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Sample records for ebaf homologue mrna

  1. Snipper, an Eri1 homologue, affects histone mRNA abundance and is crucial for normal Drosophila melanogaster development.

    Science.gov (United States)

    Alexiadis, Anastasios; Delidakis, Christos; Kalantidis, Kriton

    2017-07-01

    The conserved 3'-5' RNA exonuclease ERI1 is implicated in RNA interference inhibition, 5.8S rRNA maturation and histone mRNA maturation and turnover. The single ERI1 homologue in Drosophila melanogaster Snipper (Snp) is a 3'-5' exonuclease, but its in vivo function remains elusive. Here, we report Snp requirement for normal Drosophila development, since its perturbation leads to larval arrest and tissue-specific downregulation results in abnormal tissue development. Additionally, Snp directly interacts with histone mRNA, and its depletion results in drastic reduction in histone transcript levels. We propose that Snp protects the 3'-ends of histone mRNAs and upon its absence, histone transcripts are readily degraded. This in turn may lead to cell cycle delay or arrest, causing growth arrest and developmental perturbations. © 2017 Federation of European Biochemical Societies.

  2. Human low molecular weight neurofilament (NFL) mRNA interacts with a predicted p190RhoGEF homologue (RGNEF) in humans.

    Science.gov (United States)

    Volkening, Kathryn; Leystra-Lantz, Cheryl; Strong, Michael J

    2010-01-01

    In the mouse, p190RhoGEF is a low molecular weight neurofilament (NFL) mRNA stability factor that is involved in NF aggregate formation in neurons. A human homologue of this protein has not been described. Our objective was to identify a human homologue of p190RhoGEF, and to determine its interaction with human NFL mRNA. We used sequence homology searches to predict a human homologue (RGNEF), and RT-PCR to determine the expression of mRNA in ALS and neuropathologically normal control tissues. Gel shift assays determined the interaction of RGNEF with human NFL mRNA in vitro, while IP-RT-PCR and gel shift assays were used to confirm the interaction in tissue lysates. We determined that RGNEF is a human homologue of p190RhoGEF, and that its RNA is expressed in both brain and spinal cord. While RGNEF and NFL mRNA interact directly in vitro, interestingly they only appear to interact in ALS lysates and not in controls. These data add another player to the family of NFL mRNA stability regulators, and raise the intriguing possibility that the mechanism by which p190RhoGEF contributes to murine neuronal NF aggregate formation may be important to human ALS NF aggregate formation.

  3. Earth's Radiation Budget Variability During 2015 El Nino From CERES FLASHFlux and EBAF Data.

    Science.gov (United States)

    Sawaengphokhai, P.; Stackhouse, P. W.; Kratz, D. P.; Gupta, S. K.; Wilber, A. C.

    2015-12-01

    The Clouds and Earth's Radiant Energy System (CERES) Fast Longwave And SHortwave Radiative Fluxes (FLASHFlux) data products were introduced at the NASA Langley Research Center to address the need of the agricultural, renewable energy management, and science communities for global surface and top-of-atmosphere (TOA) radiative fluxes on a near real-time basis. This has been accomplished by enhancing the speed of CERES processing using simplified calibration and averaging techniques and fast radiation parameterizations to produce fluxes within a week of real-time. While the resulting products are not considered to be sufficiently accurate for studying long-term climate trends, they satisfy the needs for many near real-time scientific data analyses and societal applications. One of the uses of FLASHFlux data is for the evaluation of flux variability and extremes relative to climatological means. Normalizing FLASHFlux TOA fluxes with CERES Energy Balance And Filled (EBAF) TOA fluxes on a global scale, we are able to provide one-year flux change and flux anomalies relative to the EBAF TOA climatology for the "State of the Climate" report (published annually as a BAMS supplement). In this presentation, we extend our analysis to assess the seasonal variability and extremes for most of the year 2015 on a 1-degree regional scale. We also highlight the differences between FLASHFlux surface fluxes compared to the Surface EBAF flux products and assess the feasibility of normalizing the FLASHFLux surface fluxes to surface EBAF to provide surface flux anomalies on a regional scale. Using these anomalies for the TOA and possibly surface fluxes, we assess the radiative flux anomalies of the currently evolving 2015 El Nino on global and regional scales.

  4. Deep mRNA sequencing of the Tritonia diomedea brain transcriptome provides access to gene homologues for neuronal excitability, synaptic transmission and peptidergic signalling.

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    Adriano Senatore

    Full Text Available The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia, has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level.We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes. BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA.Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain.

  5. The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1: mRNA cloning, structure analysis and comparison with mammalian homologues

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    Thomas Anne VT

    2005-10-01

    Full Text Available Abstract Background Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2, the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX -producing bacteria. Results The porcine-LFA-1 CD11a (alpha subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. Conclusion Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.

  6. Estimation of the climate feedback parameter by using radiative fluxes from CERES EBAF

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    P. Björnbom

    2013-01-01

    Full Text Available Top-of-the-Atmosphere (TOA net radiative flux anomalies from Clouds and Earth's Radiant Energy Systems (CERES Energy Balanced and Filled (EBAF and surface air temperature anomalies from HadCRUT3 were compared for the time interval September 2000–May 2011. In a phase plane plot with the radiative flux anomalies lagging the temperature anomalies with 7 months the phase plane curve approached straight lines during about an eight months long period at the beginning and a five year period at the end of the interval. Both of those periods, but more clearly the latter one, could be connected to the occurrence of distinct El Niño Southern Oscillation (ENSO episodes. This result is explained by using a hypothesis stating that non-radiative forcing connected to the ENSO is dominating the temperature changes during those two periods and that there is a lag between the temperature change and the radiative flux feedback. According to the hypothesis the slopes of the straight lines equal the value of the climate feedback parameter. By linear regression based on the mentioned five year period the value of the climate feedback parameter was estimated to 5.5 ± 0.6 W m−2 K−1 (± two standard errors.

  7. An Evaluation of Satellite-Based and Re-Analysis Radiation Budget Datasets Using CERES EBAF Products

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    Gupta, Shashi; Stackhouse, Paul; Wong, Takmeng; Mikovitz, Colleen; Cox, Stephen; Zhang, Taiping

    2016-04-01

    Top-of-atmosphere (TOA) and surface radiative fluxes from CERES Energy Balanced and Filled (EBAF; Loeb et al., 2009; Kato et al. 2013) products are used to evaluate the performance of several widely used long-term radiation budget datasets. Two of those are derived from satellite observations and five more are from re-analysis products. Satellite-derived datasets are the NASA/GEWEX Surface and TOA Radiation Budget Dataset Release-3 and the ISCCP-FD Dataset. The re-analysis datasets are taken from NCEP-CFSR, ERA-Interim, Japanese Re-Analysis (JRA-55), MERRA and the newly released MERRA2 products. Close examination is made of the differences between MERRA and MERRA2 products for the purpose of identifying improvements achieved for MERRA2. Many of these datasets have undergone quality assessment under the GEWEX Radiative Flux Assessment (RFA) project. For the purposes of the present study, EBAF datasets are treated as reference and other datasets are compared with it. All-sky and clear-sky, SW and LW, TOA and surface fluxes are included in this study. A 7-year period (2001-2007) common to all datasets is chosen for comparisons of global and zonal averages, monthly and annual average timeseries, and their anomalies. These comparisons show significant differences between EBAF and the other datasets. Certain anomalies and trends observed in the satellite-derived datasets are attributable to corresponding features in satellite datasets used as input, especially ISCCP cloud properties. Comparisons of zonal averages showed significant differences especially over higher latitudes even when those differences are not obvious in the global averages. Special emphasis is placed on the analysis of the correspondence between spatial patterns of geographical distribution of the above fluxes on a 7-year average as well as on a month-by-month basis using the Taylor (2001) methodology. Results showed that for 7-year average fields correlation coefficients between spatial patterns

  8. Pepsin homologues in bacteria

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    Bateman Alex

    2009-09-01

    Full Text Available Abstract Background Peptidase family A1, to which pepsin belongs, had been assumed to be restricted to eukaryotes. The tertiary structure of pepsin shows two lobes with similar folds and it has been suggested that the gene has arisen from an ancient duplication and fusion event. The only sequence similarity between the lobes is restricted to the motif around the active site aspartate and a hydrophobic-hydrophobic-Gly motif. Together, these contribute to an essential structural feature known as a psi-loop. There is one such psi-loop in each lobe, and so each lobe presents an active Asp. The human immunodeficiency virus peptidase, retropepsin, from peptidase family A2 also has a similar fold but consists of one lobe only and has to dimerize to be active. All known members of family A1 show the bilobed structure, but it is unclear if the ancestor of family A1 was similar to an A2 peptidase, or if the ancestral retropepsin was derived from a half-pepsin gene. The presence of a pepsin homologue in a prokaryote might give insights into the evolution of the pepsin family. Results Homologues of the aspartic peptidase pepsin have been found in the completed genomic sequences from seven species of bacteria. The bacterial homologues, unlike those from eukaryotes, do not possess signal peptides, and would therefore be intracellular acting at neutral pH. The bacterial homologues have Thr218 replaced by Asp, a change which in renin has been shown to confer activity at neutral pH. No pepsin homologues could be detected in any archaean genome. Conclusion The peptidase family A1 is found in some species of bacteria as well as eukaryotes. The bacterial homologues fall into two groups, one from oceanic bacteria and one from plant symbionts. The bacterial homologues are all predicted to be intracellular proteins, unlike the eukaryotic enzymes. The bacterial homologues are bilobed like pepsin, implying that if no horizontal gene transfer has occurred the duplication

  9. Mammalian homologue of the calcium-sensitive phosphoglycoprotein, parafusin.

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    Wyroba, E; Widding Høyer, A; Storgaard, P; Satir, B H

    1995-12-01

    Three specific antipeptide antibodies and oligonucleotide probes synthesized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-sensitive phosphoglycoprotein from a unicellular eukaryote Paramecium that was recently cloned and sequenced (Subramanian et al., Proc. Natl. Acad. Sci. USA 91, 9832-9836 (1994)). Western and Southern blot analyses, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancreas, human pancreas and a murine pancreatic beta-cell line (beta TC3) arising in transgenic mice. The parafusin-specific antibodies showed cross-reaction with a protein at approximately 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the beta TC3 cells. The presence of two bands shows that parafusin homologue(s) and phosphoglucomutase are separate entities. beta TC3 cells were shown to incorporate [beta 35]UDPGlc into the parafusin homologue in a Ca(++)-sensitive manner characteristic of parafusin. Southern blot analysis revealed that the parafusin-specific probe hybridized with restriction enzyme digests of rat DNA in distinct patterns different from those observed with a phosphoglucomutase-specific probe. Rat genomic DNA and mRNA from the beta TC3 cells were used as the templates for PCR and RT-PCR using internal parafusin primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusion probe located within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.

