression of Rev induces nucleolar relocalization of some protein factors involved in Rev export. Because the function of Rev is to bind HIV RNA and facilitate transport of singly spliced and unspliced RNA to the cytoplasm, it is likely that the nucleolus plays a critical role in HIV-1 RNA export. As a test for trafficking of HIV-1 RNAs into the nucleolus, a hammerhead ribozyme that specifically cleaves HIV-1 RNA was inserted into the body of the U16 small nucleolar RNA, resulting in accumulation of the ribozyme within the nucleoli of human cells. HeLa CD4+ and T cells expressing this nucleolar localized ribozyme exhibit dramatically suppressed HIV-1 replication. The results presented here suggest a trafficking of HIV-1 RNA through the nucleoli of human cells, thus posing a different paradigm for lentiviral RNA processing. PMID:10922055

  13. Structure and epigenetics of nucleoli in comparison with non-nucleolar compartments.

    PubMed

    Bártová, Eva; Horáková, Andrea Harnicarová; Uhlírová, Radka; Raska, Ivan; Galiová, Gabriela; Orlova, Darya; Kozubek, Stanislav

    2010-05-01

    The nucleolus is a nuclear compartment that plays an important role in ribosome biogenesis. Some structural features and epigenetic patterns are shared between nucleolar and non-nucleolar compartments. For example, the location of transcriptionally active mRNA on extended chromatin loop species is similar to that observed for transcriptionally active ribosomal DNA (rDNA) genes on so-called Christmas tree branches. Similarly, nucleolus organizer region-bearing chromosomes located a distance from the nucleolus extend chromatin fibers into the nucleolar compartment. Specific epigenetic events, such as histone acetylation and methylation and DNA methylation, also regulate transcription of both rRNA- and mRNA-encoding loci. Here, we review the epigenetic mechanisms and structural features that regulate transcription of ribosomal and mRNA genes. We focus on similarities in epigenetic and structural regulation of chromatin in nucleoli and the surrounding non-nucleolar region and discuss the role of proteins, such as heterochromatin protein 1, fibrillarin, nucleolin, and upstream binding factor, in rRNA synthesis and processing. PMID:20026667

  14. Recognizing uncertainty increases robustness and reproducibility of mass spectrometry-based protein inferences

    PubMed Central

    Serang, Oliver; Moruz, Luminita; Hoopmann, Michael R.; Käll, Lukas

    2012-01-01

    Parsimony and protein grouping are widely employed to enforce economy in the number of identified proteins, with the goal of increasing the quality and reliability of protein identifications; however, in a counterintuitive manner, parsimony and protein grouping may actually decrease the reproducibility and interpretability of protein identifications. We present a simple illustration demonstrating ways in which parsimony and protein grouping may lower the reproducibility or interpretability of results. We then provide an example of a data set where a probabilistic method increases the reproducibility and interpretability of identifications made on replicate analyses of Human Du145 prostate cancer cell lines. PMID:23148905

  15. Proteomic Analysis of the Arabidopsis Nucleolus Suggests Novel Nucleolar FunctionsD⃞

    PubMed Central

    Pendle, Alison F.; Clark, Gillian P.; Boon, Reinier; Lewandowska, Dominika; Lam, Yun Wah; Andersen, Jens; Mann, Matthias; Lamond, Angus I.; Brown, John W. S.; Shaw, Peter J.

    2005-01-01

    The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance. PMID:15496452

  16. A receptor-binding protein of Campylobacter jejuni bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino-modified pseudaminic acid.

    PubMed

    Javed, Muhammad Afzal; van Alphen, Lieke B; Sacher, Jessica; Ding, Wen; Kelly, John; Nargang, Cheryl; Smith, David F; Cummings, Richard D; Szymanski, Christine M

    2015-01-01

    Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle. PMID:25354466

  17. mTOR Signaling Regulates Nucleolar Targeting of the SUMO-Specific Isopeptidase SENP3

    PubMed Central

    Raman, Nithya; Nayak, Arnab

    2014-01-01

    Ribosome biogenesis is a multistep cellular pathway that involves more than 200 regulatory components to ultimately generate translation-competent 80S ribosomes. The initial steps of this process, particularly rRNA processing, take place in the nucleolus, while later stages occur in the nucleoplasm and cytoplasm. One critical factor of 28S rRNA maturation is the SUMO-isopeptidase SENP3. SENP3 tightly interacts with the nucleolar scaffold protein NPM1 and is associated with nucleolar 60S preribosomes. A central question is how changes in energy supply feed into the regulation of ribosome maturation. Here, we show that the nutrient-sensing mTOR kinase pathway controls the nucleolar targeting of SENP3 by regulating its interaction with NPM1. We define an N-terminal domain in SENP3 as the critical NPM1 binding region and provide evidence that mTOR-mediated phosphorylation of serine/threonine residues within this region fosters the interaction of SENP3 with NPM1. The inhibition of mTOR triggers the nucleolar release of SENP3, thereby likely compromising its activity in rRNA processing. Since mTOR activity is tightly coupled to nutrient availability, we propose that this pathway contributes to the adaptation of ribosome maturation in response to the cellular energy status. PMID:25288641

  18. Properties of a purified nucleolar ribonuclease from Ehrlich ascites carcinoma cells.

    PubMed

    Eichler, D C; Tatar, T F

    1980-06-24

    A nucleolar ribonuclease specific for single-stranded ribonucleic acid (RNA) has been isolated and extensively purified from Ehrlich ascites carcinoma cells. The enzyme is optimally active at neutral pH and degrades RNA via a 2',3'-cyclic intermediate leaving 3'- or 2',3'-cyclic terminated oligonucleotides. The ribonuclease has an apparent molecular weight of 38 500 as judged by sedimentation equilibrium and is a basic protein having an isoelectric point greater than 9.0. The enzyme preferentially cleaves poly(C) over poly (U), poly(A), or poly(C).poly(I). Limit digestion products of poly(C) degratation are on the average tri-, tetra-, and pentanucleotides. In the partial digestion of yeast 5.8S rRNA, the nucleolar ribonuclease cleaves only CpA phosphodiester bonds. Spermidine, spermine, and histone I inhibit the activity of nucleolar ribonuclease. Antibodies directed toward pancreatic RNase do not cross-react with the Ehrlich nucleolar ribonuclease. PMID:7397114

  19. A Novel Strategy to Screen Bacillus Calmette-Guérin Protein Antigen Recognized by γδ TCR

    PubMed Central

    Xi, XueYan; Zhang, XiaoYan; Wang, Bei; Wang, Ji; Huang, He; Cui, LianXian; Han, XiQin; Li, Liang; He, Wei; Zhao, ZhenDong

    2011-01-01

    Background Phosphoantigen was originally identified as the main γδ TCR-recognized antigen that could activate γδ T cells to promote immune protection against mycobacterial infection. However, new evidence shows that the γδ T cells activated by phosphoantigen can only provide partial immune protection against mycobacterial infection. In contrast, whole lysates of Mycobacterium could activate immune protection more potently, implying that other γδ TCR-recognized antigens that elicit protective immune responses. To date, only a few distinct mycobacterial antigens recognized by the γδ TCR have been characterized. Methodology/Principal Findings In the present study, we established a new approach to screen epitopes or protein antigens recognized by the γδ TCR using Bacillus Calmette-Guérin- (BCG-) specific γ TCR transfected cells as probes to pan a 12-mer random-peptide phage-displayed library. Through binding assays and functional analysis, we identified a peptide (BP3) that not only binds to the BCG-specific γδ TCR but also effectively activates γδ T cells isolated from human subjects inoculated with BCG. Importantly, the γδ T cells activated by peptide BP3 had a cytotoxic effect on THP-1 cells infected with BCG. Moreover, the oxidative stress response regulatory protein (OXYS), a BCG protein that matches perfectly with peptide BP3 according to bioinformatics analysis, was confirmed as a ligand for the γδ TCR and was found to activate γδ T cells from human subjects inoculated with BCG. Conclusions/Significance In conclusion, our study provides a novel strategy to identify epitopes or protein antigens for the γδ TCR, and provides a potential means to screen mycobacterial vaccines or candidates for adjuvant. PMID:21526117

  20. Mycobacterium tuberculosis pellicles express unique proteins recognized by the host humoral response

    PubMed Central

    Kerns, Patrick W.; Ackart, David F.; Basaraba, Randall J.; Leid, Jeff; Shirtliff, Mark E.

    2014-01-01

    Mycobacterium tuberculosis (MTB) causes both acute and chronic infections in humans characterized by tolerance to antibiotics and reactivation to cause secondary tuberculosis. These characteristics have led to renewed interested in the in vitro pellicle, or biofilm mode of growth, where bacteria grow to produce a thick aggregate at the air-liquid interface and exhibit increased phenotypic resistance to antibiotics. We infected guinea pigs with the virulent H37Rv strain of MTB for 60 days at which point we collected blood. To identify antigenic proteins, membrane protein extracts of MTB H37Ra pellicles and shaken cultures grown for 3, 5, or 7 weeks were probed with the infected animals’ sera after the proteins were separated by two-dimensional gel electrophoresis (2DGE). Antigenic proteins were then identified using MALDI-TOF/TOF mass spectrometry peptide mass fingerprinting. Antigenic pellicle proteins were compared across the three timepoints to identify those that were produced consistently during pellicle growth. They were also compared to those membrane proteins identified from harvested shaken cultures to determine pellicle-specific versus universally-expressed proteins. Using this technique we identified 44 distinct antigenic proteins, nine of which were pellicle-specific. The sequence of antigenic pellicle-specific proteins was checked for sequence conservation across 15 sequenced MTB clinical isolates, three other members of the MTB complex, as well as Mycobacterium avium and Mycobacterium smegmatis. The antigenic pellicle-specific protein Rv0097 was found to have very high sequence conservation within the MTB complex but not with related mycobacteria while FabG4 was highly conserved in all mycobacteria analyzed. These conserved pellicle-specific proteins represent targets for the development of future diagnostic tests and vaccines. PMID:24453174

  1. Cloning of a leucine-zipper protein recognized by the sera of patients with antibody-associated paraneoplastic cerebellar degeneration.

    PubMed Central

    Fathallah-Shaykh, H; Wolf, S; Wong, E; Posner, J B; Furneaux, H M

    1991-01-01

    Antibody-associated paraneoplastic cerebellar degeneration (the Yo syndrome) is an uncommon disorder in which an immune response is specifically directed against tumor tissue and the cerebellum. Screening of a lambda expression library has resulted in the isolation of cDNA clones that encode the major antigen recognized by serum from these patients. The fusion protein produced by the cDNA clones provides the basis of a simple diagnostic assay for this neurological syndrome. The occurrence of leucine-zipper and zinc-finger motifs in the predicted open reading frame suggests that this protein plays a role in the regulation of gene expression. Images PMID:2014264

  2. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.

    PubMed

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-08-27

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  3. Isolation of a gene encoding a Chlamydia sp. strain TWAR protein that is recognized during infection of humans.

    PubMed

    Campbell, L A; Kuo, C C; Thissen, R W; Grayston, J T

    1989-01-01

    Chlamydia sp. strain TWAR is a unique Chlamydia sp. that causes acute respiratory disease. A gene bank consisting of TWAR isolate AR-39 DNA in pUC19 was screened with anti-AR-39 rabbit immune sera. Two positive clones were isolated that contained 7.3-kilobase (pLC1) and 14.9-kilobase (pLC2) plasmids. Restriction mapping and hybridization studies showed that both pLC1 and pLC2 contained a common 4.2-kilobase PstI fragment. Plasmids were used as templates of in vitro transcription-translation. All three plasmids had a novel protein product of ca. 75 kilodaltons not found in the vector alone. Western blots showed that this protein reacted with anti-TWAR rabbit immune sera and with human immune serum from an individual who had proven TWAR infection. Whole-cell lysates of TWAR demonstrated a protein having the same molecular weight and immunoreactivity as the recombinant gene product. This protein was also recognized by rabbit immune serum against Chlamydia psittaci or Chlamydia trachomatis. Southern hybridizations with the cloned fragment as a probe of digests of other Chlamydia spp. showed weakly hybridizing fragments. These results suggest that we have isolated a gene encoding a protein recognized during human TWAR infection that contains some sequences shared among Chlamydia spp. PMID:2909493

  4. Human CD8+ herpes simplex virus-specific cytotoxic T-lymphocyte clones recognize diverse virion protein antigens.

    PubMed Central

    Tigges, M A; Koelle, D; Hartog, K; Sekulovich, R E; Corey, L; Burke, R L

    1992-01-01

    The role of the HLA class I-restricted, CD8+, herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL) in the control of human HSV infections is controversial because previous reports suggest that a substantial portion of the antigen-specific lytic response is mediated by CD4+ cells. To address this question directly, we isolated HSV-specific CD8+ CTL clones from a patient with recurrent genital herpes. These CTL were cloned by coculturing responder peripheral blood mononuclear cells (PBMC) with phytohemagglutinin-stimulated PBMC that had been infected with live HSV-2 and then irradiated prior to the addition of responder cells. After 1 week, CTL were cloned by limiting dilution using phytohemagglutinin stimulation and allogeneic feeder PBMC. Seven clones were isolated; all seven clones were CD8+ CD4- CD3+ DRbright, six lysed only HSV-2-infected targets, and one lysed both HSV-1- and HSV-2-infected targets. Antigen presentation was restricted by two to three different HLA class I loci. To determine the antigens recognized by these HSV-specific CTL, target cells were infected with HSV in the presence of acyclovir, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, or cycloheximide in a series of drug block/release protocols to limit the repertoire of viral gene expression to select transcriptional classes. Five of the clones exhibited a different pattern of cytotoxicity, suggesting that each recognized a distinct HSV antigen. One of the clones appears to be directed against an immediate-early antigen; six of the clones recognize virion proteins. Five of these clones recognized internal virion proteins that could be introduced into target cells by HSV infection in the absence of virus gene expression. Antigen specificity was further tested by using vaccinia virus vectors that express glycoproteins gD2 and gB2 or the tegument protein VP16. One clone lysed vaccinia virus/gD2-infected target cells; the remaining clones did not recognize any of these gene

  5. Identification of Staphylococcus aureus Proteins Recognized by the Antibody-Mediated Immune Response to a Biofilm Infection

    PubMed Central

    Brady, Rebecca A.; Leid, Jeff G.; Camper, Anne K.; Costerton, J. William; Shirtliff, Mark E.

    2006-01-01

    Staphylococcus aureus causes persistent, recurrent infections (e.g., osteomyelitis) by forming biofilms. To survey the antibody-mediated immune response and identify those proteins that are immunogenic in an S. aureus biofilm infection, the tibias of rabbits were infected with methicillin-resistant S. aureus to produce chronic osteomyelitis. Sera were collected prior to infection and at 14, 28, and 42 days postinfection. The sera were used to perform Western blot assays on total protein from biofilm grown in vitro and separated by two-dimensional gel electrophoresis. Those proteins recognized by host antibodies in the harvested sera were identified via matrix-assisted laser desorption ionization-time of flight analysis. Using protein from mechanically disrupted total and fractionated biofilm protein samples, we identified 26 and 22 immunogens, respectively. These included a cell surface-associated β-lactamase, lipoprotein, lipase, autolysin, and an ABC transporter lipoprotein. Studies were also performed using microarray analyses and confirmed the biofilm-specific up-regulation of most of these genes. Therefore, although the biofilm antigens are recognized by the immune system, the biofilm infection can persist. However, these proteins, when delivered as vaccines, may be important in directing the immune system toward an early and effective antibody-mediated response to prevent chronic S. aureus infections. Previous works have identified S. aureus proteins that are immunogenic during acute infections, such as sepsis. However, this is the first work to identify these immunogens during chronic S. aureus biofilm infections and to simultaneously show the global relationship between the antigens expressed during an in vivo infection and the corresponding in vitro transcriptomic and proteomic gene expression levels. PMID:16714572

  6. Evidence of the extrathymic development of tyrosinase-related protein-2-recognizing CD8+ T cells with low avidity

    PubMed Central

    Harada, Mamoru; Yamada, Hisakata; Tatsugami, Katsunori; Nomoto, Kikuo

    2001-01-01

    The majority of the human tumour-associated antigens characterized to date are derived from non–mutated self-proteins. However, nothing is known about the development of autoreactive and tumour-associated antigen-recognizing T cells. Tyrosinase-related protein (TRP)-2 is a non-mutated melanocyte differentiation antigen and TRP-2-recognizing CD8+ T cells are known to show responses to melanoma both in humans and mice. In addition, TRP-2-reactive T cells with low avidity have been suggested to be readily induced from the spleen cells of naïve mice. On the other hand, recent reports suggest that self antigen-reactive CD8+ T cells can be positively selected in the periphery. In this study, we tested the possibility that TRP-2-reactive CD8+ T cells in naïve mice could develop via the extrathymic pathway. As a consequence, TRP-2-reactive CD8+ T cell precursors in naïve C57BL/6 mice were suggested to express both interleukin-2 (IL-2) receptor β chain (IL-2Rβ) and CD44 molecules, in a manner similar to that of extrathymically developed T cells. Furthermore, IL-2Rβ+ CD44+ CD8+ T cells were detected in the adult thymectomized and bone marrow-reconstituted mice, and functional TRP-2-reactive T cells were generated from their spleen cells. Overall, these results suggest that low avidity CD8+ T cells recognizing TRP-2 can be developed extrathymically. PMID:11576222

  7. Single Molecular Film for Recognizing Biological Molecular Interaction: DNA-Protein Interaction and Enzyme Reaction

    NASA Astrophysics Data System (ADS)

    Kurihara, Kazue

    Protein-protein and protein-substrate interactions play essential roles in biological functions. Surface forces measurement and atomic force microscopy, which directly measure the interaction forces as a function of the surface separation, enable us to quantitatively evaluate these interactions [1-3]. We have employed the surface forces measurement [4] and colloidal probe atomic force microscopy [5] to study interactions involved in specific molecular recognition of DNA-protein and enzyme-substrate reaction. Studied are interactions between nucleic acid bases (adenine and thymine) [6], Spo0A-DB (the DNA-binding site of a transcription factor Spo0A), and DNA [7,8], those between subunits I and II of heptaprenyl diphosphate (HepPP) synthase in the presence of a substrate ((E,E)-farnesyl diphosphate, FPP) and a cofactor (Mg2+) [9-11], and the selectivity of the substrates in this enzymatic reaction [12]. Keys of our approach are the preparation of well-defined samples and the appropriate analysis. We have modified he substrate surfaces with these proteins using the Langmuir-Blodgett (LB) method. This chapter reviews the LB modification method and subsequent demonstrations of biological specific interactions employing this approach.

  8. rDNA genetic imbalance and nucleolar chromatin restructuring is induced by distant hybridization between Raphanus sativus and Brassica alboglabra.

    PubMed

    Long, Hong; Chen, Chunli; Wang, Bing; Feng, Yanni

    2015-01-01

    The expression of rDNA in hybrids inherited from only one progenitor refers to nucleolar dominance. The molecular basis for choosing which genes to silence remains unclear. We report genetic imbalance induced by distant hybridization correlates with formation of rDNA genes (NORs) in the hybrids between Raphanus sativus L. and Brassica alboglabra Bailey. Moreover, increased CCGG methylation of rDNA in F1 hybrids is concomitant with Raphanus-derived rDNA gene silencing and rDNA transcriptional inactivity revealed by nucleolar configuration restriction. Newly formed rDNA gene locus occurred through chromosomal in F1 hybrids via chromosomal imbalance. NORs are gained de novo, lost, and/or transposed in the new genome. Inhibition of methyltransferases leads to changes in nucleolar architecture, implicating a key role of methylation in control of nucleolar dominance and vital nucleolar configuration transition. Our findings suggest that gene imbalance and methylation-related chromatin restructuring is important for rDNA gene silencing that may be crucial for synthesis of specific proteins. PMID:25723542

  9. Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain

    PubMed Central

    Mao, Xiaoyan; Zhang, Tiancheng; Ji, Guanghui; Shi, Xin; Xia, Tian; Lu, Weijia; Zhang, Dapeng; Dai, Jianxin; Guo, Yajun

    2012-01-01

    Background Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed. Methods and Findings We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo. Conclusions The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes. PMID:22952786

  10. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  11. Nuclease-resistant c-di-AMP derivatives that differentially recognize RNA and protein receptors

    PubMed Central

    Meehan, Robert E.; Torgerson, Chad D.; Gaffney, Barbara L.; Jones, Roger A.; Strobel, Scott A.

    2016-01-01

    The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3’-5’-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activate specific pathways and mediate phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogs, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position, more than a carbonyl at the C6 position. We also identified phosphate-modified analogs that bind both the ydaO RNA and GdpP protein with high affinity, while symmetrically-modified ribose analogs exhibited a substantial decrease in ydaO affinity, but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogs could be useful as chemical tools to specifically target subsections of the second-messenger signaling pathways. PMID:26789423

  12. Nuclease-Resistant c-di-AMP Derivatives That Differentially Recognize RNA and Protein Receptors.

    PubMed

    Meehan, Robert E; Torgerson, Chad D; Gaffney, Barbara L; Jones, Roger A; Strobel, Scott A

    2016-02-16

    The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3'-5'-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activates specific pathways and mediates phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogues, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position more than a carbonyl at the C6 position. We also identified phosphate-modified analogues that bind both the ydaO RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues exhibited a substantial decrease in ydaO affinity but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways. PMID:26789423

  13. Nuclear protein LEDGF/p75 recognizes supercoiled DNA by a novel DNA-binding domain

    PubMed Central

    Tsutsui, Kimiko M.; Sano, Kuniaki; Hosoya, Osamu; Miyamoto, Tadashi; Tsutsui, Ken

    2011-01-01

    Lens epithelium-derived growth factor (LEDGF) or p75 is a co-activator of general transcription and also involved in insertion of human immunodeficiency virus type I (HIV-1) cDNA into host cell genome, which occurs preferentially to active transcription units. These phenomena may share an underlying molecular mechanism in common. We report here that LEDGF/p75 binds negatively supercoiled DNA selectively over unconstrained DNA. We identified a novel DNA-binding domain in the protein and termed it ‘supercoiled DNA-recognition domain’ (SRD). Recombinant protein fragments containing SRD showed a preferential binding to supercoiled DNA in vitro. SRD harbors a characteristic cluster of lysine and glutamic/aspartic acid residues. A polypeptide mimicking the cluster (K9E9K9) also showed this specificity, suggesting that the cluster is an essential element for the supercoil recognition. eGFP-tagged LEDGF/p75 expressed in the nucleus distributed partially in transcriptionally active regions that were identified by immunostaining of methylated histone H3 (H3K4me3) or incorporation of Br-UTP. This pattern of localization was observed with SRD alone but abolished if the protein lacked SRD. Thus, these results imply that LEDGF/p75 guides its binding partners, including HIV-1 integrase, to the active transcription site through recognition of negative supercoils generated around it. PMID:21345933

  14. A Genetic Cascade of let-7-ncl-1-fib-1 Modulates Nucleolar Size and rRNA Pool in Caenorhabditis elegans

    PubMed Central

    Chiou, Pey-Tsyr; Chen, Po-Hsiang; Lee, Ching-Ming; Chu, Yu-De; Yu, Hsiang; Hsiung, Kuei-Ching; Tsai, Yi-Tzang; Lee, Chi-Chang; Chang, Yu-Sun; Chan, Shih-Peng; Tan, Bertrand Chin-Ming; Lo, Szecheng J.

    2015-01-01

    Ribosome biogenesis takes place in the nucleolus, the size of which is often coordinated with cell growth and development. However, how metazoans control nucleolar size remains largely unknown. Caenorhabditis elegans provides a good model to address this question owing to distinct tissue distribution of nucleolar sizes and a mutant, ncl-1, which exhibits larger nucleoli than wild-type worms. Here, through a series of loss-of-function analyses, we report that the nucleolar size is regulated by a circuitry composed of microRNA let-7, translation repressor NCL-1, and a major nucleolar pre-rRNA processing protein FIB-1/fibrillarin. In cooperation with RNA binding proteins PUF and NOS, NCL-1 suppressed the translation of FIB-1/fibrillarin, while let-7 targeted the 3’UTR of ncl-1 and inhibited its expression. Consequently, the abundance of FIB-1 is tightly controlled and correlated with the nucleolar size. Together, our findings highlight a novel genetic cascade by which post-transcriptional regulators interplay in developmental control of nucleolar size and function. PMID:26492166

  15. Anti-HmuY Antibodies Specifically Recognize Porphyromonas gingivalis HmuY Protein but Not Homologous Proteins in Other Periodontopathogens

    PubMed Central

    Śmiga, Michał; Bielecki, Marcin; Olczak, Mariusz; Smalley, John W.; Olczak, Teresa

    2015-01-01

    Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium. PMID:25658942

  16. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    PubMed

    Śmiga, Michał; Bielecki, Marcin; Olczak, Mariusz; Smalley, John W; Olczak, Teresa

    2015-01-01

    Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium. PMID:25658942

  17. Epitope mapping of monoclonal antibody 4C8 recognizing the protein huntingtin.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Roos, Raymund A C; Van Ommen, Gert-Jan B; Dorsman, Josephine C

    2005-10-01

    Huntington's disease is a dominantly inherited, devastating neurodegenerative disorder, caused by a polyglutamine expansion (>37) in the N-terminal region of huntingtin, a protein of unknown function. In patients and normal individuals, N-terminal fragments of huntingtin are found, and the N-terminal fragments of mutant huntingtin are cytotoxic. The functions of wild-type huntingtin and the mechanisms underlying the toxic effects of mutant huntingtin are still ill defined. To get more insight into these topics, monoclonal antibodies (MAbs) are indispensable tools. Antibodies raised against the N-terminus are especially important. Among these, the 4C8 mouse MAb has been extensively used in various approaches. In this study, we have mapped the epitope of 4C8 to a 15-amino acid (aa) region spanning from aa 443 to 457 of the human protein, and found that mutation of three consecutive glutamic acids present in this region disrupts the recognition by 4C8. These results allow a more accurate interpretation of the results obtained by usage of the 4C8 antibody and broaden the utility of this antibody. PMID:16225422

  18. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein.

    PubMed

    Hicar, Mark D; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U; Kalams, Spyros A; Doranz, Benjamin J; Spearman, Paul; Crowe, James E

    2016-01-01

    Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

  19. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

    PubMed Central

    Hicar, Mark D.; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U.; Kalams, Spyros A.; Doranz, Benjamin J.; Spearman, Paul; Crowe, James E.

    2016-01-01

    Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

  20. OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins.

    PubMed

    Holbrook, Lisa-Marie; Kwong, Lai-Shan; Metcalfe, Clive L; Fenouillet, Emmanuel; Jones, Ian M; Barclay, A Neil

    2016-01-01

    In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents. PMID:26986548

  1. New insights into nucleolar structure and function

    PubMed Central

    Lam, Yun Wah

    2015-01-01

    The nucleolus is a non-membrane-bound nuclear organelle found in all eukaryotes. It is the quintessential ‘RNA-seeded’ nuclear body, forming around specific chromosomal features called nucleolar organizing regions that contain arrays of ribosomal DNA. Assembly is triggered by activation of RNA polymerase I-mediated transcription and regulated in mammalian cells in a cell cycle-dependent manner. Although the nucleolus is best known for its role in coordinating ribosome biogenesis, biochemical and proteomic analyses have revealed a much wider functional complexity than previously appreciated, including roles in cell cycle regulation, DNA damage sensing and repair, pre-mRNA processing, telomere metabolism, processing of non-coding RNAs, and coordination of the cellular response to various stresses. Despite these advances, much remains to be learned about the full range of biological processes that occur within, or involve, this organelle and how its assembly/disassembly and functional reorganization in response to various stimuli are regulated. Here, we review the impact of recent studies that provide major insights into these fundamental questions, and we highlight the therapeutic potential of targeting nucleolar pathways. PMID:26097721

  2. Development of monoclonal antibodies specifically recognizing the endogenous sterile alpha motif and HD domain 1 protein in porcine cell lines.

    PubMed

    Yang, Shen; Zhou, Yan-Jun; Zhan, Yuan; Yu, Ling-Xue; Jiang, Yi-Feng; Tong, Wu; Tong, Guang-Zhi

    2014-10-01

    The sterile alpha motif and HD domain 1 (SAMHD1) protein has been identified as a novel innate immunity restriction factor that participates in processes crucial to the viral life cycle. In the present study, we describe a procedure to generate monoclonal antibodies (MAbs) against porcine SAMHD1 and investigate its characteristics to analyze the expression of endogenous SAMHD1. The open reading frame of porcine SAMHD1 was cloned into the prokaryotic expression vector pCold-TF DNA to construct a recombinant plasmid pcold-pSAMHD1 and induce expression of recombinant porcine SAMHD1 protein by IPTG in Escherichia coli Rosetta. The purified recombinant porcine SAMHD1 protein was used to prepare MAbs of SAMHD1. After subcloning five times hybridoma cell clones expressing SAMHD1, MAbs were generated. Western blot analysis and indirect immunofluorescence assay showed that the overexpressed porcine SAMHD1 in 293T cells and endogenous SAMHD1 protein in porcine cell lines could be specifically recognized by the MAbs produced in this study. In conclusion, specific MAbs of porcine SAMHD1 are reported, and these MAbs provide a valuable tool for further studies of SAMHD1-mediated signaling in virus-infected cells to elucidate the underlying antiviral mechanism. PMID:25358004

  3. Development of Monoclonal Antibodies Specifically Recognizing the Endogenous Sterile Alpha Motif and HD Domain 1 Protein in Porcine Cell Lines

    PubMed Central

    Yang, Shen; Zhou, Yan-jun; Zhan, Yuan; Yu, Ling-xue; Jiang, Yi-feng; Tong, Wu

    2014-01-01

    The sterile alpha motif and HD domain 1 (SAMHD1) protein has been identified as a novel innate immunity restriction factor that participates in processes crucial to the viral life cycle. In the present study, we describe a procedure to generate monoclonal antibodies (MAbs) against porcine SAMHD1 and investigate its characteristics to analyze the expression of endogenous SAMHD1. The open reading frame of porcine SAMHD1 was cloned into the prokaryotic expression vector pCold-TF DNA to construct a recombinant plasmid pcold-pSAMHD1 and induce expression of recombinant porcine SAMHD1 protein by IPTG in Escherichia coli Rosetta. The purified recombinant porcine SAMHD1 protein was used to prepare MAbs of SAMHD1. After subcloning five times hybridoma cell clones expressing SAMHD1, MAbs were generated. Western blot analysis and indirect immunofluorescence assay showed that the overexpressed porcine SAMHD1 in 293T cells and endogenous SAMHD1 protein in porcine cell lines could be specifically recognized by the MAbs produced in this study. In conclusion, specific MAbs of porcine SAMHD1 are reported, and these MAbs provide a valuable tool for further studies of SAMHD1-mediated signaling in virus-infected cells to elucidate the underlying antiviral mechanism. PMID:25358004

  4. Expression of expanded CAG transcripts triggers nucleolar stress in Huntington's disease.

    PubMed

    Tsoi, Ho; Chan, Ho Yin Edwin

    2013-06-01

    Polyglutamine (polyQ) diseases, including several types of spinocerebellar ataxias and Huntington's disease (HD), are dominantly inherited neurodegenerative disorders caused by the expansion of the glutamine-coding CAG repeat in the open reading frame of the disease gene. Apart from being translated to produce toxic elongated polyQ domain-containing disease proteins, transcribed expanded CAG RNAs per se also exert toxicity in polyQ degeneration. In the R6/2 HD transgenic mouse model, expanded mutant Huntingtin (Htt) transcripts were found to physically interact with nucleolin (NCL), a nucleolar protein that plays a crucial role in ribosome biogenesis. We further demonstrated that mutant Htt transcripts deprived NCL from binding onto the Upstream Control Element (UCE) of the ribosomal RNA (rRNA) promoter. This resulted in UCE hypermethylation which abolished the binding of the transcription factor Upstream Binding Factor to UCE and subsequently led to down-regulation of pre-45s rRNA transcription. We also found that the p53/mitochondria-dependent nucleolar stress cell death pathway was activated in polyQ diseases. Ribosomal RNA transcription dysfunction has been reported in other types of neurodegenerative disorders including Alzheimer's disease; it is anticipated that nucleolar stress is one common pathogenic signaling mechanism shared by different forms of neurodegeneration. PMID:23315009

  5. Guanine nucleotide depletion inhibits pre-ribosomal RNA synthesis and causes nucleolar disruption.

    PubMed

    Huang, Min; Ji, Yanshan; Itahana, Koji; Zhang, Yanping; Mitchell, Beverly

    2008-01-01

    Inosine monophosphate dehydrogenase (IMPDH) is a pivotal enzyme in the de novo pathway of guanine nucleotide biosynthesis. Inhibitors of this enzyme decrease intracellular guanine nucleotide levels by 50-80% and have potential as anti-neoplastic agents. Both mycophenolic acid (MPA) and AVN-944 are highly specific inhibitors of IMPDH that cause cell cycle arrest or apoptosis in lymphocytes and leukemic cell lines. We have examined the mechanisms by which these two agents cause cytotoxicity. Both MPA and AVN-944 inhibit the growth of K562 cells, and induce apoptosis in Raji B and CCRF-CEM T cells. Both compounds strikingly inhibit RNA synthesis within 2 h of exposure. Depletion of guanine nucleotides by MPA and AVN-944 also causes an early and near-complete reduction in levels of the 45S precursor rRNA synthesis and the concomitant translocation of nucleolar proteins including nucleolin, nucleophosmin, and nucleostemin from the nucleolus to the nucleoplasm. This efflux correlates temporally with the sustained induction of p53 in cell lines with wild-type p53. We conclude that inhibition of IMPDH causes a primary reduction in rRNA synthesis and secondary nucleolar disruption and efflux of nucleolar proteins that most likely mediate cell cycle arrest or apoptosis. The ability of AVN-944 to induce apoptosis in a number of leukemic cell lines supports its potential utility in the treatment of hematologic malignancies. PMID:17462731

  6. Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses.

    PubMed

    Sun, Encheng; Zhao, Jing; Liu, Nihong; Yang, Tao; Xu, Qingyuan; Qin, Yongli; Bu, Zhigao; Yang, Yinhui; Lunt, Ross A; Wang, Linfa; Wu, Donglai

    2012-01-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines

  7. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors.

    PubMed

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-07-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8(+) T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8(+) T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8(+) T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8(+) T-cell clones are highly focused on their index peptide sequence and that 'CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8(+) T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  8. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

    PubMed Central

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-01-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  9. Conserved epitopes on HIV-1, FIV and SIV p24 proteins are recognized by HIV-1 infected subjects

    PubMed Central

    Roff, Shannon R; Sanou, Missa P; Rathore, Mobeen H; Levy, Jay A; Yamamoto, Janet K

    2015-01-01

    Cross-reactive peptides on HIV-1 and FIV p24 protein sequences were studied using peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected long-term survivors (LTS; >10 y of infection without antiretroviral therapy, ART), short-term HIV-1 infected subjects not on ART, and ART-treated HIV-1 infected subjects. IFNγ-ELISpot and CFSE-proliferation analyses were performed with PBMC using overlapping HIV-1 and FIV p24 peptides. Over half of the HIV-1 infected subjects tested (22/31 or 71%) responded to one or more FIV p24 peptide pools by either IFNγ or T-cell proliferation analysis. PBMC and T cells from infected subjects in all 3 HIV+ groups predominantly recognized one FIV p24 peptide pool (Fp14) by IFNγ production and one additional FIV p24 peptide pool (Fp9) by T-cell proliferation analysis. Furthermore, evaluation of overlapping SIV p24 peptide sequences identified conserved epitope(s) on the Fp14/Hp15-counterpart of SIV, Sp14, but none on Fp9-counterpart of SIV, Sp9. The responses to these FIV peptide pools were highly reproducible and persisted throughout 2–4 y of monitoring. Intracellular staining analysis for cytotoxins and phenotyping for CD107a determined that peptide epitopes from Fp9 and Fp14 pools induced cytotoxic T lymphocyte-associated molecules including perforin, granzyme B, granzyme A, and/or expression of CD107a. Selected FIV and corresponding SIV epitopes recognized by HIV-1 infected patients indicate that these protein sequences are evolutionarily conserved on both SIV and HIV-1 (e.g., Hp15:Fp14:Sp14). These studies demonstrate that comparative immunogenicity analysis of HIV-1, FIV, and SIV can identify evolutionarily-conserved T cell-associated lentiviral epitopes, which could be used as a vaccine for prophylaxis or immunotherapy. PMID:25844718

  10. The anti-trp RNA-binding attenuation protein (Anti-TRAP), AT, recognizes the tryptophan-activated RNA binding domain of the TRAP regulatory protein.

    PubMed

    Valbuzzi, Angela; Gollnick, Paul; Babitzke, Paul; Yanofsky, Charles

    2002-03-22

    In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of genes involved in tryptophan metabolism in response to the accumulation of l-tryptophan. Tryptophan-activated TRAP negatively regulates expression by binding to specific mRNA sequences and either promoting transcription termination or blocking translation initiation. Conversely, the accumulation of uncharged tRNA(Trp) induces synthesis of an anti-TRAP protein (AT), which forms a complex with TRAP and inhibits its activity. In this report, we investigate the structural features of TRAP required for AT recognition. A collection of TRAP mutant proteins was examined that were known to be partially or completely defective in tryptophan binding and/or RNA binding. Analyses of AT interactions with these proteins were performed using in vitro transcription termination assays and cross-linking experiments. We observed that TRAP mutant proteins that had lost the ability to bind RNA were no longer recognized by AT. Our findings suggest that AT acts by competing with messenger RNA for the RNA binding domain of TRAP. B. subtilis AT was also shown to interact with TRAP proteins from Bacillus halodurans and Bacillus stearothermophilus, implying that the structural elements required for AT recognition are conserved in the TRAP proteins of these species. Analyses of AT interaction with B. stearothermophilus TRAP at 60 degrees C demonstrated that AT is active at this elevated temperature. PMID:11786553

  11. HPV oncoprotein E6 is a structure-dependent DNA-binding protein that recognizes four-way junctions.

    PubMed

    Ristriani, T; Masson, M; Nominé, Y; Laurent, C; Lefevre, J F; Weiss, E; Travé, G

    2000-03-10

    E6 is an oncoprotein implicated in cervical cancers, produced by "high-risk" human papillomaviruses. E6 is thought to promote tumorigenesis by stimulating cellular degradation of the tumour suppressor p53, but it might display other activities. Sequence similarity was recently detected between E6 and endonuclease VII, a protein of phage T4 that recognizes and cleaves four-way DNA junctions. Here, we purified recombinant E6 proteins and demonstrated that high-risk E6 s bind selectively to four-way junctions in a structure-dependent manner. Several residues in the C-terminal zinc-binding domain, the region of E6 similar to endonuclease VII, are necessary for the junction-binding activity. E6 binds to the junction as a monomer. Comparative electrophoresis shows that E6-bound junctions migrate in an extended square conformation. Magnesium inhibits the electrophoretic migration of the complexes but does not seem to influence their formation at equilibrium. This work is the first demonstration of specific binding of purified active E6 to a well-characterized DNA ligand, and suggests new modes of action of E6 in oncogenesis. PMID:10698626

  12. Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase

    PubMed Central

    Sato, Brian K.; Schulz, Daniel; Do, Phong H.