  10. Crystal chemistry of sartorite homologues and related sulfosalts

    DEFF Research Database (Denmark)

    Berlepsch, Peter; Makovicky, Emil; Balic-Zunic, Tonci

    2001-01-01

    sartorite homologues, sulfosalt, crystal chemistry, coordination polyhedra, bond-pairs, crankshaft chains......sartorite homologues, sulfosalt, crystal chemistry, coordination polyhedra, bond-pairs, crankshaft chains...

  11. Crystal chemical formula for sartorite homologues

    DEFF Research Database (Denmark)

    Makovicky, Emil; Topa, Dan

    2015-01-01

    The members of the sartorite homologous series are complex sulfides Me 2+ 8N–16–2X Me 3+ 16+X Me + XS8N+8where Me 2+ is Pb and Me 3+ is As and Sb, whereas Me + is Ag and/or Tl. This paper presents calculation formulae for the homologue order N and for the separate substitution percentages for Tl ...... + (As,Sb) ↔ 2Pb and Ag + (As,Sb) ↔ 2Pb substitutions. This enables one to evaluate the crystal chemistry and build a systematic classification of the sartorite homologues...

  12. IDENTIFICATION OF HUMAN MURR1, THE HOMOLOGUE OF MOUSE IMPRINTED Murr1 GENE

    Institute of Scientific and Technical Information of China (English)

    Zhang Zhongming; Wang Youdong; Hitomi Yatsuki; Keiichiro Joh; Tsuyoshi Iwasaka; Tsunehiro Mukai

    2006-01-01

    Objective To identify the mRNA sequence, genetic construction, imprinting status, and expression profile of human MURR1 gene, the homologue of mouse imprinted Murr1 gene. Methods The MURR1 mRNA sequence was identified by colony hybridization screening of human cDNA library and the 5'-RACE analyses; Absence of U2AF1-RS1 gene within MURR1 was confirmed by Southern Blotting; Expression profile of MURR1 was examined by Northern Blotting; The imprinting status of MURR1 were revealed by SNP investigation and RT-PCR followed by sequencings and RFLP analyses. Results The full-length mRNA sequence of MURR1 spans 711 bp, transcribed from 3 exons, encodes predicted MURR1 protein of 190 amino acids. The gene was expressed in all the 12 kinds of human adult tissues and 6 kinds of fetal tissues. It showed biallelic expression in all 32 investigated samples including 6 kinds of human fetal tissues and 8 adult brains. Unlike mouse imprinted U2af1-rs1 gene existing in the intron of Murr1, the human U2AF1-RS1 gene was not located in the MURR1 locus. Conclusion Human MURR1 gene is not imprinted and the non-imprinting is possible due to the absence of human homologue of mouse U2af1-rs1 within MURR1 locus.

  13. Phosphatase and tensin homologue deleted on chromosome 10

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    Imran Haruna Abdulkareem

    2013-01-01

    Full Text Available Phosphatase and tensin homologue deleted on chromosome 10 (PTEN is a tumor suppressor gene deleted or mutated in many human cancers such as glioblastoma, spinal tumors, prostate, bladder, adrenals, thyroid, breast, endometrium, and colon cancers. They result from loss of heterozygosity (LOH for the PTEN gene on chromosome 10q23. Previous studies reported that various drugs, chemicals, and foods can up-regulate PTEN mRNA and protein expression in different cell lines, and they may be useful in the future prevention and/or treatment of these cancers. PTEN has also been observed to have prognostic significance and is gradually being accepted as an independent prognostic factor. This will help in monitoring disease progression and/or recurrence, with a view to improving treatment outcomes and reducing the associated morbidity and mortality from these cancers. Neprilysin (NEP is a zinc-dependent metallopeptidase that cleaves and inactivates some biologically active peptides thus switching off signal transduction at the cell surface. Decreased NEP expression in many cancers has been reported. NEP can form a complex with PTEN and enhance PTEN recruitment to the plasma membrane as well as stabilize its phosphatase activity. MicroRNA-21 (miR-21 post-transcriptionally down-regulates the expression of PTEN and stimulates growth and invasion in non-small cell lung cancer (NSCLC (lung Ca, suggesting that this may be a potential therapeutic target in the future treatment of NSCLC. PTEN is a tumor suppressor gene associated with many human cancers. This has diagnostic, therapeutic, and prognostic significance in the management of many human cancers, and may be a target for new drug development in the future.

  14. Characterization of the C. elegans erlin homologue

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    Hoegg Maja B

    2012-01-01

    Full Text Available Abstract Background Erlins are highly conserved proteins associated with lipid rafts within the endoplasmic reticulum (ER. Biochemical studies in mammalian cell lines have shown that erlins are required for ER associated protein degradation (ERAD of activated inositol-1,4,5-trisphosphate receptors (IP3Rs, implying that erlin proteins might negatively regulate IP3R signalling. In humans, loss of erlin function appears to cause progressive intellectual disability, motor dysfunction and joint contractures. However, it is unknown if defects in IP3R ERAD are the underlying cause of this disease phenotype, whether ERAD of activated IP3Rs is the only function of erlin proteins, and what role ERAD plays in regulating IP3R-dependent processes in the context of an intact animal or embryo. In this study, we characterize the erlin homologue of the nematode Caenorhabditis elegans and examine erlin function in vivo. We specifically set out to test whether C. elegans erlin modulates IP3R-dependent processes, such as egg laying, embryonic development and defecation rates. We also explore the possibility that erlin might play a more general role in the ERAD pathway of C. elegans. Results We first show that the C. elegans erlin homologue, ERL-1, is highly similar to mammalian erlins with respect to amino acid sequence, domain structure, biochemical properties and subcellular location. ERL-1 is present throughout the C. elegans embryo; in adult worms, ERL-1 appears restricted to the germline. The expression pattern of ERL-1 thus only partially overlaps with that of ITR-1, eliminating the possibility of ERL-1 being a ubiquitous and necessary regulator of ITR-1. We show that loss of ERL-1 does not affect overall phenotype, or alter brood size, embryonic development or defecation cycle length in either wild type or sensitized itr-1 mutant animals. Moreover we show that ERL-1 deficient worms respond normally to ER stress conditions, suggesting that ERL-1 is not an

  15. Is the Prosthetic Homologue Necessary for Embodiment?

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    Dornfeld, Chelsea; Swanston, Michelle; Cassella, Joseph; Beasley, Casey; Green, Jacob; Moshayev, Yonatan; Wininger, Michael

    2016-01-01

    Embodiment is the process by which patients with limb loss come to accept their peripheral device as a natural extension of self. However, there is little guidance as to how exacting the prosthesis must be in order for embodiment to take place: is it necessary for the prosthetic hand to look just like the absent hand? Here, we describe a protocol for testing whether an individual would select a hand that looks like their own from among a selection of five hands, and whether the hand selection (regardless of homology) is consistent across multiple exposures to the same (but reordered) set of candidate hands. Pilot results using healthy volunteers reveals that hand selection is only modestly consistent, and that selection of the prosthetic homologue is atypical (61 of 192 total exposures). Our protocol can be executed in minutes, and makes use of readily available equipment and softwares. We present both a face-to-face and a virtual protocol, for maximum flexibility of implementation. PMID:28066228

  16. Is the prosthetic homologue necessary for embodiment?

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    Chelsea Dornfeld

    2016-12-01

    Full Text Available Embodiment is the process by which patients with limb loss come to accept their peripheral device as a natural extension of self. However, there is little guidance as to how exacting the prosthesis must be in order for embodiment to take place: is it necessary for the prosthetic hand to look just like the absent hand? Here, we describe a protocol for testing whether an individual would select a hand that looks like their own from among a selection of 5 hands, and whether the hand selection (regardless of homology is consistent across multiple exposures to the same (but reordered set of candidate hands. Pilot results using healthy volunteers reveals that hand selection is only modestly consistent, and that selection of the prosthetic homologue is atypical (61 of 192 total exposures. Our protocol can be executed in minutes, and makes use of readily available equipment and softwares. We present both a face-to-face and a virtual protocol, for maximum flexibility of implementation.

  17. Cloning of rat sp56, the homologue of mouse sperm ZP3 receptor-sp56

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3 (mZP3) receptor. Up to date, its homologue has only been cloned from guinea pig, namely AM67. Based on the cDNA sequence of mouse sp56, we designed a pair of primer to amplify its homologue from rat testis cDNA. Using RT-PCR,two fragments of 743 bp and 938 bp were amplified. The PCR products show very high homology to mouse sp56. However, the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56. Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues, Northern blot shows that a ~2.0 kb mRNA expresses specifically in testis. Employed the RACE method, two full cDNA sequences of rat sp56 were obtained. A Mr ~42 KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method. Rat sp56was localized on rat sperm head by the indirect immunofiuorescence method. Rat sp56 immunoreactivitywas detected from the early pachytene spermatocytes and throughout the spermatogenesis. Its cloning willfurther our understanding of the mechanism of the sperm-egg recognition and binding.

  18. Isolation of a cotton NADP(H oxidase homologue induced by drought stress

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    NEPOMUCENO ALEXANDRE LIMA

    2000-01-01

    Full Text Available The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L. genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription.