    2009-01-01

    Summary Quality control pathways such as ER-associated degradation (ERAD) employ a small number of factors to specifically recognize a wide variety of protein substrates. Delineating the mechanisms of substrate selection is a principle goal in studying quality control. The Hrd1p ubiquitin ligase mediates ERAD of numerous misfolded proteins including soluble, lumenal ERAD-L and membrane-anchored ERAD-M substrates. We tested if the Hrd1p multi-spanning membrane domain was involved in ERAD-M specificity. In this work, we have identified site-directed membrane domain mutants of Hrd1p impaired only for ERAD-M and normal for ERAD-L. Furthermore, other Hrd1p variants were specifically deficient for degradation of individual ERAD-M substrates. Thus, the Hrd1p transmembrane region bears determinants of high specificity in the ERAD-M pathway. From in vitro and interaction studies, we suggest a model in which the Hrd1p membrane domain employs intra-membrane residues to evaluate substrate misfolding, leading to selective ubiquitination of appropriate ERAD-M clients. PMID:19394298

  13. Nucleolar exit of RNF8 and BRCA1 in response to DNA damage

    SciTech Connect

    Guerra-Rebollo, Marta; Mateo, Francesca; Franke, Kristin; Huen, Michael S.Y.; Lopitz-Otsoa, Fernando; Rodriguez, Manuel S.; Plans, Vanessa; Thomson, Timothy M.

    2012-11-01

    The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to {gamma}-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did {gamma}-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage. -- Highlights: Black-Right-Pointing-Pointer RNF8 and BRCA1 are associated with the nucleolus of undamaged cells. Black-Right-Pointing-Pointer Upon {gamma}-radiation, RNF8 and BRCA1 are translocated from the nucleolus to damage foci. Black-Right-Pointing-Pointer The ribosomal protein RPSA anchors RNF8 to the nucleolus. Black-Right-Pointing-Pointer RNF8 may play previously unsuspected roles in protein synthesis.

  14. Recognizing Cataracts

    MedlinePlus

    ... link, please review our exit disclaimer . Subscribe Recognizing Cataracts Watch for Vision Changes as You Age As ... cause of impaired eyesight later in life is cataracts. A cataract is a clouding of the lens ...

  15. Gag Protein Epitopes Recognized by CD4+ T-Helper Lymphocytes from Equine Infectious Anemia Virus-Infected Carrier Horses

    PubMed Central

    Lonning, S. M.; Zhang, W.; McGuire, T. C.

    1999-01-01

    Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV. PMID:10196322

  16. Synthetic antibodies and peptides recognizing progressive multifocal leukoencephalopathy-specific point mutations in polyomavirus JC capsid viral protein 1.

    PubMed

    Chen, Gang; Gorelik, Leonid; Simon, Kenneth J; Pavlenco, Alevtina; Cheung, Anne; Brickelmaier, Margot; Chen, Ling Ling; Jin, Ping; Weinreb, Paul H; Sidhu, Sachdev S

    2015-01-01

    Polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare and frequently fatal brain disease that afflicts a small fraction of the immune-compromised population, including those affected by AIDS and transplantation recipients on immunosuppressive drug therapy. Currently there is no specific therapy for PML. The major capsid viral protein 1 (VP1) involved in binding to sialic acid cell receptors is believed to be a key player in pathogenesis. PML-specific mutations in JCV VP1 sequences present at the binding pocket of sialic acid cell receptors, such as L55F and S269F, abolish sialic acid recognition and might favor PML onset. Early diagnosis of these PML-specific mutations may help identify patients at high risk of PML, thus reducing the risks associated with immunosuppressive therapy. As a first step in the development of such early diagnostic tools, we report identification and characterization of affinity reagents that specifically recognize PML-specific mutations in VP1 variants using phage display technology. We first identified 2 peptides targeting wild type VP1 with moderate specificity. Fine-tuning via selection of biased libraries designed based on 2 parental peptides yielded peptides with different, yet still moderate, bindinspecificities. In contrast, we had great success in identifying synthetic antibodies that recognize one of the PML-specific mutations (L55F) with high specificity from the phage-displayed libraries. These peptides and synthetic antibodies represent potential candidates for developing tailored immune-based assays for PML risk stratification in addition to complementing affinity reagents currently available for the study of PML and JCV. PMID:25879139

  17. The code for directing proteins for translocation across ER membrane: SRP cotranslationally recognizes specific features of a signal sequence.

    PubMed

    Nilsson, IngMarie; Lara, Patricia; Hessa, Tara; Johnson, Arthur E; von Heijne, Gunnar; Karamyshev, Andrey L

    2015-03-27

    The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP-signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the N(ε)-(5-azido-2 nitrobenzoyl)-Lys-tRNA(amb) amber suppressor. A homogeneous population of SRP-ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP-signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP-signal sequence affinity and targeting to the translocon. PMID:24979680

  18. Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains

    PubMed Central

    Dattoli, Ada A.; Hink, Mark A.; DuBuc, Timothy Q.; Teunisse, Bram J.; Goedhart, Joachim; Röttinger, Eric; Postma, Marten

    2015-01-01

    SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus. PMID:26190255

  19. Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

    PubMed

    Dattoli, Ada A; Hink, Mark A; DuBuc, Timothy Q; Teunisse, Bram J; Goedhart, Joachim; Röttinger, Eric; Postma, Marten

    2015-01-01

    SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus. PMID:26190255

  20. Nonmyofilament-Associated Troponin T3 Nuclear and Nucleolar Localization Sequence and Leucine Zipper Domain Mediate Muscle Cell Apoptosis

    PubMed Central

    Zhang, Tan; Birbrair, Alexander; Delbono, Osvaldo

    2013-01-01

    Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 isoform is localized in the muscle nucleus, and either its full-length or COOH-terminus leads to muscle cell apoptosis. Here, we further explored the mechanism by which it enters the nucleus and promotes cytotoxicity. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK) and the basic lysine and arginine residues might play an important role in the nuclear retention and nucleolar enrichment of KLKRQK-DsRed fusion proteins. Deleting this domain or substituting lysine and arginine residues (KLAAQK) resulted in a dramatic loss of TnT3 nuclear and nucleolar localization. In contrast, the GATAKGKVGGRWK domain-DsRed construct localized exclusively in the cytoplasm, indicating that a nuclear exporting sequence is possibly localized in this region. Additionally, we identified a classical DNA-binding Leucine Zipper Domain (LZD) which is conserved among TnT isoforms and species. Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. We conclude that TnT3 contains both a nuclear localization signal and a DNA binding domain, which may mediate nuclear/nucleolar signaling and muscle cell apoptosis. PMID:23378072

  1. The structure and function of small nucleolar ribonucleoproteins

    PubMed Central

    Reichow, Steve L.; Hamma, Tomoko; Ferré-D'Amaré, Adrian R.; Varani, Gabriele

    2007-01-01

    Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequence-specific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein–RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose-2′-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the core archaeal sRNP proteins have been determined and their assembly pathways delineated. Furthermore, the recent structure of an H/ACA complex has revealed the organization of a complete sRNP. Combined with current biochemical data, these structures offer insight into the highly homologous eukaryotic snoRNPs. PMID:17284456

  2. Molecular determinants of nucleolar translocation of RNA helicase A

    SciTech Connect

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-10-15

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role.

  3. The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis

    PubMed Central

    Jeon, Young; Park, Yong-Joon; Cho, Hui Kyung; Jung, Hyun Ju; Ahn, Tae-Kyu; Kang, Hunseung; Pai, Hyun-Sook

    2015-01-01

    Nucleostemin is a nucleolar GTP-binding protein that is involved in stem cell proliferation, embryonic development, and ribosome biogenesis in mammals. Plant nucleostemin-like 1 (NSN1) plays a role in embryogenesis, and apical and floral meristem development. In this study, a nucleolar function of NSN1 in the regulation of ribosome biogenesis was identified. Green fluorescent protein (GFP)-fused NSN1 localized to the nucleolus, which was primarily determined by its N-terminal domain. Recombinant NSN1 and its N-terminal domain (NSN1-N) bound to RNA in vitro. Recombinant NSN1 expressed GTPase activity in vitro. NSN1 silencing in Arabidopsis thaliana and Nicotiana benthamiana led to growth retardation and premature senescence. NSN1 interacted with Pescadillo and EBNA1 binding protein 2 (EBP2), which are nucleolar proteins involved in ribosome biogenesis, and with several ribosomal proteins. NSN1, NSN1-N, and EBP2 co-fractionated primarily with the 60S ribosomal large subunit in vivo. Depletion of NSN1 delayed 25S rRNA maturation and biogenesis of the 60S ribosome subunit, and repressed global translation. NSN1-deficient plants exhibited premature leaf senescence, excessive accumulation of reactive oxygen species, and senescence-related gene expression. Taken together, these results suggest that NSN1 plays a crucial role in plant growth and senescence by modulating ribosome biogenesis. PMID:26163696

  4. Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion protein glycoforms: an immunoblot and immunohistochemical study at the light and electron microscopic levels.

    PubMed

    Matucci, Andrea; Zanusso, Gianluigi; Gelati, Matteo; Farinazzo, Alessia; Fiorini, Michele; Ferrari, Sergio; Andrighetto, Giancarlo; Cestari, Tiziana; Caramelli, Maria; Negro, Alessandro; Morbin, Michela; Chiesa, Roberto; Monaco, Salvatore; Tridente, Giuseppe

    2005-03-15

    The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrP(Sc)) of the cellular prion protein (PrP(C)) in different mammalian species is important for molecular diagnostics, PrP(Sc) typing and future immunotherapy. We obtained a panel of anti-PrP monoclonal antibodies in PrP(0/0) knock-out mice immunized with recombinant human PrP(23-231). Two mAbs, recognizing PrP epitopes in the alpha-helix 1 (mAb SA65) and alpha-helix 2 (mAb SA21) regions, immunoreacted with PrP(C) and PrP(Sc) and its proteolytic product, PrP27-30, from human, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monoglycosylated PrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupied or highly glycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slower migrating form of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highly efficient in detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAbs provide tools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the role of different PrP glycoforms in human and animal prion diseases. PMID:15763182

  5. Nucleolar p14ARF Overexpression in Reed-Sternberg Cells in Hodgkin’s Lymphoma

    PubMed Central

    García, Juan F.; Villuendas, Raquel; Sánchez-Beato, Margarita; Sánchez-Aguilera, Abel; Sánchez, Lydia; Prieto, Ignacio; Piris, Miguel A.

    2002-01-01

    The development of human cancers is frequently associated with the silencing of the two major tumor suppressor pathways represented by retinoblastoma protein and p53. As the incidence of p53 mutations is significantly lower in Hodgkin’s lymphoma than in other neoplasias, we investigated whether the malfunction of other proteins in this pathway could be responsible for its inactivation. Because the existence of nucleolar complexes between p14ARF and Hdm2 has been described as having a critical effect on p53 function by inhibiting its degradation, we analyzed the expression and subcellular localization of these proteins in 52 cases and in Hodgkin’s cell lines. Two of four cell lines revealed loss of p14ARF expression secondary to gene promoter methylation, this being mutually exclusive with p53 mutations (1 of 4), illustrating the existence of selective pressure to inactivate the p53 pathway. The majority of Hodgkin’s samples showed a strong nucleolar expression of p14ARF that was not associated with Hdm2. They also showed the existence of Hdm2/p53 complexes, and the absence of complexes containing either p14ARF/Hdm2 or p14ARF/p53. The different localization of Hdm2 (nucleoplasm) and p14ARF (nucleoli) observed in Hodgkin’s tumors and cell lines is associated with the presence of short alternatively spliced transcripts of Hdm2 lacking the ARF-binding region and the nuclear export signal. The absence of these p14ARF/Hdm2 nucleolar complexes could be sufficient to inactivate the pathway and may explain the low frequency of p53 mutations in this tumor. PMID:11839577

  6. Cytotoxic T lymphocytes that recognize decameric peptide sequences of retinoblastoma binding protein 1 (RBP-1) associated with human breast cancer

    PubMed Central

    Takahashi, T; Cao, J; Hoon, D S B; Irie, R F

    1999-01-01

    Retinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB). We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250–256) recognized by human antibodies and overexpressed by breast cancer cells. In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells. These decamers, GLQKASIFLK (247–256) and KASIFLKTRV (250–259), have anchor motifs for both HLA-A2 and HLA-A3. Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml−1 interleukin-2, 25 IU ml−1 interleukin-7 and 500 IU ml−1 granulocyte–macrophage colony-stimulating factor. Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide. Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors. Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors. No HLA-A2−, A3− CTL line showed specific cytotoxicity against these target cells. These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line. RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer. © 1999 Cancer Research Campaign PMID:10496363

  7. Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.

    PubMed

    Nakamoto, K; Ito, A; Watabe, K; Koma, Y; Asada, H; Yoshikawa, K; Shinomura, Y; Matsuzawa, Y; Nojima, H; Kitamura, Y

    2001-10-01

    F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus. PMID:11583964

  8. Upconversion nanophosphor: an efficient phosphopeptides-recognizing matrix and luminescence resonance energy transfer donor for robust detection of protein kinase activity.

    PubMed

    Liu, Chenghui; Chang, Lijuan; Wang, Honghong; Bai, Jie; Ren, Wei; Li, Zhengping

    2014-06-17

    Protein kinases play important regulatory roles in intracellular signal transduction pathways. The aberrant activities of protein kinases are closely associated with the development of various diseases, which necessitates the development of practical and sensitive assays for monitoring protein kinase activities as well as for screening of potential kinase-targeted drugs. We demonstrate here a robust luminescence resonance energy transfer (LRET)-based protein kinase assay by using NaYF4:Yb,Er, one of the most efficient upconversion nanophosphors (UCNPs), as an autofluorescence-free LRET donor and a tetramethylrhodamine (TAMRA)-labeled substrate peptide as the acceptor. Fascinatingly, besides acting as the LRET donor, NaYF4:Yb,Er UCNPs also serve as the phosphopeptide-recognizing matrix because the intrinsic rare earth ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by protein kinases over the unphosphorylated ones. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read format without any requirement of surface modification, substrate immobilization, separation, or washing steps, showing great potential in protein kinases-related clinical diagnosis and drug discovery. To the best of our knowledge, this is the first report by use of rare earth-doped UCNPs as both the phospho-recognizing and signal reporting elements for protein kinase analysis. PMID:24871878

  9. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    SciTech Connect

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera; Hofmann, Wilma A.

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

  10. Methylation of nucleolar RNA in HeLa cells studied by autoradiography

    SciTech Connect

    Cervera, J.; Martinez, A.; Renau-Piqueras, J.

    1984-01-01

    Methylation of nucleolar RNA was studied by autoradiography in HeLa cells using L-(methyl-/sup 3/H)methionine and S-adenosyl-L-(methyl-/sup 3/H)methionine as radioactive precursors. Pulse-labeling experiments show that nucleolar RNA methylation occurs on the newly synthesized RNA at the nucleolar fibrillar RNP component and mostly on the fibrillar ring of fibrillar centers, where pre-rRNA is being synthesized. Pulse-chase experiments show a shift of silver grains from the nucleolar fibrillar RNP component to the nucleolar granular component first and then to the cytoplasm. Labeling of nucleolar RNA via specific methylation permits the study of intranucleolar processing of pre-rRNA and confirms the sequence of labeling of the two nucleolar RNP components observed with radioactive uridine.

  11. PNRC accumulates in the nucleolus by interaction with B23/nucleophosmin via its nucleolar localization sequence.

    PubMed

    Wang, Yuanzhong; Chen, Bin; Li, Yuping; Zhou, Dujin; Chen, Shiuan

    2011-01-01

    PNRC (proline-rich nuclear receptor coregulatory protein) was primarily identified as a coactivator of nuclear receptors (NRs) by our laboratory, which enhances NR-mediated transcription by RNA polymerase II. Recent study has shown that PNRC also stimulates RNA polymerase III-dependent transcription through interaction with the subunit RPC39 of RNA polymerase III. Here, we report that PNRC accumulates in the nucleolus and its depletion by small interfering RNA (siRNA) impairs pre-rRNA transcription by RNA polymerase I. We identified the sequence at position 94-101 ((94)PKKRRKKK(101)) of PNRC as its nucleolar localization sequence (NoLS). Fusion of this sequence to GFP directed GFP to the nucleolus. Characterization of the NoLS revealed that the stretches of six successive basic residues are sufficient to function as a NoLS. Through co-immunoprecipitation assay, we demonstrated that the NoLS is necessary and sufficient to mediate the association of PNRC with B23/nucleophosmin. Moreover, B23 depletion by siRNA disrupted the accumulation of PNRC in the nucleolus. Together, our study indicates that PNRC is a novel nucleolar protein that might be involved in regulation of pre-rRNA synthesis, and it localizes to the nucleolus by interaction with B23 via its NoLS. Our study also suggests that the stretches of six successive basic residues (lysine and/or arginine) could function as NoLS. PMID:20888865

  12. Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture

    PubMed Central

    Groen, Christopher M.; Jayo, Asier; Parsons, Maddy; Tootle, Tina L.

    2015-01-01

    Fascin, a highly conserved actin-bundling protein, localizes and functions at new cellular sites in both Drosophila and multiple mammalian cell types. During Drosophila follicle development, in addition to being cytoplasmic, Fascin is in the nuclei of the germline-derived nurse cells during stages 10B–12 (S10B–12) and at the nuclear periphery during stage 13 (S13). This localization is specific to Fascin, as other actin-binding proteins, Villin and Profilin, do not exhibit the same subcellular distribution. In addition, localization of fascin1 to the nucleus and nuclear periphery is observed in multiple mammalian cell types. Thus the regulation and function of Fascin at these new cellular locations is likely to be highly conserved. In Drosophila, loss of prostaglandin signaling causes a global reduction in nuclear Fascin and a failure to relocalize to the nuclear periphery. Alterations in nuclear Fascin levels result in defects in nucleolar morphology in both Drosophila follicles and cultured mammalian cells, suggesting that nuclear Fascin plays an important role in nucleolar architecture. Given the numerous roles of Fascin in development and disease, including cancer, our novel finding that Fascin has functions within the nucleus sheds new light on the potential roles of Fascin in these contexts. PMID:25808493

  13. Endogenous MCM7 MicroRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy

    PubMed Central

    Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L.; DiGiusto, David L.

    2012-01-01

    Abstract Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications. PMID:22834872

  14. Genotoxicity evaluation of environmental pollutants using analysis of nucleolar alterations.

    PubMed

    Mazzeo, Dânia Elisa Christofoletti; Marin-Morales, Maria Aparecida

    2015-07-01

    Nucleolar alterations resulting from the action of either chemical or physical agents can serve as important genotoxicity biomarkers. In this study, the efficiency of AgNOR banding technique to identify the presence of nucleoli in micronucleus and assess nucleolar alterations in aberrant cells of Allium cepa was evaluated. Seeds of this plant were exposed to both water samples from a river that receives untreated urban effluent and to the trifluralin herbicide (0.84 mg/L concentration), both analyzed in two different seasons (summer and winter seasons). Samples induced significant frequencies of chromosomal and nuclear aberrations and micronuclei, as observed in cells submitted to conventional chromosomal staining. The herbicide caused a significant increase in the number of nucleoli and micronuclei, interpreted as due to the elimination of excessive nucleolar material resulting from polyploidization. The use of the AgNOR technique enabled the identification of both the presence of the nucleolus in some micronuclei and the nucleolar organizer region (NOR) behavior of aberrant cells. The NOR-banding technique showed to be an efficient tool for studying the genotoxic effects caused by a xenobiotics and a complex environmental sample. PMID:25639248

  15. Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

    PubMed

    Gaspin, C; Cavaillé, J; Erauso, G; Bachellerie, J P

    2000-04-01

    Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into

  16. Nucleolar damage correlates with neurotoxicity induced by different platinum drugs

    PubMed Central

    McKeage, M J; Hsu, T; Screnci, D; Haddad, G; Baguley, B C

    2001-01-01

    Platinum-based drugs are very useful in cancer therapy but are associated with neurotoxicity in the clinic. To investigate the mechanism of neurotoxicity, dorsal root ganglia of rats treated with various platinum drugs were studied. Cell body, nuclear and nucleolar dimensions of dorsal root ganglia sensory nerve cells were measured to determine morphological toxicity. Sensory nerve conduction velocity was measured to determine functional toxicity. After a single dose of oxaliplatin (10 mg kg−1), no significant change in nuclear and cell body diameter was seen but decreased nucleolar size was apparent within a few hours of treatment. Changes in nucleolar size were maximal at 24 hours, recovered very slowly and showed a non-linear dependence on oxaliplatin dose (r2= 0.99). Functional toxicity was delayed in onset until 14 days after a single dose of oxaliplatin but eventually recovered 3 months after treatment. Multiple doses of cisplatin, carboplatin, oxaliplatin, R, R -ormaplatin and S, S -ormaplatin were also associated with time-dependent reduction in nucleolar size. A linear correlation was obtained between the rate of change in nucleolar size during multiple dose treatment with the series of platinum drugs and the time taken for the development of altered sensory nerve conduction velocity (r2= 0.86;P< 0.024). Damage to the nucleolus of ganglionic sensory neurons is therefore linked to the neurotoxicity of platinum-based drugs, possibly through mechanisms resulting in the inhibition of rRNA synthesis. © 2001 Cancer Research Campaign  http://www.bjcancer.com PMID:11710838

  17. Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

    PubMed Central

    Gagnon, Keith T.; Zhang, Xinxin; Qu, Guosheng; Biswas, Shyamasri; Suryadi, Jimmy; Brown, Bernard A.; Maxwell, E. Stuart

    2010-01-01

    The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs. PMID:19926724

  18. Nucleolar localization signals of LIM kinase 2 function as a cell-penetrating peptide.

    PubMed

    Kobayashi, Nahoko; Niwa, Mikio; Hao, Yang; Yoshida, Tetsuhiko

    2010-12-01

    LIM Kinase 2 (LIMK2) is a LIM domain-containing protein kinase which regulates actin polymerization thorough phosphorylation of the actin depolymerizing factor cofilin. It is also known to function as a shuttle between the cytoplasm and nucleus in endothelial cells. A basic amino acid-rich motif in LIMK2 was previously identified to be responsible for this shuttling function, as a nucleolar localization signal (NoLS). Here it is shown that this nucleolar localization signal sequence also has the characteristic function of a cell-penetrating peptide (CPP). We synthesized LIMK2 NoLS-conjugated peptides and a protein and analyzed their cell-penetrating abilities in various types of cells. The BC-box motif of the Von Hippel-Lindau (VHL) protein was used for the peptide. This motif previously has been reported to be involved in the neural differentiation of bone marrow stromal cells and skin-derived precursor cells. Green fluorescence protein (GFP) was used as a large biologically active biomolecule for the protein. The LIMK2 NoLS-conjugated peptides and protein translocated across the cell membranes of fibroblast cells, neural stem cells, and even iPS cells. These results suggest that LIMK2 NoLS acts as a cell-penetrating peptide and its cell-penetrating ability is not restricted by cell type. Moreover, from an in vivo assay using a mouse brain, it was confirmed that NoLS has potential for transporting biomolecules across the blood-brain barrier. PMID:20937035

  19. Expression and characterization of a functional single-chain variable fragment (scFv) protein recognizing MCF7 breast cancer cells in E. coli cytoplasm.

    PubMed

    Mahgoub, Ilham Omer

    2012-08-01

    Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production. PMID:22552770

  20. The Nucleolar Channel System of Human Endometrium Is Related to Endoplasmic Reticulum and R-Rings

    PubMed Central

    Kittur, Nupur; Zapantis, Gregory; Aubuchon, Mira; Santoro, Nanette; Bazett-Jones, David P.

    2007-01-01

    The nucleolar channel system (NCS) is a well-established ultrastructural hallmark of the postovulation endometrium. Its transient presence has been associated with human fertility. Nevertheless, the biogenesis, composition, and function of these intranuclear membrane cisternae are unknown. Membrane systems with a striking ultrastructural resemblance to the NCS, termed R-rings, are induced in nuclei of tissue culture cells by overexpression of the central repeat domain of the nucleolar protein Nopp140. Here we provide a first molecular characterization of the NCS and compare the biogenesis of these two enigmatic organelles. Like the R-rings, the NCS consists of endoplasmic reticulum harboring the marker glucose-6-phosphatase. R-ring formation initiates at the nuclear envelope, apparently by a calcium-mediated Nopp140-membrane interaction, as supported by the calcium-binding ability of Nopp140, the inhibition of R-ring formation by calcium chelators, and the concentration of Nopp140 and complexed calcium in R-rings. Although biogenesis of the NCS may initiate similarly, the reduced presence of complexed calcium and Nopp140 suggests the involvement of additional factors. PMID:17429075

  1. Requirement of phosphatase of regenerating liver-3 for the nucleolar localization of nucleolin during the progression of colorectal carcinoma.

    PubMed

    Semba, Shuho; Mizuuchi, Eri; Yokozaki, Hiroshi

    2010-10-01

    Phosphatase of regenerating liver-3 (PRL-3) is a protein tyrosine phosphatase (PTP) that is frequently overexpressed in liver metastases of colorectal carcinomas (CRCs). The PTP activity of the PRL-3 protein is indispensable for the promotion of distant metastasis of CRC; however, little is known about the effect of PRL-3 on cell growth. In this study, we investigated a novel protein that can connect to PRL-3 to modulate the proliferation of CRC cells. In CRC-derived SW480 cells, transduction of ectopic wild-type PRL-3, but not the C104S catalytic "dead" mutant, up-regulated cell proliferation and increased the population of cells at the S and G(2) /M phases. Also, inhibition of PTP activity of the PRL-3 protein by treatment with the PRL-3 inhibitor suppressed cell proliferation in a dose-dependent manner as well as PRL-3 knockdown by RNA interference. Using a comparative study of monodimensional gel electrophoresis of immunoprecipitates from PRL-3-transfected SW480 cells and subsequent mass spectrometry analysis, nucleolar-specific protein nucleolin (NCL) was identified as a novel PRL-3-binding protein. We confirmed physiological interaction between PRL-3 and NCL, and found that PRL-3 phosphatase activity was associated with the suppression of the phospho-NCL levels and nucleolar assembly of NCL protein. In CRC cases, nucleolar NCL expression was correlated not only with higher levels of PRL-3 expression but also with frequent incidence of lymph node metastasis and a higher clinicopathologic stage. These findings suggest that NCL is involved in PRL-3-mediated cancer progression/metastasis signaling, which plays an important role in the acceleration of CRC growth. PMID:20860603

  2. Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

    PubMed Central

    Lin, Hong-En; Tsai, Wen-Yang; Liu, I-Ju; Li, Pi-Chun; Liao, Mei-Ying; Tsai, Jih-Jin; Wu, Yi-Chieh; Lai, Chih-Yun; Lu, Chih-Hsuan; Huang, Jyh-Hsiung; Chang, Gwong-Jen; Wu, Han-Chung; Wang, Wei-Kung

    2012-01-01

    Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. PMID:22235356

  3. Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprotein, MEQ.

    PubMed Central

    Liu, J L; Lee, L F; Ye, Y; Qian, Z; Kung, H J

    1997-01-01

    Marek's disease virus (MDV) is one of the most oncogenic herpesviruses and induces T lymphomas in chickens within weeks after infection. Only a limited number of viral transcripts are detected in MDV tumor samples and cell lines. One of the major transcripts encodes MEQ, a 339-amino-acid bZIP protein which is homologous to the Jun/Fos family of transcription factors. The C-terminal half of MEQ contains proline-rich repeats and, when fused to the DNA-binding domain of a yeast transcription factor, Gal4 (residues 1 to 147), exhibits transactivation function. MEQ can dimerize with itself and with c-Jun. The MEQ-c-Jun heterodimers bind to an AP-1-like enhancer within the MEQ promoter region with greater affinity than do homodimers of either protein, and they transactivate MEQ expression. Here we show that MEQ is expressed in the nucleus but, interestingly, with a predominant fraction in the nucleoli and coiled bodies. This makes MEQ the first bZIP protein to be identified in the nucleoli. MEQ contains two stretches of basic residues, designated basic region 1 (BR1) and basic region 2 (BR2). Using a series of deletion mutants, we have mapped the primary nuclear localization signal (NLS) and the sole nucleolar localization signal (NoLS) to the BR2 region. BR1 was shown to provide an auxiliary signal in nuclear translocation. To demonstrate that BR2 is an authentic NoLS, BR2 was fused to cytoplasmic v-Raf (delta gag) kinase. The BR2-Raf fusion protein was observed to migrate into the nucleoplasm and the nucleolus. The BR2 region can be further divided into two long arginine-lysine stretches, BR2N and BR2C, which are separated by the five amino acids Asn-Arg-Asp-Ala-Ala (NRDAA). We provide evidence that the requirement for nuclear translocation is less stringent than that for nucleolar translocation, as either BR2N or BR2C alone is sufficient to translocate the cytoplasmic v-Raf (delta gag) into the nucleus, but only in combination can they translocate v-Raf (delta gag

  4. Detection on immunoblot of new proteins from the microsomal fraction recognized by anti-liver-kidney microsome antibodies type 1.

    PubMed

    Ballot, E; Desbos, A; Auger, C; Monier, J C

    1996-09-01

    Previous studies have demonstrated that sera from patients with autoimmune hepatitis type 1 contain antibodies which react with proteins other than the endoplasmic reticulum integral membrane protein of apparent Mr 50,000, now known to be a cytochrome P450 of the IID subfamily. Sera from 141 patients found by immunofluorescence to be positive for anti-liver-kidney microsome antibodies type 1, and sera from 50 blood donors used as controls, were analyzed by immunoblotting experiments on rat liver microsomes, microsomal subfractions, and also microsomes subjected to various treatments, as described in the text. These fractions were characterized morphologically by electronic microscopy and biochemically by different enzymatic activities. Five bands were found to be stained more often by the patients' sera than by the controls' and with a statistically significant difference in frequency. These antigenic proteins were located at apparent Mr 62,000, 58,000, 50,000, 40,000, and 35,000. The 50,000 protein was of course more often stained than the others. Antibodies against these antigens belonged essentially to the IgG1 subclass. For some of them, subcellular localization and membrane topography are discussed. Interestingly, the 58,000 protein is not an integral membrane protein. PMID:8811045

  5. A novel DNA binding motif for yeast zinc cluster proteins: the Leu3p and Pdr3p transcriptional activators recognize everted repeats.

    PubMed Central

    Hellauer, K; Rochon, M H; Turcotte, B

    1996-01-01

    The Gal4, Put3, and Ppr1 yeast zinc cluster proteins bind as homodimers to DNA sequences composed of palindromic CGG triplets. Spacing between the triplets specifies the target site for a given zinc cluster protein. In addition, Hap1p, another zinc cluster protein, also recognizes CGG triplets but only when oriented as a direct repeat. Unexpectedly, our results show that Leu3p, another member of this family, also recognizes CGG triplets but oriented in opposite directions and spaced by 4 nucleotides (an everted repeat or inverted palindrome: CCG-N4-CGG). This constitutes a novel DNA motif for zinc cluster proteins. Moreover, the presence of this motif was shown to be essential for in vivo activation by Leu3p of a minimal reporter containing one copy of a target site for this activator. We also provide evidence that another member of this family, Pdr3p, binds to an everted repeat spaced by 0 nucleotides (CCGCGG). Thus, our results show that three CGG motifs are used by members of the zinc cluster family: palindromes, direct repeats, and everted repeats. PMID:8887639

  6. Identification of immunoreactive proteins from the dog heartworm (Dirofilaria immitis) differentially recognized by the sera from dogs with patent or occult infections.

    PubMed

    Oleaga, Ana; Pérez-Sánchez, Ricardo; Pagés, Elaine; Marcos-Atxutegi, Cristina; Simón, Fernando

    2009-08-01

    Heartworm disease caused by Dirofilaria immitis affects canine and feline hosts. Moreover, the parasite can infect humans, causing pulmonary dirofilariosis. Most affected dogs have patent infections with circulating microfilariae in peripheral blood, although infected dogs sometimes develop occult infections characterized by the absence of microfilariae. Microfilaremic infections (mf+) are associated with a predominant Th2-type immune response, whereas a Th1-type response predominates in amicrofilaremic infections (mf-), suggesting a role for this response in the suppression of circulating microfilariae. However, nothing is known about the molecules involved in the immune regulation of these infections. The objective of the present work was to identify the parasite proteins recognized differentially by the immune response of dogs with patent or occult infections, using two-dimensional electrophoresis and mass spectrometry. Nineteen proteins of D. immitis were identified, of which 6 were immunoreactive against serum samples from both mf+ and mf- dogs, while another two groups of 6 and 7 different proteins were differentially recognized by sera from mf+ or mf- dogs, respectively. The results point to the existence of differential antigen recognition in patent and occult infections due to D. immitis. Several proteins that could be involved in the immune regulation of these infections are identified. Additionally, the findings seem to suggest that some antigens of D. immitis, together with Wolbachia antigens, could contribute to the stimulation of the Th1-type response. PMID:19450730

  7. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    SciTech Connect

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.; Lurz, Rudi; Calendar, Richard; Klumpp, Jochen; Loessner, Martin J.

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  8. Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1.

    PubMed

    Zhang, Lisha; Kars, Ilona; Essenstam, Bert; Liebrand, Thomas W H; Wagemakers, Lia; Elberse, Joyce; Tagkalaki, Panagiota; Tjoitang, Devlin; van den Ackerveken, Guido; van Kan, Jan A L

    2014-01-01

    Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis. PMID:24259685

  9. Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES11[W

    PubMed Central

    Zhang, Lisha; Kars, Ilona; Essenstam, Bert; Liebrand, Thomas W.H.; Wagemakers, Lia; Elberse, Joyce; Tagkalaki, Panagiota; Tjoitang, Devlin; van den Ackerveken, Guido; van Kan, Jan A.L.

    2014-01-01

    Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis. PMID:24259685

  10. Identification and purification of a novel 120-kDa protein that recognizes the cAMP-responsive element

    SciTech Connect

    Andrisani, O.; Dixon, J.E. )

    1990-02-25

    The TGACGTCA (CRE) motif required for function by a number of cellular (somatostatin, enkephalin, alpha-human chorionic gonadotropin) and viral (Ad5 E1A-inducible, HTLV-1 TAX-inducible) genes is the site of interaction of multiple sequence-specific complexes. A protocol has been developed for the fractionation and purification of these activities. We report here the purification from HeLa nuclear extracts of a novel 120-kDa polypeptide which by Southwestern blots, gel retardation, and UV cross-linking assays displays CRE-specific binding. The CRE-affinity purified 120-kDa protein displays properties distinct from those of the 43-kDa CREB/ATF polypeptide. The 120-kDa protein is readily phosphorylated in vitro by protein kinase C but not by protein kinase A, suggesting that this molecule may mediate cellular signals distinct from the cAMP-responsive pathway. In vitro transcription-complementation assays utilizing the purified 120-kDa protein failed to transactivate the cAMP-responsive somatostatin promoter suggesting that the mode of action of this 120-kDa polypeptide may require an activation step distinct from the cAMP-signaling pathway.

  11. Synthetic peptides derived from the Wilms' tumor 1 protein sensitize human T lymphocytes to recognize chronic myelogenous leukemia cells.

    PubMed

    Müller, Ludmila; Knights, Ashley; Pawelec, Graham

    2003-01-01

    The Wilms' tumour 1 (WT1) molecule was screened in silico for the presence of 15-mer sequences predicted to bind HLA-DRB1(*)0401 (www.syfpeithi.de). Two peptides with the highest binding scores were synthesized (WT12e, PQQMGSDVRDLNALL and WT331, NKRYFKLSHLQMHSR). In vitro sensitization experiments using PBMC and the 15-mer peptides yielded peptide-specific responses against both WT12e and WT331 from six of seven healthy donors. Moreover, four of four different primary CML cell preparations were directly recognized by five different T cell lines, as assessed by IFN-gamma release. These responses were to a great extent blocked by anti-DR monoclonal antibody. These results suggest that WT1 peptides can be selected that are immunogenic for class II-restricted T-cell responses to native tumor cells, and indicate that they may find application in active immunotherapy of CML. PMID:12692522

  12. Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration

    SciTech Connect

    Furneaux, H.M.; Dropcho, E.J.; Barbut, D.; Chen, Yaotseng; Rosenblum, M.K.; Old, L.J.; Posner, J.B. )

    1989-04-01

    Paraneoplastic cerebellar degeneration is a neurological disorder of unknown cause occurring in patients with an identified or occult cancer. An autoimmune etiology is likely since autoantibodies directed against the Purkinje cells of the cerebellum have been found in the serum and cerebrospinal fluid of some patients. Two Purkinje cell-specific antigens are recognized by these autoantibodies, a major antigen of 62 kDa (CDR 62, cerebellar degeneration-related 62-kDa protein) and a minor antigen of 34 kDa (CDR 34). Previous studies have described the isolation and characterization of a human cerebellar cDNA that encodes an epitope recognized by sera from patients with paraneoplastic cerebellar degeneration. The authors have now established by two independent methods that this gene is uniquely expressed in Purkinje cells of the cerebellum and corresponds to the minor antigen CDR 34. This antigen is also expressed in tumor tissue from a patient with paraneoplastic cerebellar degeneration.

  13. Phages harboring specific peptides that recognize the N protein of the porcine reproductive and respiratory syndrome virus distinguish the virus from other viruses.

    PubMed

    Ren, Xiaofeng; Wang, Mingcui; Yin, Jiechao; Li, Guangxing

    2010-05-01

    The aim of the current study was to develop a novel diagnostic test for detecting porcine reproductive and respiratory syndrome virus (PRRSV) using phage display technology. The N gene of PRRSV isolate HH08 was cloned following reverse transcription-PCR. Sequence comparison indicated that the N gene shared 96.4% homology to that of North American PRRSV (isolate VR2332) and 35.5% with that of European PRRSV (isolate LV), indicating that the PRRSV isolate was related to the North American PRRSV genotype. The bacterially expressed N protein was used as a target in a biopanning process using a phage display random peptide library. Seven phages expressing different peptides had a specific binding activity with the N protein. The putative binding motifs were identified by DNA sequencing. More importantly, the selected phages harboring specific peptides that recognize the N protein of PRRSV were able to efficiently distinguish PRRSV from other viruses in enzyme-linked immunosorbent assays. PMID:20237096

  14. Early effects of altered gravity environments on plant cell growth and cell proliferation: Characterization of morphofunctional nucleolar types in an Arabidopsis cell culture system

    NASA Astrophysics Data System (ADS)

    Manzano, Ana Isabel; Herranz, Raul; Manzano, Aránzazu; Van Loon, Jack; Medina, Francisco Javier

    2016-02-01

    Changes in the cell growth rate of an in vitro cellular system in Arabidopsis thaliana induced by short exposure to an altered gravity environment have been estimated by a novel approach. The method consisted of defining three structural nucleolar types which are easy and reliable indicators of the ribosome biogenesis activity and, consequently, of protein biosynthesis, a parameter strictly correlated to cell growth in this cellular system. The relative abundance of each nucleolar type was statistically assessed in different conditions of gravity. Samples exposed to simulated microgravity for 200 min showed a significant decrease in nucleolar activity compared to 1g controls, whereas samples exposed to hypergravity (2g) for the same period showed nucleolar activity slightly increased,. These effects could be considered as an early cellular response to the environmental alteration, given the short duration of the treatment. The functional significance of the structural data was validated by a combination of several different well-known parameters, using microscopical, flow cytometry, qPCR and proteomic approaches, which showed that the decreased cell growth rate was decoupled from an increased cell proliferation rate under simulated microgravity, and the opposite trend was observed under hypergravity. Actually, not all parameters tested showed the same quantitative changes, indicating that the response to the environmental alteration is time-dependent. These results are in agreement with previous observations in root meristematic cells and they show the ability of plant cells to produce a response to gravity changes, independently of their integration into plant organs.

  15. Antibodies from women urogenitally infected with C. trachomatis predominantly recognized the plasmid protein pgp3 in a conformation-dependent manner

    PubMed Central

    Li, Zhongyu; Zhong, Youmin; Lei, Lei; Wu, Yimou; Wang, Shiping; Zhong, Guangming

    2008-01-01

    Background C. trachomatis organisms carry a cryptic plasmid that encodes 8 open reading frames designated as pORF1 to 8. It is not clear whether all 8 pORFs are expressed during C. trachomatis infection in humans and information on the functionality of the plasmid proteins is also very limited. Results When antibodies from women urogenitally infected with C. trachomatis were reacted with the plasmid proteins, all 8 pORFs were positively recognized by one or more human antibody samples with the recognition of pORF5 protein (known as pgp3) by most antibodies and with the highest titers. The antibody recognition of the pORFs was blocked by C. trachomatis-infected HeLa but not normal HeLa cell lysates. The pgp3 fusion protein-purified human IgG detected the endogenous pgp3 in the cytosol of C. trachomatis-infected cells with an intracellular distribution pattern similar to that of CPAF, a chlamydial genome-encoded protease factor. However, the human antibodies no longer recognized pgp3 but maintained recognition of CPAF when both antigens were linearized or heat-denatured. The pgp3 conformation is likely maintained by the C-terminal 75% amino acid sequence since further deletion blocked the binding by the human antibodies and two conformation-dependent mouse monoclonal antibodies. Conclusion The plasmid-encoded 8 proteins are both expressed and immunogenic with pgp3 as the most immunodominant antigen during chlamydial infection in humans. More importantly, the human anti-pgp3 antibodies are highly conformation-dependent. These observations have provided important information for further understanding the function of the plasmid-encoded proteins and exploring the utility of pgp3 in chlamydial diagnosis and vaccination. PMID:18541036

  16. The shared genomic architecture of human nucleolar organizer regions

    PubMed Central

    Floutsakou, Ioanna; Agrawal, Saumya; Nguyen, Thong T.; Seoighe, Cathal; Ganley, Austen R.D.; McStay, Brian

    2013-01-01

    The short arms of the five acrocentric human chromosomes harbor sequences that direct the assembly and function of the nucleolus, one of the key functional domains of the nucleus, yet they are absent from the current human genome assembly. Here we describe the genomic architecture of these human nucleolar organizers. Sequences distal and proximal to ribosomal gene arrays are conserved among the acrocentric chromosomes, suggesting they are sites of frequent recombination. Although previously believed to be heterochromatic, characterization of these two flanking regions reveals that they share a complex genomic architecture similar to other euchromatic regions of the genome, but they have distinct genomic characteristics. Proximal sequences are almost entirely segmentally duplicated, similar to the regions bordering centromeres. In contrast, the distal sequence is predominantly unique to the acrocentric short arms and is dominated by a very large inverted repeat. We show that the distal element is localized to the periphery of the nucleolus, where it appears to anchor the ribosomal gene repeats. This, combined with its complex chromatin structure and transcriptional activity, suggests that this region is involved in nucleolar organization. Our results provide a platform for investigating the role of NORs in nucleolar formation and function, and open the door for determining the role of these regions in the well-known empirical association of nucleoli with pathology. PMID:23990606

  17. A Phytophthora sojae Glycoside Hydrolase 12 Protein Is a Major Virulence Factor during Soybean Infection and Is Recognized as a PAMP[OPEN

    PubMed Central

    Ma, Zhenchuan; Song, Tianqiao; Zhu, Lin; Ye, Wenwu; Wang, Yang; Shao, Yuanyuan; Dong, Suomeng; Zhang, Zhengguang; Dou, Daolong; Zheng, Xiaobo; Tyler, Brett M.; Wang, Yuanchao

    2015-01-01

    We identified a glycoside hydrolase family 12 (GH12) protein, XEG1, produced by the soybean pathogen Phytophthora sojae that exhibits xyloglucanase and β-glucanase activity. It acts as an important virulence factor during P. sojae infection but also acts as a pathogen-associated molecular pattern (PAMP) in soybean (Glycine max) and solanaceous species, where it can trigger defense responses including cell death. GH12 proteins occur widely across microbial taxa, and many of these GH12 proteins induce cell death in Nicotiana benthamiana. The PAMP activity of XEG1 is independent of its xyloglucanase activity. XEG1 can induce plant defense responses in a BAK1-dependent manner. The perception of XEG1 occurs independently of the perception of ethylene-inducing xylanase. XEG1 is strongly induced in P. sojae within 30 min of infection of soybean and then slowly declines. Both silencing and overexpression of XEG1 in P. sojae severely reduced virulence. Many P. sojae RXLR effectors could suppress defense responses induced by XEG1, including several that are expressed within 30 min of infection. Therefore, our data suggest that PsXEG1 contributes to P. sojae virulence, but soybean recognizes PsXEG1 to induce immune responses, which in turn can be suppressed by RXLR effectors. XEG1 thus represents an apoplastic effector that is recognized via the plant’s PAMP recognition machinery. PMID:26163574

  18. The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

    SciTech Connect

    Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.

    2009-09-02

    The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N{sub x})AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5{prime} splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine 'ladder.' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

  19. A Phytophthora sojae Glycoside Hydrolase 12 Protein Is a Major Virulence Factor during Soybean Infection and Is Recognized as a PAMP.

    PubMed

    Ma, Zhenchuan; Song, Tianqiao; Zhu, Lin; Ye, Wenwu; Wang, Yang; Shao, Yuanyuan; Dong, Suomeng; Zhang, Zhengguang; Dou, Daolong; Zheng, Xiaobo; Tyler, Brett M; Wang, Yuanchao

    2015-07-01

    We identified a glycoside hydrolase family 12 (GH12) protein, XEG1, produced by the soybean pathogen Phytophthora sojae that exhibits xyloglucanase and β-glucanase activity. It acts as an important virulence factor during P. sojae infection but also acts as a pathogen-associated molecular pattern (PAMP) in soybean (Glycine max) and solanaceous species, where it can trigger defense responses including cell death. GH12 proteins occur widely across microbial taxa, and many of these GH12 proteins induce cell death in Nicotiana benthamiana. The PAMP activity of XEG1 is independent of its xyloglucanase activity. XEG1 can induce plant defense responses in a BAK1-dependent manner. The perception of XEG1 occurs independently of the perception of ethylene-inducing xylanase. XEG1 is strongly induced in P. sojae within 30 min of infection of soybean and then slowly declines. Both silencing and overexpression of XEG1 in P. sojae severely reduced virulence. Many P. sojae RXLR effectors could suppress defense responses induced by XEG1, including several that are expressed within 30 min of infection. Therefore, our data suggest that PsXEG1 contributes to P. sojae virulence, but soybean recognizes PsXEG1 to induce immune responses, which in turn can be suppressed by RXLR effectors. XEG1 thus represents an apoplastic effector that is recognized via the plant's PAMP recognition machinery. PMID:26163574

  20. Comparative genomics of Fusarium oxysporum f. sp. melonis reveals the secreted protein recognized by the Fom-2 resistance gene in melon.

    PubMed

    Schmidt, Sarah Maria; Lukasiewicz, Joanna; Farrer, Rhys; van Dam, Peter; Bertoldo, Chiara; Rep, Martijn

    2016-01-01

    Development of resistant crops is the most effective way to control plant diseases to safeguard food and feed production. Disease resistance is commonly based on resistance genes, which generally mediate the recognition of small proteins secreted by invading pathogens. These proteins secreted by pathogens are called 'avirulence' proteins. Their identification is important for being able to assess the usefulness and durability of resistance genes in agricultural settings. We have used genome sequencing of a set of strains of the melon wilt fungus Fusarium oxysporum f. sp. melonis (Fom), bioinformatics-based genome comparison and genetic transformation of the fungus to identify AVRFOM2, the gene that encodes the avirulence protein recognized by the melon Fom-2 gene. Both an unbiased and a candidate gene approach identified a single candidate for the AVRFOM2 gene. Genetic complementation of AVRFOM2 in three different race 2 isolates resulted in resistance of Fom-2-harbouring melon cultivars. AvrFom2 is a small, secreted protein with two cysteine residues and weak similarity to secreted proteins of other fungi. The identification of AVRFOM2 will not only be helpful to select melon cultivars to avoid melon Fusarium wilt, but also to monitor how quickly a Fom population can adapt to deployment of Fom-2-containing cultivars in the field. PMID:26305378

  1. Polyclonal Antibodies Recognizing the AmoB Protein of Ammonia Oxidizers of the β-Subclass of the Class Proteobacteria

    PubMed Central

    Pinck, Claudia; Coeur, Caroline; Potier, Patrick; Bock, Eberhard

    2001-01-01

    A 41-kDa protein of Nitrosomonas eutropha was purified, and the N-terminal amino acid sequence was found to be nearly identical with the sequence of AmoB, a subunit of ammonia monooxygenase. This protein was used to develop polyclonal antibodies, which were highly specific for the detection of the four genera of ammonia oxidizers of the β-subclass of Proteobacteria (Nitrosomonas, including Nitrosococcus mobilis, which belongs phylogenetically to Nitrosomonas; Nitrosospira; Nitrosolobus; and Nitrosovibrio). In contrast, the antibodies did not react with ammonia oxidizers affiliated with the γ-subclass of Proteobacteria (Nitrosococcus oceani and Nitrosococcus halophilus). Moreover, methane oxidizers (Methylococcus capsulatus, Methylocystis parvus, and Methylomonas methanica) containing the related particulate methane monooxygenase were not detected. Quantitative immunoblot analysis revealed that total cell protein of N. eutropha consisted of approximately 6% AmoB, when cells were grown using standard conditions (mineral medium containing 10 mM ammonium). This AmoB amount was shown to depend on the ammonium concentration in the medium. About 14% AmoB of total protein was found when N. eutropha was grown with 1 mM ammonium, whereas 4% AmoB was detected when 100 mM ammonium were used. In addition, the cellular amount of AmoB was influenced by the absence of the substrate. Cells starved for more than 2 months contained nearly twice as much AmoB as actively growing cells, although these cells possessed low ammonia-oxidizing activity. AmoB was always present and could even be detected in cells of Nitrosomonas after 1 year of ammonia starvation. PMID:11133435

  2. Three different proteins recognize a multifunctional determinant that controls replication initiation, fork arrest and transcription in Tetrahymena.

    PubMed

    Mohammad, M; Saha, S; Kapler, G M

    2000-02-01

    Type I elements regulate the initiation of DNA replication, elongation of replication forks and transcription of the Tetrahymena thermophila rDNA minichromosome. Previous studies identified a 24 kDa protein, ssA-TIBF, which binds the A-rich strand of type I elements. Here we describe two additional type I element binding activities (native mol. wt approximately 65 and approximately 250 kDa) that interact with DNA via previously unidentified 32 and 110 kDa polypeptides. The 65 kDa activity was purified to homogeneity and consists of a homodimer of a 32 kDa polypeptide. In contrast to the other type I element binding factors, the 65 kDa activity partitions preferentially to the nuclear fraction during isolation. Levels of the 65 kDa activity increase dramatically in starved cells, raising the possibility that it might negatively regulate replication or transcription. By comparison, the other two binding activities were elevated slightly during macronuclear development, when the rDNA was undergoing DNA replication. Previous studies indicate that the initiation of rDNA replication is regulated by long range interactions between dispersed type I elements. Competitive DNA binding or cooperative protein-protein interactions between the factors described here may play a regulatory role in replication or expression of the rDNA minichromosome. PMID:10637338

  3. Nucleolar Integrity Is Required for the Maintenance of Long-Term Synaptic Plasticity

    PubMed Central

    Allen, Kim D.; Gourov, Andrei V.; Harte, Christopher; Gao, Peng; Lee, Clarice; Sylvain, Darlene; Splett, Joshua M.; Oxberry, William C.; van de Nes, Paula S.; Troy-Regier, Matthew J.; Wolk, Jason; Alarcon, Juan M.; Hernández, A. Iván

    2014-01-01

    Long-term memory (LTM) formation requires new protein synthesis and new gene expression. Based on our work in Aplysia, we hypothesized that the rRNA genes, stimulation-dependent targets of the enzyme Poly(ADP-ribose) polymerase-1 (PARP-1), are primary effectors of the activity-dependent changes in synaptic function that maintain synaptic plasticity and memory. Using electrophysiology, immunohistochemistry, pharmacology and molecular biology techniques, we show here, for the first time, that the maintenance of forskolin-induced late-phase long-term potentiation (L-LTP) in mouse hippocampal slices requires nucleolar integrity and the expression of new rRNAs. The activity-dependent upregulation of rRNA, as well as L-LTP expression, are poly(ADP-ribosyl)ation (PAR) dependent and accompanied by an increase in nuclear PARP-1 and Poly(ADP) ribose molecules (pADPr) after forskolin stimulation. The upregulation of PARP-1 and pADPr is regulated by Protein kinase A (PKA) and extracellular signal-regulated kinase (ERK)—two kinases strongly associated with long-term plasticity and learning and memory. Selective inhibition of RNA Polymerase I (Pol I), responsible for the synthesis of precursor rRNA, results in the segmentation of nucleoli, the exclusion of PARP-1 from functional nucleolar compartments and disrupted L-LTP maintenance. Taken as a whole, these results suggest that new rRNAs (28S, 18S, and 5.8S ribosomal components)—hence, new ribosomes and nucleoli integrity—are required for the maintenance of long-term synaptic plasticity. This provides a mechanistic link between stimulation-dependent gene expression and the new protein synthesis known to be required for memory consolidation. PMID:25089620

  4. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  5. A commercial human protamine-2 antibody used in several studies to detect mouse protamine-2 recognizes mouse transition protein-2 but not protamine-2.

    PubMed

    Eckhardt, Matthias; Wang-Eckhardt, Lihua

    2015-11-01

    The exchange of histones for transition proteins (TNPs) and finally protamines is an essential process during spermatogenesis that enables the strong condensation of chromatin during sperm formation. Research on this process obviously depends on the availability of specific antibodies recognizing these nuclear proteins. A commercial antibody generated against human protamine-2 (PRM2) has been described to cross-react with mouse PRM2 and in fact has been used in several studies to detect mouse PRM2. Some inconsistent results obtained with this goat-derived antibody prompted us to re-examine its specificity. In immunofluorescence experiments with epididymal sperm, only a low percentage of sperm nuclei were stained by this antibody, whereas a mouse monoclonal anti- PRM2 antibody stained most sperm, as expected. Western blot analysis of basic nuclear proteins from spermatids and sperm separated by acid urea (AU) gel electrophoresis revealed that the goat anti- PRM2 antiserum binds to mouse TNP2 but not mouse PRM2. Epitope mapping using glutathione-S-transferase-fusion proteins with peptide sequences conserved in human PRM2 and mouse TNP2 identified the tetrapeptide arginyl-lysyl-arginyl-threonine as an epitope of the goat anti- PRM2 antiserum. Our findings underline the importance of using AU gel electrophoresis to confirm specificities of antibodies directed against basic nuclear proteins, which are not well separated, and may show abnormal migration behaviour, in SDS-polyacrylamide gel electrophoresis. PMID:26268249

  6. Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qbeta replicase efficiently binds small RNAs by recognizing pyrimidine residues.

    PubMed

    Preuss, R; Dapprich, J; Walter, N G

    1997-10-31

    Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qbeta was studied utilizing a fluorometric assay. A DNA oligonucleotide probe of sequence 5'-d(TTTTTCC) was 5'-end-labeled with pyrene. In this construct, the proximal thymine residues efficiently quench the fluorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state fluorescence increases by two orders of magnitude, the fluorescence anisotropy increases, and a long fluorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state fluorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely reflects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qbeta replicase. The effect was utilized to access binding affinities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16 degrees C, equilibrium dissociation constants for different template midi- and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qbeta replicase during replication, exhibited discriminative affinities, while their hybrid bound less efficiently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qbeta replicase does not require a 2' hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A.T base-pair, while that with cytosine is retained after Watson-Crick base-pairing. These findings can explain the affinities of RNA-Qbeta replicase interactions reported here and in earlier investigations. The sensitivity of the described fluorometric assay allows

  7. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    DOEpatents

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  8. [Studies on changes in nucleolar organizer region of human promyelocytic leukemia cells (HL-60) treated with retinoic acid].

    PubMed

    Xie, R L; Wang, Y Q

    1989-12-01

    Changes of nucleolar organizer region in HL-60 cells after treated with retinoic acid (RA) were studied with techniques of silver-staining nucleolar organizer region (Ag-NOR) in metaphase karyotypes, Brachet's reaction and with our improved TEM techniques for studying silver-stained active nucleolar organizer region (Ag-aNOR) in interphase nucleoli. Number of Ag-NOR in HL-60 cells is 4.5/cell on average. The Ag-NOR number of cells treated with RA showed no remarkable difference from that of control group. Ag-aNOR number treated with RA was reduced obviously as compared with that of control group. Meanwhile, the changes of nucleolus number showed by Brachet's reaction were in accordance with those of Ag-aNOR. Therefore, it may be concluded: (1). Though the number of active rRNA genes did not changed after the differentiation of HL-60 cells induced by RA, their expression was clearly inhibited: (2). The relationship between the changes of Brachet-No and Ag-aNOR is in positive correlation (r = 0.98, p less than 0.01). EM examination of Ag-aNOR of HL-60 cells reveals that Ag-protein (RNA polymerase I) only presented in fibrillar centers (FC) and the dense fibrillar components (DFC) of nucleolus. In addition, in control group, large amount of Ag-protein, FC, DFC and granular components (GC) were observed, and there were many large nucleoli in a nucleus, meanwhile, the cells of the treated group tended to be mature, with a decrease in the amount of Ag-protein, FC, DFC and GC accordingly, and the nucleoli reduced both in size and number significantly.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2626898

  9. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    SciTech Connect

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. )

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  10. Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin.

    PubMed

    Weisemann, Jasmin; Stern, Daniel; Mahrhold, Stefan; Dorner, Brigitte G; Rummel, Andreas

    2016-01-01

    Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A-C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain HC of BoNT/A. Here, we deciphered the BoNT/A-SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C > SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A-SV2C interaction is of different nature compared to BoNT/B-Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone-backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A-SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. PMID:27196927

  11. Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin

    PubMed Central

    Weisemann, Jasmin; Stern, Daniel; Mahrhold, Stefan; Dorner, Brigitte G.; Rummel, Andreas

    2016-01-01

    Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A‐C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain HC of BoNT/A. Here, we deciphered the BoNT/A‐SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C >> SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A‐SV2C interaction is of different nature compared to BoNT/B‐Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone–backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A‐SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. PMID:27196927

  12. Fibronectin Binding to the Salmonella enterica Serotype Typhimurium ShdA Autotransporter Protein Is Inhibited by a Monoclonal Antibody Recognizing the A3 Repeat

    PubMed Central

    Kingsley, Robert A.; Abi Ghanem, Daad; Puebla-Osorio, Nahum; Keestra, A. Marijke; Berghman, Luc; Bäumler, Andreas J.

    2004-01-01

    ShdA is a large outer membrane protein of the autotransporter family whose passenger domain binds the extracellular matrix proteins fibronectin and collagen I, possibly by mimicking the host ligand heparin. The ShdA passenger domain consists of ∼1,500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence: an N-terminal nonrepeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, called type A and type B. The repeat region bound bovine fibronectin with an affinity similar to that for the complete ShdA passenger domain, while the nonrepeat region exhibited comparatively low fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger domain to fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3, and B9. Furthermore, a monoclonal antibody that specifically recognized an epitope in a recombinant protein containing the A3 repeat inhibited binding of ShdA to fibronectin. PMID:15262930

  13. Fibronectin binding to the Salmonella enterica serotype Typhimurium ShdA autotransporter protein is inhibited by a monoclonal antibody recognizing the A3 repeat.

    PubMed

    Kingsley, Robert A; Abi Ghanem, Daad; Puebla-Osorio, Nahum; Keestra, A Marijke; Berghman, Luc; Bäumler, Andreas J

    2004-08-01

    ShdA is a large outer membrane protein of the autotransporter family whose passenger domain binds the extracellular matrix proteins fibronectin and collagen I, possibly by mimicking the host ligand heparin. The ShdA passenger domain consists of approximately 1,500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence: an N-terminal nonrepeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, called type A and type B. The repeat region bound bovine fibronectin with an affinity similar to that for the complete ShdA passenger domain, while the nonrepeat region exhibited comparatively low fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger domain to fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3, and B9. Furthermore, a monoclonal antibody that specifically recognized an epitope in a recombinant protein containing the A3 repeat inhibited binding of ShdA to fibronectin. PMID:15262930

  14. Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21

    PubMed Central

    Zhang, Cuiyan; Li, Xiaotong; Adelmant, Guillaume; Dobbins, Jessica; Geisen, Christoph; Oser, Matthew G.; Wucherpfenning, Kai W.; Marto, Jarrod A.; Kaelin, William G.

    2015-01-01

    EP300-interacting inhibitor of differentiation 1 (EID1) belongs to a protein family implicated in the control of transcription, differentiation, DNA repair, and chromosomal maintenance. EID1 has a very short half-life, especially in G0 cells. We discovered that EID1 contains a peptidic, modular degron that is necessary and sufficient for its polyubiquitylation and proteasomal degradation. We found that this degron is recognized by an Skp1, Cullin, and F-box (SCF)-containing ubiquitin ligase complex that uses the F-box Only Protein 21 (FBXO21) as its substrate recognition subunit. SCFFBXO21 polyubiquitylates EID1 both in vitro and in vivo and is required for the efficient degradation of EID1 in both cycling and quiescent cells. The EID1 degron partially overlaps with its retinoblastoma tumor suppressor protein-binding domain and is congruent with a previously defined melanoma-associated antigen-binding motif shared by EID family members, suggesting that binding to retinoblastoma tumor suppressor and melanoma-associated antigen family proteins could affect the polyubiquitylation and turnover of EID family members in cells. PMID:26631746

  15. TBMS1 exerts its cytotoxicity in NCI-H460 lung cancer cells through nucleolar stress-induced p53/MDM2-dependent mechanism, a quantitative proteomics study.

    PubMed

    Lin, Yingying; Xie, Guobin; Xia, Ji; Su, Dan; Liu, Jie; Jiang, Fuquan; Xu, Yang

    2016-02-01

    Tubeimoside-1 (TBMS1) exerts its anticancer effects by inducing G2/M arrest and apoptosis of cancer cells. However, the precise molecular mechanism of its anti-tumor effects has not been fully elucidated, especially the signaling pathways involved in the early stage of TBMS1 stimulation. In this study, we employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics approach and identified 439 proteins that exhibit significant differential expressions in NCI-H460 lung cancer cells upon exposure to TBMS1. Gene ontology and network analysis using DAVID and STRING on-line tools revealed that several nucleolar stress (ribosomal biogenesis) response proteins were differentially regulated by TBMS1. Functional validation demonstrated that TBMS1-induced NCI-H460 cell cytotoxicity involved nucleolar stress-induced p53/murine double minute clone 2 (MDM2), mTOR, and NF-κB signaling pathways. PMID:26549658

  16. Identification of a Novel Streptococcal Adhesin P (SadP) Protein Recognizing Galactosyl-α1–4-galactose-containing Glycoconjugates

    PubMed Central

    Kouki, Annika; Haataja, Sauli; Loimaranta, Vuokko; Pulliainen, Arto T.; Nilsson, Ulf J.; Finne, Jukka

    2011-01-01

    Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1–4-galactose (Galα1–4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1–4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1–4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1–4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO3 (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1–4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure. PMID:21908601

  17. Small molecule BMH-compounds that inhibit RNA polymerase I and cause nucleolar stress.

    PubMed

    Peltonen, Karita; Colis, Laureen; Liu, Hester; Jäämaa, Sari; Zhang, Zhewei; Af Hällström, Taija; Moore, Henna M; Sirajuddin, Paul; Laiho, Marikki

    2014-11-01

    Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9- and BMH-22-mediated nucleolar stress was detected in ex vivo-cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting. PMID:25277384

  18. Morphometric Analysis of Recognized Genes for Autism Spectrum Disorders and Obesity in Relationship to the Distribution of Protein-Coding Genes on Human Chromosomes.

    PubMed

    McGuire, Austen B; Rafi, Syed K; Manzardo, Ann M; Butler, Merlin G

    2016-01-01

    Mammalian chromosomes are comprised of complex chromatin architecture with the specific assembly and configuration of each chromosome influencing gene expression and function in yet undefined ways by varying degrees of heterochromatinization that result in Giemsa (G) negative euchromatic (light) bands and G-positive heterochromatic (dark) bands. We carried out morphometric measurements of high-resolution chromosome ideograms for the first time to characterize the total euchromatic and heterochromatic chromosome band length, distribution and localization of 20,145 known protein-coding genes, 790 recognized autism spectrum disorder (ASD) genes and 365 obesity genes. The individual lengths of G-negative euchromatin and G-positive heterochromatin chromosome bands were measured in millimeters and recorded from scaled and stacked digital images of 850-band high-resolution ideograms supplied by the International Society of Chromosome Nomenclature (ISCN) 2013. Our overall measurements followed established banding patterns based on chromosome size. G-negative euchromatic band regions contained 60% of protein-coding genes while the remaining 40% were distributed across the four heterochromatic dark band sub-types. ASD genes were disproportionately overrepresented in the darker heterochromatic sub-bands, while the obesity gene distribution pattern did not significantly differ from protein-coding genes. Our study supports recent trends implicating genes located in heterochromatin regions playing a role in biological processes including neurodevelopment and function, specifically genes associated with ASD. PMID:27164088

  19. Morphometric Analysis of Recognized Genes for Autism Spectrum Disorders and Obesity in Relationship to the Distribution of Protein-Coding Genes on Human Chromosomes

    PubMed Central

    McGuire, Austen B.; Rafi, Syed K.; Manzardo, Ann M.; Butler, Merlin G.

    2016-01-01

    Mammalian chromosomes are comprised of complex chromatin architecture with the specific assembly and configuration of each chromosome influencing gene expression and function in yet undefined ways by varying degrees of heterochromatinization that result in Giemsa (G) negative euchromatic (light) bands and G-positive heterochromatic (dark) bands. We carried out morphometric measurements of high-resolution chromosome ideograms for the first time to characterize the total euchromatic and heterochromatic chromosome band length, distribution and localization of 20,145 known protein-coding genes, 790 recognized autism spectrum disorder (ASD) genes and 365 obesity genes. The individual lengths of G-negative euchromatin and G-positive heterochromatin chromosome bands were measured in millimeters and recorded from scaled and stacked digital images of 850-band high-resolution ideograms supplied by the International Society of Chromosome Nomenclature (ISCN) 2013. Our overall measurements followed established banding patterns based on chromosome size. G-negative euchromatic band regions contained 60% of protein-coding genes while the remaining 40% were distributed across the four heterochromatic dark band sub-types. ASD genes were disproportionately overrepresented in the darker heterochromatic sub-bands, while the obesity gene distribution pattern did not significantly differ from protein-coding genes. Our study supports recent trends implicating genes located in heterochromatin regions playing a role in biological processes including neurodevelopment and function, specifically genes associated with ASD. PMID:27164088

  20. Conformation-dependent recognition of a protein by T-lymphocytes: apomyoglobin-specific T-cell clone recognizes conformational changes between apomyoglobin and myoglobin

    NASA Technical Reports Server (NTRS)

    Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

    1988-01-01

    A T-cell clone specific to apomyoglobin was generated. It was prepared from a T-cell culture obtained by in vitro driving of lymph node cells with apomyoglobin from SJL mice that have been primed in vivo with apomyoglobin. In proliferative assays, the T-cell clone responded to apomyoglobin but did not recognize native myoglobin or any of the synthetic peptides corresponding to the six T sites of myoglobin. The demonstration that a T-cell clone can be isolated, whose specificity is directed entirely to apomyoglobin and not to its counterpart myoglobin, with an identical amino acid composition, indicates the importance of the three-dimensional structure in the presentation of the protein to T cells.

  1. Alu element-containing RNAs maintain nucleolar structure and function.

    PubMed

    Caudron-Herger, Maïwen; Pankert, Teresa; Seiler, Jeanette; Németh, Attila; Voit, Renate; Grummt, Ingrid; Rippe, Karsten

    2015-11-12

    Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production. PMID:26464461

  2. Cyclopentenyl cytosine induces senescence in breast cancer cells through the nucleolar stress response and activation of p53.

    PubMed

    Huang, Min; Whang, Patrick; Lewicki, Patrick; Mitchell, Beverly S

    2011-07-01

    The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated β-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G(2)- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G(1) phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death. PMID:21464199

  3. Cyclopentenyl Cytosine Induces Senescence in Breast Cancer Cells through the Nucleolar Stress Response and Activation of p53S⃞

    PubMed Central

    Huang, Min; Whang, Patrick; Lewicki, Patrick

    2011-01-01

    The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated β-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G2- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G1 phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death. PMID:21464199

  4. Protein factors in Blastocladiella emersonii cell extracts recognize similar sequence elements in the promoters of the genes encoding cAMP-dependent protein kinase subunits.

    PubMed

    de Oliveira, J C; Marques, M V; Gomes, S L

    1997-08-01

    Blastocladiella emersonii contains a single cAMP-dependent protein kinase (PKA), which is similar to the mammalian type II isoforms. Its activity is regulated during development by changes in the levels of the catalytic (C) and regulatory (R) subunits, which occur in parallel with changes in levels of the corresponding mRNAs, suggesting coordinate transcriptional control of the expression of both subunits. Both R and C mRNA levels are low in vegetative cells, rise sharply during sporulation and decrease to basal levels again after germination. To investigate sequence elements common to both Blastocladiella R and C gene promoters, which might be involved in the coordinate regulation of these genes, their 5'-flanking regions were analyzed by gel mobility shift and DNase I footprinting assays. We determined that different DNA-protein complexes are generated when fragments of the R and C gene promoters are incubated with extracts from cells expressing (sporulating cells) or not expressing (vegetative cells) both subunits, and competition experiments suggested that similar protein factors bind to both promoters. DNase I footprinting experiments have indicated that a sequence common to both R and C promoters, and similar to mammalian E-boxes, binds factors present in extracts from vegetative and sporulating cells, whereas sequences flanking the E-boxes in both promoters showed a change in the pattern of DNase I digestion only when the vegetative cell extract was used. This result suggests that the composition of the protein complexes binding to these regions changes during sporulation. PMID:9294034

  5. Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution.

    PubMed Central

    Lima, Susana; Hildenbrand, Jayne; Korostelev, Andrei; Hattman, Stanley; Li, Hong

    2002-01-01

    The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com. PMID:12166647

  6. A beta-complex statistical four body contact potential combined with a hydrogen bond statistical potential recognizes the correct native structure from protein decoy sets.

    PubMed

    Sánchez-González, Gilberto; Kim, Jae-Kwan; Kim, Deok-Soo; Garduño-Juárez, Ramón

    2013-08-01

    We present a new four-body knowledge-based potential for recognizing the native state of proteins from their misfolded states. This potential was extracted from a large set of protein structures determined by X-ray crystallography using BetaMol, a software based on the recent theory of the beta-complex (β-complex) and quasi-triangulation of the Voronoi diagram of spheres. This geometric construct reflects the size difference among atoms in their full Euclidean metric; property not accounted for in a typical 3D Delaunay triangulation. The ability of this potential to identify the native conformation over a large set of decoys was evaluated. Experiments show that this potential outperforms a potential constructed with a classical Delaunay triangulation in decoy discrimination tests. The addition of a statistical hydrogen bond potential to our four-body potential allows a significant improvement in the decoy discrimination, in such a way that we are able to predict successfully the native structure in 90% of cases. PMID:23568277

  7. Regulation of protein biosynthesis by non-lymphoid cells requires the participation of receptors, which recognize the same protein through a center analogous to the antibody active center

    SciTech Connect

    Kul'berg, A.Y.; Ivanovska, N.D.; Tarkhanova, I.A.

    1986-09-01

    This paper studies the mechanism for regulating the biosynthesis of one of the complement components (anti-idiotypic antibodies CI /SUB q/ ) by macrophages. The experiments were conducted on mouse resident peritoneal macrophages cultivated in medium containing C 14-glycine. The synthesis of CI /SUB q/ was evaluated according to the content of protein which was bound by rabbit antibodies against mouse CI /SUB q/ immobilized on bromocyan-Sepharose 4B. The study of the kinetics of the biosynthesis of CI /SUB q/ by propagated macrophages shows that the biosynthesis was initially recorded and in the subsequent period the culture contained no other cells apart from macrophages.

  8. Antibodies against a novel nucleolar and cytoplasmic antigen (p105-p42) present in the sera of patients with a subset of rheumatoid arthritis (RA) with signs of scleroderma

    PubMed Central

    Labrador, M; Algueró, A; Díaz, C; Geli, C; Pérez, E; García-Valero, J; Rodriguez-Sanchez, J L; Gelpí, C

    1998-01-01

    We identified three patients (two of them relatives) with RA and signs of scleroderma whose sera contained a high titre of IgG class antibodies against the nucleoli and the nucleoplasm of cells of different mammalian origins. Sera from these patients uniformly immunoprecipitated four polypeptides, from a 35S-methionine-labelled HeLa cell extract, whose mol. wts were 120, 105, 95 and 42 kD. Of these, the 95-kD protein was highly phosphorylated. By immunoblotting, these sera reacted with 105-, 95- and 42-kD proteins and affinity-purified antibodies from these, demonstrating that 105- and 95-kD proteins shared cross-reactive epitopes. Moreover, affinity-purified antibodies from each of these proteins immunoprecipitated the whole complex. Localization studies using immunoelectron microscopy and in vivo actinomycin-D-treated cells demonstrated that the 105-, 95- and 42-kD proteins were present in the granular component of the nucleolus and the nucleoplasm. In addition, the 105- and 95-kD were present in free polyribosomes as well as ribosomes attached to endoplasmic reticulum. Pulse/chase experiments strongly suggested that the complex was accomplished shortly after a 10-min pulse. It was preferentially present in the nucleus after a 2 h chase and in both nucleus and cytoplasm after a 5 h chase. We conclude that a protein complex with a main nucleolar distribution is a new autoantigen (p105-p42) recognized by autoantibodies present in the serum of a subgroup of patients with RA and scleroderma signs. These antibodies could be useful as diagnostic markers and as tools for further studies involving the biology of the nucleolus. PMID:9822291

  9. The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors.

    PubMed

    Tafforeau, Lionel; Zorbas, Christiane; Langhendries, Jean-Louis; Mullineux, Sahra-Taylor; Stamatopoulou, Vassiliki; Mullier, Romain; Wacheul, Ludivine; Lafontaine, Denis L J

    2013-08-22

    Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents. PMID:23973377

  10. Is one-time carbon monoxide intoxication harmless? Evaluation by argyrophilic nucleolar-organizing regions staining method.