  19. Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

    Institute of Scientific and Technical Information of China (English)

    QiFan; LijiaAn; LiwangCui

    2005-01-01

    The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodiumfalclparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.

  20. Detection of a Yersinia pestis gene homologue in rodent samples

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    Timothy A. Giles

    2016-08-01

    Full Text Available A homologue to a widely used genetic marker, pla, for Yersinia pestis has been identified in tissue samples of two species of rat (Rattus rattus and Rattus norvegicus and of mice (Mus musculus and Apodemus sylvaticus using a microarray based platform to screen for zoonotic pathogens of interest. Samples were from urban locations in the UK (Liverpool and Canada (Vancouver. The results indicate the presence of an unknown bacterium that shares a homologue for the pla gene of Yersinia pestis, so caution should be taken when using this gene as a diagnostic marker.

  1. Characterization of the POU5F1 Homologue in Nile Tilapia: From Expression Pattern to Biological Activity.

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    Xiaohuan, Huang; Yang, Zhao; Linyan, Liu; Zhenhua, Fan; Linyan, Zhou; Zhijian, Wang; Ling, Wei; Deshou, Wang; Jing, Wei

    2016-09-15

    POU5F1 (OCT4) is a crucial transcription factor for induction and maintenance of cellular pluripotency, as well as survival of germ cells in mammals. However, the homologues of POU5F1 in teleost fish, including zebrafish and medaka, now named Pou5f3, exhibit considerable differences in expression pattern and pluripotency-maintaining activity. To what extent the POU5F1 homologues are conserved in vertebrates has been unclear. In this study, we report that the POU5F1 homologue from the Nile tilapia (Oreochromis niloticus), OnPou5f3, displays an expression pattern and biological activity somewhat different from those in zebrafish or medaka. The expression of Onpou5f3 at both mRNA and protein levels was abundant in early development embryos until blastula stages, barely detectable as proceeding, and then displayed a transiently strong expression domain in the brain region during neurula stages similar to zebrafish but not medaka. Afterward, OnPou5f3 appeared as germline-restricted (including primordial germ cells and female and male gonad germ cells) expression just like medaka. Notably, OnPou5f3 depletion through morpholino oligos caused blastula blockage or lethality and failure of survival and proliferation of blastula cell-derived cells. These findings indicate that equivalent POU5F1-like expression and activity of Pou5f3 might be conserved accompanying with species-specific expression pattern during evolution. Our study provides insight into the evolutionary conservation of the POU5F1 homologues across vertebrates.

  2. Lipopolysaccharide-induced expression of IP-10 mRNA in rat brain and in cultured rat astrocytes and microglia

    NARCIS (Netherlands)

    Ren, LQ; Gourmala, N; Boddeke, HWGM; Gebicke-Haerter, PJ

    1998-01-01

    Using mRNA differential display technique, we have found a differentially expressed band in rat brain, designated HAP(2)G1, which was the strongest one induced in response to peripheral administration of lipopolysaccharide (LPS). Sequence analysis showed that HAP(2)G1 cDNA is the rat homologue of th

  3. Isolation and characterization of an AGAMOUS homologue from cocoa

    NARCIS (Netherlands)

    Chaidamsari, T.; Sugiarit, H.; Santoso, D.; Angenent, G.C.; Maagd, de R.A.

    2006-01-01

    We report the cloning of a cDNA from TcAG, an AG (Arabidopsis thaliana MADS-box C-type transcription factor gene AGAMOUS) homologue from cocoa (Theobroma cacao L.). TcAG was in the cocoa flower expressed primarily in stamens and ovaries, comparable to AG in Arabidopsis. Additionally, we found that T

  4. Characterization and expression analysis of an allograft inflammatory factor-1 homologue in yellow grouper (Epinephelus awoara)

    Institute of Scientific and Technical Information of China (English)

    WANG Li; SHI Dawei; WU Xinzhong

    2008-01-01

    Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic calcium-binding protein involved in iullammatory response-related dis-eases in mammals.Previously an identified AIF-1 gene was simply reported in yellow grouper.The characterization of AIF-1 gene and its expression at the gene and protein level are further described.Yellow grouper AIF-1 is composed of 147 amino acids,and 64% ~ 84% identical to other homologues.Basal level AIF-I mRNA expression was noted in spleen,anterior kid-ney and kidney,using reverse-transcription polymerase chain reaction (RT-PCR).After stimulation of LPS,the AIF-1 mRNA expression was up-regulated in tissues examined:spleen,anterior kidney,kidney,heart and liver,but not in muscle.The re-combinant AIF-1 protein was expressed in Escherichia coli,and then purified for the development of antiserum.Western blotting analysis revealed a band with a molecular mass of about 17 ku.

  5. Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA

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    Goldman Gustavo H

    2010-01-01

    Full Text Available Abstract Background Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and ΔAfcrzA mutant strains. Results We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively. Decreased mRNA abundance in the ΔcrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 μM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride. Conclusion We have performed a transcriptional profiling analysis of the A. fumigatus ΔAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin

  6. Monolayer structures of alkyl aldehydes: Odd-membered homologues

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, T.K. [BP Institute, Department of Chemistry, University of Cambridge, Cambridge (United Kingdom); Clarke, S.M., E-mail: stuart@bpi.cam.ac.u [BP Institute, Department of Chemistry, University of Cambridge, Cambridge (United Kingdom); Bhinde, T. [BP Institute, Department of Chemistry, University of Cambridge, Cambridge (United Kingdom); Castro, M.A.; Millan, C. [Instituto Ciencia de los Materiales de Sevilla, Departamento de Quimica Inorganica (CSIC-Universidad de Sevilla) (Spain); Medina, S. [Centro de Investigacion, Tecnologia e Innovacion de la Universidad de Sevilla (CITIUS), Sevilla (Spain)

    2011-03-01

    Crystalline monolayers of three aldehydes with an odd number of carbon atoms in the alkyl chain (C{sub 7}, C{sub 9} and C{sub 11}) at low coverages are observed by a combination of X-ray and neutron diffraction. Analysis of the diffraction data is discussed and possible monolayer crystal structures are proposed; although unique structures could not be ascertained for all molecules. We conclude that the structures are flat on the surface, with the molecules lying in the plane of the layer. The C{sub 11} homologue is determined to have a plane group of either p2, pgb or pgg, and for the C{sub 7} homologue the p2 plane group is preferred.

  7. Characterization of mouse Dach2, a homologue of Drosophila dachshund.

    Science.gov (United States)

    Davis, R J; Shen, W; Sandler, Y I; Heanue, T A; Mardon, G

    2001-04-01

    The Drosophila genes eyeless, eyes absent, sine oculis and dachshund cooperate as components of a network to control retinal determination. Vertebrate homologues of these genes have been identified and implicated in the control of cell fate. We present the cloning and characterization of mouse Dach2, a homologue of dachshund. In situ hybridization studies demonstrate Dach2 expression in embryonic nervous tissues, sensory organs and limbs. This pattern is similar to mouse Dach1, suggesting a partially redundant role for these genes during development. In addition, we determine that Dach2 expression in the forebrain of Pax6 mutants and dermamyotome of Pax3 mutants is not detectably altered. Finally, genetic mapping experiments place mouse Dach2 on the X chromosome between Xist and Esx1. The identification of human DACH2 sequences at Xq21 suggests a possible role for this gene in Allan-Herndon syndrome, Miles-Carpenter syndrome, X-linked cleft palate and/or Megalocornea.

  8. In vitro dentine remineralization with a potential salivary phosphoprotein homologue.

    Science.gov (United States)

    Romero, Maria Jacinta Rosario H; Nakashima, Syozi; Nikaido, Toru; Sadr, Alireza; Tagami, Junji

    2016-08-01

    Advantages of introducing a salivary phosphoprotein homologue under standardized in vitro conditions to simulate the mineral-stabilizing properties of saliva have been proposed. This study longitudinally investigates the effects of casein, incorporated as a potential salivary phosphoprotein homologue in artificial saliva (AS) solutions with/without fluoride (F) on in vitro dentine lesion remineralization. Thin sections of bovine root dentine were demineralized and allocated randomly into 6 groups (n=18) having equivalent mineral loss (ΔZ) after transverse microradiography (TMR). The specimens were remineralized using AS solutions containing casein 0μg/ml, F 0ppm (C0-F0); casein 0μg/ml, F 1ppm (C0-F1); casein 10μg/ml, F 0ppm (C10-F0); casein 10μg/ml, F 1ppm (C10-F1); casein 100μg/ml, F 0ppm (C100-F0) or casein 100μg/ml, F 1ppm (C100-F1) for 28days with TMR taken every 7 days. Surface mineral precipitation, evident in group C0-F1, was apparently inhibited in groups with casein incorporation. Repeated measures ANOVA with Bonferroni correction revealed higher ΔZ for non-F and non-casein groups than for their counterparts (p<0.001). Subsequent multiple comparisons showed that mineral gain was higher (p<0.001) with 10μg/ml casein than with 100μg/ml when F was present in the earlier stages of remineralization, with both groups achieving almost complete remineralization after 28 days. Casein is a potential salivary phosphoprotein homologue that could be employed for in vitro dentine remineralization studies. Concentration related effects may be clinically significant and thus must be further examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    Energy Technology Data Exchange (ETDEWEB)

    Ding, J.H.; Yang, B.Z.; Liu, H.M. [Duke Univ. Medical Center, Durham, NC (United States)] [and others

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  10. Characterization of a chaperone ClpB homologue of Paracoccidioides brasiliensis.

    Science.gov (United States)

    Jesuino, Rosália S A; Azevedo, Maristela O; Felipe, M Sueli S; Pereira, Maristela; De Almeida Soares, Célia M

    2002-08-01

    We report the cloning and sequence analysis of a genomic clone encoding a Paracoccidioides brasiliensis ClpB chaperone homologue (PbClpB). The clpb gene was identified in a lambda Dash II library. Sequencing of Pbclpb revealed a long open reading frame capable of encoding a 792 amino acid, 87.9 kDa protein, pI of 5.34. The predicted polypeptide contains several consensus motifs of the ClpB proteins. Canonical sequences such as two putative nucleotide-binding sites, chaperonins ClpA/B signatures and highly conserved casein kinase phosphorylation domains are present. ClpB is 69% to 49% identical to members of the ClpB family from several organisms from prokaryotes to eukaryotes. The transcript of PbclpB was detected as a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phase of the fungus. A 89 kDa protein was also detected in yeast cells of P. brasiliensis.