    PubMed

    Çolakoğlu, S; Saritas, A; Eroz, R; Oktay, M; Yaykasli, K O; Akoz, A; Kaya, E; Kandis, H

    2015-01-01

    In carbon monoxide (CO) poisoning, CO affects the oxygen-carrying capacity of the hemoglobin molecule. Nucleolar-organizing regions (NORs) are genetic loci on chromosomes that are composed of ribosomal DNA and proteins. NORs can be stained with silver. A total of 18 rats were exposed to CO in three different concentrations (1000, 3000, and 5000 ppm) with 6 rats as controls. The animals were euthanized 7 days after CO intoxication. Lung tissues were taken, embedded in paraffin blocks, and sectioned at 5 μm thickness. Argyrophilic nucleolar-organizing region (AgNOR) staining was carried out. One hundred nuclei per individual were evaluated, and total AgNOR number per total nuclear number and total AgNOR area per nuclear area (TAA/NA) for each nucleus were analyzed. The CO exposure groups had significantly higher TAA/NA values and AgNOR numbers than the control group (p < 0.05). Although the differences between 1000 ppm and the other two CO-exposed groups were meaningful (p < 0.05) in the TAA/NA values, there were no differences among the CO exposure groups for the AgNOR number (p > 0.05). The increase in TAA/NA value depends on the increase in the CO exposure. Significant correlations between both the AgNOR values and histopathological scoring methods were found. Therefore, AgNOR staining method may be used as an indirect indicator for evaluating the degree of cell damage rate. PMID:24812150

  11. Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

    PubMed

    Faleri, Agnese; Santini, Laura; Brier, Sébastien; Pansegrau, Werner; Lo Surdo, Paola; Scarselli, Maria; Buricchi, Francesca; Volpini, Gianfranco; Genovese, Alessia; van der Veen, Stijn; Lea, Susan; Tang, Christoph M; Savino, Silvana; Pizza, Mariagrazia; Finco, Oretta; Norais, Nathalie; Masignani, Vega

    2014-04-01

    Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes. PMID:24371123

  12. Deep Sequencing Analysis of Nucleolar Small RNAs: RNA Isolation and Library Preparation.

    PubMed

    Bai, Baoyan; Laiho, Marikki

    2016-01-01

    The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments. PMID:27576723

  13. An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

    PubMed Central

    McGrath, K E; Smothers, J F; Dadd, C A; Madireddi, M T; Gorovsky, M A; Allis, C D

    1997-01-01

    An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo. Images PMID:9017598

  14. The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI).

    PubMed

    Thiebaut, F; Rigaut, J P; Feren, K; Reith, A

    1984-01-01

    Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in "doublets" as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase. PMID:6085018

  15. A 47-kDa human nuclear protein recognized by antikinetochore autoimmune sera is homologous with the protein encoded by RCC1, a gene implicated in onset of chromosome condensation.

    PubMed Central

    Bischoff, F R; Maier, G; Tilz, G; Ponstingl, H

    1990-01-01

    Several autoimmune sera from patients with Raynaud phenomenon decorated mammalian kinetochores and bound to a 47-kDa protein on immunoblots of nuclear lysates. Antibody affinity-purified from immunoblots of the 47-kDa band recognized kinetochores, but due to crossreaction with an 18-kDa protein, localization remains elusive. We used one of these sera to purify the antigen from HeLa cells synchronized in mitosis as a noncovalent complex with a 25-kDa protein. The antigen was released from DNA by intercalation with 25 mM chloroquine. Ion-exchange chromatography yielded the pure complex with an apparent molecular size of 68 kDa, which was separated into its components by gel filtration in 6 M guanidinium chloride. Upon two-dimensional gel electrophoresis the 47-kDa protein gave two main spots of pI 6.6 and 6.7, respectively. Posttranslational modification is indicated by additional antigenic spots, by lack of a free alpha-amino group, and by chromatographic behavior of peptides on reversed-phase chromatography. The amino acid sequence for 205 residues of the 47-kDa protein has been established. This sequence is highly homologous with the translated reading frame of RCC1, a gene reportedly involved in regulating onset of mammalian chromosome condensation. Images PMID:2236072

  16. Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection.

    PubMed

    Shang, Shaobin; Siddiqui, Sarah; Bian, Yao; Zhao, Jie; Wang, Chyung-Ru

    2016-06-01

    MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules. PMID:27272249

  17. Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection

    PubMed Central

    Shang, Shaobin; Siddiqui, Sarah; Bian, Yao; Zhao, Jie; Wang, Chyung-Ru

    2016-01-01

    MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules. PMID:27272249

  18. Dynamic localization of tripartite motif-containing 22 in nuclear and nucleolar bodies

    SciTech Connect

    Sivaramakrishnan, Gayathri; Sun, Yang; Tan, Si Kee; Lin, Valerie C.L.

    2009-05-01

    Tripartite motif-containing 22 (TRIM22) exhibits antiviral and growth inhibitory properties, but there has been no study on the localization and dynamics of the endogenous TRIM22 protein. We report here that TRIM22 is dramatically induced by progesterone in MDA-MB-231-derived ABC28 cells and T47D cells. This induction was associated with an increase in TRIM22 nuclear bodies (NB), and an even more prominent increase in nucleolar TRIM22 bodies. Distinct endogenous TRIM22 NB were also demonstrated in several other cell lines including MCF7 and HeLa cells. These TRIM22 NB resemble Cajal bodies, co-localized with these structures and co-immunoprecipitated with p80-coilin. However, IFN{gamma}-induced TRIM22 in HeLa and MCF7 cells did not form NB, implying the forms and distribution of TRIM22 are regulated by specific cellular signals. This notion is also supported by the observation that TRIM22 NB undergoes dynamic cell-cycle dependent changes in distribution such that TRIM22 NB started to form in early G0/G1 but became dispersed in the S-phase. In light of its potential antiviral and antitumor properties, the findings here provide an interesting gateway to study the relationship between the different forms and functions of TRIM22.

  19. Study of nucleolar behavior during spermatogenesis in Martarega brasiliensis (Heteroptera, Notonectidae).

    PubMed

    Pereira, L L V; Alevi, K C C; Moreira, F F F; Barbosa, J F; Silistino-Souza, E R; Silva Júnior, F C; Souza-Firmino, T S; Banho, C A; Itoyama, M M

    2015-01-01

    Few cytogenetic studies have been undertaken using aquatic heteropterans and the nucleolar behavior of these insects has been described in only four species, Limnogonus aduncus, Brachymetra albinerva, Halobatopsis platensis, and Cylindrostethus palmaris. The nucleolus is a cellular structure related to biosynthetic activity and it exhibits a peculiar behavior in the heteropterans of the Triatominae subfamily; it persists during all stages of meiosis. Thus, this study aims to analyze spermatogenesis in Martarega brasiliensis, with an emphasis on nucleolar behavior. Twenty M. brasiliensis adult males were used and collected from the Municipal reservoir in the city of São José do Rio Preto, São Paulo, Brazil. The species were fixed in methanol:acetic acid (3:1), then dissected, and the testicles were extracted, torn apart, and impregnated with silver ions. During prophase, the nuclei of M. brasiliensis were composed of the nucleolus and nucleolar corpuscles, which varied in number from one to four, emphasizing that this insect has great synthetic activity during meiosis. The analysis of cells in metaphase I showed that M. brasiliensis presents a nucleolar organizing region in at least one autosome. Furthermore, the phenomenon of nucleolar persistence was not observed. All spermatids presented nucleolar markings that varied in number and position according to the stage of elongation. Moreover, it was also possible to highlight the presence of a vesicle in spermatids. Thus, this paper describes the nucleolar behavior of M. brasiliensis and highlights important characteristics during spermatogenesis, thus, increasing the knowledge about the biology of these aquatic heteropterans. PMID:26345830

  20. Regulatory Role of Small Nucleolar RNAs in Human Diseases

    PubMed Central

    Stepanov, Grigory A.; Filippova, Julia A.; Komissarov, Andrey B.; Kuligina, Elena V.; Richter, Vladimir A.; Semenov, Dmitry V.

    2015-01-01

    Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2′-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs. PMID:26060813

  1. Regulatory role of small nucleolar RNAs in human diseases.

    PubMed

    Stepanov, Grigory A; Filippova, Julia A; Komissarov, Andrey B; Kuligina, Elena V; Richter, Vladimir A; Semenov, Dmitry V

    2015-01-01

    Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2'-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs. PMID:26060813

  2. Small nucleolar RNAs as new biomarkers in chronic lymphocytic leukemia

    PubMed Central

    2013-01-01

    Background Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs are non-coding RNAs involved in the maturation of other RNA molecules. Alterations of sno/scaRNA expression may play a role in cancerogenesis. This study elucidates the patterns of sno/scaRNA expression in 211 chronic lymphocytic leukemia (CLL) patients (Binet stage A) also in comparison with those of different normal B-cell subsets. Methods The patterns of sno/scaRNA expression in highly purified CD19+ B-cells of 211 CLL patients and in 18 normal B-cell samples - 6 from peripheral blood, and 12 from tonsils (4 germinal center, 2 marginal zone, 3 switched memory and 3 naïve B-cells) - were analyzed on the Affymetrix GeneChip® Human Gene 1.0 ST array. Results CLLs display a sno/scaRNAs expression profile similar to normal memory, naïve and marginal-zone B-cells, with the exception of a few down-regulated transcripts (SNORA31, -6, -62, and -71C). Our analyses also suggest some heterogeneity in the pattern of sno/scaRNAs expression which is apparently unrelated to the major biological (ZAP-70 and CD38), molecular (IGHV mutation) and cytogenetic markers. Moreover, we found that SNORA70F was significantly down-regulated in poor prognostic subgroups and this phenomenon was associated with the down-regulation of its host gene COBLL1. Finally, we generated an independent model based on SNORA74A and SNORD116-18 expression, which appears to distinguish two different prognostic CLL groups. Conclusions These data extend the view of sno/scaRNAs deregulation in cancer and may contribute to discover novel biomarkers associated with the disease and potentially useful to predict the clinical outcome of early stage CLL patients. PMID:24004562

  3. Loss of Nucleolar Histone Chaperone NPM1 Triggers Rearrangement of Heterochromatin and Synergizes with a Deficiency in DNA Methyltransferase DNMT3A to Drive Ribosomal DNA Transcription*

    PubMed Central

    Holmberg Olausson, Karl; Nistér, Monica; Lindström, Mikael S.

    2014-01-01

    Nucleoli are prominent nuclear structures assembled and organized around actively transcribed ribosomal DNA (rDNA). The nucleolus has emerged as a platform for the organization of chromatin enriched for repressive histone modifications associated with repetitive DNA. NPM1 is a nucleolar protein required for the maintenance of genome stability. However, the role of NPM1 in nucleolar chromatin dynamics and ribosome biogenesis remains unclear. We found that normal fibroblasts and cancer cells depleted of NPM1 displayed deformed nucleoli and a striking rearrangement of perinucleolar heterochromatin, as identified by immunofluorescence staining of trimethylated H3K9, trimethylated H3K27, and heterochromatin protein 1γ (HP1γ/CBX3). By co-immunoprecipitation we found NPM1 associated with HP1γ and core and linker histones. Moreover, NPM1 was required for efficient tethering of HP1γ-enriched chromatin to the nucleolus. We next tested whether the alterations in perinucleolar heterochromatin architecture correlated with a difference in the regulation of rDNA. U1242MG glioma cells depleted of NPM1 presented with altered silver staining of nucleolar organizer regions, coupled to a modest decrease in H3K9 di- and trimethylation at the rDNA promoter. rDNA transcription and cell proliferation were sustained in these cells, indicating that altered organization of heterochromatin was not secondary to inhibition of rDNA transcription. Furthermore, knockdown of DNA methyltransferase DNMT3A markedly enhanced rDNA transcription in NPM1-depleted U1242MG cells. In summary, this study highlights a function of NPM1 in the spatial organization of nucleolus-associated heterochromatin. PMID:25349213

  4. Postembryonic Establishment of Megabase-Scale Gene Silencing in Nucleolar Dominance

    PubMed Central

    Pontes, Olga; Lawrence, Richard J.; Silva, Manuela; Preuss, Sasha; Costa-Nunes, Pedro; Earley, Keith; Neves, Nuno; Viegas, Wanda; Pikaard, Craig S.

    2007-01-01

    Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor's rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana –derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generation. PMID:17987131

  5. The identification and characterization of epitopes in the 30-34 kDa Trypanosoma cruzi proteins recognized by antibodies in the serum samples of chagasic patients.

    PubMed

    Verissimo da Costa, Giovani Carlo; Lery, Leticia Miranda Santos; da Silva, Manuela Leal; Moura, Hércules; Peralta, Regina Helena Saramago; von Krüger, Wanda Maria Almeida; Bisch, Paulo Mascarello; Barr, John R; Peralta, José Mauro

    2013-03-27

    Trypanosoma cruzi proteins with molecular weight between 30 and 34 kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34 kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools. PMID:23159400

  6. Immune Cells in Blood Recognize Tumors

    Cancer.gov

    NCI scientists have developed a novel strategy for identifying immune cells circulating in the blood that recognize specific proteins on tumor cells, a finding they believe may have potential implications for immune-based therapies.

  7. Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture.

    PubMed

    Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P H

    2014-05-01

    Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. PMID:24609384

  8. Recognizing Computational Science

    NASA Astrophysics Data System (ADS)

    Bland-Hawthorn, J.

    2006-08-01

    There are prestigious international awards that recognize the role of theory and experiment in science and mathematics, but there are no awards of a similar stature that explicitly recognize the role of computational science in a scientific field. In 1945, John von Neumann noted that "many branches of both pure and applied mathematics are in great need of computing instruments to break the present stalemate created by the failure of the purely analytical approach to nonlinear problems." In the past few decades, great strides in mathematics and in the applied sciences can be linked to computational science.

  9. Monoclonal antibodies to a rat nestin fusion protein recognize a 220-kDa polypeptide in subsets of fetal and adult human central nervous system neurons and in primitive neuroectodermal tumor cells.

    PubMed Central

    Tohyama, T.; Lee, V. M.; Rorke, L. B.; Marvin, M.; McKay, R. D.; Trojanowski, J. Q.

    1993-01-01

    Nestin is the major intermediate filament protein of embryonic central nervous system (CNS) progenitor cells. To identify proteins involved in early stages of lineage commitment in the developing human CNS we generated monoclonal antibodies to a TrpE-rat nestin fusion protein. This resulted in a monoclonal antibody (designated NST11) that did not recognize authentic human nestin, but did recognize a novel neuron-specific human polypeptide expressed in a subset of embryonic and adult CNS neurons as well as in medulloblastomas. NST11 immunoreactivity was abundant in developing spinal cord motor neurons, but was extinguished in these neurons by 17 weeks gestation. NST11 also labeled Purkinje cells at 17 weeks gestation, but Purkinje cells continued to express the NST11 antigen throughout gestation as well as in the adult cerebellum, and NST11 immunoreactivity was more abundant in Purkinje cells than in any other human CNS neurons. No NST11 immunoreactivity was detected in cells of the adult human peripheral nervous system or in a variety of adult non-neural human tissues. Further, NST11 almost exclusively stained cerebellar medulloblastomas. In Western blots of immature and mature human cerebral and cerebellar extracts, NST11 did not bind human nestin, but did detect an immunoband with a molecular weight of 220 kd. A similar immunoband was detected in medulloblastoma-derived cell lines with a neuron-like phenotype. These findings suggest that the NST11 monoclonal antibody recognizes a novel protein expressed by a subpopulation of immature and mature human CNS neurons, medulloblastomas, and medulloblastoma-derived cell lines. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7686344

  10. Comparative genomics of eukaryotic small nucleolar RNAs reveals deep evolutionary ancestry amidst ongoing intragenomic mobility

    PubMed Central

    2012-01-01

    Background Small nucleolar (sno)RNAs are required for posttranscriptional processing and modification of ribosomal, spliceosomal and messenger RNAs. Their presence in both eukaryotes and archaea indicates that snoRNAs are evolutionarily ancient. The location of some snoRNAs within the introns of ribosomal protein genes has been suggested to belie an RNA world origin, with the exons of the earliest protein-coding genes having evolved around snoRNAs after the advent of templated protein synthesis. Alternatively, this intronic location may reflect more recent selection for coexpression of snoRNAs and ribosomal components, ensuring rRNA modification by snoRNAs during ribosome synthesis. To gain insight into the evolutionary origins of this genetic organization, we examined the antiquity of snoRNA families and the stability of their genomic location across 44 eukaryote genomes. Results We report that dozens of snoRNA families are traceable to the Last Eukaryotic Common Ancestor (LECA), but find only weak similarities between the oldest eukaryotic snoRNAs and archaeal snoRNA-like genes. Moreover, many of these LECA snoRNAs are located within the introns of host genes independently traceable to the LECA. Comparative genomic analyses reveal the intronic location of LECA snoRNAs is not ancestral however, suggesting the pattern we observe is the result of ongoing intragenomic mobility. Analysis of human transcriptome data indicates that the primary requirement for hosting intronic snoRNAs is a broad expression profile. Consistent with ongoing mobility across broadly-expressed genes, we report a case of recent migration of a non-LECA snoRNA from the intron of a ubiquitously expressed non-LECA host gene into the introns of two LECA genes during the evolution of primates. Conclusions Our analyses show that snoRNAs were a well-established family of RNAs at the time when eukaryotes began to diversify. While many are intronic, this association is not evolutionarily stable across

  11. Detection on immunoblot of new proteins from the soluble fraction of the cell recognized either by anti-liver-kidney microsome antibodies type 1 or by anti-liver cytosol antibodies type 1--relationship with hepatitis C virus infection.

    PubMed

    Ballot, E; Desbos, A; Monier, J C

    1996-09-01

    Antibodies directed against liver cytosol protein, called anti-liver cytosol type 1 (LC1 Ab), have been described by both immunofluorescence (IF) and immunodiffusion techniques in sera from patients with autoimmune hepatitis (AIH). They have never been found in association with antibodies directed against the hepatitis C virus (HCV), unlike the anti-liver-kidney microsome antibodies type 1 (LKM1 Ab), the serological marker of AIH type 2. This suggests that there are two subgroups of AIH type 2, i.e., HCV-related and non-HCV-related. In this study, immunoblotting experiments were performed using proteins from the soluble phase of the rat liver cell; 141 sera which tested positive for LKM1 Ab by IF, 24 identified as having LC1 Ab by IF, and 50 from blood donors as controls were analyzed. Three bands were stained by LC1 Ab sera more often than by the control sera, and with a statistically significant frequency. These 3 proteins were located at apparent Mr 50,000, 55,000, and 60,000. The LKM1 Ab-positive sera as defined by IF stained six bands with a statistically significant frequency compared to the controls. Their apparent Mr were 35,000, 39,000, 47,000, 50,000, 55,000, and 60,000. LKM1 Ab-positive sera which were anti-HCV negative recognized a 60,000 protein belonging to the soluble phase of the cell, with a statistically significant frequency compared to LKM1 Ab-positive sera which were anti-HCV positive. This 60,000 protein was also recognized by LC1 Ab-positive sera, which were almost always anti-HCV negative. The presence of antibodies against a 60,000 protein from the soluble phase of the cell is discussed in terms of the anti-HCV serological markers found in the sera from patients with AIH. PMID:8811044

  12. Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA.

    PubMed Central

    Hughes, J M; Ares, M

    1991-01-01

    Multiple processing events are required to convert a single eukaryotic pre-ribosomal RNA (pre-rRNA) into mature 18S (small subunit), 5.8S and 25-28S (large subunit) rRNAs. We have asked whether U3 small nucleolar RNA is required for pre-rRNA processing in vivo by depleting Saccharomyces cerevisiae of U3 by conditional repression of U3 synthesis. The resulting pattern of accumulation and depletion of specific pre-rRNAs indicates that U3 is required for multiple events leading to the maturation of 18S rRNA. These include an initial cleavage within the 5' external transcribed spacer, resembling the U3 dependent initial processing event of mammalian pre-rRNA. Formation of large subunit rRNAs is unaffected by U3 depletion. The similarity between the effects of U3 depletion and depletion of U14 small nucleolar RNA and the nucleolar protein fibrillarin (NOP1) suggests that these could be components of a single highly conserved processing complex. Images PMID:1756730

  13. Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

    PubMed

    Ries, W; Hotzy, C; Schocher, I; Sleytr, U B; Sára, M

    1997-06-01

    The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi. PMID:9190804

  14. On recognizing ignorance

    NASA Technical Reports Server (NTRS)

    Greene, Richard J.

    1988-01-01

    How an expert system reasons about its own ability to deal with a particular problem is studied. Ideally, an expert system ought to rapidly recognize that a particular problem is beyond its abilities and defer to another, perhaps human, expert. This capability is extremely important in domains where expert systems may control life critical processes such as air traffic control, medicine, strategic defense, and manned space exploration. The methods used by knowledge engineers to infuse an expert system with knowledge of its own limitations is surveyed. A computability theory is employed to analyze the general problem of meta-knowledge and to give insight into the efficacy of specific solutions.

  15. Preventing and Recognizing Embezzlement.

    PubMed

    Phairas, Debra

    2016-01-01

    It is estimated that approximately one in six physicians will be the victim of embezzlement at least once during his or her lifetime. This may be due to the trusting nature of physicians, a lack of business training about separating duties in transactions involving money, or employees' feeling overworked, underpaid, or underappreciated. The best protection against embezzlement is prevention. This article informs the reader of the steps to take to prevent stealing in the medical office, how to recognize if it is occurring, and how to obtain restitution or prosecution. PMID:27039633

  16. Formation of nucleolar polymorphisms in trisomic chickens and subsequent microevolution of rRNA gene clusters in diploids.

    PubMed

    Delany, M E; Muscarella, D E; Bloom, S E

    1991-01-01

    Variations in nucleolar size are common in animals and man, yet the basis and significance of this variation are not well understood. In this report, we describe the generation de novo of individuals that express nucleolar size variations (polymorphisms) and the underlying basis for this phenotype in a vertebrate animal system (Gallus domesticus). Individuals that express nucleolar size polymorphisms were produced from mating chickens trisomic for the nucleolar organizer (NO) chromosome; 10%-18% of progeny demonstrated nucleolar polymorphisms. These progeny were incorporated into a diploid genetic line in which the polymorphic trait was observed to segregate in Mendelian fashion. An even more dramatic nucleolar size polymorphism (one macro- plus one micronucleolus) evolved in one diploid family over the course of only two generations. These individuals were used to ascertain that the polymorphic-nucleoli phenotype was expressed in tissues derived from the three primary embryonic cell layers in embryos and neonates. Image analysis was conducted on cells of these birds to quantitate the size differences between macro- and micronucleoli (5 mu2 versus 1 mu2, respectively). Finally, these birds were studied with the technique of in situ hybridization, which showed that gene number differences between homologous NO chromosomes (i.e., heterozygosity for rRNA gene copy number), underlies the polymorphic-nucleoli phenotype. Thus, the chicken emerges as an experimental system through which heterozygosity for the rRNA gene copy number can be induced, easily identified, transmitted, and expressed in all somatic tissues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2061593

  17. Recognizing musical text

    NASA Astrophysics Data System (ADS)

    Clarke, Alastair T.; Brown, B. M.; Thorne, M. P.

    1993-08-01

    This paper reports on some recent developments in a software product that recognizes printed music notation. There are a number of computer systems available which assist in the task of printing music; however the full potential of these systems cannot be realized until the musical text has been entered into the computer. It is this problem that we address in this paper. The software we describe, which uses computationally inexpensive methods, is designed to analyze a music score, previously read by a flat bed scanner, and to extract the musical information that it contains. The paper discusses the methods used to recognize the musical text: these involve sampling the image at strategic points and using this information to estimate the musical symbol. It then discusses some hard problems that have been encountered during the course of the research; for example the recognition of chords and note clusters. It also reports on the progress that has been made in solving these problems and concludes with a discussion of work that needs to be undertaken over the next five years in order to transform this research prototype into a commercial product.

  18. Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

    PubMed

    Perrera, Claudia; Colombo, Riccardo; Valsasina, Barbara; Carpinelli, Patrizia; Troiani, Sonia; Modugno, Michele; Gianellini, Laura; Cappella, Paolo; Isacchi, Antonella; Moll, Jurgen; Rusconi, Luisa

    2010-04-16

    Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis. PMID:20177074

  19. Correlative Light and Electron Microscopy of Nucleolar Transcription in Saccharomyces cerevisiae.

    PubMed

    Normand, Christophe; Berthaud, Maxime; Gadal, Olivier; Léger-Silvestre, Isabelle

    2016-01-01

    Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller's spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated "pipeline" for imaging. PMID:27576708

  20. Identification and Functional Characterization of Three NoLS (Nucleolar Localisation Signals) Mutations of the CDC73 Gene

    PubMed Central

    Baorda, Filomena; Alfarano, Michela; Chetta, Massimiliano; Muscarella, Lucia Anna; Battista, Claudia; Copetti, Massimiliano; Kotzot, Dieter; Kapelari, Klaus; Al-Abdulrazzaq, Dalia; Perlman, Kusiel; Sochett, Etienne; Cole, David E. C.; Pellegrini, Fabio; Canaff, Lucie; Hendy, Geoffrey N.; D’Agruma, Leonardo; Zelante, Leopoldo; Carella, Massimo; Scillitani, Alfredo; Guarnieri, Vito

    2013-01-01

    Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein. PMID:24340015

  1. Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

    SciTech Connect

    Wang, Chengliang; Zhang, Qiongdi; Hang, Tianrong; Tao, Yue; Ma, Xukai; Wu, Minhao; Zhang, Xuan Zang, Jianye

    2015-08-28

    The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

  2. Nucleotide sequence analysis of CDR3 elements of a panel of anti-peptide monoclonal antibodies recognizing parathyroid hormone-related protein.

    PubMed Central

    Rapley, R; Flora, P S; Walsh, D J; Walker, M R

    1993-01-01

    Nucleotide sequences of heavy (VH) and light (VL) chain variable region complementarity determining regions have been determined from in vitro amplified mRNA isolated from a panel of monoclonal antibodies (mAb) raised to a synthetic 34mer peptide representing the N-terminal portion of human parathyroid hormone-related protein (PTHrP or parathyrin) reported to contain an immunodominant epitope. These mAb vary in affinity for the synthetic peptide and native PTHrP (Ka between 5.9 x 10(8) and 1.9 x 10(11)l/M). All 10 mAb studied were found were found to utilized restricted VH2, V kappa 2, JH4 and J kappa 1 family genes. Significant differences in the length and sequence of D elements were found; however 9/10 mAb utilize members of the DSP2 family. Significantly, two broad ranges of affinity could be determined based on the presence of Asp or Ala at residue 101 in JH. Images Figure 2 PMID:8478021

  3. Human immunoglobulin G recognizing fibrinogen-binding surface proteins is protective against both Staphylococcus aureus and Staphylococcus epidermidis infections in vivo.

    PubMed

    Vernachio, John H; Bayer, Arnold S; Ames, Brenda; Bryant, Dawn; Prater, Bradley D; Syribeys, Peter J; Gorovits, Elena L; Patti, Joseph M

    2006-02-01

    A human donor-selected immunoglobulin G for intravenous injection (IGIV) product with elevated titers against the staphylococcal fibrinogen-binding MSCRAMM proteins ClfA and SdrG (INH-A21) was tested in vitro and in vivo. INH-A21 contained a significantly increased ability to inhibit the fibrinogen-binding activity of recombinant forms of both ClfA and SdrG. Evaluation of the opsonizing potential of INH-A21 was evaluated using fluorescently labeled bacteria; this assay indicated an increase in phagocytic activity compared to normal IGIV. The prophylactic efficacy of INH-A21 against an intraperitoneal challenge of methicillin-resistant Staphylococcus epidermidis (MRSE) was evaluated in a neonatal rat model. INH-A21 was also evaluated for prophylactic and therapeutic efficacy in a rabbit model of catheter-induced aortic valve infective endocarditis caused by either MRSE or methicillin-resistant Staphylococcus aureus (MRSA). Results from the in vivo models demonstrated potent prophylactic and therapeutic efficacy against both MRSE and MRSA. These data suggest that INH-A21 may be an important tool for the prevention and treatment of staphylococcal infections, especially in high-risk populations. PMID:16436704

  4. Human Immunoglobulin G Recognizing Fibrinogen-Binding Surface Proteins Is Protective against both Staphylococcus aureus and Staphylococcus epidermidis Infections In Vivo

    PubMed Central

    Vernachio, John H.; Bayer, Arnold S.; Ames, Brenda; Bryant, Dawn; Prater, Bradley D.; Syribeys, Peter J.; Gorovits, Elena L.; Patti, Joseph M.

    2006-01-01

    A human donor-selected immunoglobulin G for intravenous injection (IGIV) product with elevated titers against the staphylococcal fibrinogen-binding MSCRAMM proteins ClfA and SdrG (INH-A21) was tested in vitro and in vivo. INH-A21 contained a significantly increased ability to inhibit the fibrinogen-binding activity of recombinant forms of both ClfA and SdrG. Evaluation of the opsonizing potential of INH-A21 was evaluated using fluorescently labeled bacteria; this assay indicated an increase in phagocytic activity compared to normal IGIV. The prophylactic efficacy of INH-A21 against an intraperitoneal challenge of methicillin-resistant Staphylococcus epidermidis (MRSE) was evaluated in a neonatal rat model. INH-A21 was also evaluated for prophylactic and therapeutic efficacy in a rabbit model of catheter-induced aortic valve infective endocarditis caused by either MRSE or methicillin-resistant Staphylococcus aureus (MRSA). Results from the in vivo models demonstrated potent prophylactic and therapeutic efficacy against both MRSE and MRSA. These data suggest that INH-A21 may be an important tool for the prevention and treatment of staphylococcal infections, especially in high-risk populations. PMID:16436704

  5. Recognizing People in Motion.

    PubMed

    Yovel, Galit; O'Toole, Alice J

    2016-05-01

    Natural movements of the face and body, as well as voice, provide converging cues to a person's identity. To date, person recognition has been studied primarily with static images of faces. Face recognition, however, is part of a larger system, whose preeminent goal is to efficiently recognize dynamic familiar people in unconstrained environments. We present a comprehensive framework for understanding person recognition as it happens in the real world. In this framework, dynamic information plays the central role in binding multi-modal information from the face, body, and the voice to achieve robust and highly accurate recognition. The superior temporal sulcus (STS) integrates multisensory, dynamic information from the whole person for recognition, thereby complementing its role in social cognition. PMID:27016844

  6. Recognizing the Trends

    NASA Technical Reports Server (NTRS)

    Marley, Mark Scott

    2016-01-01

    Solar system planetary science has traditionally focused on understanding in depth individual planets. While there have been some efforts at synergy, most studies have focused on understanding the details of individual planets. Now that we are in the era of exoplanet science, with thousands of known planets and hundreds that have been characterized to varying degrees, the systematics of planetary science are becoming apparent. This also means that, for the first time, what had previously been seen as individual quirks of solar system planets are instead being recognized as part of the normal range of planetary behavior. In my talk I will consider a number of such characteristics and explain how we are now starting to understand their true context. In particular I will discuss the atmospheric composition, clouds, hazes, and winds of giant planets, trace gasses in the atmosphere of Venus, and the presence and absence of atmospheres on various terrestrial worlds.

  7. Recognizing outstanding achievements

    NASA Astrophysics Data System (ADS)

    Speiss, Fred

    One function of any professional society is to provide an objective, informed means for recognizing outstanding achievements in its field. In AGU's Ocean Sciences section we have a variety of means for carrying out this duty. They include recognition of outstanding student presentations at our meetings, dedication of special sessions, nomination of individuals to be fellows of the Union, invitations to present Sverdrup lectures, and recommendations for Macelwane Medals, the Ocean Sciences Award, and the Ewing Medal.Since the decision to bestow these awards requires initiative and judgement by members of our section in addition to a deserving individual, it seems appropriate to review the selection process for each and to urge you to identify those deserving of recognition.

  8. Uptake and cellular distribution of nucleolar targeting peptides (NrTPs) in different cell types.

    PubMed

    Rodrigues, Margarida; Andreu, David; Santos, Nuno C

    2015-03-01

    Nucleolar targeting peptides (NrTPs) are a family of cell penetrating peptides (CPPs) derived from crotamine, a rattlesnake venom toxin. They were named NrTPs for their remarkable nucleolus-homing properties and have been studied for their potential as drug delivery vehicles. Live cell microscopy experiments were conducted to monitor NrTP uptake and distribution in different cell types, including primary cells (PBMCs and erythrocytes) and different immortalized cell lines (HeLa, BHK21, BV-173, and MOLT-4). Uptake dependence on cell type (primary vs. immortalized, suspension vs. adherent, cancer vs. healthy cells), peptide concentration and cell viability were evaluated. To gain further insight on the internalization mechanism, uptake kinetics was also monitored. Results showed the uptake and distribution pattern as strongly dependent on peptide sequence, peptide concentration and membrane constituents. Under similar conditions, NrTP6 is more internalized than NrTP1, NrTP2 and NrTP5. Additionally, while internalization of NrTP7 and NrTP8 may cause cytotoxicity, NrTP6 is noncytotoxic. Higher peptide concentrations can be correlated to nucleolar targeting, although even at low concentrations a residual number of cells reveal positive nucleolar labeling. NrTPs were successfully internalized into all cell types tested except erythrocytes. PMID:25620660

  9. Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

    PubMed Central

    Jarząb, Anna; Witkowska, Danuta; Ziomek, Edmund; Dąbrowska, Anna; Szewczuk, Zbigniew; Gamian, Andrzej

    2013-01-01

    Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections. PMID:23940590

  10. Shigella flexneri 3a outer membrane protein C epitope is recognized by human umbilical cord sera and associated with protective activity.

    PubMed

    Jarząb, Anna; Witkowska, Danuta; Ziomek, Edmund; Dąbrowska, Anna; Szewczuk, Zbigniew; Gamian, Andrzej

    2013-01-01

    Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections. PMID:23940590

  11. Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

    PubMed Central

    Greco, Anna; Arata, Loredana; Soler, Eric; Gaume, Xavier; Couté, Yohann; Hacot, Sabine; Callé, Aleth; Monier, Karine; Epstein, Alberto L.; Sanchez, Jean-Charles; Bouvet, Philippe

    2012-01-01

    Herpes simplex virus type 1 (HSV-1) infection induces profound nucleolar modifications at the functional and organizational levels, including nucleolar invasion by several viral proteins. One of these proteins is US11, which exhibits several different functions and displays both cytoplasmic localization and clear nucleolar localization very similar to that of the major multifunctional nucleolar protein nucleolin. To determine whether US11 interacts with nucleolin, we purified US11 protein partners by coimmunoprecipitations using a tagged protein, Flag-US11. From extracts of cells expressing Flag-US11 protein, we copurified a protein of about 100 kDa that was further identified as nucleolin. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. This association was confirmed in HSV-1-infected cells. We found an increase in the nucleolar accumulation of US11 in nucleolin-depleted cells, thereby revealing that nucleolin could play a role in US11 nucleocytoplasmic trafficking through one-way directional transport out of the nucleolus. Since nucleolin is required for HSV-1 nuclear egress, the interaction of US11 with nucleolin may participate in the outcome of infection. PMID:22130536

  12. Nucleolin interacts with US11 protein of herpes simplex virus 1 and is involved in its trafficking.

    PubMed

    Greco, Anna; Arata, Loredana; Soler, Eric; Gaume, Xavier; Couté, Yohann; Hacot, Sabine; Callé, Aleth; Monier, Karine; Epstein, Alberto L; Sanchez, Jean-Charles; Bouvet, Philippe; Diaz, Jean-Jacques

    2012-02-01

    Herpes simplex virus type 1 (HSV-1) infection induces profound nucleolar modifications at the functional and organizational levels, including nucleolar invasion by several viral proteins. One of these proteins is US11, which exhibits several different functions and displays both cytoplasmic localization and clear nucleolar localization very similar to that of the major multifunctional nucleolar protein nucleolin. To determine whether US11 interacts with nucleolin, we purified US11 protein partners by coimmunoprecipitations using a tagged protein, Flag-US11. From extracts of cells expressing Flag-US11 protein, we copurified a protein of about 100 kDa that was further identified as nucleolin. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. This association was confirmed in HSV-1-infected cells. We found an increase in the nucleolar accumulation of US11 in nucleolin-depleted cells, thereby revealing that nucleolin could play a role in US11 nucleocytoplasmic trafficking through one-way directional transport out of the nucleolus. Since nucleolin is required for HSV-1 nuclear egress, the interaction of US11 with nucleolin may participate in the outcome of infection. PMID:22130536

  13. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.

    PubMed Central

    Ramprasad, M P; Fischer, W; Witztum, J L; Sambrano, G R; Quehenberger, O; Steinberg, D

    1995-01-01

    We have previously reported the partial purification of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that binds oxidatively modified low density lipoprotein (OxLDL) and phosphatidylserine-rich liposomes. We have now identified that protein as macrosialin, a previously cloned macrophage-restricted membrane protein in the lysosomal-associated membrane protein family (mouse homologue of human CD68). Early in the course of purification of the 94- to 97-kDa protein, a new OxLDL-binding band at 190-200 kDa appeared and copurified with the 94- to 97-kDa protein. The HPLC pattern of tryptic peptides from this higher molecular mass ligand-binding band closely matched that derived from the 94- to 97-kDa band. Specifically, the same three macrosialin-derived tryptic peptides (9, 9, and 15 residues) were present in the purified 94- to 97-kDa band and in the 190- to 200-kDa band and antisera raised against peptide sequences in macrosialin recognized both bands. An antiserum against macrosialin precipitated most of the 94- to 97-kDa OxLDL-binding material. We conclude that the binding of OxLDL to mouse macrophage membranes is in part attributable to macrosialin. Our previous studies show that OxLDL competes with oxidized red blood cells and with apoptotic thymocytes for binding to mouse peritoneal macrophages. Whether macrosialin plays a role in recognition of OxLDL and oxidatively damaged cells by intact macrophages remains uncertain. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568176

  14. Response to copper bromide exposure in Vicia sativa L. seeds: analysis of genotoxicity, nucleolar activity and mineral profile.

    PubMed

    Bellani, Lorenza M; Muccifora, Simonetta; Giorgetti, Lucia

    2014-09-01

    Copper bromide (CuBr2) effects on seed germination and plantlet development of Vicia sativa L. are evaluated through mitotic index, chromosome aberrations, nucleolar activity and mineral profile. CuBr2 induces a significant presence of micronuclei, sticky and c-metaphases, anaphase bridges and chromosome breaks. Increased number of nucleoli and scattering of AgNOR proteins from the nucleolus in the nuclear surface at CuBr2 1mM and in the cytoplasm at CuBr2 5mM, goes along with the decrease of root growth. In V. sativa embryo the content of many macro and micronutrients increases up to copper 1mM in agreement with reserve mobilization while at CuBr2 5mM some elements are present in lower amount. We hypothesize that inhibitory effects observed at 5mM are due either to a nutrient shortage or to a direct influence of copper on root cell division, evidenced by low mitotic index, high occurrence of chromosome aberrations and loss of material from the nucleolus. PMID:25011121

  15. UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity

    PubMed Central

    Sobol, Margarita; Yildirim, Sukriye; Philimonenko, Vlada V; Marášek, Pavel; Castaño, Enrique; Hozák, Pavel

    2013-01-01

    To maintain growth and division, cells require a large-scale production of rRNAs which occurs in the nucleolus. Recently, we have shown the interaction of nucleolar phosphatidylinositol 4,5-bisphosphate (PIP2) with proteins involved in rRNA transcription and processing, namely RNA polymerase I (Pol I), UBF, and fibrillarin. Here we extend the study by investigating transcription-related localization of PIP2 in regards to transcription and processing complexes of Pol I. To achieve this, we used either physiological inhibition of transcription during mitosis or inhibition by treatment the cells with actinomycin D (AMD) or 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB). We show that PIP2 is associated with Pol I subunits and UBF in a transcription-independent manner. On the other hand, PIP2/fibrillarin colocalization is dependent on the production of rRNA. These results indicate that PIP2 is required not only during rRNA production and biogenesis, as we have shown before, but also plays a structural role as an anchor for the Pol I pre-initiation complex during the cell cycle. We suggest that throughout mitosis, PIP2 together with UBF is involved in forming and maintaining the core platform of the rDNA helix structure. Thus we introduce PIP2 as a novel component of the NOR complex, which is further engaged in the renewed rRNA synthesis upon exit from mitosis. PMID:24513678

  16. Transcription-dependent nucleolar cap localization and possible nuclear function of DExH RNA helicase RHAU

    SciTech Connect

    Iwamoto, Fumiko; Stadler, Michael; Chalupnikova, Katerina; Oakeley, Edward; Nagamine, Yoshikuni

    2008-04-01

    RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments.

  17. How legumes recognize rhizobia

    PubMed Central

    Via, Virginia Dalla; Zanetti, María Eugenia; Blanco, Flavio

    2016-01-01

    ABSTRACT Legume plants have developed the capacity to establish symbiotic interactions with soil bacteria (known as rhizobia) that can convert N2 to molecular forms that are incorporated into the plant metabolism. The first step of this relationship is the recognition of bacteria by the plant, which allows to distinguish potentially harmful species from symbiotic partners. The main molecular determinant of this symbiotic interaction is the Nod Factor, a diffusible lipochitooligosaccharide molecule produced by rhizobia and perceived by LysM receptor kinases; however, other important molecules involved in the specific recognition have emerged over the years. Secreted exopolysaccharides and the lipopolysaccharides present in the bacterial cell wall have been proposed to act as signaling molecules, triggering the expression of specific genes related to the symbiotic process. In this review we will briefly discuss how transcriptomic analysis are helping to understand how multiple signaling pathways, triggered by the perception of different molecules produced by rhizobia, control the genetic programs of root nodule organogenesis and bacterial infection. This knowledge can help to understand how legumes have evolved to recognize and establish complex ecological relationships with particular species and strains of rhizobia, adjusting gene expression in response to identity determinants of bacteria. PMID:26636731

  18. Analysis of Mass Spectrometry Data for Nucleolar Proteomics Experiments.

    PubMed

    Nicolas, Armel; Bensaddek, Dalila; Lamond, Angus I

    2016-01-01

    With recent advances in experiment design, sample preparation, separation and instruments, mass spectrometry (MS)-based quantitative proteomics is becoming increasingly more popular. This has the potential to usher a new revolution in biology, in which the protein complement of cell populations can be described not only with increasing coverage, but also in all of its dimensions with unprecedented precision. Indeed, while earlier proteomics studies aimed solely at identifying as many as possible of the proteins present in the sample, newer, so-called Next Generation Proteomics studies add to this the aim of determining and quantifying the protein variants present in the sample, their mutual associations within complexes, their posttranslational modifications, their variation across the cell-cycle or in response to stimuli or perturbations, and their subcellular distribution. This has the potential to make MS proteomics much more useful for researchers, but will also mean that researchers with no background in MS will increasingly be confronted with the less-than trivial challenges of preparing samples for MS analysis, then processing and interpreting the results. In Chapter 20 , we described a workflow for isolating the protein contents of a specific SILAC-labeled organelle sample (the nucleolus) and processing it into peptides suitable for bottom-up MS analysis. Here, we complete this workflow by describing how to use the freely available MaxQuant software to convert the spectra stored in the Raw files into peptide- and protein-level information. We also briefly describe how to visualize the data using the free R scripting language. PMID:27576726

  19. Potent inhibitors of cyclin-dependent kinase 2 induce nuclear accumulation of wild-type p53 and nucleolar fragmentation in human untransformed and tumor-derived cells.

    PubMed

    David-Pfeuty, T

    1999-12-01

    The cdk2 gene has been identified as a human cdc2/CDC28-related gene that encodes a protein kinase essential for the G1/S transition in mammalian cells, but not for the G2/M transition, which requires Cdk1, another p34cdc2/CDC28 homolog. Novel potential functions of Cdk2 have been uncovered by using two potent and specific inhibitors of its kinase activity, roscovitine and olomoucine, on human wt p53-expresser untransformed and tumor-derived cells. At concentrations equal or superior to respectively 30- and 20-fold their in vitro IC50 values for cyclin B/Cdk1, cyclin A/Cdk2 and cyclin E/Cdk2, the Cdk inhibitors precipitately induce a dramatic nuclear accumulation of wt p53 and a delocalization of nucleolin from the nucleolus in all interphase cells, whatever their cell cycle status, acting in this way like the DNA-damaging drug, mitomycin C (7 microg/ml). These early events are soon followed by a nucleolar fragmentation in both normal and tumor cells in the presence of the Cdk inhibitors but not in the presence of the DNA-damaging drug. Yet, treatment with either type of compounds eventually triggers rapidly the death of the tumor cells and, much more slowly, that of the normal cells. The Cdk inhibitors, however, stimulate cell death from any stage of the cell cycle, whereas the DNA-damaging drug kills more efficaciously S phase cells. These observations provide a hint that the Cdk2 kinase might be involved in controlling the nuclear levels of the tumor suppressor wt p53 protein and in maintaining the nucleolar integrity and function, linking in this way the cell division cycle machinery to survival functions and overall cell metabolism via the control of nucleocytoplasmic transport and of ribosome production. PMID:10602500

  20. Drosophila Mbm Is a Nucleolar Myc and Casein Kinase 2 Target Required for Ribosome Biogenesis and Cell Growth of Central Brain Neuroblasts

    PubMed Central

    Hovhanyan, Anna; Herter, Eva K.; Pfannstiel, Jens; Gallant, Peter

    2014-01-01

    Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy-sensing organs into growth-promoting processes, such as sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila melanogaster. Yet the molecular function of Mbm has so far been unknown. Here we show that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which, among other functions, relays information from nutrient-dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by casein kinase 2 (CK2), which has an impact on localization of the protein. We conclude that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which, together with CK2-mediated signals, enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth. PMID:24615015

  1. Combined ligand-observe 19F and protein-observe 15N,1H-HSQC NMR suggests phenylalanine as the key Δ-somatostatin residue recognized by human protein disulfide isomerase

    PubMed Central

    Richards, Kirsty L.; Rowe, Michelle L.; Hudson, Paul B.; Williamson, Richard A.; Howard, Mark J.

    2016-01-01

    Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (Kd) of the peptide-ligand Δ-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using 19F ligand-observe and 15N,1H-HSQC protein-observe NMR methods. Phe residues in Δ-somatostatin are hypothesised as important for recognition by hPDI therefore, step-wise peptide Phe-to-Ala changes were progressively introduced and shown to raise the Kd from 103 + 47 μM until the point where binding was abolished when all Phe residues were modified to Ala. The largest step-changes in Kd involved the F11A peptide modification which implies the C-terminus of Δ-somatostatin is a prime recognition region. Furthermore, this study also validated the combined use of 19F ligand-observe and complimentary 15N,1H-HSQC titrations to monitor interactions from the protein’s perspective. 19F ligand-observe NMR was ratified as mirroring 15N protein-observe but highlighted the advantage that 19F offers improved Kd precision due to higher spectrum resolution and greater chemical environment sensitivity. PMID:26786784

  2. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

    PubMed Central

    Smith, Corey L.; Matheson, Timothy D.; Trombly, Daniel J.; Sun, Xiaoming; Campeau, Eric; Han, Xuemei; Yates, John R.; Kaufman, Paul D.

    2014-01-01

    Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus. PMID:25057015

  3. Recognizing apathy in Alzheimer's disease.

    PubMed

    Lerner, Alan J; Strauss, Milton; Sami, Susie A

    2007-11-01

    Apathy has been increasingly recognized as a neuropsychiatric symptom in many neurologic disorders. In this paper, we review the clinical features of apathy in Alzheimer's disease. We also review screening, the differential diagnosis including depression, medical illnesses, and mild cognitive impairment, and treating modalities and issues. It must also be recognized that apathy per se almost never occurs as an isolated syndrome, so it must be viewed in the context of an individual's entire behavioral and cognitive status. PMID:17999565

  4. The mechanisms determining the nucleolar-organizing regions inactivation of domestic horse chromosomes.

    PubMed

    Slota, E; Wnuk, M; Bugno, M; Pienkowska-Schelling, A; Schelling, C; Bratus, A; Kotylak, Z

    2007-06-01

    Cytogenetic investigations of the nucleolar-organizing regions (NORs) show that there is variation in the transcriptional activity of rDNA in many organisms. As a consequence, genetic polymorphism of these regions has been detected. The aim of the present study was to evaluate the hypothetic genetic mechanisms determining the NORs polymorphism of the domestic horse chromosomes. Molecular cytogenetic analyses were carried out on Hucul horses and the following techniques were used: fluorescence in situ hybridization (FISH), telomere primed in situ synthesis (PRINS), in situ nick-translation with HpaII, silver staining (AgNOR) and C-banding technique (CBG). The obtained results suggest that variation in the number and size of silver deposits is related to the number of rDNA copies, DNA methylation and the localization of ribosomal DNA loci in telomeric regions. Moreover, we have found that chromosome pairs 28 and 31 are characterized by higher variation in the NORs number. PMID:17550359

  5. Nucleolar organizer region variants as a risk factor for Down syndrome.

    PubMed Central

    Jackson-Cook, C K; Flannery, D B; Corey, L A; Nance, W E; Brown, J A

    1985-01-01

    An unusual nucleolar organizer region (NOR) heteromorphism was noted among 13 of 41 parents in whom nondisjunction leading to trisomy 21 was known to have occurred. In contrast, only one of these double NOR (dNOR) variants was found among the 41 normal spouses and none were seen among 50 control individuals. In two dNOR(+) families, a second child with trisomy 21 was conceived. In both families, the extra chromosome in each child was contributed by the parent who carried the dNOR variant and resulted from a recurrent meiosis I error. Our data suggest that the dNOR heteromorphism may play a role in meiotic nondisjunction and could be associated with as much as a 20-fold increased risk for having offspring with trisomy 21. Images Fig. 1 PMID:2934977

  6. Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

    PubMed Central

    Yamada, Hiroko; Horigome, Chihiro; Okada, Takafumi; Shirai, Chiharu; Mizuta, Keiko

    2007-01-01

    We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth. PMID:17804645

  7. Autoantibody to Th ribonucleoprotein (nucleolar 7-2 RNA protein particle) in patients with systemic sclerosis

    SciTech Connect

    Okano, Y.; Medsger, T.A. Jr. )

    1990-12-01

    We studied sera of 371 consecutive new patients with systemic sclerosis (SSc; scleroderma) who were first evaluated during 1984-1988. All sera were tested for antinuclear antibodies by immunofluorescence staining using HEp-2 cells as substrate. We excluded 219 sera showing dark nucleoli and screened for antibodies to Th in the remaining 152 sera by immunoprecipitation of a 32P-labeled HeLa cell extract. Fifteen (4.0%) of 371 sera were anti-Th+. Anti-Th antibodies were present in 14 (8.4%) of 167 SSc patients with limited cutaneous involvement, in 1 of 167 with diffuse cutaneous involvement, and in 0 of 37 with SSc overlap syndrome. Among 244 controls with other connective tissue diseases, anti-Th was detected in only 3 patients, all having primary Raynaud's phenomenon of less than 2 years duration. In the subgroup with SSc with limited cutaneous involvement, the 14 anti-Th+ patients had a significantly greater frequency of puffy fingers, small bowel involvement, and hypothyroidism, and a significantly lower frequency of arthralgia and/or arthritis. Their cumulative survival rate from the time of onset of symptoms was lower than that for anti-Th- patients (78% versus 91% at 10 years), primarily due to 3 deaths from pulmonary arterial hypertension (2 from primary pulmonary hypertension and 1 from pulmonary hypertension secondary to pulmonary interstitial fibrosis). Serum anti-Th antibodies are present almost exclusively in patients with SSc with limited cutaneous involvement or in those with primary Raynaud's phenomenon whose disease may evolve to SSc with limited cutaneous involvement, and these antibodies may identify those patients who are at greater risk for reduced survival.

  8. A Global Identification and Analysis of Small Nucleolar RNAs and Possible Intermediate-Sized Non-Coding RNAs in Oryza sativa

    PubMed Central

    Liu, Ting-Ting; Zhu, Danmeng; Chen, Wei; Deng, Xing Wang

    2013-01-01

    Accumulating evidence suggests that non-coding RNAs (ncRNAs) are both widespread and functionally important in many eukaryotic organisms. In this study, we employed a special size fractionation and cDNA library construction method followed by 454 deep sequencing to systematically profile rice intermediate-size ncRNAs. Our analysis resulted in the identification of 1349 ncRNAs in total, including 754 novel ncRNAs of an unknown functional category. Chromosome distribution of all identified ncRNAs showed no strand bias, and displayed a pattern similar to that observed in protein-coding genes with few chromosome dependencies. More than half of the ncRNAs were centered around the plus-strand of the 5’ and 3’ termini of the coding regions. The majority of the novel ncRNAs were rice specific, while 78% of the small nucleolar RNAs (snoRNAs) were conserved. Tandem duplication drove the expansion of over half of the snoRNA gene families. Furthermore, 90% of the snoRNA candidates were shown to produce small RNAs between 20–30 nt, 80% of which were associated with ARGONAUT proteins generally, and AGO1b in particular. Overall, our findings provide a comprehensive view of an intermediate-size non-coding transcriptome in a monocot species, which will serve as a useful platform for an in-depth analysis of ncRNA functions. PMID:22986792

  9. H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification

    PubMed Central

    Wu, Haihong; Feigon, Juli

    2007-01-01

    During the biogenesis of eukaryotic ribosomal RNA (rRNA) and spliceosomal small nuclear RNA (snRNA), uridines at specific sites are converted to pseudouridines by H/ACA ribonucleoprotein particles (RNPs). Each H/ACA RNP contains a substrate-specific H/ACA RNA and four common proteins, the pseudouridine synthase Cbf5, Nop10, Gar1, and Nhp2. The H/ACA RNA contains at least one pseudouridylation (ψ) pocket, which is complementary to the sequences flanking the target uridine. In this article, we show structural evidence that the ψ pocket can form the predicted base pairs with substrate RNA in the absence of protein components. We report the solution structure of the complex between an RNA hairpin derived from the 3′ ψ pocket of human U65 H/ACA small nucleolar RNA (snoRNA) and the substrate rRNA. The snoRNA–rRNA substrate complex has a unique structure with two offset parallel pairs of stacked helices and two unusual intermolecular three-way junctions, which together organize the substrate for docking into the active site of Cbf5. The substrate RNA interacts on one face of the snoRNA in the complex, forming a structure that easily could be accommodated in the H/ACA RNP, and explains how successive substrate RNAs could be loaded onto and unloaded from the H/ACA RNA in the RNP. PMID:17412831

  10. Genome-wide analysis of small nucleolar RNAs of Leishmania major reveals a rich repertoire of RNAs involved in modification and processing of rRNA.

    PubMed

    Eliaz, Dror; Doniger, Tirza; Tkacz, Itai Dov; Biswas, Viplov Kumar; Gupta, Sachin Kumar; Kolev, Nikolay G; Unger, Ron; Ullu, Elisabetta; Tschudi, Christian; Michaeli, Shulamit

    2015-01-01

    Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications. PMID:25970223

  11. Recognizing Prefixes in Scientific Quantities

    ERIC Educational Resources Information Center

    Sokolowski, Andrzej

    2015-01-01

    Although recognizing prefixes in physical quantities is inherent for practitioners, it might not be inherent for students, who do not use prefixes in their everyday life experiences. This deficiency surfaces in AP Physics exams. For example, readers of an AP Physics exam reported "a common mistake of incorrectly converting nanometers to…

  12. Do You Recognize This Parent?

    ERIC Educational Resources Information Center

    Wallace, Edna

    1997-01-01

    Suggests effective ways to work with parents who may be permissive, busy, detached, overprotective, or negative. Recommends that child care professionals be sensitive and understanding, recognize other demands on parents' time and communicate competitively with them, use terms parents understand, accept various levels of parental involvement, be…

  13. Recognizing and Managing Interpersonal Conflict.

    ERIC Educational Resources Information Center

    Deane, Nancy; Hovland, Michael

    1993-01-01

    Practical advice is offered, to managers and supervisors at any level, on recognizing and analyzing interpersonal conflicts, managing such conflicts and making them productive, and ensuring that performance reviews result in progress for both supervisor and employee. Conflict is seen as inevitable, an opportunity to take action, and manageable.…

  14. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

    SciTech Connect

    Zhang, Heyu; Ma, Xi; Shi, Taiping; Song, Quansheng; Zhao, Hongshan; Ma, Dalong

    2010-01-01

    NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

  15. Regulation of the Nucleolar DNA-Dependent RNA Polymerase by Amino Acids in Ehrlich Ascites Tumor Cells

    PubMed Central

    Franze-Fernández, M. T.; Pogo, A. O.

    1971-01-01

    Experiments were performed to ascertain the degree to which the amount of amino acids might be one of the regulatory factors that control the activity of the nucleolar RNA polymerase. Assays of the enzymatic activity were done with isolated nuclei from cells incubated with low and high concentrations of amino acids. Soon after the cells were exposed to a medium enriched in amino acids, a rapid increase of nucleolar RNA polymerase activity occurred. A similar result was obtained in cells incubated with lower concentrations of amino acids. However, the rate of ribosomal RNA synthesized was regularly much higher in cells incubated in a medium enriched with amino acids than in a medium low in amino acids. Apparently, the amino acids only controlled ribosomal RNA synthesis. Thus, neither maturation, processing, and transport of nuclear precursors into cytoplasmic ribosomal RNA, nor the synthesis of rapidly labeled RNA was affected. PMID:4108870

  16. Localization by high resolution in situ hybridization of the ribosomal minichromosomes during the nucleolar cycle of Physarum polycephalum.

    PubMed

    Puvion-Dutilleul, F; Pierron, G

    1992-12-01

    We have used biotinylated rDNA probes to localize by in situ hybridization the extrachromosomal genes for ribosomal RNA in the slime mold Physarum polycephalum. We established conditions that allow for highly specific hybridization at the ultrastructural level and determined that the 60-kb palindromic rDNA molecules are confined to the nucleolus in interphase. Our study definitively locates these extrachromosomal genes in mitosis in the form of thin DNA fibers contained within nucleolar remnants. We further show that these rDNA minichromosomes do not condense and that they segregate as entities independent of the condensed chromosomal DNA. In telophase, these minichromosomes migrate from the poles toward the equatorial region of the nucleus in a direction opposite that of the chromosomes. Our results illustrate the discontinuous nature of the nucleolar organizing region in Physarum. PMID:1459200

  17. Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

    PubMed Central

    Narayanan, Aarthi; Speckmann, Wayne; Terns, Rebecca; Terns, Michael P.

    1999-01-01

    Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs. PMID:10397754

  18. Nucleolar disruption and cajal body disassembly are nuclear hallmarks of DNA damage-induced neurodegeneration in purkinje cells.

    PubMed

    Baltanás, Fernando C; Casafont, Iñigo; Weruaga, Eduardo; Alonso, José R; Berciano, María T; Lafarga, Miguel

    2011-07-01

    The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration. PMID:21054627

  19. Recognizing Body Dysmorphic Disorder (Dysmorphophobia)

    PubMed Central

    Varma, Anukriti; Rastogi, Rajesh

    2015-01-01

    Dysmorphophobia is a psychiatric condition which frequently presents in the clinics of dermatologists and plastic surgeons. This disorder (also called body dysmorphic disorder) is troublesome to the patient whilst being confusing for the doctor. This commonly undiagnosed condition can be detected by a few simple steps. Timely referral to a psychiatrist benefits most patients suffering from it. This article describes with a case vignette, how to recognize body dysmorphic disorder presenting in the dermatological or aesthetic surgery set up. Diagnostic criteria, eitiology, approach to patient, management strategy and when to refer are important learning points. The importance of recognizing this disorder timely and referring the patient to the psychiatrist for appropriate treatment is crucial. This article covers all aspects of body dysmorphic disorder relevant to dermatologists and plastic surgeons and hopes to be useful in a better understanding of this disorder. PMID:26644741

  20. Recognizing Prefixes in Scientific Quantities

    NASA Astrophysics Data System (ADS)

    Sokolowski, Andrzej

    2015-09-01

    Although recognizing prefixes in physical quantities is inherent for practitioners, it might not be inherent for students, who do not use prefixes in their everyday life experiences. This deficiency surfaces in AP Physics exams. For example, readers of an AP Physics exam reported "a common mistake of incorrectly converting nanometers to meters." Similar students' mistakes were reported also by AP Chemistry readers "as in previous years, students still had difficulty converting kJ to J." While traditional teaching focuses on memorizing the symbols of prefixes, little attention is given to helping learners recognize a prefix in a given quantity. I noticed in my teaching practice that by making the processes of identifying prefixes more explicit, students make fewer mistakes on unit conversion. Thus, this paper presents an outline of a lesson that focuses on prefix recognition. It is designed for a first-year college physics class; however, its key points can be addressed to any group of physics students.

  1. Recognizing Body Dysmorphic Disorder (Dysmorphophobia).

    PubMed

    Varma, Anukriti; Rastogi, Rajesh

    2015-01-01

    Dysmorphophobia is a psychiatric condition which frequently presents in the clinics of dermatologists and plastic surgeons. This disorder (also called body dysmorphic disorder) is troublesome to the patient whilst being confusing for the doctor. This commonly undiagnosed condition can be detected by a few simple steps. Timely referral to a psychiatrist benefits most patients suffering from it. This article describes with a case vignette, how to recognize body dysmorphic disorder presenting in the dermatological or aesthetic surgery set up. Diagnostic criteria, eitiology, approach to patient, management strategy and when to refer are important learning points. The importance of recognizing this disorder timely and referring the patient to the psychiatrist for appropriate treatment is crucial. This article covers all aspects of body dysmorphic disorder relevant to dermatologists and plastic surgeons and hopes to be useful in a better understanding of this disorder. PMID:26644741

  2. Estimation of argyrophilic nucleolar organizer regions in different grades of oral submucous fibrosis

    PubMed Central

    Girish, KL; Kumaraswamy, KL; Balan, Usha; Jose, Maji

    2015-01-01

    Context: Assessment of potential for malignant transformation of oral submucous fibrosis (OSF) through clinical or light microscopic examination of hematoxylin and eosin stained tissue sections is not totally satisfactory. The search is for such a tissue marker that will differentiate those cases of OSF, which carry a higher risk for malignant transformation. During the past few years, numerous workers have validated the usefulness of enumerating argyrophilic nucleolar organizer regions (AgNORs) in predicting the malignant potential of lesions. The present study was carried out to validate the diagnostic potential of this marker. Objectives of the Study: Quantitative and qualitative assessment of AgNORs in different grades of OSF and to compare the count of AgNORs in different grades of OSF and normal mucosa. Materials and Methods: AgNORs were investigated in tissue specimens from 90 patients diagnosed with different histopathological grades of OSF. AgNORs were identified in tissue specimens stained with silver nitrate, using light microscope. AgNORs were counted as small, large and total count to analyze them both quantitatively and qualitatively. Statistical Analysis Used: Results were subjected to statistical analysis for obtaining significance value (P value) by unpaired Student's t-test. Results: The mean total count of AgNORs was 2.464 ± 0.101, 4.358 ± 0.108, 3.704 ± 0.106 and 3.279 ± 0.161 in normal mucosa, Grades I, II and III of OSF, respectively. A qualitative difference was observed in the presentation of AgNORs in different grades of OSF. Mean value of small nucleolar organizer regions (NORs) decreased while the mean of large NORs increased as the grade of OSF increased. Conclusions: The study findings suggest that AgNORs are increased in OSF and they can serve as a reliable tool adjunct to histopathological diagnosis. Their ease of demonstration and high specificity to cellular proliferation make them the best available histopathological marker in

  3. Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing.

    PubMed

    Falaleeva, Marina; Pages, Amadis; Matuszek, Zaneta; Hidmi, Sana; Agranat-Tamir, Lily; Korotkov, Konstantin; Nevo, Yuval; Eyras, Eduardo; Sperling, Ruth; Stamm, Stefan

    2016-03-22

    C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes. PMID:26957605

  4. Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells

    PubMed Central

    Lirussi, Lisa; Antoniali, Giulia; Vascotto, Carlo; D'Ambrosio, Chiara; Poletto, Mattia; Romanello, Milena; Marasco, Daniela; Leone, Marilisa; Quadrifoglio, Franco; Bhakat, Kishor K.; Scaloni, Andrea; Tell, Gianluca

    2012-01-01

    Apurinic/apyrimidinic endonuclease 1 (APE1) is the main abasic endonuclease in the base excision repair (BER) pathway of DNA lesions caused by oxidation/alkylation in mammalian cells; within nucleoli it interacts with nucleophosmin and rRNA through N-terminal Lys residues, some of which (K27/K31/K32/K35) may undergo acetylation in vivo. Here we study the functional role of these modifications during genotoxic damage and their in vivo relevance. We demonstrate that cells expressing a specific K-to-A multiple mutant are APE1 nucleolar deficient and are more resistant to genotoxic treatment than those expressing the wild type, although they show impaired proliferation. Of interest, we find that genotoxic treatment induces acetylation at these K residues. We also find that the charged status of K27/K31/K32/K35 modulates acetylation at K6/K7 residues that are known to be involved in the coordination of BER activity through a mechanism regulated by the sirtuin 1 deacetylase. Of note, structural studies show that acetylation at K27/K31/K32/K35 may account for local conformational changes on APE1 protein structure. These results highlight the emerging role of acetylation of critical Lys residues in regulating APE1 functions. They also suggest the existence of cross-talk between different Lys residues of APE1 occurring upon genotoxic damage, which may modulate APE1 subnuclear distribution and enzymatic activity in vivo. PMID:22918947

  5. NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape

    PubMed Central

    Holmberg Olausson, Karl; Elsir, Tamador; Moazemi Goudarzi, Kaveh; Nistér, Monica; Lindström, Mikael S.

    2015-01-01

    Glioblastoma (grade IV glioma) is the most common and aggressive adult brain tumor. A better understanding of the biology of glioblastoma cells is crucial to identify molecular targets stimulating cell death. NPM1 (nucleophosmin) is a multifunctional chaperone that plays an important role in cancer development. Herein, NPM1 was analyzed by immunohistochemistry in human astrocytic gliomas. NPM1 was detected in all tumors but with a significantly higher staining intensity in grade IV than in low grade tumors. Depletion of NPM1 had only modest effects on the viability of U251MG, U1242MG, and U343MGa Cl2:6 glioma cells, despite alterations in nucleolar morphology. Glioma cell cultures depleted of NPM1 exposed to micromolar levels of actinomycin D were more prone to cell death (apoptosis) compared to cultures retaining NPM1. We had previously found that NPM1 binds to linker histone H1.5. Here we could show that silencing of H1.5 triggered glioma cell apoptosis as evidenced by a marked increase in both the numbers of cleaved caspase-3+ cells and in the amounts of cleaved PARP. Enforced expression of NPM1 suppressed apoptosis in H1.5 depleted glioma cells. Although our studies would suggest little effectiveness of targeting NPM1 alone there could be potential using it as a combination treatment. PMID:26559910

  6. Nucleolar organizer regions in a chronic stress and oral cancer model

    PubMed Central

    RUZ, IVONNE ANDREA MUÑOZ; OSSA, DANIEL ANDRÉS DROGUETT; TORRES, WENDY KARINA DONOSO; KEMMERLING, ULRIKE; ROJAS, BERNARDO ARTURO VENEGAS; MARTÍNEZ, CÉSAR ANDRÉS RIVERA

    2011-01-01

    This study aimed to examine the role of chronic restraint stress (RS) on oral squamous cell carcinomas induced by 4-nitroquinoline-1-oxide (4-NQO) in CF-1 mouse tongues, measured by the expression of argyrophilic staining of nucleolar organizer regions (AgNOR). Thirty one samples of lingual epithelial tissue of CF-1 mice with a diagnosis of oral squamous cell carcinoma (OSSC) were assigned to two experimental groups: the RS/4-NQO group, where animals received RS and induction of oral chemical carcinogenesis (n=17); and the 4-NQO group, where animals received induction of chemical carcinogenesis without restraint stress (n=14). The mean number and distribution pattern of AgNOR were recorded. The mean AgNOR number per cell was found to be slightly higher in the 4-NQO group. AgNOR in the RS/4-NQO group revealed a higher tendency to be arranged in a clumped distribution compared to the 4-NQO group. No statistically significant difference was found between the groups. In conclusion, the induction of chronic restraint stress in CF-1 mice does not increase the number or affect the distribution pattern of AgNORs in OSSC induced by 4-NQO. PMID:22740947

  7. Relationship between interphasic nucleolar organizer regions and growth rate in two neuroblastoma cell lines.

    PubMed Central

    Derenzini, M.; Pession, A.; Farabegoli, F.; Trerè, D.; Badiali, M.; Dehan, P.

    1989-01-01

    The relationship between the quantity of silver-stained interphasic nucleolar organizer regions (NORs) and nuclear synthetic activity, caryotype, and growth rate was studied in two established neuroblastoma cell lines (CHP 212 and HTB 10). Statistical analysis of silver-stained NORs revealed four times as many in CHP 212 cells compared with HTB 10 cells. No difference was observed in the ribosomal RNA synthesis between the two cell lines. The caryotype index was 1.2 for CHP 212 and 1.0 for HTB 10 cells. The number of chromosomes carrying NORs and the quantity of ribosomal genes was found to be the same for the two cell lines. Doubling time of CHP 212 cells was 20 hours compared with 54 hours for HTB 10 cells. In CHP 212 cells bindering of cell duplication by serum deprivation induced a progressive lowering (calculated at 48, 72, and 96 hours) of the quantity of silver-stained interphasic NORs. Recovery of duplication by new serum addition induced, after 24 hours, an increase of the quantity of silver-stained interphasic NORs up to control levels. In the light of available data, these results indicate that the quantity of interphasic NORs is strictly correlated only to the growth rate of the cell. Images Figure 2 Figure 3 Figure 4 PMID:2705511

  8. Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer

    PubMed Central

    Barfeld, Stefan J.; Fazli, Ladan; Persson, Margareta; Marjavaara, Lisette; Urbanucci, Alfonso; Kaukoniemi, Kirsi M.; Rennie, Paul S.; Ceder, Yvonne; Chabes, Andrei; Visakorpi, Tapio; Mills, Ian G.

    2015-01-01

    The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses. PMID:25869206

  9. Fractal Analysis and the Diagnostic Usefulness of Silver Staining Nucleolar Organizer Regions in Prostate Adenocarcinoma

    PubMed Central

    Stepan, Alex; Simionescu, Cristiana; Pirici, Daniel; Ciurea, Raluca; Margaritescu, Claudiu

    2015-01-01

    Pathological diagnosis of prostate adenocarcinoma often requires complementary methods. On prostate biopsy tissue from 39 patients including benign nodular hyperplasia (BNH), atypical adenomatous hyperplasia (AAH), and adenocarcinomas, we have performed combined histochemical-immunohistochemical stainings for argyrophilic nucleolar organizer regions (AgNORs) and glandular basal cells. After ascertaining the pathology, we have analyzed the number, roundness, area, and fractal dimension of individual AgNORs or of their skeleton-filtered maps. We have optimized here for the first time a combination of AgNOR morphological denominators that would reflect best the differences between these pathologies. The analysis of AgNORs' roundness, averaged from large composite images, revealed clear-cut lower values in adenocarcinomas compared to benign and atypical lesions but with no differences between different Gleason scores. Fractal dimension (FD) of AgNOR silhouettes not only revealed significant lower values for global cancer images compared to AAH and BNH images, but was also able to differentiate between Gleason pattern 2 and Gleason patterns 3–5 adenocarcinomas. Plotting the frequency distribution of the FDs for different pathologies showed clear differences between all Gleason patterns and BNH. Together with existing morphological classifiers, AgNOR analysis might contribute to a faster and more reliable machine-assisted screening of prostatic adenocarcinoma, as an essential aid for pathologists. PMID:26366372

  10. Antibodies to a nuclear/nucleolar antigen in patients with polymyositis overlap syndromes.

    PubMed

    Reichlin, M; Maddison, P J; Targoff, I; Bunch, T; Arnett, F; Sharp, G; Treadwell, E; Tan, E M

    1984-01-01

    A precipitating antigen-antibody system has been characterized that occurs in patients with polymyositis. At least half of the patients not only have polymyositis but also have scleroderma. The proposed name for this antigen found in calf thymus extract (CTE) is PM-Scl, to indicate the almost universal presence of polymyositis and the frequent occurrence of scleroderma in the patients who make antibodies to this antigen. The antigen is probably nucleolar since all sera which precipitate with the PM-Scl antigen stain the nucleoli of Hep2 cells by indirect immunofluorescence. The PM-Scl immune system is a distinctive one different from the other known precipitins that occur in patients with polymyositis and dermatomyositis including Jo1, nRNP, and Mi. This PM-Scl antigen and its antibody represent one system which constitutes part of the reactions previously designated as PM1. Interlaboratory exchange of sera and extracts have established the unique nature of this reaction which occurs in patients with inflammatory myopathy. PMID:6699138

  11. Analysis of small nucleolar RNAs in sputum for lung cancer diagnosis

    PubMed Central

    Guarnera, Maria A.; Zhan, Min; Fang, HongBin; Stass, Sanford A.; Jiang, Feng

    2016-01-01

    Molecular analysis of sputum presents a noninvasive approach for diagnosis of lung cancer. We have shown that dysregulation of small nucleolar RNAs (snoRNAs) plays a vital role in lung tumorigenesis. We have also identified six snoRNAs whose changes are associated with lung cancer. Here we investigated if analysis of the snoRNAs in sputum could provide a potential tool for diagnosis of lung cancer. Using qRT-PCR, we determined expressions of the six snoRNAs in sputum of a training set of 59 lung cancer patients and 61 cancer-free smokers to develop a biomarker panel, which was validated in a testing set of 67 lung cancer patients and 69 cancer-free smokers for the diagnostic performance. The snoRNAs were robustly measurable in sputum. In the training set, a panel of two snoRNA biomarkers (snoRD66 and snoRD78) was developed, producing 74.58% sensitivity and 83.61% specificity for identifying lung cancer. The snoRNA biomarkers had a significantly higher sensitivity (74.58%) compared with sputum cytology (45.76%) (P < 0.05). The changes of the snoRNAs were not associated with stage and histology of lung cancer (All P >0.05). The performance of the biomarker panel was confirmed in the testing cohort. We report for the first time that sputum snoRNA biomarkers might be useful to improve diagnosis of lung cancer. PMID:26246471

  12. Nucleolar organizer regions as discriminators for the diagnosis of well-differentiated adenocarcinoma of the prostate.

    PubMed

    Schned, A R

    1993-10-01

    This study sought to determine whether nucleolar organizer region (NOR) counts might be of diagnostic use specifically in the frequently difficult identification of well-differentiated prostate cancer. Total silver-stained NOR (AgNOR) counts on sections were done in 50 consecutive nuclei from 22 cases of well-differentiated adenocarcinoma composed exclusively of Gleason grades 1 or 2 and in 50 consecutive nuclei in foci of benign glands on the same slide. Malignant nuclei contained a mean of 4.84 AgNORs; benign nuclei, 3.39 AgNORs. This difference was statistically highly significant (P < .0001), but there was considerable overlap of mean AgNOR counts between malignant and benign groups. Thus, to obtain a rule requiring a specificity of greater than 95% using the mean AgNOR count as a definition of malignancy, the maximum sensitivity possible was only 32%. Despite the overlap in mean AgNOR counts, however, in all but one case the mean count for malignant nuclei was greater than that for benign nuclei. This suggests that the diagnostic potential of AgNOR counts might be better realized by a comparison of counts between a suspicious focus and an adjacent focus of benign glands. PMID:8215819

  13. Activity patterns of nucleolar organizer region during spermatogenesis of different curimatid species (Characiformes: Curimatidae).

    PubMed

    Sampaio, Tatiane R; Pires, Larissa B; da Rosa, Renata; Dias, Ana Lúcia

    2014-02-01

    The nucleolus is an important nuclear structure where transcription of ribosomal DNA (rDNA) takes place. During mitotic division, the nucleolus passes through different processes that inactivate rDNA transcription; in meiosis, its reassembly takes place during telophase II. The objective of this study was to identify the activity patterns and localization of nucleolar organizer regions (NORs) during meiotic division in fish species of the family Curimatidae. For this analysis, the meiotic division in five curimatid species was studied using silver nitrate impregnation, fluorescent in situ hybridization (FISH), and base-specific fluorochrome staining. Silver nitrate staining indicated the presence of a nucleolus in interphase nuclei, one chromosome pair in the spermatogonial metaphases, and one bivalent at the pachytene stage. No Ag-NORs were identified for cells at the diplotene, diakinesis, metaphase I, or metaphase II stages; however, FISH confirmed the presence of Ag-NORs in the nuclei, in spermatogonia, and at the pachytene phase. FISH identified this region during the other stages of meiosis, as did fluorochrome CMA3 staining, which revealed fluorescent marks corresponding to NORs during all stages of meiosis analyzed. The gene activity and localization of this ribosomal sequence during the different stages involved will also be discussed. PMID:24702069

  14. Nucleolar Organizer Regions of Oral Epithelial Cells in Crack Cocaine Users

    PubMed Central

    Carvalho de M. Thiele, Magna; Carlos Bohn, Joslei; Lima Chaiben, Cassiano; Trindade Grégio, Ana Maria; Ângela Naval Machado, Maria; Adilson Soares de Lima, Antonio

    2013-01-01

    Background: The health risks of crack cocaine smoking on the oral mucosa has not been widely researched and documented. Objective: The purpose of this study was to analyze the proliferative activity of oral epithelial cells exposed to crack cocaine smoke using silver nucleolar organizer region (AgNOR) staining. Methods: Oral smears were collected from clinically normal-appearing buccal mucosa by liquid-based exfoliative cytology of 60 individuals (30 crack cocaine users and 30 healthy controls matched for age and gender) and analyzed for cytomorphologic and cytomorphometric techniques. Results: Crack cocaine users consumed about 13.3 heat-stable rocks per day and the time consumption of the drug was of 5.2 (± 3.3) years. Mean values of AgNOR counting for case and control groups were 5.18 ± 1.83 and 3.38 ± 1.02 (P<0.05), respectively. AgNOR area and percentage of AgNOR-occupied nuclear area were increased in comparison with the control (P<0.05). There was no statistically significant difference in the mean values of the nuclear area between the groups (P>0.05). Conclusion: This study revealed that crack cocaine smoke increases the rate of cellular proliferation in cells of normal buccal mucosa. PMID:23567853

  15. Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells

    SciTech Connect

    Berry, Andrea M.; Flock, Kelly E.; Loh, Horace H.; Ko, Jane L. . E-mail: kojane@shu.edu

    2006-11-03

    Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis.