  11. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  12. A novel lipocalin homologue from the venom gland of Deinagkistrodon acutus similar to mammalian lipocalins

    Directory of Open Access Journals (Sweden)

    CB Wei

    2012-01-01

    Full Text Available Lipocalins are involved in a variety of functions including retinol transport, cryptic coloration, olfaction, pheromone transport, prostaglandin synthesis, regulation of the immune response and cell homeostatic mediation. A full-length cDNA clone (named d-lipo, isolated from the venom gland cDNA library of Deinagkistrodon acutus, contained an insert of 664 bp including an open reading frame that encodes a lipocalin homologue of 177 amino acids. Comparison of d-lipo and other related proteins revealed an overall amino acid identity of less than 21.5%. Primary structures of d-lipo carried three structurally conserved regions (SCR showing homologies to those of lipocalins. The first conserved Cys residue - the essential amino acid residue for the catalytic activity and unique to lipocalin-type prostaglandin D synthase (L-PGDS in the lipocalin protein family - was identified in d-lipo at amino acid position 58. Phylogenetic tree analysis showed that d-lipo was in-between the large L-PGDS cluster and the small von Ebner's-gland proteins (VEGP cluster. Moreover, d-lipo gene presented a high-level expression in the venom gland and a low-level expression in the brain and its expression was significantly increased under pathological conditions, suggesting a possible relationship between d-lipo mRNA expression and the venom gland inflammatory disease. This is also the first report of a lipocalin homologous gene identified in the venom gland of a snake.

  13. Cloning of an agr homologue of Staphylococcus saprophyticus.

    Science.gov (United States)

    Sakinc, Türkan; Kulczak, Pawel; Henne, Karsten; Gatermann, Sören G

    2004-08-01

    An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.

  14. Alkylresorcinol content and homologue composition in durum wheat (Triticum durum) kernels and pasta products.

    Science.gov (United States)

    Landberg, Rikard; Kamal-Eldin, Afaf; Andersson, Roger; Aman, Per

    2006-04-19

    The total alkylresorcinol (AR) content and relative homologue composition of 21 durum wheat (Triticum durum) kernel samples, as well as 5 pasta products and the corresponding flour mixtures, were determined. Durum wheat contained on average 455 microg/g ARs, and the average relative homologue composition was C17:0 (0.4%), C19:0 (14%), C21:0 (58%), C23:0 (21%), and C25:0 (6.5%). The homologue composition was found to be relatively consistent among samples, with durum wheat being different from common wheat by having a higher proportion of the longer homologues. No differences in content or homologue composition were observed in pasta products compared to flour ingredients, showing that alkylresorcinols are stable during pasta processing. The ratio of the homologues C17:0 to C21:0 was wheat products, which is different from those of common wheat (0.1) and rye (0.9).

  15. Structure of a DsbF homologue from Corynebacterium diphtheriae.

    Science.gov (United States)

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-09-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein.

  16. Characterization of the cDNA encoding a BPI/LBP homologue in venom gland of the hundred-pace snake Deinagkistrodon acutus

    Directory of Open Access Journals (Sweden)

    Jianrao HU, Mingfu CAO, Jiong Chen

    2009-10-01

    Full Text Available Bactericidal/permeability-increasing protein (BPI and LPS-binding protein (LBP play an important role in host defence. Current evidence shows that BPI/LBP may be widely existed in different cells and tissue types of animals. A full-length cDNA clone encoding a BPI/LBP homologue (dBPI, 1757bp in size, was characterized in venom gland of the hundred-pace snake Deinagkistrodon acutus. Its deduced amino acid sequence of 417 residues had 13.8%–21.5% identity to BPI like 1(BPIL1 and BPI like 3(BPIL3 of other animals. Conserved cysteine residues which are involved in disulfide bond formation between the final strand of the N-terminal beta sheet and the long alpha helix of BPI are identified as Cys146-Cys183 of dBPI. Phylogenetic tree analysis showed that the BPI/LBP homologues formed five large clusters and dBPI was in a large cluster including BPIL1 and BPIL3. dBPI mRNA shows a tissue specific expression in venom gland. This is the first study to identify the cDNA encoding BPI/LBP homologues from reptiles [Current Zoology 55 (5: –2009].

  17. Characterization of the cDNA encoding a BPI/LBP homologue in venom gland of the hundred-pace snake Deinagkistrodon acutus

    Institute of Scientific and Technical Information of China (English)

    Jianrao HU; Mingfu CAO; Jiong CHEN

    2009-01-01

    Bactericidal/permeability-increasing protein (BPI) and LPS-binding protein (LBP) play an important role in host defence. Current evidence shows that BPI/LBP may be widely existed in different cells and tissue types of animals. A full-length cDNA clone encoding a BPI/LBP homologue (dBPI), 1757 bp in size, was characterized in venom gland of the hundred-pace snake Deinagkistrodon acutus. Its deduced amino acid sequence of 417 residues had 13.8% - 21.5% identity to BPI like 1 (BPIL1) and BPI like 3 (BPIL3) of other animals. Conserved cysteine residues which are involved in disulfide bond formation between the final strand of the N-terminal beta sheet and the long alpha helix of BPI are identified as Cys146-Cys183 of dBPI. Phylogenetic tree analysis showed that the BPI/LBP homologues formed five large clusters and dBPl was in a large cluster including BPIL1 and BPIL3. dBPI mRNA shows a tissue specific expression in venom gland. This is the fast study to identify the cDNA encoding BPI/LBP homologues from reptiles [Current Zoology 55 (5) : 376 - 382, 2009].

  18. Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse

    Directory of Open Access Journals (Sweden)

    Green Carla B

    2001-05-01

    Full Text Available Abstract Background Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. Results cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. Conclusion The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

  19. Histamine homologues discriminating between two functional H3 receptor assays. Evidence for H3 receptor heterogeneity?.

    Science.gov (United States)

    Leurs, R; Kathmann, M; Vollinga, R C; Menge, W M; Schlicker, E; Timmerman, H

    1996-03-01

    We studied several histamine homologues as potential ligands for the histamine H3 receptor in two binding assays ([125l]iodophenpropit and N alpha-[3H]methylhistamine binding to rat brain cortex membranes) and two functional H3 receptor models (inhibition of the neurogenic contraction in the guinea pig jejunum and of [3H]noradrenaline release in mouse brain cortex slices). The histamine homologues acted all as competitive H3 antagonists at the guinea pig jejunum. The potency in this model and/or the affinity for N alpha-[3H]methylhistamine binding was higher for the butylene (pA2 = 7.7; pKi = 9.4) and pentylene homologue (impentamine, pA2 = 8.4; pKi = 9.1) than for the propylene, hexylene and octylene homologues (pA2 = 5.9-7.8; pKi = 6.1-7.6). In the mouse brain cortex the propylene, butylene and pentylene homologues acted as partial agonists (alpha = 0.3-0.6) and the hexylene and octylene homologues acted as antagonists. [125I]Iodophenpropit binding was displaced monophasically by the propylene, hexylene and octylene homologues and biphasically by the butylene and pentylene homologues. Biphasic displacement curves were converted to monophasic ones by 10 microM guanosine-5'-O-(3-thiotriphosphate. In conclusion, the homologue of histamine with five methylene groups is a more potent H3 receptor antagonist in the guinea pig jejunum than the other homologues tested. Furthermore, the propylene, butylene and pentylene homologues can discriminate between the two functional H3 receptor models in the guinea pig jejunum and mouse brain. These data are discussed in relation to the efficiency of receptor coupling and receptor heterogeneity.

  20. Structure of a bacterial homologue of vitamin K epoxide reductase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A. (Harvard-Med); (HHMI)

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  1. Isolation and characterization of the human MRE11 homologue

    Energy Technology Data Exchange (ETDEWEB)

    Petrini, J.H.J.; Walsh, M.E.; DiMare, C. [Harvard Medical School, Boston, MA (United States)] [and others

    1995-09-01

    Mutation of the Saccharomyces cerevisiae RAD52 epistasis group gene, MRE11, blocks meiotic recombination, confers profound sensitivity to double-strand break damage, and has a hyperrecombinational phenotype in mitotic cells. We isolated a highly conserved human MRE11 homologue using a two-hybrid screen for DNA ligase I-interacting proteins. Human MRE11 shares approximately 50% identity with its yeast counterpart over the N-terminal half of the protein. MRE11 is expressed at the highest levels in proliferating tissues, but is also observed in other tissues. The MRE11 locus maps to human chromosome 11q21 in a region frequently associated with cancer-related chromosomal abnormalities. A MRE11-related locus was found on chromosome 7q11.2-q11.3. 60 refs., 4 figs.

  2. Molecular cloning, sequence characterization and expression analysis of a CD63 homologue from the coleopteran beetle, Tenebrio molitor.

    Science.gov (United States)

    Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

    2013-10-15

    CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic "Cys-Cys-Gly" motif and "Cys188" residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

  3. Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

    Directory of Open Access Journals (Sweden)

    Yeon Soo Han

    2013-10-01

    Full Text Available CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63 was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

  4. miR-210 Targets Iron-Sulfur Cluster Scaffold Homologue in Human Trophoblast Cell Lines

    Science.gov (United States)

    Lee, Deug-Chan; Romero, Roberto; Kim, Jung-Sun; Tarca, Adi L.; Montenegro, Daniel; Pineles, Beth L.; Kim, Ernest; Lee, JoonHo; Kim, Sun Young; Draghici, Sorin; Mittal, Pooja; Kusanovic, Juan Pedro; Chaiworapongsa, Tinnakorn; Hassan, Sonia S.; Kim, Chong Jai

    2011-01-01

    This study was performed to assess the biological significance of miR-210 in preeclampsia and small-for-gestational-age (SGA) pregnancies. Placental miR-210 expression was evaluated by quantitative RT-PCR (RT-qPCR) in the following groups: i) appropriate-for-gestational-age pregnancies (n = 72), ii) preeclampsia (n = 52), iii) SGA (n = 66), and iv)preeclampsia with SGA (n = 31). The effects of hypoxia (1% O2) on miR-210 and iron-sulfur cluster scaffold homologue (ISCU) expressions and miR-210 binding to ISCU 3′ UTR were examined in Swan 71 and BeWo cell lines. Perls' reaction (n = 229) and electron microscopy (n = 3) were conducted to verify siderosis of trophoblasts. miR-210 expression was increased in preeclampsia and SGA cases and was decreased with birth weight and gestational age. In both cell lines, miR-210 was induced by hypoxia, whereas ISCU expression was decreased. The luciferase assay confirmed miR-210 binding to ISCU mRNA 3′ UTR. RNA interference knockdown of ISCU expression in Swan 71, but not in BeWo, cells resulted in autophagosomal and siderosomal iron accumulation and a fourfold decrease of Matrigel invasion (P = 0.004). Placental ISCU expression was decreased in preeclampsia (P = 0.002) and SGA (P = 0.002) cases. Furthermore, hemosiderin-laden trophoblasts were more frequent in the placental bed of preterm preeclampsia and/or SGA births than in control cases (48.7% versus 17.9%; P = 0.004). Siderosis of interstitial trophoblasts is a novel pathological feature of preeclampsia and SGA. The findings herein suggest that ISCU down-regulation by miR-210 perturbing trophoblast iron metabolism is associated with defective placentation. PMID:21801864

  5. A La autoantigen homologue is required for the internal ribosome entry site mediated translation of giardiavirus.

    Directory of Open Access Journals (Sweden)

    Srinivas Garlapati

    Full Text Available Translation of Giardiavirus (GLV mRNA is initiated at an internal ribosome entry site (IRES in the viral transcript. The IRES localizes to a downstream portion of 5' untranslated region (UTR and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA, known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200-348aa of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.

  6. Homologue Pairing in Flies and Mammals: Gene Regulation When Two Are Involved

    Directory of Open Access Journals (Sweden)

    Manasi S. Apte

    2012-01-01

    Full Text Available Chromosome pairing is usually discussed in the context of meiosis. Association of homologues in germ cells enables chromosome segregation and is necessary for fertility. A few organisms, such as flies, also pair their entire genomes in somatic cells. Most others, including mammals, display little homologue pairing outside of the germline. Experimental evidence from both flies and mammals suggests that communication between homologues contributes to normal genome regulation. This paper will contrast the role of pairing in transmitting information between homologues in flies and mammals. In mammals, somatic homologue pairing is tightly regulated, occurring at specific loci and in a developmentally regulated fashion. Inappropriate pairing, or loss of normal pairing, is associated with gene misregulation in some disease states. While homologue pairing in flies is capable of influencing gene expression, the significance of this for normal expression remains unknown. The sex chromosomes pose a particularly interesting situation, as females are able to pair X chromosomes, but males cannot. The contribution of homologue pairing to the biology of the X chromosome will also be discussed.

  7. Homologues of sox8 and sox10 in the orange-spotted grouper Epinephelus coioides: sequences, expression patterns, and their effects on cyp19a1a promoter activities in vitro.

    Science.gov (United States)

    Liu, Qiongyou; Lu, Huijie; Zhang, Lihong; Xie, Jun; Shen, Wenying; Zhang, Weimin

    2012-09-01

    Sox8 and Sox10 are members of group E Sox proteins involved in a wide range of developmental processes including sex determination and neurogenesis in vertebrates. The orange-spotted grouper sox8a and sox10a homologues were isolated and characterized in the present study. Both sox8a and sox10a genes contain three exons and two introns, and encode putative proteins with typical structures of group E Sox. Sox8a was expressed in diverse tissues including the central nervous system and some peripheral tissues. In contrast, sox10a mRNA was detected primarily in the central nervous system. During embryogenesis, sox8a mRNA seemed to be de novo synthesized in the embryos from otic vesicle stage. However, sox10a mRNA was only detectable in juvenile fish 35 days post hatching and thereafter. The mRNA levels of sox8a in the gonads were not significantly different among ovarian developmental stages but increased in the testis. In vitro transfection assays showed that the Sox10a but not Sox8a up-regulated cyp19a1a promoter activities. Taken together, these results suggested that the sox8a may play roles in diverse tissues and during embryogenesis, whereas sox10a may be mainly involved in the neural regulation of juvenile and adult fish, and that certain Sox homologues may regulate the orange-spotted grouper cyp19a1a promoter.

  8. Phosphatase and Tensin Homologue: Novel Regulation by Developmental Signaling

    Directory of Open Access Journals (Sweden)

    Travis J. Jerde

    2015-01-01

    Full Text Available Phosphatase and tensin homologue (PTEN is a critical cell endogenous inhibitor of phosphoinositide signaling in mammalian cells. PTEN dephosphorylates phosphoinositide trisphosphate (PIP3, and by so doing PTEN has the function of negative regulation of Akt, thereby inhibiting this key intracellular signal transduction pathway. In numerous cell types, PTEN loss-of-function mutations result in unopposed Akt signaling, producing numerous effects on cells. Numerous reports exist regarding mutations in PTEN leading to unregulated Akt and human disease, most notably cancer. However, less is commonly known about nonmutational regulation of PTEN. This review focuses on an emerging literature on the regulation of PTEN at the transcriptional, posttranscriptional, translational, and posttranslational levels. Specifically, a focus is placed on the role developmental signaling pathways play in PTEN regulation; this includes insulin-like growth factor, NOTCH, transforming growth factor, bone morphogenetic protein, wnt, and hedgehog signaling. The regulation of PTEN by developmental mediators affects critical biological processes including neuronal and organ development, stem cell maintenance, cell cycle regulation, inflammation, response to hypoxia, repair and recovery, and cell death and survival. Perturbations of PTEN regulation consequently lead to human diseases such as cancer, chronic inflammatory syndromes, developmental abnormalities, diabetes, and neurodegeneration.

  9. Characterization of Major Surface Protease Homologues of Trypanosoma congolense

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    Veronica Marcoux

    2010-01-01

    Full Text Available Trypanosomes encode a family of proteins known as Major Surface Metalloproteases (MSPs. We have identified six putative MSPs encoded within the partially sequenced T. congolense genome. Phylogenic analysis indicates that T. congolense MSPs belong to five subfamilies that are conserved among African trypanosome species. Molecular modeling, based on the known structure of Leishmania Major GP63, reveals subfamily-specific structural variations around the putative active site despite conservation of overall structure, suggesting that each MSP subfamily has evolved to recognize distinct substrates. We have cloned and purified a protein encoding the amino-terminal domain of the T. congolense homologue TcoMSP-D (most closely related to Leishmania GP63. We detect TcoMSP-D in the serum of T. congolense-infected mice. Mice immunized with the amino-terminal domain of TcoMSP-D generate a persisting IgG1 antibody response. Surprisingly, a low-dose challenge of immunized mice with T. congolense significantly increases susceptibility to infection, indicating that immunity to TcoMSP-D is a factor affecting virulence.

  10. Identification and analysis of cation channel homologues in human pathogenic fungi.

    Directory of Open Access Journals (Sweden)

    David L Prole

    Full Text Available Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K(+, calcium (Ca(2+ and transient receptor potential (Trp channels, but not sodium (Na(+ channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K(+ and Trp channel subunits, and genes encoding novel homologues of voltage-gated K(v channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca(2+ channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca(2+ channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca(2+ uniporter (MCU. In contrast to humans, which express many K(+, Ca(2+ and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K(+, Ca(2+ and Trp channel homologues. Furthermore, the sequences of fungal K(+, Ca(2+, Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

  11. [Patterns of PCDD/Fs, PCBs and PCNs homologues in fly ash from cement kilns].

    Science.gov (United States)

    Zhang, Jing; Ni, Yu-Wen; Zhang, Hai-Jun; Zhang, Xue-Ping; Zhang, Qing; Chen, Ji-Ping

    2009-02-15

    The concentrations and toxic equivalent (TEQ) values of PCDD/Fs, PCBs and PCNs in fly ash collected from three types of cement kilns (vertical shaft kiln, wet-process rotary kiln and dry-process rotary kiln) and two types of waste incinerators were determined, and the patterns of homologues and congeners were compared. The results showed that the total TEQ of PCDD/Fs, PCBs and PCNs in cement kiln fly ash, which were in the range of 4.0-62, 0.069-3.9 and 0.47-2.8 ng x kg(-1) respectively, were much lower than that of fly ash from waste incinerators. In cement kiln fly ash, the predominating PCDD/Fs homologues were TCDFs, and the chief 2, 3, 7, 8-PCDD/Fs congeners were OCDD, 2, 3, 7, 8-TCDF and 1, 2, 3, 4, 6, 7, 8-HpCDF. The patterns of PCBs homologues in cement kiln fly ash were similar to those of waste incinerators in which TeCB were predominating homologues. PCB77, PCB105, PCB118 were at higher concentrations than other co-polar PCBs. Different types of cement kiln fly ash presented similar PCNs homologue patterns. The predominant homologues were TeCN, whereas OcCN were not detected. PCN 66/67 which has dioxin like toxity was the most abundant congener in all fly ash.