  16. Preventing and Recognizing Prescription Drug Abuse

    MedlinePlus

    ... Abuse » Preventing and recognizing prescription drug abuse Prescription Drug Abuse Email Facebook Twitter Preventing and recognizing prescription drug abuse To ensure proper medical care, patients should discuss ...

  17. Brandon RHA recognized for energy efficiency.

    PubMed

    Waddington, Kent; Neal, Gordon

    2002-01-01

    In a recent national competition recognizing leadership in energy efficiency and greenhouse gas education, Brandon Regional Health Authority was recognized for conscientious use of resources. PMID:12357581

  18. Recognizing Patterns in Debris Disks

    NASA Technical Reports Server (NTRS)

    Kuchner, Marc

    2009-01-01

    An extrasolar planet sculpts the famous debris dish around Fomalhaut; probably many other debris disks contain planets that we could locate if only we could better recognize their signatures in the dust that surrounds them. I will describe the latest 3-D models of debris dish dynamics / models that include planets, grain-grain collisions and even ISM-disk interactions. I will show why all these ingredients are needed to explain disk images--and what the images are telling us about planet formation.

  19. The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation

    SciTech Connect

    Nishida, Tamotsu; Yamada, Yoshiji

    2011-03-11

    Research highlights: {yields} SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. {yields} SMT3IP1 competes with p53 for binding to the central acidic domain of Mdm2. {yields} SMT3IP1 binding to Mdm2 inhibits Mdm2-mediated p53 ubiquitination and degradation. {yields} We postulate that SMT3IP1 acts as a new regulator of the p53-Mdm2 pathway. -- Abstract: SUMO (small ubiquitin-like modifier) modification plays multiple roles in several cellular processes. Sumoylation is reversibly regulated by SUMO-specific proteases. SUMO-specific proteases have recently been implicated in cell proliferation and early embryogenesis, but the underlying mechanisms remain unknown. Here, we show that a nucleolar SUMO-specific protease, SMT3IP1/SENP3, controls the p53-Mdm2 pathway. We found that SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. Overexpression of SMT3IP1 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein. In addition, SMT3IP1 bound to the acidic domain of Mdm2, which also mediates the p53 interaction, and competed with p53 for binding. Increasing expression of SMT3IP1 suppressed Mdm2-mediated p53 ubiquitination and subsequent proteasomal degradation. Interestingly, the desumoylation activity of SMT3IP1 was not necessary for p53 stabilization. These results suggest that SMT3IP1 is a new regulator of the p53-Mdm2 pathway.

  20. Antarctic skuas recognize individual humans.

    PubMed

    Lee, Won Young; Han, Yeong-Deok; Lee, Sang-Im; Jablonski, Piotr G; Jung, Jin-Woo; Kim, Jeong-Hoon

    2016-07-01

    Recent findings report that wild animals can recognize individual humans. To explain how the animals distinguish humans, two hypotheses are proposed. The high cognitive abilities hypothesis implies that pre-existing high intelligence enabled animals to acquire such abilities. The pre-exposure to stimuli hypothesis suggests that frequent encounters with humans promote the acquisition of discriminatory abilities in these species. Here, we examine individual human recognition abilities in a wild Antarctic species, the brown skua (Stercorarius antarcticus), which lives away from typical human settlements and was only recently exposed to humans due to activities at Antarctic stations. We found that, as nest visits were repeated, the skua parents responded at further distances and were more likely to attack the nest intruder. Also, we demonstrated that seven out of seven breeding pairs of skuas selectively responded to a human nest intruder with aggression and ignored a neutral human who had not previously approached the nest. The results indicate that Antarctic skuas, a species that typically inhabited in human-free areas, are able to recognize individual humans who disturbed their nests. Our findings generally support the high cognitive abilities hypothesis, but this ability can be acquired during a relatively short period in the life of an individual as a result of interactions between individual birds and humans. PMID:26939544

  1. Recognizing new medical knowledge computationally.

    PubMed Central

    Nelson, S. J.; Cole, W. G.; Tuttle, M. S.; Olson, N. E.; Sherertz, D. D.

    1993-01-01

    Can new medical knowledge be recognized computationally? We know knowledge is changing, and our knowledge-based systems will need to accommodate that change in knowledge on a regular basis if they are to stay successful. Computational recognition of these changes seems desirable. It is unlikely that low level objects in the computational universe, bits and characters, will change much over time, higher level objects of language, where meaning begins to emerge, may show change. An analysis of ten arbitrarily selected paragraphs from the Medical Knowledge Self-Assessment Program of the American College of Physicians was used as a test bed for nominal phrase recognition. While there were words not known to Meta-1.2, only 8 of the 32 concepts new to the primary author were pointed to by new words. Use of a barrier word method was successful in identifying 23 of the 32 new concepts. Use of co-occurrence (in sentences) of putative nominal phrases may reduce the amount of human effort involved in recognizing the emergence of new relationships. PMID:8130505

  2. Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery.

    PubMed

    Booth, Daniel G; Takagi, Masatoshi; Sanchez-Pulido, Luis; Petfalski, Elizabeth; Vargiu, Giulia; Samejima, Kumiko; Imamoto, Naoko; Ponting, Chris P; Tollervey, David; Earnshaw, William C; Vagnarelli, Paola

    2014-01-01

    When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells.DOI: http://dx.doi.org/10.7554/eLife.01641.001. PMID:24867636

  3. Changes in nuclear, nucleolar and cytoplasmic RNA content during growth and differentiation of root parenchyma cells in plant species with different dynamics of DNA endoreplication.

    PubMed

    Marciniak, K; Bilecka, A

    1985-01-01

    Using cytophotometric method, after staining preparations with gallocyanin RNA content was examined in nucleus, nucleolus and cytoplasm of six species of angiospermal plants in successive (1-7 mm) segments of root representing successive zones of differentiation. During the cell cycle, RNA content duplicates in the nucleus, nucleolus and cytoplasm of meristematic cells. On the other hand, during growth and differentiation of parenchyma cells in species with endoreplication the content of nucleolar RNA does not increase in proportion with DNA content. High level of endoreplication is connected with high nucleolar RNA content and low cytoplasmic RNA content. In species without endoreplication at low nucleolar RNA content, a considerable growth of cytoplasmic RNA content takes place. PMID:2417894

  4. Correlation between histologic diagnosis mean nucleolar organizer region count and prognosis in canine mammary tumors.

    PubMed

    Bostock, D E; Moriarty, J; Crocker, J

    1992-09-01

    In this study, surgically excised mammary tumors from 98 bitches were graded histologically, and the grade was compared with the mean nucleolar organizer region (NOR) count in silver-stained paraffin-embedded sections. Histologically benign tumors, papillary adenocarcinomas, and intraductal carcinomas showed relatively little variation; the mean count for each category was between three and four NOR per nucleus. There was, however, a significant increase in the NOR counts in tubular and solid carcinomas. This increase was most pronounced for tumors that showed evidence of infiltration into the surrounding connective tissues. The mean NOR count for noninfiltrative carcinomas was 5.1, and that for invasive carcinomas was 7.3 (P less than 0.03). The mean NOR count for individual carcinomas ranged from 2.0 to 12.3, and a significant correlation was found between an increased NOR count and tumor-related death during the first post-surgical year. The 39 bitches in which the tumor had an NOR count less than 8.0 had a generally favorable prognosis; only six (15%) died as a result of the original neoplasm. In contrast, 18/21 dogs (85%) with a carcinoma having an NOR count greater than 8.0 died from the tumor during the first post-surgical year. A similar, although less pronounced result was obtained specifically for invasive carcinomas, in which 3/12 (25%) tumors with an NOR count less than 6.0 resulted in the death of the host, compared with 17/20 (85%) that had an NOR count greater than 6. By using this technique, it is possible to identify a subgroup of bitches with invasive mammary carcinomas that have a very poor prognosis following apparently adequate surgical ablation of the primary tumor. PMID:1413404

  5. Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients with type-1 autoimmune hepatitis

    PubMed Central

    Costa, M; Rodríguez-Sánchez, J L; Czaja, A J; Gelpí, C

    2000-01-01

    We previously described autoantibodies against a UGA serine tRNA–protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The presence of anti‐tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47·5% of patients were positive compared with none of the control subjects. To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones (19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48·8 kD. Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35·9-kD protein. Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex. Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec. PMID:10931155

  6. [The nuclear matrix proteins (mol. mass 38 and 50 kDa) are transported by chromosomes in mitosis].

    PubMed

    Murasheva, M I; Chentsov, Iu S

    2010-01-01

    It was shown by immunofluorescence method that serum M68 and serum K43 from patients with autoimmune disease stain interphase nuclei and periphery of mitotic chromosomes of pig kidney cells. Western blotting reveals the polypeptide with mol. mass of 50 kDa in serum M68, and the polypeptide with mol. mass of 38 kDa in serum K43. In the nuclear protein matrix, the antibodies to protein with mol. mass of 38 kDa stained only nucleolar periphery, while the antibodies to the protein with mol. mass of 50 kDa stained both the nucleolar periphery and all the interphase nucleus. It shows that among all components of nuclear protein matrix (lamina, internuclear network, residual nucleoli) only nucleolar periphery contains the 38 kDa protein, while the 50 kDa protein is a part of residual nucleolar periphery and takes part in nuclear protein network formation. In the interphase cells, both proteins were in situ localized in the nuclei, but one of them with mol. mass of 50 kDa was in the form of small clearly outlined granules, while the other (38 kDa) was in the form of small bright granules against the background of diffusely stained nuclei. Both proteins were also revealed as continuous ring around nucleolar periphery. During all mitotic stages, the 50 kDa protein was seen on the chromosomal periphery as a cover, and the 38 kDa protein formed separate fragments and granules around them. After nuclear and chromosome decondensation induced by hypotonic treatment, both antibodies stain interphase nuclei in diffuse manner, but in mitotic cells they stained the surface of the swollen chromosomes. The polypeptide with mol. mass of 50 kDa maintained strong connection with chromosome periphery both in norm and under condition of decondensation induced by hypotonic treatment and at subsequent recondensation in isotonic medium. In contrast, the protein with mol. mass of 38 kDa partially lost the contact with a chromosome during recondensation appearing also in the form of granules in

  7. Structural elucidation and functional characterization of the Hyaloperonospora arabidopsidis effector protein ATR13.

    PubMed

    Leonelli, Lauriebeth; Pelton, Jeffery; Schoeffler, Allyn; Dahlbeck, Douglas; Berger, James; Wemmer, David E; Staskawicz, Brian

    2011-12-01

    The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity. PMID:22194684

  8. Principles of protein targeting to the nucleolus

    PubMed Central

    Martin, Robert M; Ter-Avetisyan, Gohar; Herce, Henry D; Ludwig, Anne K; Lättig-Tünnemann, Gisela; Cardoso, M Cristina

    2015-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution. PMID:26280391

  9. Principles of protein targeting to the nucleolus.

    PubMed

    Martin, Robert M; Ter-Avetisyan, Gohar; Herce, Henry D; Ludwig, Anne K; Lättig-Tünnemann, Gisela; Cardoso, M Cristina

    2015-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution. PMID:26280391

  10. Recognizing child maltreatment in Bangladesh.

    PubMed

    Khan, N Z; Lynch, M A

    1997-08-01

    Concern is increasing in Bangladesh over child abuse, neglect, and exploitation. Children from all walks of life are being treated at the Child Development Center (CDC) Dhaka Shishu Hospital for neurodevelopmental problems resulting from abuse and neglect. Efforts to protect children from sexual harassment result in girls being isolated at home or married at an early age. Some young brides are eventually abandoned and forced into prostitution. Early marriage reflects the lack of acknowledgement of a period of adolescence and the belief that puberty is a marker of adulthood. Many girls aged 8-16 are employed as live-in domestic servants, and many suffer sexual as well as emotional abuse. Garment factories, on the other hand, offer girls an escape from extreme poverty, domestic service, and early marriage but are threatened by forces that condemn child labor. Rather than ending such opportunities, employers should be encouraged to provide employees with educational and welfare facilities. The CDC seeks to explore the extent and depth of the problem of child abuse while recognizing the special circumstances at work in Bangladesh. It is also necessary to raise awareness of these issues and of the discrepancies between the law and cultural practices. For example, the legal marriage age of 18 years for a woman and 21 years for a man is often ignored. Additional forms of abuse receiving the attention of women's organizations and human rights groups include the trafficking of children. A network of concerned organizations should be created to work against the child abuse, neglect, and exploitation that Bangladesh has pledged to overcome by signing the UN Convention on the Rights of the Child. PMID:9280385

  11. Producing and recognizing analogical relations.

    PubMed

    Lipkens, Regina; Hayes, Steven C

    2009-01-01

    Analogical reasoning is an important component of intelligent behavior, and a key test of any approach to human language and cognition. Only a limited amount of empirical work has been conducted from a behavior analytic point of view, most of that within Relational Frame Theory (RFT), which views analogy as a matter of deriving relations among relations. The present series of four studies expands previous work by exploring the applicability of this model of analogy to topography-based rather than merely selection-based responses and by extending the work into additional relations, including nonsymmetrical ones. In each of the four studies participants pretrained in contextual control over nonarbitrary stimulus relations of sameness and opposition, or of sameness, smaller than, and larger than, learned arbitrary stimulus relations in the presence of these relational cues and derived analogies involving directly trained relations and derived relations of mutual and combinatorial entailment, measured using a variety of productive and selection-based measures. In Experiment 1 participants successfully recognized analogies among stimulus networks containing same and opposite relations; in Experiment 2 analogy was successfully used to extend derived relations to pairs of novel stimuli; in Experiment 3 the procedure used in Experiment 1 was extended to nonsymmetrical comparative relations; in Experiment 4 the procedure used in Experiment 2 was extended to nonsymmetrical comparative relations. Although not every participant showed the effects predicted, overall the procedures occasioned relational responses consistent with an RFT account that have not yet been demonstrated in a behavior-analytic laboratory setting, including productive responding on the basis of analogies. PMID:19230515

  12. A combined nuclear and nucleolar localization motif in activation-induced cytidine deaminase (AID) controls immunoglobulin class switching.

    PubMed

    Hu, Yi; Ericsson, Ida; Torseth, Kathrin; Methot, Stephen P; Sundheim, Ottar; Liabakk, Nina B; Slupphaug, Geir; Di Noia, Javier M; Krokan, Hans E; Kavli, Bodil

    2013-01-23

    Activation-induced cytidine deaminase (AID) is a DNA mutator enzyme essential for adaptive immunity. AID initiates somatic hypermutation and class switch recombination (CSR) by deaminating cytosine to uracil in specific immunoglobulin (Ig) gene regions. However, other loci, including cancer-related genes, are also targeted. Thus, tight regulation of AID is crucial to balance immunity versus disease such as cancer. AID is regulated by several mechanisms including nucleocytoplasmic shuttling. Here we have studied nuclear import kinetics and subnuclear trafficking of AID in live cells and characterized in detail its nuclear localization signal. Importantly, we find that the nuclear localization signal motif also directs AID to nucleoli where it colocalizes with its interaction partner, catenin-β-like 1 (CTNNBL1), and physically associates with nucleolin and nucleophosmin. Moreover, we demonstrate that release of AID from nucleoli is dependent on its C-terminal motif. Finally, we find that CSR efficiency correlates strongly with the arithmetic product of AID nuclear import rate and DNA deamination activity. Our findings suggest that directional nucleolar transit is important for the physiological function of AID and demonstrate that nuclear/nucleolar import and DNA cytosine deamination together define the biological activity of AID. This is the first study on subnuclear trafficking of AID and demonstrates a new level in its complex regulation. In addition, our results resolve the problem related to dissociation of deamination activity and CSR activity of AID mutants. PMID:23183374

  13. Expression of ribosomal RNA genes in lines of barley with a standard karyotype and with a translocated nucleolar organizer

    SciTech Connect

    Karag'ozov, L.K.; Ananiev, E.D.; Mateeva, Z.E.; Khadzhiolov, A.A.

    1986-10-01

    The authors have investigated the rRNA synthesis and the sensitivity of rRNA genes to the action of DNAase I in developing embryos of two forms of barley. The Frigga variety has a standard karyotype and the T/sub 506/ line is characterized by translocation of the nucleolar organizer, which leads to a reduction in the number of nucleoli observed in the telophase. The results of the investigation of rRNA synthesis in vivo and of the activity of RNA polymerase I in isolated nuclei revealed the absence of differences between the two barley forms. They have established that the genes of ribosomal RNAs possess greater sensitivity to digestion by DNAase the authors compared to that of the total nuclear DNA. They conclude that the translocation of one of the nucleolar organizers causes a delay in the appearance of its activity during the telophase, this not changing the expression of the rRNA genes in the subsequent stages of cell development.

  14. Cyclophosphamide induced generation of giant hypersegmented granulocytes in rat bone marrow: cell cycle distribution and silver nucleolar staining.

    PubMed

    Kotelnikov, V M; Pogorelov, V M; Berger, J; Kozinets, G I

    1988-01-01

    Daily injection of cyclophosphamide (20 mg/kg body weight) for 7 days resulted in accumulation of 50% of rat bone marrow granulocytes in G2. Tetraploid neutrophils were hypersegmented (7.25 +/- 0.33) in comparison with diploid ones (3.92 +/- 0.33). After 14 days of cyclophosphamide treatment tetraploid hypersegmented neutrophils could be found in peripheral blood. Diploid neutrophils in these animals were also hypersegmented (4.78 +/- 0.14 versus 3.15 +/- 0.02 in control, p less than 0.001). Nucleolar ribosomal gene activities, evaluated by morphometry of silver nucleolar grains, decreased on bone marrow granulocytes in the course of differentiation in control rats. After cyclophosphamide treatment mature granulocytes contained more silver grains than in controls which may be explained by conservation of silver binding sites of nucleoli from the stages of promyelocytes and myelocytes. These results suggest two mechanisms of hypersegmented neutrophil, generation in cyclophosphamide treated rats: the first, via maturation of myelocytes arrested in G2, and second, a direct one, without tetraploid granulocyte involvement. PMID:2465255

  15. Phase Transitions in the Nucleus: the functional implications of concentration-dependent assembly of a Liquid-like RNA/Protein Body

    NASA Astrophysics Data System (ADS)

    Zhu, Lian; Weber, Stephanie; Berry, Joel; Vaidya, Nilesh; Haataja, Mikko; Brangwynne, Clifford

    2015-03-01

    The nucleolus is a liquid-like membrane-less nuclear body which plays an important role in cell growth and size control. By modulating nucleolar component concentration through RNAi conditions that change C. elegans cell size, we find that nucleoli only assemble above a threshold concentration; moreover, the ripening dynamics of nucleated droplets are consistent with the hypothesis that the assembly of the nucleolus represents an intracellular liquid-liquid phase transition. A key question is how this phase-transition is linked to the primary function of the nucleolus, in transcribing and processing ribosomal RNA. To address this, we characterize the localization of RNA Polymerase I, a key transcriptional enzyme, into nucleolar foci as a function of nucleolar component concentration. Our results suggest that there are a small number of key disordered phosphoproteins that may serve as a link between transcription and assembly. Finally, we present preliminary results using a reduced model system consisting of purified nucleolar proteins to assess the ability of nucleolar proteins to drive liquid-liquid phase separation in vitro. These results lay the foundation for a quantitative understanding of intracellular phase transitions and their impact on biomedically-critical RNA-processing steps.

  16. The new evidence of nucleolar ultrastructural dynamic change: fibrillar centre (FC) fusion in G1 phase and regeneration in S phase.

    PubMed

    Wang, Fengcai; Ying, Chen; Shang, Guangbin; Jiao, Mingda; Hongfang, Zhang

    2013-06-01

    In higher eukaryotes ribosome production starts at the end of mitosis, increases during G1, is maximal in G2 (Sirri et al., 2000) and stops during prophase (Gébrane-Younès et al., 1997). But the mechanism of the change is still uncovered. Especially in the actively growing mammalian somatic cells usually contain one or several giant fibrillar centres (GFCs) with many tiny fibrillar centre (FCs) (Koberna et al., 2002; Raška et al., 2004; Casafont et al., 2007). The process how the giant fibrillar centre (GFC) and the many tiny fibrillar centres (FCs) were formed is unknown. The present results showed there were processes of FCs fusion in G1 phase and FCs regeneration in S phase respectively in the nucleoli of A 375 cells. A few FCs fused each other in late G1 phase when the process of nucleoli fusion was completed. In S phase, a lot of tiny FCs were regenerated from the periphery of GFC, separated and scattered into nucleolar matrix in late S phase and early G2 phase. The GFC was found to be coexisted with numerous tiny FCs in the nucleolus in G2 phase. The present study provided a new evidence of nucleolar dynamic change during interphase: fibrillar centre (FC) was not to be a stable state subunit of nucleolar compartment but a highly dynamic process that may be the bases of nucleolar morphological architecture organization and its function taking place. PMID:23602556

  17. Mean diameter of nucleolar bodies in cultured human leukemic myeloblasts is mainly related to the S and G2 phase of the cell cycle.

    PubMed

    Smetana, Karel; Kuzelova, K; Zápotocký, M; Starková, J; Hrkal, Z; Trka, J

    2007-01-01

    Mean diameter of nucleolar bodies (nucleoli without the perinucleolar chromatin) per cell was studied in human leukemic myeloblasts represented by K 562 and Kasumi 1 cell lines which originated from chronic and acute myeloid leukaemia. The measurement of mean diameter of nucleolar bodies in specimens stained for RNA was very simple. Such approach eliminated the variability of the perinucleolar chromatin discontinuous shell which might influence the measured nucleolar size as suggested by earlier studies. Ageing of K 562 myeloblasts produced a significant decrease of cells in S+G2 phase of the cell cycle accompanied by a significant reduction of mean diameter of nucleolar bodies (MDNoBs) per cell. In contrast, treatment of Kasumi 1 myeloblasts with histone deacetylase inhibitor - Trichostatin A - produced a large incidence of resistant cells in S+G2 phase which were characterised by a large increase of MDNoBs. Thus, MDNoBs in leukemic myeloblasts might be a helpful tool to estimate the incidence of cells in the S+G2 phase at the single cell level in smear preparations when the number of cells is very small. PMID:18162456

  18. The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin.

    PubMed

    Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas; Chan, Albert H; Dickson, Claire F; Fabian, Marian; Olson, John S; Gell, David A; Clubb, Robert T

    2016-03-27

    Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix. PMID:25687963

  19. Meiotic chromosomes and nucleolar behavior in testicular cells of the grassland spittlebugs Deois flavopicta, Mahanarva fimbriolata and Notozulia entreriana (Hemiptera, Auchenorrhyncha)

    PubMed Central

    2010-01-01

    Spittlebugs annually infest pastures and cause severe damage, representing a serious problem for the tropical American beef cattle industry. Spittlebugs are an important biotic constraint to forage production and there is a lack of cytogenetic data for this group of insects. For these reasons, we conducted this work, in which the spermatogenesis and nucleolar behavior of Deois flavopicta, Mahanarva fimbriolata and Notozulia entreriana were studied. The males possessed testes in the shape of a “bunch of grapes”; a variable number of testicular lobes per individual and polyploid nuclei composed of several heteropycnotic bodies. A heteropycnotic area was located in the periphery of the nucleus (prophase I); the chiasmata were terminal or interstitial; metaphases I were circular or linear and anaphase showed late migration of the sex chromosome. The chromosome complement had 2n = 19, except for N. entreriana (2n = 15); the spermatids were round with heteropycnotic material in the center and elongated with conspicuos chromatin. The analysis of testes after silver nitrate staining showed polyploid nuclei with three large and three smaller nucleolar bodies. Early prophase cells had an intensely stained nucleolar body located close to the chromatin and another less evident body located away from the chromatin. The nucleolar bodies disintegrated during diplotene. Silver staining occurred in two autosomes, in terminal and subterminal locations, the latter probably corresponding to the nucleolus organizer regions (NORs). The spermatids were round with a round nucleolar body and silver staining was observed in the medial and posterior region of the elongated part of the spermatid head. PMID:21637477

  20. A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

    PubMed Central

    Olguin-Lamas, Alejandro; Madec, Edwige; Hovasse, Agnes; Werkmeister, Elisabeth; Callebaut, Isabelle; Slomianny, Christian; Delhaye, Stephane; Mouveaux, Thomas; Schaeffer-Reiss, Christine; Van Dorsselaer, Alain; Tomavo, Stanislas

    2011-01-01

    In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome

  1. H2-M3 Major Histocompatibility Complex Class Ib-Restricted CD8 T Cells Induced by Salmonella enterica Serovar Typhimurium Infection Recognize Proteins Released by Salmonella Serovar Typhimurium

    PubMed Central

    Ugrinovic, S.; Brooks, C. G.; Robson, J.; Blacklaws, B. A.; Hormaeche, C. E.; Robinson, J. H.

    2005-01-01

    Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria. PMID:16299293

  2. Recombinant Salmonella typhimurium outer membrane protein A is recognized by synovial fluid CD8 cells and stimulates synovial fluid mononuclear cells to produce interleukin (IL)-17/IL-23 in patients with reactive arthritis and undifferentiated spondyloarthropathy.

    PubMed

    Chaurasia, S; Shasany, A K; Aggarwal, A; Misra, R

    2016-08-01

    In developing countries, one-third of patients with reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) are triggered by Salmonella typhimurium. Synovial fluid mononuclear cells (SFMCs) of patients with ReA and uSpA proliferate to low molecular weight fractions (lmwf) of outer membrane proteins (Omp) of S. typhimurium. To characterize further the immunity of Omp of Salmonella, cellular immune response to two recombinant proteins of lmwf, OmpA and OmpD of S. typhimurium (rOmpA/D-sal) was assessed in 30 patients with ReA/uSpA. Using flow cytometry, 17 of 30 patients' SF CD8(+) T cells showed significant intracellular interferon (IFN)-γ to Omp crude lysate of S. typhimurium. Of these 17, 11 showed significantly more CD8(+) CD69(+) IFN-γ T cells to rOmpA-sal, whereas only four showed reactivity to rOmpD-sal. The mean stimulation index was significantly greater in rOmpA-sal than rOmpD-sal [3·0 (1·5-6·5) versus 1·5 (1·0-2·75), P < 0·005]. Similarly, using enzyme-linked immunospot (ELISPOT) in these 17 patients, the mean spots of IFN-γ-producing SFMCs were significantly greater in rOmpA-sal than rOmpD-sal [44·9 (3·5-130·7) versus 19·25 (6-41), P < 0·05]. SFMCs stimulated by rOmpA-sal produced significantly more proinflammatory cytokines than rOmpD-sal: IFN-γ [1·44 (0·39-20·42) versus 0·72 (0·048-9·15) ng/ml, P < 0·05], interleukin (IL)-17 [28·60 (6·15-510·86) versus 11·84 (6·83-252·62) pg/ml, P < 0·05], IL-23 [70·19 (15-1161·16) versus 28·25 (> 15-241·52) pg/ml, P < 0·05] and IL-6 [59·78 (2·03-273·36) versus 10·17 (0·004-190·19) ng/ml, P < 0·05]. The rOmpA-sal-specific CD8(+) T cell response correlated with duration of current synovitis (r = 0·53, P < 0·05). Thus, OmpA of S. typhimurium is a target of SF CD8(+) T cells and drives SFMC to produce increased cytokines of the IL-17/IL-23 axis which contribute to the pathogenesis of Salmonella-triggered ReA. PMID:27060348

  3. K-Ras4B proteins are expressed in the nucleolus: Interaction with nucleolin.

    PubMed

    Birchenall-Roberts, Maria C; Fu, Tao; Kim, Soo-Gyung; Huang, Ying K; Dambach, Michael; Resau, James H; Ruscetti, Francis W

    2006-09-22

    Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin. PMID:16889753

  4. K-Ras4B proteins are expressed in the nucleolus: Interaction with nucleolin

    SciTech Connect

    Birchenall-Roberts, Maria C. . E-mail: birchena@mail.ncifcrf.gov; Fu, Tao; Kim, Soo-Gyung; Huang, Ying K.; Dambach, Michael; Resau, James H.; Ruscetti, Francis W.

    2006-09-22

    Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin.

  5. Vitrification: Machines learn to recognize glasses

    NASA Astrophysics Data System (ADS)

    Ceriotti, Michele; Vitelli, Vincenzo

    2016-05-01

    The dynamics of a viscous liquid undergo a dramatic slowdown when it is cooled to form a solid glass. Recognizing the structural changes across such a transition remains a major challenge. Machine-learning methods, similar to those Facebook uses to recognize groups of friends, have now been applied to this problem.

  6. The Developmental Dimensions of Recognizing Racist Thoughts

    ERIC Educational Resources Information Center

    Torres, Vasti

    2009-01-01

    This study focuses on understanding the developmental process that occurs when racist ideas are recognized as a part of college students' thought processes. Longitudinal data were collected from 29 Latino/a college students in order to illustrate how these students made meaning of racist thoughts when they began to recognize it. The framework of…

  7. Higher-Order Neural Networks Recognize Patterns

    NASA Technical Reports Server (NTRS)

    Reid, Max B.; Spirkovska, Lilly; Ochoa, Ellen

    1996-01-01

    Networks of higher order have enhanced capabilities to distinguish between different two-dimensional patterns and to recognize those patterns. Also enhanced capabilities to "learn" patterns to be recognized: "trained" with far fewer examples and, therefore, in less time than necessary to train comparable first-order neural networks.

  8. Teaching Students to Recognize Irony

    ERIC Educational Resources Information Center

    Milner, Joseph O.; Hawkins, Robin H.; Milner, Lucy M.

    2014-01-01

    This article exposes the problem of using declarative rather than procedural knowledge to help K--12 students recognize irony in stories. It offers commonplace procedures drawn from students' everyday language experience together with more abstract irony clues to help students recognize irony in stories and increase their story comprehension.…

  9. Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase.

    PubMed Central

    Belenguer, P; Caizergues-Ferrer, M; Labbé, J C; Dorée, M; Amalric, F

    1990-01-01

    Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin. Images PMID:2192260

  10. Automatic Target Recognizer Working Group Security Committee

    NASA Astrophysics Data System (ADS)

    Schoch, Suzanne

    1987-09-01

    The ATRWG Security Committee participates in the development of Automatic Target Recognizers (ATRs) and image processing guidelines as related to TEMPEST requirements and policies set forth by the Department of Defense (DoD).

  11. The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2013-05-31

    Highlights: •Rad52 might play a key role in the repair of DSB immediately after irradiation. •EYFP-Rad52 accumulates rapidly at DSB sites and colocalizes with Ku80. •Accumulation of Rad52 at DSB sites is independent of the core NHEJ factors. •Localization and recruitment of Rad52 to DSB sites are dependent on the Rad52 CTR. •Basic amino acids in Rad52 CTR are highly conserved among vertebrate species. -- Abstract: Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1–418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1–418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1–418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1–418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411–418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is

  12. Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells.

    PubMed

    Wojciechowski, Jacek; Horky, Marcel; Gueorguieva, Marieta; Wesierska-Gadek, Józefa

    2003-09-10

    The aim of our study was to explore the antiproliferative and pro-apoptotic action of roscovitine (ROSC) on human breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (AgNORs) and nucleolar RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of histone acetylation and phosphorylation. Inhibition of DNA synthesis and accumulation of G(2)/M-arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of Annexin binding, activation of caspases as well as of DNA fragmentation. Onset of PARP-1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF-7 cells with caspase-3 did not change the kinetics of ROSC-induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC-induced changes. Cell cycle arrest precedes the main wave of apoptosis. PMID:12845642

  13. Cell cycle-dependent translocations of a major nucleolar phosphoprotein, B23, and some characteristics of its variants.

    PubMed

    Zatsepina, O V; Todorov, I T; Philipova, R N; Krachmarov, C P; Trendelenburg, M F; Jordan, E G

    1997-05-01

    A major nucleolar phosphoprotein, B23, is thought to play several apparently unrelated roles and appears to be associated with other cell compartments besides the nucleolus. However, characteristic properties of B23 variants still remain to be established. Here, we raised a new monoclonal antibody against B23 (20B2) and used it to address the issue particularly focusing on the events during mitosis. Also, we made an attempt to generalize the data on the cell cycle-dependent translocations of B23 by the use of three mammalian cell lines (HeLa, PK, RAMT) which were found to be immunoreactive for 20B2. In all the cell strains studied, B23 was chiefly located within the nucleolus at interphase, and was associated with a few cellular domains during mitosis. They were: the nucleoplasm (at prophase before the nuclear envelope breakdown), the cytoplasm (from prometaphase until mid telophase), the perichromosomal layer (from prometaphase till early telophase), cytoplasmic B23-containing bodies (at anaphase and telophase) and prenucleolar bodies, PNBs (at telophase). On Western blots, electrophoretic mobility of B23 was found to be the same at G1, S and G2 periods of interphase, but became slower at mitosis. In situ and cell extraction experiments showed that like the nucleolar B23, B23 of the perichromosomal layer and that of PNBs was highly resistant to extraction with Triton X-100, but could be released with Triton X-100/RNase A. These findings indicated that these portions of B23 were most likely to be associated with RNA. The cytoplasmic B23 was the major intracellular variant of B23 during mitosis. It had a slightly lower electrophoretic mobility than the perichromosomal B23 and could readily be extracted with Triton X-100 without addition of RNase A, a fact indicating that the cytoplasmic B23 was mainly in an RNA-free state. Mitosis-like translocations of B23 from the nucleolus to the nucleoplasm induced by actinomycin D increased its extractability but did not affect

  14. Infants recognize the subtle happiness expression.

    PubMed

    Ichikawa, Hiroko; Kanazawa, So; Yamaguchi, Masami K

    2014-01-01

    Facial movement facilitates the recognition of facial expressions. While an intense expression is expressive enough to be recognized in a still image, a subtle expression can be recognized only in motion (Ambadar, Schooler, & Cohn, 2005, Psychological Science, 16, 403-410). The present study investigated whether infants recognize a subtle expression, and whether facial movement facilitates infants' recognition of a subtle expression. In experiment 1 4- to 7-month-old infants were tested for their spontaneous preference for a happy subtle expression rather than a neutral face, but they did not show a spontaneous preference. To confirm that infants did not recognize the static subtle expression, we conducted experiment 2 using the familiarization-novelty procedure. Infants were first familiarized with a static subtle happy expression. Following familiarization, they were presented with a pair of peak expressions of happiness and anger, but showed no significant novelty preference. In experiment 3 we presented the subtle expression dynamically. Infants were familiarized with a dynamic subtle expression and were tested for their novelty preference. The 6- to 7-month-olds showed a significant novelty preference, while 4- to 5-month-olds did not. These results suggest that infants can recognize the subtle expression only in dynamic presentation and that facial movement facilitates infants' recognition of facial expression. PMID:25109015

  15. Recognizing, Confronting, and Eliminating Workplace Bullying.

    PubMed

    Berry, Peggy Ann; Gillespie, Gordon L; Fisher, Bonnie S; Gormley, Denise K

    2016-07-01

    Workplace bullying (WPB) behaviors negatively affect nurse productivity, satisfaction, and retention, and hinder safe patient care. The purpose of this article is to define WPB, differentiate between incivility and WPB, and recommend actions to prevent WPB behaviors. Informed occupational and environmental health nurses and nurse leaders must recognize, confront, and eliminate WPB in their facilities and organizations. Recognizing, confronting, and eliminating WPB behaviors in health care is a crucial first step toward sustained improvements in patient care quality and the health and safety of health care employees. PMID:27053288

  16. Equipping African American Clergy to Recognize Depression.

    PubMed

    Anthony, Jean Spann; Morris, Edith; Collins, Charles W; Watson, Albert; Williams, Jennifer E; Ferguson, Bʼnai; Ruhlman, Deborah L

    2016-01-01

    Many African Americans (AAs) use clergy as their primary source of help for depression, with few being referred to mental health providers. This study used face-to-face workshops to train AA clergy to recognize the symptoms and levels of severity of depression. A pretest/posttest format was used to test knowledge (N = 42) about depression symptoms. Results showed that the participation improved the clergy's ability to recognize depression symptoms. Faith community nurses can develop workshops for clergy to improve recognition and treatment of depression. PMID:27610907

  17. Ommexecha virens (Thunberg, 1824) and Descampsacris serrulatum (Serville, 1831) (Orthoptera, Ommexechidae): karyotypes, constitutive heterochromatin and nucleolar organizing regions

    PubMed Central

    Carvalho, D.B.; Rocha, M.F.; Loreto, V.; Silva, A.E.B.; Souza, M.J.

    2011-01-01

    Abstract Chromosomes of Ommexecha virens and Descampsacris serrulatum (Ommexechidae) were analyzed through conventional staining, C-banding, base specific fluorochromes, silver nitrate impregnation (AgNO3), and fluorescent in situ hybridization (FISH) with probe for 45S rDNA. The two species presented diploid number 2n= 23,X0 in males and acrocentric autosomes, except the pair one that presented submetacentric morphology. The X chromosome has distinct morphology in the two analyzed species, being a medium acrocentric in Ommexecha virens and large submetacentric in Descampsacris serrulatum. The C-banding revealed pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of Descampsacris serrulatum. For Ommexecha virens it was evidenced that the blocks of CH are preferentially located in the pericentromeric area (however some bivalents presents additional blocks) or in different positions. The staining with CMA3/DA/DAPI showed GC rich CH blocks (CMA3+) in some chromosomes of the two species. The nucleolar organizer regions (NORs) were located in the bivalents L2, S9, S10 of Ommexecha virens and M5, M6, M7, S11 of Descampsacris serrulatum. The FISH for rDNA showed coincident results with the pattern of active NORs revealed by AgNO3. This work presents the first chromosomal data, obtained through differential cytogenetics techniques in Ommexechidae, contributing to a better characterization of karyotypic evolution for this grasshopper family. PMID:24260624

  18. 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

  19. Repositioning of human interphase chromosomes by nucleolar dynamics in the reverse transformation of HT1080 fibrosarcoma cells.