  12. The human homologue of macaque area V6A.

    Science.gov (United States)

    Pitzalis, S; Sereno, M I; Committeri, G; Fattori, P; Galati, G; Tosoni, A; Galletti, C

    2013-11-15

    In macaque monkeys, V6A is a visuomotor area located in the anterior bank of the POs, dorsal and anterior to retinotopically-organized extrastriate area V6 (Galletti et al., 1996). Unlike V6, V6A represents both contra- and ipsilateral visual fields and is broadly retinotopically organized (Galletti et al., 1999b). The contralateral lower visual field is over-represented in V6A. The central 20°-30° of the visual field is mainly represented dorsally (V6Ad) and the periphery ventrally (V6Av), at the border with V6. Both sectors of area V6A contain arm movement-related cells, active during spatially-directed reaching movements (Gamberini et al., 2011). In humans, we previously mapped the retinotopic organization of area V6 (Pitzalis et al., 2006). Here, using phase-encoded fMRI, cortical surface-based analysis and wide-field retinotopic mapping, we define a new cortical region that borders V6 anteriorly and shows a clear over-representation of the contralateral lower visual field and the periphery. As with macaque V6A, the eccentricity increases moving ventrally within the area. The new region contains a non-mirror-image representation of the visual field. Functional mapping reveals that, as in macaque V6A, the new region, but not the nearby area V6, responds during finger pointing and reaching movements. Based on similarity in position, retinotopic properties, functional organization and relationship with the neighboring extrastriate visual areas, we propose that the new cortical region is the human homologue of macaque area V6A.

  13. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    Energy Technology Data Exchange (ETDEWEB)

    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  14. Acute digoxin loading reduces ABCA8A mRNA expression in the mouse liver.

    Science.gov (United States)

    Wakaumi, Michi; Ishibashi, Kenichi; Ando, Hitoshi; Kasanuki, Hiroshi; Tsuruoka, Shuichi

    2005-12-01

    Human ABCA8, a new member of the ATP binding cassette (ABC) transporter family, transports certain lipophilic drugs, such as digoxin. To investigate the roles of this transporter, we cloned a mouse homologue of ABCA8, from a mouse heart cDNA library, named ABCA8a. The deduced mouse ABCA8a protein is 66% identical with that of human ABCA8 and possesses features common to the ABC superfamily. It was found that ABCA8a was mainly expressed in the liver and heart, similar to human ABCA8. We further evaluated the effect of acute digoxin (a substrate for ABCA8) intoxication on the mRNA expression of ABCA8 using northern blotting with a 3' non-coding region as a probe to avoid cross-hybridization with other ABCA genes. Following acute digoxin infusion, the mRNA expression of ABCA8 was significantly reduced in the liver 12-24 h after injection (14.7% of vehicle treatment), but not in the heart and kidney. Real-time quantitative polymerase chain reaction analysis confirmed the reduction in ABCA8a mRNA. Similar reductions in ABCA5, ABCA7, ABCA8b and ABCA9 mRNA were also observed. A comparable amount of digitoxin did not affect ABCA8a mRNA expression in the liver. The results suggest that ABCA8 may play a role in digoxin metabolism in the liver.

  15. Cloning of interleukin-10 from African clawed frog (Xenopus tropicalis), with the Finding of IL-19/20 homologue in the IL-10 locus.

    Science.gov (United States)

    Qi, Zhitao; Zhang, Qihuan; Wang, Zisheng; Zhao, Weihong; Gao, Qian

    2015-01-01

    Interleukin-10 (IL-10) is a pleiotropic cytokine that plays an important role in immune system. In the present study, the IL-10 gene of African clawed frog (Xenopus tropicalis) was first cloned, and its expression pattern and 3D structure were also analyzed. The frog IL-10 mRNA encoded 172 amino acids which possessed several conserved features found in IL-10s from other species, including five-exon/four-intron genomic structure, conserved four cysteine residues, IL-10 family motif, and six α-helices. Real-time PCR showed that frog IL-10 mRNA was ubiquitous expressed in all examined tissues, highly in some immune related tissues including kidney, spleen, and intestine and lowly in heart, stomach, and liver. The frog IL-10 mRNA was upregulated at 24 h after LPS stimulation, indicating that it plays a part in the host immune response to bacterial infection. Another IL, termed as IL-20, was identified from the frog IL-10 locus, which might be the homologue of mammalian IL-19/20 according to the analysis results of the phylogenetic tree and the sequence identities.

  16. Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC.

    Science.gov (United States)

    Borrego, C M; Garcia-Gil, L J

    1994-07-01

    A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and β-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.

  17. Solid-Phase Synthesis of Amine/Carboxyl Substituted Prolines and Proline Homologues: Scope and Limitations.

    Science.gov (United States)

    Zhou, Ziniu; Scott, William L; O'Donnell, Martin J

    2016-03-15

    A solid-phase procedure is used to synthesize racemic peptidomimetics based on the fundamental peptide unit. The peptidomimetics are constructed around proline or proline homologues variably substituted at the amine and carbonyl sites. The procedure expands the diversity of substituted peptidomimetic molecules available to the Distributed Drug Discovery (D3) project. Using a BAL-based solid-phase synthetic sequence the proline or proline homologue subunit is both constructed and incorporated into the peptidomimetic by an α-alkylation, hydrolysis and intramolecular cyclization sequence. Further transformations on solid-phase provide access to a variety of piperazine derivatives representing a class of molecules known to exhibit central nervous system activity. The procedure works well with proline cores, but with larger six- and seven-membered ring homologues the nature of the carboxylic acid acylating the cyclic amine can lead to side reactions and result in poor overall yields.

  18. Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus).

    Science.gov (United States)

    Rattanachai, Achara; Hirono, Ikuo; Ohira, Tsuyoshi; Takahashi, Yukinori; Aoki, Takashi

    2005-01-01

    A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.

  19. Expression pattern of INNER NO OUTER homologue in Nymphaea (water lily family, Nymphaeaceae).

    Science.gov (United States)

    Yamada, Toshihiro; Ito, Motomi; Kato, Masahiro

    2003-10-01

    Two homologues of INNER NO OUTER ( INO) in Nymphaea alba and N. colorata (Nymphaeaceae) were isolated and the expression pattern of the N. alba INO homologue NaINO was examined by in situ hybridization. The INO homologues obtained have a portion similar to INO in the predicted amino acid sequences between the conserved zinc finger-like and YABBY domains. In an in situ hybridization analysis, NaINO is expressed in the outer epidermis of the outer integument, inner integument, and the tip of the nucellus. The pattern observed in the outer integument is very similar to that of Arabidopsis thaliana, while the expression in the inner integument and nucellus is not observed in A. thaliana.

  20. Identification and Characterisation of the Murine Homologue of the Gene Responsible for Cystinosis, Ctns

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    Poras Isabelle

    2000-12-01

    Full Text Available Abstract Background Cystinosis is an autosomal recessive disorder characterised by an intralysosomal accumulation of cystine, and affected individuals progress to end-stage renal failure before the age of ten. The causative gene, CTNS, was cloned in 1998 and the encoded protein, cystinosin, was predicted to be a lysosomal membrane protein. Results We have cloned the murine homologue of CTNS, Ctns, and the encoded amino acid sequence is 92.6% similar to cystinosin. We localised Ctns to mouse chromosome 11 in a region syntenic to human chromosome 17 containing CTNS. Ctns is widely expressed in all tissues tested with the exception of skeletal muscle, in contrast to CTNS. Conclusions We have isolated, characterised and localised Ctns, the murine homologue of CTNS underlying cystinosis. Furthermore, our work has brought to light the existence of a differential pattern of expression between the human and murine homologues, providing critical information for the generation of a mouse model for cystinosis.

  1. CRIP homologues maintain apical cytoskeleton to regulate tubule size in C. elegans.

    Science.gov (United States)

    Tong, Xiangyan; Buechner, Matthew

    2008-05-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of Caenorhabditis elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface.

  2. CRIP Homologues Maintain Apical Cytoskeleton to Regulate Tubule Size in C. elegans

    OpenAIRE

    Tong, Xiangyan; Buechner, Matthew

    2008-01-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of C. elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 sho...

  3. Main: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 S000069 17-May-1998 (last modified) kehi Cis element in 3' end region ...of wheat (T.a.) histone H3 mRNA; 3' end formation; Also found in histone genes of other plants, yeast, etc; histone H3; mRNA

  4. Identification and cloning of a sequence homologue of dopamine β-hydroxylase

    NARCIS (Netherlands)

    Chambers, Kaylene J.; Tonkin, Leath A.; Chang, Edwin; Shelton, Dawne N.; Linskens, Maarten H.; Funk, Walter D.

    1998-01-01

    We have identified and cloned a cDNA encoding a new member of the monooxygenase family of enzymes. This novel enzyme, which we call MOX (monooxygenase X; unknown substrate) is a clear sequence homologue of the enzyme dopamine β-hydroxylase (DBH). MOX maintains many of the structural features of DBH,

  5. Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue

    DEFF Research Database (Denmark)

    Blicher, Thomas; Kastrup, Jette Sandholm; Pedersen, Lars Østergaard

    2006-01-01

    A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural...

  6. The actin homologue MreB organizes the bacterial cell membrane

    NARCIS (Netherlands)

    Strahl, H.; Burmann, F.; Hamoen, L.W.

    2014-01-01

    The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate

  7. Identification of Plant Homologues of Dual Specificity Yak1-Related Kinases

    Directory of Open Access Journals (Sweden)

    Pavel Karpov

    2014-01-01

    Full Text Available Currently, Dual Specificity YAK1-Related Kinases (MNB/DYRK were found in slime molds, protista, fungi, and animals, but the existence of plant homologues is still unclear. In the present study, we have identified 14 potential plant homologues with the previously unknown functions, based on the strong sequence similarity. The results of bioinformatics analysis revealed their correspondence to DYRK1A, DYRK1B, DYRK3, and DYRK4. For two plant homologues of animal DYRK1A from Physcomitrella patens and Arabidopsis thaliana spatial structures of catalytic domains were predicted, as well as their complexes with ADP and selective inhibitor d15. Comparative analysis of 3D-structures of the human DYRK1A and plant homologues, their complexes with the specific inhibitors, and results of molecular dynamics confirm their structural and functional similarity with high probability. Preliminary data indicate the presence of potential MNB/DYRK specific phosphorylation sites in such proteins associated with plant cytoskeleton as plant microtubule-associated proteins WVD2 and WDL1, and FH5 and SCAR2 involved in the organization and polarity of the actin cytoskeleton and some kinesin-like microtubule motor proteins.

  8. Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

    DEFF Research Database (Denmark)

    Schmitz-Abe, Klaus; Ciesielski, Szymon J; Schmidt, Paul J

    2015-01-01

    The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases, characterized by defects in mitochondrial heme synthesis, iron-sulfur cluster (Fe-S) biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homologue located in the 5q...

  9. The Retention Behaviors of Benzene and Its Alkyl Homologues in Microemulsion Electrokinetic Chromatography

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The retention behaviors of benzene and its alkyl homologues in microemulsion electrokinetic chromatography were investigated in both anionic and cationic surfactant MEEKC systems. The effects of the composition of microemulsion on retention time and selectivity were studied. A good linear relationship was obtained between log k' and the carbon number of alkyl chain.

  10. Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins

    NARCIS (Netherlands)

    Ketelaar, T.; Voss, C.; Dimmock, S.A.; Thumm, M.; Hussey, P.J.

    2004-01-01

    Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells. Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of

  11. The SPF27 homologue Num1 connects splicing and kinesin 1-dependent cytoplasmic trafficking in Ustilago maydis.

    Science.gov (United States)

    Kellner, Nikola; Heimel, Kai; Obhof, Theresa; Finkernagel, Florian; Kämper, Jörg

    2014-01-01

    The conserved NineTeen protein complex (NTC) is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.

  12. The SPF27 homologue Num1 connects splicing and kinesin 1-dependent cytoplasmic trafficking in Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Nikola Kellner

    2014-01-01

    Full Text Available The conserved NineTeen protein complex (NTC is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.

  13. Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Zou, Jun; Bird, Steve; Truckle, Jonathan; Bols, Niels; Horne, Mike; Secombes, Chris

    2004-05-01

    A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41-45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1beta in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1beta in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1beta in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout.

  14. Expression of achaete-scute homologue 2 and its correlation with serum globulin in patients with chronic hepatitis C

    Directory of Open Access Journals (Sweden)

    ZHAO Jieru

    2017-01-01

    Full Text Available ObjectiveTo investigate the correlation between peripheral CD4+CXCR5+PD-1+T cells and serum globulin in chronic hepatitis C (CHC patients and health volunteers, and to clarify the role of the expression of achaete-scute homologue 2 (Ascl2, the transcription factor in peripheral follicular helper T (Tfh cells, in the process of chronic hepatitis C virus (HCV infection. MethodsA total of 46 previously untreated CHC patients who were admitted to Tangdu Hospital, Fourth Military Medical University, from October 2015 to May 2016 were enrolled, and 32 healthy laboratory technicians and persons who underwent physical examination in our hospital were enrolled as healthy control group. Flow cytometry was used to measure the percentage of Tfh cells in CD4+ T cells and the change in its subset (CD4+CXCR5+PD-1+ T cells in peripheral blood, an automatic biochemical analyzer was used to measure the serum globulin level, and real-time PCR was used to measure the mRNA expression of Ascl2 in Tfh cells. The association between the changes in these parameters and HCV infection was analyzed. The independent samples t-test was used for the comparison of continuous data between groups, and the Pearson correlation analysis was also performed. ResultsIn CHC patients, peripheral Tfh cells were positively correlated with B lymphocytes (r=0.582 3, P=0.011 2, peripheral B lymphocytes were positively correlated with globulin (r=0.450 9, P=0.031 6, and Tfh cells were positively correlated with globulin (r=0.583 5, P=0.038 3. CHC patients had significantly higher mRNA expression of Ascl2 in peripheral Tfh cells than the healthy control group (1.019±0.666 vs 6.437±5.776, t=4.552, P=0.001 9. ConclusionTfh cells may be involved in the production of serum globulin in CHC patients, and the transcription factor Ascl2 may participate in the differentiation and development of Tfh cells in the process of HCV infection.

  15. Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1

    Institute of Scientific and Technical Information of China (English)

    Shuang-Gang Hu; Han Du; Guang-Xin Yao; Yong-Lian Zhang

    2012-01-01

    Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility.In this study,we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1.The mHong1 gene is located on chromosome 12p14,spanning five exons.The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids.The mHong1 mRNA shows similarity with HongrES1 in the expression patterns:(i) specific expression in epididymal tissue,especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence.Its protein product shows 71% similarity with HongrES 1 and contains a classical serpin domain as does HongrES1.A polyclonal antibody against mHong1 with high specificity and sensitivity was raised.Like HongrES1,the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen.The mHong1 protein shows higher glycosylation than HongrES1.Although both of them are deposited onto the sperm head surface,mHong 1 is localized to the equatorial segment,which is different from that of HongrES 1.The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein.Collectively,we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1.

  16. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  17. Natural replacement of vertically inherited lux-rib genes of Photobacterium aquimaris by horizontally acquired homologues.

    Science.gov (United States)

    Urbanczyk, Henryk; Furukawa, Takashi; Yamamoto, Yuki; Dunlap, Paul V

    2012-08-01

    We report here the first instance of a complete replacement of vertically inherited luminescence genes by horizontally acquired homologues. Different strains of Photobacterium aquimaris contain homologues of the lux-rib genes that have a different evolutionary history. Strain BS1 from the Black Sea contains a vertically inherited lux-rib operon, which presumably arose in the ancestor of this species, whereas the type strain NBRC 104633(T) , from Sagami Bay, lacks the vertically inherited lux-rib operon and instead carries a complete and functional lux-rib operon acquired horizontally from a bacterium related to Photobacterium mandapamensis. The results indicate that the horizontal acquisition of the lux genes expanded the pan-genome of P. aquimaris, but it did not influence the phylogenetic divergence of this species.

  18. Formation of Benzyl Carbanion in Collision-Induced Dissociation of Deprotonated Phenylalanine Homologues.

    Science.gov (United States)

    Sekimoto, Kanako; Matsuda, Natsuki; Takayama, Mitsuo

    2014-01-01

    The fragmentation behavior of deprotonated L-phenylalanine (Phe) and its homologues including L-homophenylalanine (HPA) and L-phenylglycine (PG) was investigated using collision-induced dissociation mass spectrometry coupled with a negative ion atmospheric pressure corona discharge ionization (APCDI) technique. The deprotonated molecules [M-H](-) fragmented to lose unique neutral species, e.g., the loss of NH3, CO2, toluene and iminoglycine for [Phe-H](-); styrene and ethenamine/CO2 for [HPA-H](-); and CO2 for [PG-H](-). All of the fragmentations observed are attributable to the formation of intermediates and/or product ions which include benzyl carbanions having resonance-stabilized structures. The carbanions are formed via proton rearrangement through a transition state or via a simple dissociation reaction. These results suggest that the principal factor governing the fragmentation behavior of deprotonated Phe homologues is the stability of the intermediate and/or product ion structures.

  19. C21orf57 is a human homologue of bacterial YbeY proteins.

    Science.gov (United States)

    Ghosal, Anubrata; Köhrer, Caroline; Babu, Vignesh M P; Yamanaka, Kinrin; Davies, Bryan W; Jacob, Asha I; Ferullo, Daniel J; Gruber, Charley C; Vercruysse, Maarten; Walker, Graham C

    2017-03-11

    The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.

  20. [Homologue pairing: initiation sites and effects on crossing over and chromosome disjunction in Drosophila melanogaster].

    Science.gov (United States)

    Chubykin, V L

    1996-01-01

    The role of homologue pairing and chromocentral association of chromosomes in recombination and segregation during cell division is discussed. Peculiarities of mitotic and meiotic chromosome pairing in Drosophila males and females are considered. On the basis of our own and published data, the presence and localization of sites of homologue pairing initiation in euchromatin are substantiated. The effects of transfer of initiation sites along a chromosome (exemplified by inversions) on chromosome pairing (asynapsis), crossing over (intrachromosomal, interchromosomal, and centromeric effects), and segregation are discussed. To record the effects of pairing sites on crossing over, a method of comparing crossing-over frequencies in an inverted region with those in a region of the same size and position with regard to the centromere on cytological maps was proposed. Chromosomes orient toward opposite division poles during paracentromeric heterochromatin pairing. This occurs after successful euchromatin pairing, during which the chromocentral circular structure is reorganized. If heterochromatin pairing is disrupted because of structural or locus mutations, nonexchange bivalents segregate randomly. In this case, chromosome coordination may occur due to proximal chiasmata or chromocentral associations between homologues.

  1. Homologue of mammalian apolipoprotein A-Ⅱ in non-mammalian vertebrates

    Institute of Scientific and Technical Information of China (English)

    Malay Choudhury; Shoji Yamada; Masaharu Komatsu; Hideki Kishimura; Seiichi Ando

    2009-01-01

    Although apolipoprotein with molecular weight 14 kDa (apo-14 kDa) is associated with fish plasma highdensity lipoproteins(HDLs),it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-Ⅱ.We have obtained the full cDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa.Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database.Fish apo14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-Ⅱ.Nevertheless,phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-Ⅱ.In addition,in silico cloning of the TGI,Ensembl,or NCBI database revealed apoA-Ⅱs in dog,chicken,green anole lizard,and African clawed frog whose sequences had not so far been available,suggesting both apoA-Ⅰ and apoA-Ⅱas fundamental constituents of vertebrate HDLs.

  2. Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

    Directory of Open Access Journals (Sweden)

    Oehme Felix

    2001-03-01

    Full Text Available Abstract Background Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress. Results We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [32P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction. Conclusions A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases.

  3. Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis.

    Science.gov (United States)

    Huang, Nien-Chen; Jane, Wann-Neng; Chen, Jychian; Yu, Tien-Shin

    2012-10-01

    Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote-control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti-florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS homologue (ATC), an Arabidopsis FT homologue, may act in a non-cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short-day-induced floral inhibitor. Cell type-specific expression showed that companion cells and apex that express ATC are sufficient to inhibit floral initiation. Histochemical analysis showed that the promoter activity of ATC was mainly found in vasculature but under the detection limit in apex, a finding that suggests that ATC may move from the vasculature to the apex to influence flowering. Consistent with this notion, Arabidopsis seedling grafting experiments demonstrated that ATC moved over a long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and affect the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT homologues to systemically influence floral initiation.