    PubMed

    Krystosek, A

    1998-05-25

    An experimental system which should be valuable for studying the role of spatial positioning of the nuclear genome in human cell function has been developed. Reverse transformation of the malignant HT1080 fibrosarcoma cell line upon treatment with 8-chloro-cAMP results in growth inhibition, cytoskeletal reorganization, changes in nuclear shape and chromatin accessibility, and formation of prominent nucleoli. Fluorescent in situ hybridization was used to study DNA positioning during nuclear remodeling. Morphometric analysis of the hybridization sites for both repetitive sequences and "painting probes" for whole chromosomes indicated dispersal of acrocentric chromosomes in untreated cells and a highly organized central location of these ribosome gene-containing chromosomes in association with one or a few large nucleoli in nondividing treated cells. The results suggest that there was a directed movement of interphase chromosomes during a response which normalized a malignant cell line. These large-scale repositionings may serve two functions in restoring a normal transcriptional setup to the nucleus. First, ribosome genes are placed in the nucleolus, their transcriptional suborganelle. Second, nucleolar anchorings together with additional perinucleolar centromeric associations orient the domain shapes of entire chromosomes, installing gene-rich chromosomal regions into pockets of (accessible) DNAse I-sensitive chromatin populated by spliceosomes. PMID:9633529

  20. Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

    SciTech Connect

    Kakuk, Annamaria; Friedlaender, Elza; Vereb, Gyoergy; Lisboa, Duarte; Bagossi, Peter; Toth, Gabor; Gergely, Pal; Vereb, Gyoergy

    2008-08-01

    PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence {sup 916}NFNHIHKRIRRVADKYLSG{sup 934} comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin {alpha}/{beta} mechanism employing importins {alpha}1 and {alpha}3 but not {alpha}5. This transport was inhibited by wheat germ agglutinin and GTP{gamma}S. The sequence {sup 1414}SKKTNRGSQLHKYYMKRRTL{sup 1433}, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin {alpha}1/{beta} or {alpha}3/{beta} complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

  1. The expression pattern of small nucleolar and small Cajal body-specific RNAs characterizes distinct molecular subtypes of multiple myeloma

    PubMed Central

    Ronchetti, D; Todoerti, K; Tuana, G; Agnelli, L; Mosca, L; Lionetti, M; Fabris, S; Colapietro, P; Miozzo, M; Ferrarini, M; Tassone, P; Neri, A

    2012-01-01

    Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is now emerging that altered sno/scaRNAs expression may have a pathological role in cancer. This study elucidates the patterns of sno/scaRNAs expression in multiple myeloma (MM) by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCLs) and 4 normal controls. Overall, a global sno/scaRNAs downregulation was found in MMs and, even more, in sPCLs compared with normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the translocation/cyclin D4 (TC4) MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11, which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. Finally, integrative analyses with available gene expression and genome-wide data revealed the occurrence of significant sno/scaRNAs/host genes co-expression and the putative influence of allelic imbalances on specific snoRNAs expression. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information to the bio-molecular complexity of plasma cell dyscrasias. PMID:23178508

  2. Human autoreactive T cells recognize CD1b and phospholipids

    PubMed Central

    Van Rhijn, Ildiko; van Berlo, Twan; Hilmenyuk, Tamara; Cheng, Tan-Yun; Wolf, Benjamin J.; Tatituri, Raju V. V.; Uldrich, Adam P.; Napolitani, Giorgio; Cerundolo, Vincenzo; Altman, John D.; Willemsen, Peter; Huang, Shouxiong; Rossjohn, Jamie; Besra, Gurdyal S.; Brenner, Michael B.; Godfrey, Dale I.; Moody, D. Branch

    2016-01-01

    In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids. PMID:26621732

  3. How Should a Speech Recognizer Work?

    ERIC Educational Resources Information Center

    Scharenborg, Odette; Norris, Dennis; ten Bosch, Louis; McQueen, James M.

    2005-01-01

    Although researchers studying human speech recognition (HSR) and automatic speech recognition (ASR) share a common interest in how information processing systems (human or machine) recognize spoken language, there is little communication between the two disciplines. We suggest that this lack of communication follows largely from the fact that…

  4. Great Apes' Capacities to Recognize Relational Similarity

    ERIC Educational Resources Information Center

    Haun, Daniel B. M.; Call, Josep

    2009-01-01

    Recognizing relational similarity relies on the ability to understand that defining object properties might not lie in the objects individually, but in the relations of the properties of various object to each other. This aptitude is highly relevant for many important human skills such as language, reasoning, categorization and understanding…

  5. Recognizing Materials using Perceptually Inspired Features

    PubMed Central

    Sharan, Lavanya; Liu, Ce; Rosenholtz, Ruth; Adelson, Edward H.

    2013-01-01

    Our world consists not only of objects and scenes but also of materials of various kinds. Being able to recognize the materials that surround us (e.g., plastic, glass, concrete) is important for humans as well as for computer vision systems. Unfortunately, materials have received little attention in the visual recognition literature, and very few computer vision systems have been designed specifically to recognize materials. In this paper, we present a system for recognizing material categories from single images. We propose a set of low and mid-level image features that are based on studies of human material recognition, and we combine these features using an SVM classifier. Our system outperforms a state-of-the-art system [Varma and Zisserman, 2009] on a challenging database of real-world material categories [Sharan et al., 2009]. When the performance of our system is compared directly to that of human observers, humans outperform our system quite easily. However, when we account for the local nature of our image features and the surface properties they measure (e.g., color, texture, local shape), our system rivals human performance. We suggest that future progress in material recognition will come from: (1) a deeper understanding of the role of non-local surface properties (e.g., extended highlights, object identity); and (2) efforts to model such non-local surface properties in images. PMID:23914070

  6. Recognizing Dynamic Faces in Malaysian Chinese Participants.

    PubMed

    Tan, Chrystalle B Y; Sheppard, Elizabeth; Stephen, Ian D

    2016-03-01

    High performance level in face recognition studies does not seem to be replicable in real-life situations possibly because of the artificial nature of laboratory studies. Recognizing faces in natural social situations may be a more challenging task, as it involves constant examination of dynamic facial motions that may alter facial structure vital to the recognition of unfamiliar faces. Because of the incongruences of recognition performance, the current study developed stimuli that closely represent natural social situations to yield results that more accurately reflect observers' performance in real-life settings. Naturalistic stimuli of African, East Asian, and Western Caucasian actors introducing themselves were presented to investigate Malaysian Chinese participants' recognition sensitivity and looking strategies when performing a face recognition task. When perceiving dynamic facial stimuli, participants fixated most on the nose, followed by the mouth then the eyes. Focusing on the nose may have enabled participants to gain a more holistic view of actors' facial and head movements, which proved to be beneficial in recognizing identities. Participants recognized all three races of faces equally well. The current results, which differed from a previous static face recognition study, may be a more accurate reflection of observers' recognition abilities and looking strategies. PMID:26562869

  7. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  8. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  9. Effects of Age, Sex and Multiple Endocrine Neoplasia Type-II on Silver Stained Nucleolar Organizer Regions

    PubMed Central

    Butler, Merlin G.; Lane, John R.

    2016-01-01

    Summary Silver stained nucleolar organizer regions (AgNORs) were studied in phytohemagglutinin (PHA)-stimulated lymphocytes from 55 Caucasian control individuals (34 females with average age of 24 years and age range 19 weeks gestation to 87 years; 21 males with average age of 31 years and age range 29 weeks gestation to 72 years) and 13 individuals (7 females, 6 males; average age 38.8 years with age range 25—58 years) with multiple endocrine neoplasia-type II (MEN-II), an autosomal dominant malignancy with increased chromosome breakage. For the first time, AgNORs were examined in lymphocytes from normal fetuses and patients with MEN-II in order to determine the effects of age, sex or malignancy on the number of AgNORs. No significant difference in the average number of AgNORs were found in fetal cells (8.2 ± S.D. 0.7/cell) when compared with cells from older individuals including those over 65 years of age (8.0 ± S.D. 0.8/cell). There was a statistically significant negative correlation (P< 0.05) between the modal number of AgNORs on G but not D chromosomes in both males and females. A negative correlation was also found between the mean number of AgNORs and age but was not statistically significant. The average number of AgNORs in the MEN-II individuals was 8.5 ± S.D. 0.7/cell, which was not significantly different than 8.2 ± S.D. 0.7/cell observed in age-matched control subjects. PMID:2471022

  10. Integrated analysis of the prostate cancer small-nucleolar transcriptome reveals SNORA55 as a driver of prostate cancer progression.

    PubMed

    Crea, Francesco; Quagliata, Luca; Michael, Agnieszka; Liu, Hui Hsuan; Frumento, Paolo; Azad, Arun A; Xue, Hui; Pikor, Larissa; Watahiki, Akira; Morant, Rudolf; Eppenberger-Castori, Serenella; Wang, Yuwei; Parolia, Abhijit; Lennox, Kim A; Lam, Wan L; Gleave, Martin; Chi, Kim N; Pandha, Hardev; Wang, Yuzhuo; Helgason, Cheryl D

    2016-05-01

    Metastasis is the primary cause of death in prostate cancer (PCa) patients. Small nucleolar RNAs (snoRNAs) have long been considered "housekeeping" genes with no relevance for cancer biology. Emerging evidence has challenged this assumption, suggesting that snoRNA expression is frequently modulated during cancer progression. Despite this, no study has systematically addressed the prognostic and functional significance of snoRNAs in PCa. We performed RNA Sequencing on paired metastatic/non-metastatic PCa xenografts derived from clinical specimens. The clinical significance of differentially expressed snoRNAs was further investigated in two independent primary PCa cohorts (131 and 43 patients, respectively). The snoRNA demonstrating the strongest association with clinical outcome was quantified in PCa patient-derived serum samples and its functional relevance was investigated in PCa cells via gene expression profiling, pathway analysis and gene silencing. Our comparison revealed 21 differentially expressed snoRNAs in the metastatic vs. non-metastatic xenografts. Of those, 12 were represented in clinical databases and were further analyzed. SNORA55 emerged as a predictor of shorter relapse-free survival (results confirmed in two independent databases). SNORA55 was reproducibly detectable in serum samples from PCa patients. SNORA55 silencing in PCa cell lines significantly inhibited cell proliferation and migration. Pathway analysis revealed that SNORA55 expression is significantly associated with growth factor signaling and pro-inflammatory cytokine expression in PCa. Our results demonstrate that SNORA55 up-regulation predicts PCa progression and that silencing this non-coding gene affects PCa cell proliferation and metastatic potential, thus positioning it as both a novel biomarker and therapeutic target. PMID:26809501

  11. Recognizing Patterns In Log-Polar Coordinates

    NASA Technical Reports Server (NTRS)

    Weiman, Carl F. R.

    1992-01-01

    Log-Hough transform is basis of improved method for recognition of patterns - particularly, straight lines - in noisy images. Takes advantage of rotational and scale invariance of mapping from Cartesian to log-polar coordinates, and offers economy of representation and computation. Unification of iconic and Hough domains simplifies computations in recognition and eliminates erroneous quantization of slopes attributable to finite spacing of Cartesian coordinate grid of classical Hough transform. Equally efficient recognizing curves. Log-Hough transform more amenable to massively parallel computing architectures than traditional Cartesian Hough transform. "In-place" nature makes it possible to apply local pixel-neighborhood processing.

  12. Recognizing signs and symptoms of bipolar disorder.

    PubMed

    Carbray, Julie A; Iennaco, Joanne DeSanto

    2015-11-01

    Psychiatric mental health nurses and advanced practice nurses play an important role in the assessment and care of patients with bipolar disorder. Using appropriate rating scales and diagnostic criteria can aid in the assessment of patients who present with a variety of symptoms. In this game-based CME activity, you will assume the role of a psychiatric mental health advanced practice nurse who must recognize the signs and symptoms of bipolar disorder and select appropriate treatment for a 20-year-old patient with suicidal thoughts. PMID:26646046

  13. Recognizing women in the archaeological record

    SciTech Connect

    Bumsted, M.P.

    1987-01-01

    Primary sexual characteristics are usually absent in the archaeological record. The recovered secondary sex markers in bone morphology or mortuary context reflect the lifelong integrated biocultural experience of the individual man or woman. Internal patterns of variability within and between sexes can be recognized but are too frequently masked by traditional descriptive and univariate analyses. Fortunately, a more detailed picture of life experience is gained by analyzing chemical composition (isotopic and elemental) of hard tissues using an analytical anthropology approach and by examining the variation in novel ways. 7 figs.

  14. Recognizing and treating patients with envenomations.

    PubMed

    Hurt, John B; Maday, Kristopher R

    2016-07-01

    Venomous spiders and snakes are found throughout the United States, and clinicians often encounter patients with suspected spider or snakebites. Due to the significant morbidity and mortality that can be related to a particular envenomation, clinicians must be able to recognize the species of spiders and snakes that are capable of delivering a venomous bite. Through proper species identification, recognition of the specific signs and symptoms that specific venom produces, and understanding the treatment guidelines for the envenomation, clinicians can properly diagnosis, treat, and manage patients with venomous bites. PMID:27351646

  15. Recognizing focus in noise filled sentences

    NASA Astrophysics Data System (ADS)

    Xu, Ching X.; Xu, Yi

    2003-04-01

    This study is designed to help identify the intrinsic constituents of focus. Twelve four-word Mandarin sentences were recorded by a native speaker five times, each time either with focus on one of the words, or without any focus. Then, one, two or three words in each sentence produced by the speaker were replaced by pink noise. The noise-filled sentences were presented to subjects along with the text. The subjects' task was to determine if the sentence had a focus, and if yes, on which word. Ten native Mandarin speakers participated as subjects. Their performance was compared across noise replacement conditions. It was found that, when both on-focus and post-focus words were present, focus could be recognized consistently. When only the focused word was present, focus could be recognized fairly well unless the focus position was sentence final, in which case it was not very distinct from no focus. When post-focus word(s) was(were) left intact while focused words were replaced by noise, focus could still be detected successfully, but its exact localization was sometimes judged wrong. These results seem to support the dual-component hypothesis about focus. Further implications of the findings will be discussed.

  16. Recognizing connotative meaning in military chat communications

    NASA Astrophysics Data System (ADS)

    Budlong, Emily R.; Walter, Sharon M.; Yilmazel, Ozgur

    2009-05-01

    Over the last five to seven years the use of chat in military contexts has expanded quite significantly, in some cases becoming a primary means of communicating time-sensitive data to decision makers and operators. For example, during humanitarian operations with Joint Task Force-Katrina, chat was used extensively to plan, task, and coordinate predeployment and ongoing operations. The informal nature of chat communications allows the relay of far more information than the technical content of messages. Unlike formal documents such as newspapers, chat is often emotive. "Reading between the lines" to understand the connotative meaning of communication exchanges is now feasible, and often important. Understanding the connotative meaning of text is necessary to enable more useful automatic intelligence exploitation. The research project described in this paper was directed at recognizing user connotations of uncertainty and urgency. The project built a matrix of speech features indicative of these categories of meaning, developed data mining software to recognize them, and evaluated the results.

  17. How do infants recognize joint attention?

    PubMed

    Gustafsson, Erik; Brisson, Julie; Beaulieu, Christelle; Mainville, Marc; Mailloux, Dominique; Sirois, Sylvain

    2015-08-01

    The emergence of joint attention is still a matter of vigorous debate. It involves diverse hypotheses ranging from innate modules dedicated to intention reading to more neuro-constructivist approaches. The aim of this study was to assess whether 12-month-old infants are able to recognize a "joint attention" situation when observing such a social interaction. Using a violation-of-expectation paradigm, we habituated infants to a "joint attention" video and then compared their looking time durations between "divergent attention" videos and "joint attention" ones using a 2 (familiar or novel perceptual component)×2 (familiar or novel conceptual component) factorial design. These results were enriched with measures of pupil dilation, which are considered to be reliable measures of cognitive load. Infants looked longer at test events that involved novel speaker and divergent attention but no changes in infants' pupil dilation were observed in any conditions. Although looking time data suggest that infants may appreciate discrepancies from expectations related to joint attention behavior, in the absence of clear evidence from pupillometry, the results show no demonstration of understanding of joint attention, even at a tacit level. Our results suggest that infants may be sensitive to relevant perceptual variables in joint attention situations, which would help scaffold social cognitive development. This study supports a gradual, learning interpretation of how infants come to recognize, understand, and participate in joint attention. PMID:26036712

  18. Decreased argyrophilic nucleolar organiser region (AgNOR) expression in Purkinje cells: first signal of neuronal damage in sudden fetal and infant death

    PubMed Central

    Lavezzi, Anna M; Alfonsi, Graziella; Pusiol, Teresa; Matturri, Luigi

    2016-01-01

    Aims The nucleolus is an important cellular component involved in the biogenesis of the ribosome. This study was performed in order to validate the introduction of the argyrophilic nucleolar organiser region (AgNOR) stain technique, specific for the nucleoli detection, in neuropathological studies on sudden fetal and infant death. Methods In a wide set of fetuses and infants, aged from 27 gestational weeks to eight postnatal months and dead from both known and unknown causes, an in-depth neuropathological study usually applied at the Lino Rossi Research Center of the Milan University was implemented by the AgNOR method. Results Peculiar abnormalities of the nucleoli, as partial or total disruption above all in Purkinje cells (PCs), were exclusively found in victims of sudden fetal and infant death, and not in controls. The observed nucleolar alterations were frequently related to nicotine absorption in pregnancy. Conclusions We conclude that these findings represent early hallmarks of PC degeneration, contributing to the pathophysiology of sudden perinatal death. PMID:26567317

  19. Nucleolar introns from Physarum flavicomum contain insertion elements that may explain how mobile group I introns gained their open reading frames.

    PubMed Central

    Vader, A; Naess, J; Haugli, K; Haugli, F; Johansen, S

    1994-01-01

    Comparison of two group I intron sequences in the nucleolar genome of the myxomycete Physarum flavicomum to their homologs in the closely related Physarum polycephalum revealed insertion-like elements. One of the insertion-like elements consists of two repetitive sequence motifs of 11 and 101 bp in five and three copies, respectively. The smaller motif, which flanks the larger, resembles a target duplication and indicates a relationship to transposons or retroelements. The insertion-like elements are found in the peripheral loops of the RNA structure; the positions occupied by the ORFs of mobile nucleolar group I introns. The P. flavicomum introns are 1184 and 637 bp in size, located in the large subunit ribosomal RNA gene, and can be folded into group I intron structures at the RNA level. However, the intron 2s from both P. flavicomum and P. polycephalum contain an unusual core region that lacks the P8 segment. None of the introns are able to self-splice in vitro. Southern analysis of different isolates indicates that the introns are not optional in myxomycetes. Images PMID:7984404

  20. 46 CFR 162.039-5 - Recognized laboratory.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 162.039-5 Section 162.039-5... Recognized laboratory. (a) A recognized laboratory is one which is regularly engaged in the examination... motorboats. The following laboratories are recognized, and the semiportable fire extinguishers bearing...

  1. 46 CFR 160.048-8 - Recognized laboratory.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.048-8 Section 160.048-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  2. 46 CFR 160.048-8 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 160.048-8 Section 160.048-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  3. 46 CFR 162.039-5 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 162.039-5 Section 162.039-5... Recognized laboratory. (a) A recognized laboratory is one which is regularly engaged in the examination... motorboats. The following laboratories are recognized, and the semiportable fire extinguishers bearing...

  4. 46 CFR 160.048-8 - Recognized laboratory.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratory. 160.048-8 Section 160.048-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  5. 46 CFR 160.048-8 - Recognized laboratory.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratory. 160.048-8 Section 160.048-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  6. 46 CFR 160.077-9 - Recognized laboratory.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratory. 160.077-9 Section 160.077-9... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  7. 46 CFR 160.048-8 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 160.048-8 Section 160.048-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  8. 46 CFR 160.076-19 - Recognized laboratories.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratories. 160.076-19 Section 160.076-19... Recognized laboratories. The approval and production oversight functions that this subpart requires to be conducted by a recognized laboratory must be conducted by an independent laboratory recognized by the...

  9. 46 CFR 160.077-9 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 160.077-9 Section 160.077-9... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  10. 46 CFR 160.049-8 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 160.049-8 Section 160.049-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  11. 46 CFR 162.039-5 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 162.039-5 Section 162.039-5... Recognized laboratory. (a) A recognized laboratory is one which is regularly engaged in the examination... motorboats. The following laboratories are recognized, and the semiportable fire extinguishers bearing...

  12. 46 CFR 160.076-19 - Recognized laboratories.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratories. 160.076-19 Section 160.076-19... Recognized laboratories. The approval and production oversight functions that this subpart requires to be conducted by a recognized laboratory must be conducted by an independent laboratory recognized by the...

  13. 46 CFR 160.076-19 - Recognized laboratories.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratories. 160.076-19 Section 160.076-19... Recognized laboratories. The approval and production oversight functions that this subpart requires to be conducted by a recognized laboratory must be conducted by an independent laboratory recognized by the...

  14. 46 CFR 162.039-5 - Recognized laboratory.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratory. 162.039-5 Section 162.039-5... Recognized laboratory. (a) A recognized laboratory is one which is regularly engaged in the examination... motorboats. The following laboratories are recognized, and the semiportable fire extinguishers bearing...

  15. 46 CFR 160.077-9 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 160.077-9 Section 160.077-9... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  16. 46 CFR 160.077-9 - Recognized laboratory.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratory. 160.077-9 Section 160.077-9... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  17. 46 CFR 162.039-5 - Recognized laboratory.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratory. 162.039-5 Section 162.039-5... Recognized laboratory. (a) A recognized laboratory is one which is regularly engaged in the examination... motorboats. The following laboratories are recognized, and the semiportable fire extinguishers bearing...

  18. 46 CFR 160.049-8 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 160.049-8 Section 160.049-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  19. Long-term analysis to objectify the tumour grading by means of automated microscopic image analysis of the nucleolar organizer regions (AgNORs) in the case of breast carcinoma

    PubMed Central

    2013-01-01

    Background Apart from a number of cases of inaccurate prognosis in regard to individual patients, the inter- and intra-observer variability of the classical, histological prognosis parameters have been under repeated discussion. For this reason, a long-term analysis was carried out in regard to overall survival by means of automated microscopic image analysis of the nucleolar organizer regions (AgNORs) to objectify tumour grading in the case of breast carcinoma. This consists of a selective representation of argyrophilic proteins that are associated with the nucleolus organising regions. Methods The evaluation included 244 female patients with an average age of 59.3 years. The characterisation of the histological sections was carried out on the basis of the AMBA/R system. With this software the histometric characterisation level was evaluated in terms of the nucleolus organizer regions. The post-observation data were obtained from the clinical register and were complemented by mortality data from the cancer registers and by data supplied by the residents’ registration office of Berlin. Results The average post-observation period was 106.6 months. With the Cox-Regression the influence of the co-variables (conventional prognosis parameters and AgNOR parameters) were examined. In the model, only the parameters pN, G and various AgNOR parameters remain present. Conclusion There is a strong correlation between survival and selected AgNOR parameters. These could replace the conventional grading as the standard measure for the mitosis rate together with the pleomorphism level. Instead of the-time consuming AMBA/R system originally used, a new implementation of AgNOR quantification with modern VM systems could be applied. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1449591192859058. PMID:23566354

  20. Recognizing familial myeloid leukemia in adults

    PubMed Central

    Nickels, Eric M.; Soodalter, Jesse; Churpek, Jane E.

    2013-01-01

    Germline testing for familial cases of myeloid leukemia in adults is becoming more common with the recognition of multiple genetic syndromes predisposing people to bone marrow disease. Currently, Clinical Laboratory Improvement Amendments approved testing exists for several myeloid leukemia predisposition syndromes: familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML), caused by mutations in RUNX1; familial AML with mutated CEBPA; familial myelodysplastic syndrome and acute leukemia with mutated GATA2; and the inherited bone marrow failure syndromes, including dyskeratosis congenita, a disease of abnormal telomere maintenance. With the recognition of additional families with a genetic component to their leukemia, new predisposition alleles will likely be identified. We highlight how to recognize and manage these cases as well as outline the characteristics of the major known syndromes. We look forward to future research increasing our understanding of the scope of inherited myeloid leukemia syndromes. PMID:23926458

  1. Shuttle inspection team recognized for 'eagle eyes.'

    NASA Technical Reports Server (NTRS)

    2000-01-01

    NASA Administrator Daniel Goldin (left) applauds the Space Shuttle ice and debris inspection team who were recognized for their keen safety observations prior to the launch of Space Shuttle Discovery. Standing next to Goldin are (left to right) D. Scott Otto, with Lockheed Martin Space Services Company; John B. Blue, Thomas F. Ford and Michael Barber, with United Space Alliance; Gregory N. Katnik and Jorge E. Rivera, with NASA. Katnick and Rivera received the agency's Exceptional Achievement Medal; Barber, Blue, Ford and Otto received the NASA Public Service Medal. While scanning the launch pad before launch, the team found a stray 4-inch pin near the Shuttle's external fuel tank that could have caused damage during launch. Discovery was safely launched the next day, on Oct. 11.

  2. Obstacle detection by recognizing binary expansion patterns

    NASA Technical Reports Server (NTRS)

    Baram, Yoram; Barniv, Yair

    1993-01-01

    This paper describes a technique for obstacle detection, based on the expansion of the image-plane projection of a textured object, as its distance from the sensor decreases. Information is conveyed by vectors whose components represent first-order temporal and spatial derivatives of the image intensity, which are related to the time to collision through the local divergence. Such vectors may be characterized as patterns corresponding to 'safe' or 'dangerous' situations. We show that essential information is conveyed by single-bit vector components, representing the signs of the relevant derivatives. We use two recently developed, high capacity classifiers, employing neural learning techniques, to recognize the imminence of collision from such patterns.

  3. Position, rotation, and intensity invariant recognizing method

    DOEpatents

    Ochoa, Ellen; Schils, George F.; Sweeney, Donald W.

    1989-01-01

    A method for recognizing the presence of a particular target in a field of view which is target position, rotation, and intensity invariant includes the preparing of a target-specific invariant filter from a combination of all eigen-modes of a pattern of the particular target. Coherent radiation from the field of view is then imaged into an optical correlator in which the invariant filter is located. The invariant filter is rotated in the frequency plane of the optical correlator in order to produce a constant-amplitude rotational response in a correlation output plane when the particular target is present in the field of view. Any constant response is thus detected in the output The U.S. Government has rights in this invention pursuant to Contract No. DE-AC04-76DP00789 between the U.S. Department of Energy and AT&T Technologies, Inc.

  4. Proliferative activity of urothelial neoplasms: comparison of BrdU incorporation, Ki67 expression, and nucleolar organiser regions.

    PubMed Central

    Limas, C; Bigler, A; Bair, R; Bernhart, P; Reddy, P

    1993-01-01

    AIMS: To evaluate the proliferative activity of urothelial neoplasms, compare it with that of the normal urinary tract epithelium, and determine its relation to morphological grade and presence of invasion. METHODS: Multiple biopsy specimens from 53 individuals--eight normal controls, five patients with severe urothelial atypia, and 40 with transitional cell carcinomas (TCCs)--were studied using in vitro bromodeoxyuridine (BrdU) incorporation, Ki67 antigen expression, and quantitation of the nucleolar organiser regions (NORs). RESULTS: The percentage of nuclei labelled by BrdU (BrdU index) correlated well with the percentage of nuclei expressing the Ki67 antigen (Ki67 index). These proliferation indices were very low (less than 0.1% in 60% of samples) in the urothelium of normal controls and the morphologically unremarkable epithelium of patients with TCCs. Non-invasive TCCs had increased proliferation (BrdU index 6.32 (SD 0.8)%, Ki67 index 5.04 (0.6)% but lagged behind the invasive tumours (BrdU 20.9 (3.2)%, Ki67 18.6 (2.8)%). The average NOR count was 1.57 (0.03) in morphological normal epithelium, which increased progressively with grade in non-invasive TCCs, but varied greatly in invasive tumours and did not correlate with the proliferation indices. The spectrum of values for both proliferation indices and NORs was particularly wide in grade 2 TCCs. Severe atypias without exophytic growth had an increase in BrdU and Ki67 indices comparable with that found in grade 3-4 invasive TCCs; these also had the highest NORs per nucleus. CONCLUSIONS: The growth potential of urothelial neoplasms is an important indicator of their aggressive course. In particular, growth indices over 10% are strongly associated with the presence of invasion. Papillary grade 2 TCCs show heterogeneity in their growth characteristics which may relate to their diverse clinical course. The mitotic count underestimates the growth potential of papillary TCCs and the addition of proliferation

  5. How can we recognize continuous quality improvement?

    PubMed Central

    Rubenstein, Lisa; Khodyakov, Dmitry; Hempel, Susanne; Danz, Margie; Salem-Schatz, Susanne; Foy, Robbie; O'Neill, Sean; Dalal, Siddhartha; Shekelle, Paul

    2014-01-01

    Objective Continuous quality improvement (CQI) methods are foundational approaches to improving healthcare delivery. Publications using the term CQI, however, are methodologically heterogeneous, and labels other than CQI are used to signify relevant approaches. Standards for identifying the use of CQI based on its key methodological features could enable more effective learning across quality improvement (QI) efforts. The objective was to identify essential methodological features for recognizing CQI. Design Previous work with a 12-member international expert panel identified reliably abstracted CQI methodological features. We tested which features met rigorous a priori standards as essential features of CQI using a three-phase online modified-Delphi process. Setting Primarily United States and Canada. Participants 119 QI experts randomly assigned into four on-line panels. Intervention(s) Participants rated CQI features and discussed their answers using online, anonymous and asynchronous discussion boards. We analyzed ratings quantitatively and discussion threads qualitatively. Main outcome measure(s) Panel consensus on definitional CQI features. Results Seventy-nine (66%) panelists completed the process. Thirty-three completers self-identified as QI researchers, 18 as QI practitioners and 28 as both equally. The features ‘systematic data guided activities,’ ‘designing with local conditions in mind’ and ‘iterative development and testing’ met a priori standards as essential CQI features. Qualitative analyses showed cross-cutting themes focused on differences between QI and CQI. Conclusions We found consensus among a broad group of CQI researchers and practitioners on three features as essential for identifying QI work more specifically as ‘CQI.’ All three features are needed as a minimum standard for recognizing CQI methods. PMID:24311732

  6. Nucleic acid recognizing Toll-like receptors and autoimmunity.

    PubMed

    von Landenberg, Philipp; Bauer, Stefan

    2007-12-01

    The understanding of autoimmune diseases experienced an impressive boost since the Toll-like receptors (TLRs) have been identified as possible key players in autoimmune pathophysiology. Although these receptors recognize a variety of structures derived from viruses, bacteria, and fungi leading to subsequent initiation of the relevant immune responses, recent data support the idea that TLRs are crucial in the induction and perpetuation of certain autoimmune diseases, especially the systemic lupus erythematosus (SLE). In this review, we will summarize recent data on involvement of TLRs in the development of autoimmune diseases. We will focus on TLRs 7, 8, and 9 that were originally identified as receptors specific for bacterial and viral RNA/DNA, but more recent in vitro and in vivo studies have linked these receptors to the detection of host RNA, DNA, and RNA-associated or DNA-associated proteins in the context of autoimmunity. PMID:18060756

  7. NO66, a Highly Conserved Dual Location Protein in the Nucleolus and in a Special Type of Synchronously Replicating ChromatinD⃞

    PubMed Central

    Eilbracht, Jens; Reichenzeller, Michaela; Hergt, Michaela; Schnölzer, Martina; Heid, Hans; Stöhr, Michael; Franke, Werner W.; Schmidt-Zachmann, Marion S.

    2004-01-01

    It has recently become clear that the nucleolus, the most prominent nuclear subcompartment, harbors diverse functions beyond its classic role in ribosome biogenesis. To gain insight into nucleolar functions, we have purified amplified nucleoli from Xenopus laevis oocytes using a novel approach involving fluorescence-activated cell sorting techniques. The resulting protein fraction was analyzed by mass spectrometry and used for the generation of monoclonal antibodies directed against nucleolar components. Here, we report the identification and molecular characterization of a novel, ubiquitous protein, which in most cell types appears to be a constitutive nucleolar component. Immunolocalization studies have revealed that this protein, termed NO66, is highly conserved during evolution and shows in most cells analyzed a dual localization pattern, i.e., a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalizations with proteins Ki-67, HP1α, and PCNA, respectively, have further shown that the staining pattern of NO66 overlaps with certain clusters of late replicating chromatin. Biochemical experiments have revealed that protein NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes. We propose that in addition to its role in ribosome biogenesis protein NO66 has functions in the replication or remodeling of certain heterochromatic regions. PMID:14742713

  8. Recognizing and identifying people: A neuropsychological review.

    PubMed

    Barton, Jason J S; Corrow, Sherryse L

    2016-02-01

    Recognizing people is a classic example of a cognitive function that involves multiple processing stages and parallel routes of information. Neuropsychological data have provided important evidence for models of this process, particularly from case reports; however, the quality and extent of the data varies widely between studies. In this review we first discuss the requirements and logical basis of the types of neuropsychological evidence to support conclusions about the modules in this process. We then survey the adequacy of the current body of reports to address two key issues. First is the question of which cognitive operation generates a sense of familiarity: the current debate revolves around whether familiarity arises in modality-specific recognition units or later amodal processes. Key evidence on this point comes from the search for dissociations between familiarity for faces, voices and names. The second question is whether lesions can differentially affect the abilities to link diverse sources of person information (e.g., face, voice, name, biographic data). Dissociations of these linkages may favor a 'distributed-only' model of the organization of semantic knowledge, whereas a 'person-hub' model would predict uniform impairments of all linkages. While we conclude that there is reasonable evidence for dissociations in name, voice and face familiarity in regards to the first question, the evidence for or against dissociated linkages between information stores in regards to the second question is tenuous at best. We identify deficiencies in the current literature that should motivate and inform the design of future studies. PMID:26773237

  9. Arabic word recognizer for mobile applications

    NASA Astrophysics Data System (ADS)

    Khanna, Nitin; Abdollahian, Golnaz; Brame, Ben; Boutin, Mireille; Delp, Edward J.

    2011-03-01

    When traveling in a region where the local language is not written using a "Roman alphabet," translating written text (e.g., documents, road signs, or placards) is a particularly difficult problem since the text cannot be easily entered into a translation device or searched using a dictionary. To address this problem, we are developing the "Rosetta Phone," a handheld device (e.g., PDA or mobile telephone) capable of acquiring an image of the text, locating the region (word) of interest within the image, and producing both an audio and a visual English interpretation of the text. This paper presents a system targeted for interpreting words written in Arabic script. The goal of this work is to develop an autonomous, segmentation-free Arabic phrase recognizer, with computational complexity low enough to deploy on a mobile device. A prototype of the proposed system has been deployed on an iPhone with a suitable user interface. The system was tested on a number of noisy images, in addition to the images acquired from the iPhone's camera. It identifies Arabic words or phrases by extracting appropriate features and assigning "codewords" to each word or phrase. On a dictionary of 5,000 words, the system uniquely mapped (word-image to codeword) 99.9% of the words. The system has a 82% recognition accuracy on images of words captured using the iPhone's built-in camera.

  10. Development of NATO's recognized environmental picture

    NASA Astrophysics Data System (ADS)

    Teufert, John F.; Trabelsi, Mourad

    2006-05-01

    An important element for the fielding of a viable, effective NATO Response Force (NRF) is access to meteorological, oceanographic, geospatial data (GEOMETOC) and imagery. Currently, the available GEOMETOC information suffers from being very fragmented. NATO defines the Recognised Environmental Picture as controlled information base for GEOMETOC data. The NATO REP proposes an architecture that is both flexible and open. The focus lies on enabling a network-centric approach. The key into achieving this is relying on using open, well recognized standards that apply to both the data exchange protocols and the data formats. Communication and information exchange based on open standards enables system interoperability. Diverse systems, each with unique, specialized contributions to an increased understanding of the battlespace, can now cooperate to a manageable information sphere. By clearly defining responsibilities in the generation of information, a reduction in data transfer overhead is achieved . REP identifies three main stages in the dissemination of GEOMETOC data. These are Collection, Fusion (and Analysis) and Publication. A REP architecture has been successfully deployed during the NATO Coalition Warrior Interoperability Demonstration (CWID) in Lillehammer, Norway during June 2005. CWID is an annual event to validate and improve the interoperability of NATO and national Consultation and command, control, communications, computers, intelligence, surveillance, and reconnaissance (C4ISR) systems. With a test case success rate of 84%, it was able to provide relevant GEOMETOC support to the main NRF component headquarters. In 2006, the REP architecture will be deployed and validated during the NATO NRF Steadfast live exercises.

  11. Artificial Immune System for Recognizing Patterns

    NASA Technical Reports Server (NTRS)

    Huntsberger, Terrance

    2005-01-01

    A method of recognizing or classifying patterns is based on an artificial immune system (AIS), which includes an algorithm and a computational model of nonlinear dynamics inspired by the behavior of a biological immune system. The method has been proposed as the theoretical basis of the computational portion of a star-tracking system aboard a spacecraft. In that system, a newly acquired star image would be treated as an antigen that would be matched by an appropriate antibody (an entry in a star catalog). The method would enable rapid convergence, would afford robustness in the face of noise in the star sensors, would enable recognition of star images acquired in any sensor or spacecraft orientation, and would not make an excessive demand on the computational resources of a typical spacecraft. Going beyond the star-tracking application, the AIS-based pattern-recognition method is potentially applicable to pattern- recognition and -classification processes for diverse purposes -- for example, reconnaissance, detecting intruders, and mining data.

  12. Sternocostoclavicular Hyperostosis: An Ill-Recognized Disease.

    PubMed

    Roed, Bolette; Kristensen, Tatiana; Thorsen, Søren; Poulsen Bloch, Klaus; Afzelius, Pia

    2016-01-01

    Sternocostoclavicular hyperostosis (SCCH) is an ill-recognized, rarely diagnosed disease. Today, SCCH is widely considered part of the synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO) syndrome. SCCH develops over years with intermittent attacks of pain, swelling, and reddening of the sternocostoclavicular region. The disease causes progressive hyperostosis, fusion of the sternocostoclavicular joints, and soft tissue ossification. SCCH is chronic, non-malignant, and occurs predominantly bilaterally in middle-aged women. The incidence of the disease is unknown. We present a case of isolated SCCH, where chest radiographs showed a clear development of bilateral disease over the course of more than a decade. Whole-body bone scintigraphy was performed and was suggestive of SCCH. The diagnosis was established as late as 14 years from the onset of symptoms. During this period, the patient underwent several inconclusive examinations, resulting in a delay of diagnosis and in prolonged and aggravated symptoms. With this case report, we want to draw attention to SCCH and the importance of early diagnosis of the disease. PMID:27527220

  13. Overview: recognizing the problem of magnesium deficiency

    SciTech Connect

    Seelig, M.S.

    1988-01-01

    The magnesium content of the usual American diet is less than the recommended dietary allowance. Excesses of some macro- and micro-nutrients interact with Mg, increasing its requirements. Marginal deficiency of Mg is not associated with hypomagnesemia, is not characterized by typical manifestations, as is thus difficult to diagnose. Serum or plasma Mg levels are held within narrow limits unless tissue levels are very low, or renal function is poor. Vulnerability to Mg deficiency increases during growth and development, pregnancy, when under physical or psychological stress, and during illness or its treatment that interferes with absorption or causes loss of Mg. Evidence of biochemical changes of early Mg deficiency is rarely sought, although the roles of Mg in many enzyme systems are recognized. The effects of Mg deficiency on metabolism, even in disorders caused by vitamin dependencies in which Mg is a co-factor, are largely unexplored. Deficiency of Mg is diagnosed confidently when the laboratory reports hypomagnesemia in patients with convulsions or arrhythmias. Without these signs, Mg levels are not often ordered, even in the presence of neuromuscular irritability such as respond to Mg repletion. Because Mg supplementation or Mg-sparing drugs protect against premature or ectopic heart beats and sudden death, to which diuretic-treated hypertensive patients are at risk, it is increasingly being advised that their Mg status be determined.

  14. Position, rotation, and intensity invariant recognizing method

    DOEpatents

    Ochoa, E.; Schils, G.F.; Sweeney, D.W.

    1987-09-15

    A method for recognizing the presence of a particular target in a field of view which is target position, rotation, and intensity invariant includes the preparing of a target-specific invariant filter from a combination of all eigen-modes of a pattern of the particular target. Coherent radiation from the field of view is then imaged into an optical correlator in which the invariant filter is located. The invariant filter is rotated in the frequency plane of the optical correlator in order to produce a constant-amplitude rotational response in a correlation output plane when the particular target is present in the field of view. Any constant response is thus detected in the output plane to determine whether a particular target is present in the field of view. Preferably, a temporal pattern is imaged in the output plane with a optical detector having a plurality of pixels and a correlation coefficient for each pixel is determined by accumulating the intensity and intensity-square of each pixel. The orbiting of the constant response caused by the filter rotation is also preferably eliminated either by the use of two orthogonal mirrors pivoted correspondingly to the rotation of the filter or the attaching of a refracting wedge to the filter to remove the offset angle. Detection is preferably performed of the temporal pattern in the output plane at a plurality of different angles with angular separation sufficient to decorrelate successive frames. 1 fig.

  15. METHOD AND MEANS FOR RECOGNIZING COMPLEX PATTERNS

    DOEpatents

    Hough, P.V.C.

    1962-12-18

    This patent relates to a method and means for recognizing a complex pattern in a picture. The picture is divided into framelets, each framelet being sized so that any segment of the complex pattern therewithin is essentially a straight line. Each framelet is scanned to produce an electrical pulse for each point scanned on the segment therewithin. Each of the electrical pulses of each segment is then transformed into a separate strnight line to form a plane transform in a pictorial display. Each line in the plane transform of a segment is positioned laterally so that a point on the line midway between the top and the bottom of the pictorial display occurs at a distance from the left edge of the pictorial display equal to the distance of the generating point in the segment from the left edge of the framelet. Each line in the plane transform of a segment is inclined in the pictorial display at an angle to the vertical whose tangent is proportional to the vertical displacement of the generating point in the segment from the center of the framelet. The coordinate position of the point of intersection of the lines in the pictorial display for each segment is determined and recorded. The sum total of said recorded coordinate positions being representative of the complex pattern. (AEC)

  16. Recognizing Disguised Faces: Human and Machine Evaluation

    PubMed Central

    Dhamecha, Tejas Indulal; Singh, Richa; Vatsa, Mayank; Kumar, Ajay

    2014-01-01

    Face verification, though an easy task for humans, is a long-standing open research area. This is largely due to the challenging covariates, such as disguise and aging, which make it very hard to accurately verify the identity of a person. This paper investigates human and machine performance for recognizing/verifying disguised faces. Performance is also evaluated under familiarity and match/mismatch with the ethnicity of observers. The findings of this study are used to develop an automated algorithm to verify the faces presented under disguise variations. We use automatically localized feature descriptors which can identify disguised face patches and account for this information to achieve improved matching accuracy. The performance of the proposed algorithm is evaluated on the IIIT-Delhi Disguise database that contains images pertaining to 75 subjects with different kinds of disguise variations. The experiments suggest that the proposed algorithm can outperform a popular commercial system and evaluates them against humans in matching disguised face images. PMID:25029188

  17. Dogs recognize dog and human emotions.

    PubMed

    Albuquerque, Natalia; Guo, Kun; Wilkinson, Anna; Savalli, Carine; Otta, Emma; Mills, Daniel

    2016-01-01

    The perception of emotional expressions allows animals to evaluate the social intentions and motivations of each other. This usually takes place within species; however, in the case of domestic dogs, it might be advantageous to recognize the emotions of humans as well as other dogs. In this sense, the combination of visual and auditory cues to categorize others' emotions facilitates the information processing and indicates high-level cognitive representations. Using a cross-modal preferential looking paradigm, we presented dogs with either human or dog faces with different emotional valences (happy/playful versus angry/aggressive) paired with a single vocalization from the same individual with either a positive or negative valence or Brownian noise. Dogs looked significantly longer at the face whose expression was congruent to the valence of vocalization, for both conspecifics and heterospecifics, an ability previously known only in humans. These results demonstrate that dogs can extract and integrate bimodal sensory emotional information, and discriminate between positive and negative emotions from both humans and dogs. PMID:26763220

  18. Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

    PubMed Central

    2013-01-01

    Background For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity. Results During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity. Conclusion Specific activity calculated from the level of

  19. Replication factor C recognizes 5'-phosphate ends of telomeres.

    PubMed

    Uchiumi, F; Ohta, T; Tanuma, S

    1996-12-01

    Telomere structure is suggested to be important for chromosome and cell integrity and thereby for cell senescence and immortality. In a search for cDNA encoding proteins that bind specifically to telomere repeat sequences, we used random primer-labeled telomere probes to screen a lambda gt11 Jurkat cDNA library. The clone obtained encodes the central region of the large subunit of replication factor C (RFC), a known activator of DNA polymerase delta. Electrophoretic mobility shift analyses of the binding ability of RFC-glutathione S-transferase (GST) fusion protein to telomere probes revealed that RFC recognizes preferentially 5'-phosphoryl (P) groups but not 3'-hydroxyl (OH) groups at the ends of double-stranded telomere repeats. This structure-specific binding of RFC is supported by the observations that it binds to 3'-OH/5'-P ends in telomere repeats produced by DNase gamma, but not to those produced by 3'-P/5'-OH ends for DNase alpha. These findings suggest a novel function for RFC in telomere stability or turnover. PMID:8954124

  20. Novel peptide recognized by RhoA GTPase.

    PubMed

    Drulis-Fajdasz, Dominika; Jelen, Filip; Oleksy, Arkadiusz; Otlewski, Jacek

    2006-01-01

    A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein. PMID:17019437

  1. Immobilization of fluorous oligosaccharide recognized by influenza virus on polytetrafluoroethylene filter.

    PubMed

    Tojino, Mami; Mori, Masako; Kasuya, Maria Carmelita Z; Hatanaka, Kenichi; Kawaguchi, Atsushi; Nagata, Kyosuke; Shirai, Takashi; Mizuno, Mamoru

    2012-01-15

    The lactoside with PEG-fluorous tag was introduced to BHK-21(C-13) cells to generate a GM3-type oligosaccharide (Siaα2-3Galβ1-4Glc). The GM3-type oligosaccharide obtained was easily immobilized by spotting onto commercially available polytetrafluoroethylene (PTFE) filter through non-covalent fluorous affinity and simply assessed by dot blot method using the interaction of carbohydrate- with proteins which recognize sialic acid such as virus membrane proteins. PMID:22177082

  2. 46 CFR 42.05-60 - Recognized classification society.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Recognized classification society. 42.05-60 Section 42... society. The term recognized classification society means the American Bureau of Shipping or other classification society recognized by the Commandant, as provided in 46 U.S.C. 5107, and who also may be...

  3. 46 CFR 90.10-35 - Recognized classification society.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Recognized classification society. 90.10-35 Section 90... classification society. The term recognized classification society means the American Bureau of Shipping or other classification society recognized by the Commandant....

  4. 46 CFR 160.076-19 - Recognized laboratories.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratories. 160.076-19 Section 160.076-19... Recognized laboratories. (a) PFDs. The following laboratories are recognized under § 159.010-9 of this... Laboratories, Inc., 12 Laboratory Drive, P.O. Box 13995, Research Triangle Park, NC 27709-3995, (919)...

  5. 46 CFR 164.019-17 - Recognized laboratory.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 164.019-17 Section 164.019-17...: SPECIFICATIONS AND APPROVAL MATERIALS Personal Flotation Device Components § 164.019-17 Recognized laboratory. (a) General. A laboratory may be designated as a recognized laboratory under this subpart if it is—...

  6. 46 CFR 160.047-7 - Recognized laboratory.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.047-7 Section 160.047-7... and Child § 160.047-7 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  7. 46 CFR 160.064-7 - Recognized laboratory.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.064-7 Section 160.064-7...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Marine Buoyant Devices § 160.064-7 Recognized laboratory. (a) A... laboratory. The following laboratories are recognized under § 159.010-7 of this part, to perform testing...

  8. 46 CFR 164.012-12 - Recognized laboratory.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 164.012-12 Section 164.012-12...: SPECIFICATIONS AND APPROVAL MATERIALS Interior Finishes for Merchant Vessels § 164.012-12 Recognized laboratory. A recognized laboratory is one which is operated as a nonprofit public service and is...

  9. 46 CFR 164.019-17 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 164.019-17 Section 164.019-17...: SPECIFICATIONS AND APPROVAL MATERIALS Personal Flotation Device Components § 164.019-17 Recognized laboratory. (a) General. A laboratory may be designated as a recognized laboratory under this subpart if it is—...

  10. 46 CFR 160.047-7 - Recognized laboratory.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratory. 160.047-7 Section 160.047-7... and Child § 160.047-7 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  11. 46 CFR 160.064-7 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 160.064-7 Section 160.064-7...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Marine Buoyant Devices § 160.064-7 Recognized laboratory. (a) A... laboratory. The following laboratories are recognized under § 159.010-7 of this part, to perform testing...

  12. 46 CFR 164.019-17 - Recognized laboratory.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratory. 164.019-17 Section 164.019-17...: SPECIFICATIONS AND APPROVAL MATERIALS Personal Flotation Device Components § 164.019-17 Recognized laboratory. (a) General. A laboratory may be designated as a recognized laboratory under this subpart if it is—...

  13. 46 CFR 160.047-7 - Recognized laboratory.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratory. 160.047-7 Section 160.047-7... and Child § 160.047-7 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  14. 46 CFR 164.019-17 - Recognized laboratory.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratory. 164.019-17 Section 164.019-17...: SPECIFICATIONS AND APPROVAL MATERIALS Personal Flotation Device Components § 164.019-17 Recognized laboratory. (a) General. A laboratory may be designated as a recognized laboratory under this subpart if it is—...

  15. 46 CFR 160.064-7 - Recognized laboratory.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratory. 160.064-7 Section 160.064-7...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Marine Buoyant Devices § 160.064-7 Recognized laboratory. (a) A... laboratory. The following laboratories are recognized under § 159.010-7 of this part, to perform testing...

  16. 46 CFR 164.012-12 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 164.012-12 Section 164.012-12...: SPECIFICATIONS AND APPROVAL MATERIALS Interior Finishes for Merchant Vessels § 164.012-12 Recognized laboratory. A recognized laboratory is one which is operated as a nonprofit public service and is...

  17. 46 CFR 164.012-12 - Recognized laboratory.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratory. 164.012-12 Section 164.012-12...: SPECIFICATIONS AND APPROVAL MATERIALS Interior Finishes for Merchant Vessels § 164.012-12 Recognized laboratory. A recognized laboratory is one which is operated as a nonprofit public service and is...

  18. 46 CFR 160.064-7 - Recognized laboratory.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Recognized laboratory. 160.064-7 Section 160.064-7...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Marine Buoyant Devices § 160.064-7 Recognized laboratory. (a) A... laboratory. The following laboratories are recognized under § 159.010-7 of this part, to perform testing...

  19. 46 CFR 164.012-12 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 164.012-12 Section 164.012-12...: SPECIFICATIONS AND APPROVAL MATERIALS Interior Finishes for Merchant Vessels § 164.012-12 Recognized laboratory. A recognized laboratory is one which is operated as a nonprofit public service and is...

  20. 46 CFR 160.064-7 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 160.064-7 Section 160.064-7...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Marine Buoyant Devices § 160.064-7 Recognized laboratory. (a) A... laboratory. The following laboratories are recognized under § 159.010-7 of this part, to perform testing...

  1. 46 CFR 164.019-17 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 164.019-17 Section 164.019-17...: SPECIFICATIONS AND APPROVAL MATERIALS Personal Flotation Device Components § 164.019-17 Recognized laboratory. (a) General. A laboratory may be designated as a recognized laboratory under this subpart if it is—...

  2. 46 CFR 160.047-7 - Recognized laboratory.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Recognized laboratory. 160.047-7 Section 160.047-7... and Child § 160.047-7 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  3. 46 CFR 160.076-19 - Recognized laboratories.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratories. 160.076-19 Section 160.076-19... Recognized laboratories. (a) PFDs. The following laboratories are recognized under § 159.010-9 of this... Laboratories, Inc., 12 Laboratory Drive, P.O. Box 13995, Research Triangle Park, NC 27709-3995, (919)...

  4. 46 CFR 164.012-12 - Recognized laboratory.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Recognized laboratory. 164.012-12 Section 164.012-12...: SPECIFICATIONS AND APPROVAL MATERIALS Interior Finishes for Merchant Vessels § 164.012-12 Recognized laboratory. A recognized laboratory is one which is operated as a nonprofit public service and is...

  5. 46 CFR 160.047-7 - Recognized laboratory.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Recognized laboratory. 160.047-7 Section 160.047-7... and Child § 160.047-7 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  6. Trafficking motifs in the SARS-coronavirus nucleocapsid protein

    SciTech Connect

    You, Jae-Hwan; Reed, Mark L.; Hiscox, Julian A. . E-mail: j.a.hiscox@leeds.ac.uk

    2007-07-13

    The severe acute respiratory syndrome-coronavirus nucleocapsid (N) protein is involved in virus replication and modulation of cell processes. In this latter respect control may in part be achieved through the sub-cellular localisation of the protein. N protein predominately localises in the cytoplasm (the site of virus replication and assembly) but also in the nucleus/nucleolus. Using a combination of live-cell and confocal microscopy coupled to mutagenesis we identified a cryptic nucleolar localisation signal in the central part of the N protein. In addition, based on structural comparison to the avian coronavirus N protein, a nuclear export signal was identified in the C-terminal region of the protein.

  7. Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP) with nucleolin.

    PubMed

    Taha, Mohamed S; Nouri, Kazem; Milroy, Lech G; Moll, Jens M; Herrmann, Christian; Brunsveld, Luc; Piekorz, Roland P; Ahmadian, Mohammad R

    2014-01-01

    Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa) in the cytosol, and a low molecular weight complex (∼200 kDa) in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs) in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG) domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs. PMID:24658146

  8. Argyrophilic nucleolar organizer regions (AgNORs) in interphases and metaphases of normal and neoplastic gill cells of Macoma balthica (Bivalvia: Tellinidae) from the Gulf of Gdansk, Baltic Sea.

    PubMed

    Smolarz, K; Wolowicz, M; Thiriot-Quiévreux, C

    2003-10-24

    Chromosome analysis of gill cells of different populations of Macoma balthica (L.) from the Bay of Gdansk (Baltic Sea) revealed 2 clam categories, 1 with neoplastic features and 1 without. Silver-staining was performed on interphase and metaphase cells of both categories. The mean argyrophilic nucleolar organizer region (AgNOR) count per abnormal interphase cell was significantly higher than in normal interphase cells. Normal silver-stained metaphases had 3 nucleolar organizer region (NOR) chromosome phenotypes. The location of the NORs in the most frequent phenotype (55.6% in 54 metaphases scored) was interstitial on the largest metacentric chromosome pair, Pair No. 1. Abnormal silver-stained metaphases had a higher number of active NOR sites. Different phenotypes were observed (frequency greater than 10% for 67 metaphases scored); 2 were similar to those in normal metaphases and 5 were ectopic. The higher activity of AgNORs observed in abnormal cells confirmed the diagnosis of malignant neoplasia. PMID:14667039

  9. 15 CFR 286.9 - Maintaining recognized status.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... INSTITUTE OF STANDARDS AND TECHNOLOGY, DEPARTMENT OF COMMERCE ACCREDITATION AND ASSESSMENT PROGRAMS NATIONAL VOLUNTARY CONFORMITY ASSESSMENT SYSTEM EVALUATION (NVCASE) PROGRAM § 286.9 Maintaining recognized...

  10. 15 CFR 286.9 - Maintaining recognized status.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... INSTITUTE OF STANDARDS AND TECHNOLOGY, DEPARTMENT OF COMMERCE ACCREDITATION AND ASSESSMENT PROGRAMS NATIONAL VOLUNTARY CONFORMITY ASSESSMENT SYSTEM EVALUATION (NVCASE) PROGRAM § 286.9 Maintaining recognized...

  11. 15 CFR 286.9 - Maintaining recognized status.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... INSTITUTE OF STANDARDS AND TECHNOLOGY, DEPARTMENT OF COMMERCE ACCREDITATION AND ASSESSMENT PROGRAMS NATIONAL VOLUNTARY CONFORMITY ASSESSMENT SYSTEM EVALUATION (NVCASE) PROGRAM § 286.9 Maintaining recognized...

  12. 15 CFR 286.9 - Maintaining recognized status.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... INSTITUTE OF STANDARDS AND TECHNOLOGY, DEPARTMENT OF COMMERCE ACCREDITATION AND ASSESSMENT PROGRAMS NATIONAL VOLUNTARY CONFORMITY ASSESSMENT SYSTEM EVALUATION (NVCASE) PROGRAM § 286.9 Maintaining recognized...

  13. 15 CFR 286.9 - Maintaining recognized status.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... INSTITUTE OF STANDARDS AND TECHNOLOGY, DEPARTMENT OF COMMERCE ACCREDITATION AND ASSESSMENT PROGRAMS NATIONAL VOLUNTARY CONFORMITY ASSESSMENT SYSTEM EVALUATION (NVCASE) PROGRAM § 286.9 Maintaining recognized...

  14. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing.

    PubMed

    Schludi, Martin H; May, Stephanie; Grässer, Friedrich A; Rentzsch, Kristin; Kremmer, Elisabeth; Küpper, Clemens; Klopstock, Thomas; Arzberger, Thomas; Edbauer, Dieter

    2015-10-01

    A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly

  15. Fibroblast growth factor 3, a protein with a dual subcellular fate, is interacting with human ribosomal protein S2

    SciTech Connect

    Antoine, Marianne; Reimers, Kerstin; Wirz, Werner; Gressner, Axel M.; Mueller, Robert; Kiefer, Paul . E-mail: pkiefer@ukaachen.de

    2005-12-16

    The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.

  16. The yeast ERAD-C ubiquitin ligase Doa10 recognizes an intramembrane degron

    PubMed Central

    Habeck, Gregor; Ebner, Felix A.; Shimada-Kreft, Hiroko

    2015-01-01

    Aberrant endoplasmic reticulum (ER) proteins are eliminated by ER-associated degradation (ERAD). This process involves protein retrotranslocation into the cytosol, ubiquitylation, and proteasomal degradation. ERAD substrates are classified into three categories based on the location of their degradation signal/degron: ERAD-L (lumen), ERAD-M (membrane), and ERAD-C (cytosol) substrates. In Saccharomyces cerevisiae, the membrane proteins Hrd1 and Doa10 are the predominant ERAD ubiquitin-protein ligases (E3s). The current notion is that ERAD-L and ERAD-M substrates are exclusively handled by Hrd1, whereas ERAD-C substrates are recognized by Doa10. In this paper, we identify the transmembrane (TM) protein Sec61 β-subunit homologue 2 (Sbh2) as a Doa10 substrate. Sbh2 is part of the trimeric Ssh1 complex involved in protein translocation. Unassembled Sbh2 is rapidly degraded in a Doa10-dependent manner. Intriguingly, the degron maps to the Sbh2 TM region. Thus, in contrast to the prevailing view, Doa10 (and presumably its human orthologue) has the capacity for recognizing intramembrane degrons, expanding its spectrum of substrates. PMID:25918226

  17. 40 CFR 745.88 - Recognized test kits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Recognized test kits. 745.88 Section 745.88 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Residential Property Renovation § 745.88 Recognized test kits....

  18. When Do Infants Begin Recognizing Familiar Words in Sentences?

    ERIC Educational Resources Information Center

    DePaolis, Rory A.; Vihman, Marilyn M.; Keren-Portnoy, Tamar

    2014-01-01

    Previous studies have shown that by 11 but not by 10 months infants recognize words that have become familiar from everyday life independently of the experimental setting. This study explored the ability of 10-, 11-, and 12- month-old infants to recognize familiar words in sentential context, without experimental training. The headturn preference…

  19. Prospective Teacher Learning: Recognizing Evidence of Conceptual Understanding

    ERIC Educational Resources Information Center

    Bartell, Tonya Gau; Webel, Corey; Bowen, Brian; Dyson, Nancy

    2013-01-01

    This study examined prospective teachers' (PSTs) ability to recognize evidence of children's conceptual understanding of mathematics in three content areas before and after an instructional intervention designed to support this ability. It also investigates the role PSTs' content knowledge plays in their ability to recognize children's…

  20. Small nucleolar RNA clusters in trypanosomatid Leptomonas collosoma. Genome organization, expression studies, and the potential role of sequences present upstream from the first repeated cluster.

    PubMed

    Liang, Xue-hai; Ochaion, Avivit; Xu, Yu-xin; Liu, Qing; Michaeli, Shulamit

    2004-02-13

    Trypanosomatid small nucleolar RNA (snoRNA) genes are clustered in the genome. snoRNAs are transcribed polycistronically and processed into mature RNAs. In this study, we characterized four snoRNA clusters in Leptomonas collosoma. All of the clusters analyzed carry both C/D and H/ACA RNAs. The H/ACA RNAs are composed of a single hairpin, a structure typical to trypanosome and archaea guide RNAs. Using deletion and mutational analysis of a tagged C/D snoRNA situated within the snoRNA cluster, we identified 10-nucleotide flanking sequences that are essential for processing snoRNA from its precursor. Chromosome walk was performed on a snoRNA cluster, and a sequence of 700 bp was identified between the first repeat and the upstream open reading frame. Cloning of this sequence in an episome vector enhanced the expression of a tagged snoRNA gene in an orientation-dependent manner. However, continuous transcript spanning of this region was detected in steady-state RNA, suggesting that snoRNA transcription also originates from an upstream-long polycistronic transcriptional unit. The 700-bp fragment may therefore represent an example of many more elements to be discovered that enhance transcription along the chromosome, especially when transcription from the upstream gene is reduced or when enhanced transcription is needed. PMID:14645367

  1. A Case-Control Study of Oral Epithelial Proliferative Markers among Sudanese Toombak Dippers Using Micronuclei Assay, Argyrophilic Nucleolar Organizer Region, Papanicolaou and Crystal Violet Methods

    PubMed Central

    Anass, M. Abbas; G. Ahmed, Hussain

    2013-01-01

    The use of Toombak has been reported to play a major role in the etiology of oral cancer in Sudan. The cellular proliferative activity on the oral epithelium of 210 Toombak dippers was assessed by applying the micronuclei frequency, mean argyrophilic nucleolar organizer region (AgNOR) counts, Papanicolaou method, and 1% crystal violet stain. Participants were divided into 3 groups: 200 were apparently healthy individuals, 100 were Toombak users (cases), 100 were non-tobacco users (control) and 10 were patients with oral squamous cell carcinomas. Cytological atypia was identified among 4 (4%). Toombak users and was not found among the control group (P<0.04). The micronuclei frequencies were higher in Toombak users (1.026) than in the control group (0.356) (P<0.0001). The mean AgNOR counts in Toombak users (2.423) were higher than control group (1.303) (P<0.0001). Neither Toombak users nor control group showed mitotic figures in 1% crystal violet method. The results of this research showed that Toombak dipping is a high risk factor for increase in the cellular proliferation in the oral mucosa. The cytological proliferative marker methods used are useful for screening Toombak users. PMID:24179643

  2. A small nucleolar guide RNA functions both in 2′-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA

    PubMed Central

    Jády, Beáta E.; Kiss, Tamás

    2001-01-01

    In eukaryotes, two distinct classes of small nucleolar RNAs (snoRNAs), namely the fibrillarin-associated box C/D snoRNAs and the Gar1p-associated box H/ACA snoRNAs, direct the site-specific 2′-O-ribose methylation and pseudouridylation of ribosomal RNAs (rRNAs), respectively. We have identified a novel evolutionarily conserved snoRNA, called U85, which possesses the box elements of both classes of snoRNAs and associates with both fibrillarin and Gar1p. In vitro and in vivo pseudouridylation and 2′-O-methylation experiments provide evidence that the U85 snoRNA directs 2′-O-methylation of the C45 and pseudouridylation of the U46 residues in the invariant loop 1 of the human U5 spliceosomal RNA. The U85 is the first example of a snoRNA that directs modification of an RNA polymerase II-transcribed spliceosomal RNA and that functions both in RNA pseudouridylation and 2′-O-methylation. PMID:11157760

  3. Plant dicistronic tRNA–snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z

    PubMed Central

    Kruszka, Katarzyna; Barneche, Fredy; Guyot, Romain; Ailhas, Jérôme; Meneau, Isabelle; Schiffer, Steffen; Marchfelder, Anita; Echeverría, Manuel

    2003-01-01

    Small nucleolar RNAs (snoRNAs) guiding modifications of ribosomal RNAs and other RNAs display diverse modes of gene organization and expression depending on the eukaryotic system: in animals most are intron encoded, in yeast many are monocistronic genes and in plants most are polycistronic (independent or intronic) genes. Here we report an unprecedented organization: plant dicistronic tRNA–snoRNA genes. In Arabidopsis thaliana we identified a gene family encoding 12 novel box C/D snoRNAs (snoR43) located just downstream from tRNAGly genes. We confirmed that they are transcribed, probably from the tRNA gene promoter, producing dicistronic tRNAGly–snoR43 precursors. Using transgenic lines expressing a tagged tRNA–snoR43.1 gene we show that the dicistronic precursor is accurately processed to both snoR43.1 and tRNAGly. In addition, we show that a recombinant RNase Z, the plant tRNA 3′ processing enzyme, efficiently cleaves the dicistronic precursor in vitro releasing the snoR43.1 from the tRNAGly. Finally, we describe a similar case in rice implicating a tRNAMet-e expressed in fusion with a novel C/D snoRNA, showing that this mode of snoRNA expression is found in distant plant species. PMID:12554662

  4. Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

    SciTech Connect

    Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki . E-mail: yigarash@pharm.hokudai.ac.jp

    2006-05-12

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.

  5. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA

    PubMed Central

    Mitrea, Diana M; Cika, Jaclyn A; Guy, Clifford S; Ban, David; Banerjee, Priya R; Stanley, Christopher B; Nourse, Amanda; Deniz, Ashok A; Kriwacki, Richard W

    2016-01-01

    The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus. DOI: http://dx.doi.org/10.7554/eLife.13571.001 PMID:26836305

  6. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA.

    PubMed

    Mitrea, Diana M; Cika, Jaclyn A; Guy, Clifford S; Ban, David; Banerjee, Priya R; Stanley, Christopher B; Nourse, Amanda; Deniz, Ashok A; Kriwacki, Richard W

    2016-01-01

    The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus. PMID:26836305

  7. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA

    DOE PAGESBeta

    Mitrea, Diana M.; Cika, Jaclyn A.; Guy, Clifford S.; Ban, David; Banerjee, Priya R.; Stanley, Christopher B.; Nourse, Amanda; Deniz, Ashok A.; Kriwacki, Richard W.

    2016-02-02

    The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidicmore » tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.« less

  8. ADEQUACY OF DISINFECTION FOR CONTROL OF NEWLY RECOGNIZED WATERBORNE PATHOGENS

    EPA Science Inventory

    Agents recently recognized as causes or potential causes of waterborne outbreaks include pathogenic bacteria (Campylobacter jejuni, Yersinia enterocoliticia), viruses (rotavirus, Norwalk virus and other poorly defined viral agents) and Giardia lamblia, a protozoan agent. Although...

  9. ORNL, partners officially recognized for discovery of elements 115, 117

    ScienceCinema

    Roberto, Jim

    2016-01-15

    The International Union for Pure and Applied Chemistry has announced formal verification of four new chemical elements, recognizing the Department of Energy?s Oak Ridge National Laboratory and its collaborators for the discovery of elements 115 and 117.

  10. Drinking to Excess: Recognize and Treat Alcohol Problems

    MedlinePlus

    ... disclaimer . Subscribe Drinking to Excess Recognize and Treat Alcohol Problems Some people enjoy an occasional glass of ... while watching a football game. Most people drink alcohol moderately, within their limits. Others overdo it occasionally. ...

  11. 3-minute diagnosis: Researchers develop new method to recognize pathogens

    ScienceCinema

    Beer, Reg

    2014-05-30

    Imagine knowing precisely why you feel sick ... before the doctor's exam is over. Lawrence Livermore researcher Reg Beer and his engineering colleagues have developed a new method to recognize disease-causing pathogens quicker than ever before.

  12. 3-minute diagnosis: Researchers develop new method to recognize pathogens

    SciTech Connect

    Beer, Reg

    2014-01-06

    Imagine knowing precisely why you feel sick ... before the doctor's exam is over. Lawrence Livermore researcher Reg Beer and his engineering colleagues have developed a new method to recognize disease-causing pathogens quicker than ever before.

  13. Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription.

    PubMed Central

    Newman, D R; Kuhn, J F; Shanab, G M; Maxwell, E S

    2000-01-01

    The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) essential for ribosome biogenesis. The box C/D snoRNA family possesses conserved nucleotide boxes C and D that are multifunctional elements required for snoRNA processing, snoRNA transport to the nucleolus, and 2'-O-methylation of ribosomal RNA. We have previously demonstrated that the assembly of an snoRNP complex is essential for processing the intronic box C/D snoRNAs and that specific nuclear proteins associate with the box C/D core motif in vitro. Using a box C/D motif derived from mouse U14 snoRNA, we have now affinity purified and defined four mouse proteins that associate with this minimal RNA substrate. These four proteins consist of two protein pairs: members of each pair are highly related in sequence. One protein pair corresponds to the essential yeast nucleolar proteins Nop56p and Nop58p. Affinity purification of mouse Nop58 confirms observations made in yeast that Nop58 is a core protein of the box C/D snoRNP complex. Isolation of Nop56 using this RNA motif defines an additional snoRNP core protein. The second pair of mouse proteins, designated p50 and p55, are also highly conserved among eukaryotes. Antibody probing of nuclear fractions revealed a predominance of p55 and p50 in the nucleoplasm, suggesting a possible role for the p50/p55 pair in snoRNA production and/or nucleolar transport. The reported interaction of p55 with TATA-binding protein (TBP) and replication A protein as well as the DNA helicase activity of p55 and p50 may suggest the coordination of snoRNA processing and snoRNP assembly with replication and/or transcriptional events in the nucleus. Homologs for both snoRNA-associated protein pairs occur in Archaea, strengthening the hypothesis that the box C/D RNA elements and their interacting proteins are of ancient evolutionary origin. PMID:10864044

  14. Recognizing the intensity of strength training exercises with wearable sensors.

    PubMed

    Pernek, Igor; Kurillo, Gregorij; Stiglic, Gregor; Bajcsy, Ruzena

    2015-12-01

    In this paper we propose a system based on a network of wearable accelerometers and an off-the-shelf smartphone to recognize the intensity of stationary activities, such as strength training exercises. The system uses a hierarchical algorithm, consisting of two layers of Support Vector Machines (SVMs), to first recognize the type of exercise being performed, followed by recognition of exercise intensity. The first layer uses a single SVM to recognize the type of the performed exercise. Based on the recognized type a corresponding intensity prediction SVM is selected on the second layer, specializing in intensity prediction for the recognized type of exercise. We evaluate the system for a set of upper-body exercises using different weight loads. Additionally, we compare the most important features for exercise and intensity recognition tasks and investigate how different sliding window combinations, sensor configurations and number of training subjects impact the algorithm performance. We perform all of the experiments for two different types of features to evaluate the feasibility of implementation on resource constrained hardware. The results show the algorithm is able to recognize exercise types with approximately 85% accuracy and 6% intensity prediction error. Furthermore, due to similar performance using different types of features, the algorithm offers potential for implementation on resource constrained hardware. PMID:26453822

  15. Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury1

    PubMed Central

    Kulik, Liudmila; Fleming, Sherry D.; Moratz, Chantal; Reuter, Jason W.; Novikov, Aleksey; Chen, Kuan; Andrews, Kathy A.; Markaryan, Adam; Quigg, Richard J.; Silverman, Gregg J.; Tsokos, George C.; Holers, V. Michael

    2010-01-01

    Intestinal ischemia-reperfusion (IR)3 injury is initiated when natural IgM antibodies recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild type C57BL/6 mice. We identified a novel IgM monoclonal antibody (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1−/− mice. Monoclonal Ab B4 was found to specifically recognize mouse annexin IV. Pre-injection of recombinant annexin IV blocked IR injury in wild type C57BL/6 mice, demonstrating the requirement for recognition of this protein in order to develop IR injury in the context of a complex natural antibody repertoire. Humans were also found to exhibit IgM natural antibodies that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target antigen that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury. PMID:19380783

  16. Characterization of Two Novel mAbs Recognizing Different Epitopes on CD43

    PubMed Central

    Kim, Soseul; Hong, Jeong won; Cho, Woon-Dong; Moon, Yoo Ri; Yoon, Sang Soon; Kim, Min-Young; Hong, Kwon Pyo; Lee, Yong-Moon; Yi, Jae Hyuk; Ham, Young Jun; Rah, Hyung Chul; Kim, Seung Ryul

    2014-01-01

    JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43. PMID:24999313

  17. "RecognizeCane" : The new concept of a cane which recognizes the most common objects and safety clues.

    PubMed

    Scherlen, Anne-Catherine; Dumas, Jean Claude; Guedj, Benjamin; Vignot, Alexandre

    2007-01-01

    This paper introduces the new concept of an electronic cane for blind people. While some systems inform the subject only of the presence of the object and its relative distance, RecognizeCane is also able to recognize most common objects and environment clues to increase the safety and confidence of the navigation process. The originality of RecognizeCane is the use of simple sensors, such as infrared, brilliance or water sensors to inform the subject of the presence, for example, of a stairway, a water puddle, a zebra crossing or a trash can. This cane does not use an embedded vision system. RecognizeCane is equipped with several sensors and microprocessors to collect sensor data and extract the desired information about the close environment by means of a dynamic analysis of output signals. PMID:18003475

  18. SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs

    PubMed Central

    Grozdanov, Petar N.; Roy, Sujayita; Kittur, Nupur; Meier, U. Thomas

    2009-01-01

    Assembly of H/ACA RNPs in yeast is aided by at least two accessory factors, Naf1p and Shq1p. Although the function of Naf1p and its human ortholog NAF1 has been delineated in detail, that of Shq1p and its putative human ortholog SHQ1 remains obscure. We demonstrate that SHQ1 indeed functions in the biogenesis of human H/ACA RNPs and we dissect its mechanism of action. Like NAF1, SHQ1 binds the major H/ACA core protein and pseudouridine synthase NAP57 (aka dyskerin) but precedes the assembly role of NAF1 at nascent H/ACA RNAs because the interaction of SHQ1 with NAP57 in vivo and in vitro precludes that of NAF1 and of the other H/ACA core proteins that are present at the sites of H/ACA RNA transcription. The N-terminal heat shock protein 20-like CS domain of SHQ1 is dispensable for NAP57 binding. Consistent with its role as an assembly factor, SHQ1 localizes to the nucleoplasm and is excluded from nucleoli and Cajal bodies, the sites of mature H/ACA RNPs. In an in vitro assembly system of functional H/ACA RNPs that is dependent on NAF1, excess recombinant SHQ1 interferes with assembly. Importantly, knockdown of cellular SHQ1 prevents accumulation of a newly synthesized H/ACA reporter RNA and generally reduces the levels of endogenous H/ACA RNAs including telomerase RNA. In summary, the sequential action of SHQ1 and NAF1 is required for functional assembly of H/ACA RNPs in vivo and in vitro. This step-wise process could serve as an efficient means of quality control during H/ACA RNP assembly. PMID:19383767

  19. Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

    PubMed Central

    Miller, M M; Goto, R M; Taylor, R L; Zoorob, R; Auffray, C; Briles, R W; Briles, W E; Bloom, S E

    1996-01-01

    Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8632997

  20. Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast.

    PubMed

    Qu, L H; Henras, A; Lu, Y J; Zhou, H; Zhou, W X; Zhu, Y Q; Zhao, J; Henry, Y; Caizergues-Ferrer, M; Bachellerie, J P

    1999-02-01

    Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII. The corresponding snoRNAs have been positively identified in yeast cells. Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested. The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA. Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p. After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated. In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated. This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs. In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor. PMID:9891049