  4. Obesity-linked homologues TfAP-2 and Twz establish meal frequency in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Michael J Williams

    2014-09-01

    Full Text Available In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK homologue Drosulfakinin (Dsk function downstream of TfAP-2 and Tiwaz (Twz to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2β and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA demonstrated that AP-2β and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2β and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2β may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.

  5. Reference: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 Ohtsubo N, Iwabuchi M The conserved 3'-flanking sequence, AATGGAAATG, ...of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA. Nucleic Acids Res 22:1052-1058 (1994) PubMed: 8152910; ...

  6. Systems perspectives on mRNA processing

    Institute of Scientific and Technical Information of China (English)

    Adrienne E McKee; Pamela A Silver

    2007-01-01

    The application of genomic technologies to the study of mRNA processing is increasingly conducted in metazoan organisms in order to understand the complex events that occur during and after transcription. Large-scale systems analyses of mRNA-protein interactions and mRNA dynamics have revealed specificity in mRNA transcription, splicing, transport, translation, and turnover, and have begun to make connections between the different layers of mRNA processing. Here, we review global studies of post-transcriptional processes and discuss the challenges facing our understanding of mRNA regulation in metazoan organisms. In parallel, we examine genome-scale investigations that have expanded our knowledge of RNA-binding proteins and the networks of mRNAs that they regulate.

  7. Achaete-scute complex homologue-1 promotes development of laryngocarcinoma via facilitating the epithelial-mesenchymal transformation.

    Science.gov (United States)

    Ma, Huaci; Du, Xiaodong; Zhang, Shu; Wang, Qiang; Yin, Yong; Qiu, Xiaoxia; Da, Peng; Yue, Huijun; Wu, Hao; Xu, Fenglei

    2017-06-01

    Laryngeal cancer is one of the most common fatal cancers among head and neck carcinomas, whose mechanism, however, remains unclear. The proneural basic-helix-loop-helix protein achaete-scute complex homologue-1, a member of the basic helix-loop-helix family, plays a very important role in many cancers. This study aims to explore the clinical value and mechanism of achaete-scute complex homologue-1 in laryngeal cancer. Methods including Cell Counting Kit-8, flow cytometry, Transwell invasion assays, and scratch assay were adopted to further explore the bio-function of achaete-scute complex homologue-1, whose expression was examined in fresh and paraffin chip of laryngeal carcinoma tissues by means of western blot and immunohistochemistry, after the interference of achaete-scute complex homologue-1; achaete-scute complex homologue-1, an overexpression in laryngeal carcinoma whose carcinogenicity potential was confirmed via western blot, was correlative with T classification (p = 0.002), histological differentiation (p = 0.000), lymph node metastasis (p = 0.000), and poor survival (p = 0.000). Multivariate analysis shows that achaete-scute complex homologue-1 overexpression is an independent prognostic factor unfavorable to laryngeal carcinoma patients (p = 0.000). Moreover, knocking down achaete-scute complex homologue-1 expression could significantly suppress the proliferation, migration, and invasion of laryngeal carcinoma cell in vitro and disorder epithelial-mesenchymal transformation-associated protein expression. Achaete-scute complex homologue-1 plays an important role in the genesis and progression of laryngeal carcinoma and may act as a potential biomarker for therapeutic target and prognostic prediction.

  8. Functional Characterization of Aspergillus nidulans ypkA, a Homologue of the Mammalian Kinase SGK

    Science.gov (United States)

    Colabardini, Ana Cristina; Brown, Neil Andrew; Savoldi, Marcela; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2013-01-01

    The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the

  9. Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK.

    Science.gov (United States)

    Colabardini, Ana Cristina; Brown, Neil Andrew; Savoldi, Marcela; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2013-01-01

    The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the

  10. Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK.

    Directory of Open Access Journals (Sweden)

    Ana Cristina Colabardini

    Full Text Available The serum- and glucocorticoid-regulated protein kinase (SGK is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene and niiA (from the nitrate reductase gene. Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets

  11. Importance of MutL homologue MLH1 and MutS homologue MSH2 expression in Turkish patients with sporadic colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Sibel Erdamar; Esra Ucaryilmaz; Gokhan Demir; Tayfun Karahasanoglu; Gulen Dogusoy; Ahmet Dirican; Suha Goksel

    2007-01-01

    AIM: To assess the incidence of MLH1 (the human MutL homologue) and MSH2 (the human MutS homologue)protein expression in Turkish patients with sporadic colorectal cancers and to compare their survival and clinicopathological features.METHODS: We validated the tissue microarray technology in 77 colorectal carcinomas by analyzing the immunohistochemical expression of proteins involved in two main pathways of colorectal carcinogenesis: p53 protein for loss of heterozygosity tumors; MLH1 and MSH2 proteins for microsatellite instability (MSI).RESULTS: Our analysis showed that 29 (39.2%) had loss of MLH1 expression, 5 (6.8%) had loss of MSH2 expression and 2 cases had loss of expression of both proteins. We found that 60% of MSH2-negative tumors were located in the right side of the colon; all MSH2-negative cases were women. In addition, the loss of MSH2 expression was correlated with low p53 expression. Neither MLH1 nor MSH2 expressions were associated with prognosis, although there seemed a tendency of longer survival (71.7 ± 8.65 mo vs 47.08 ± 5.26 mo) for the patients with MLH1-negative versus MLH1-positive carcinomas. There were not significant differences in overall and recurrence-free survival among MLH1/MSH2-positive and -negative cases.CONCLUSION: Our data supports that Turkish patients with MLH1- and MSH2-defective tumors have some distinct features from each other. Although prognostic importance remains controversial, immunohistochemical analysis of mismatch repair genes may be used as a routine histopathological examination of sporadic colorectal carcinomas.

  12. Molecular cloning and mRNA expression of duck invariant chain.

    Science.gov (United States)

    Zhong, Dalian; Yu, Weiyi; Bao, Min; Xu, Zhiben; Li, Lin; Liu, Jing

    2006-04-15

    In the present study we identified a duck invariant chain (Ii) cDNA, named duck Ii-1, by RT-PCR and RACE. It was 1190 bp in length and contained a 669 bp open reading frame. An alternative transcript encoding a thyroglobulin (Tg)-containing form of Ii, named duck Ii-2, was also found in duck. The putative amino acid sequence of duck Ii-1 showed an 82% similarity to chicken Ii-1 and about 60% similarity to its mammalian homologues. The similarity of the Tg domain between duck and chicken Ii-2 was 96%, and about 70% between duck and mammalian Ii. The result of RT-PCR showed that Ii mRNA was extensively expressed in various tissues. High levels of both Ii-1 and Ii-2 mRNA were observed in the spleen and bursa of Fabricius. The predicted three-dimensional (3D) structures of duck Ii trimerization and Tg domain are similar to the corresponding regions of human Ii analyzed by comparative protein modeling. These findings indicate that the two isoforms of duck Ii, which strongly expressed in the major immune organs, share structural identity with human Ii.

  13. Vaccination against Bm86 Homologues in Rabbits Does Not Impair Ixodes ricinus Feeding or Oviposition.

    Directory of Open Access Journals (Sweden)

    Jeroen Coumou

    Full Text Available Human tick-borne diseases that are transmitted by Ixodes ricinus, such as Lyme borreliosis and tick borne encephalitis, are on the rise in Europe. Diminishing I. ricinus populations in nature can reduce tick exposure to humans, and one way to do so is by developing an anti-vector vaccine against tick antigens. Currently, there is only one anti-vector vaccine available against ticks, which is a veterinary vaccine based on the tick antigen Bm86 in the gut of Rhipicephalus microplus. Bm86 vaccine formulations cause a reduction in the number of Rhipicephalus microplus ticks that successfully feed, i.e. lower engorgement weights and a decrease in the number of oviposited eggs. Furthermore, Bm86 vaccines reduce transmission of bovine Babesia spp. Previously two conserved Bm86 homologues in I. ricinus ticks, designated as Ir86-1 and Ir86-2, were described. Here we investigated the effect of a vaccine against recombinant Ir86-1, Ir86-2 or a combination of both on Ixodes ricinus feeding. Recombinant Ixodes ricinus Bm86 homologues were expressed in a Drosophila expression system and rabbits were immunized with rIr86-1, rIr86-2, a combination of both or ovalbumin as a control. Each animal was infested with 50 female adults and 50 male adults Ixodes ricinus and tick mortality, engorgement weights and egg mass were analyzed. Although serum IgG titers against rIr86 proteins were elicited, no effect was found on tick feeding between the rIr86 vaccinated animals and ovalbumin vaccinated animals. We conclude that vaccination against Bm86 homologues in Ixodes ricinus is not an effective approach to control Ixodes ricinus populations, despite the clear effects of Bm86 vaccination against Rhipicephalus microplus.

  14. The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry.

    Science.gov (United States)

    Iwata, Hikaru; Gaston, Amèlia; Remay, Arnaud; Thouroude, Tatiana; Jeauffre, Julien; Kawamura, Koji; Oyant, Laurence Hibrand-Saint; Araki, Takashi; Denoyes, Béatrice; Foucher, Fabrice

    2012-01-01

    Flowering is a key event in plant life, and is finely tuned by environmental and endogenous signals to adapt to different environments. In horticulture, continuous flowering (CF) is a popular trait introduced in a wide range of cultivated varieties. It played an essential role in the tremendous success of modern roses and woodland strawberries in gardens. CF genotypes flower during all favourable seasons, whereas once-flowering (OF) genotypes only flower in spring. Here we show that in rose and strawberry continuous flowering is controlled by orthologous genes of the TERMINAL FLOWER 1 (TFL1) family. In rose, six independent pairs of CF/OF mutants differ in the presence of a retrotransposon in the second intron of the TFL1 homologue. Because of an insertion of the retrotransposon, transcription of the gene is blocked in CF roses and the absence of the floral repressor provokes continuous blooming. In OF-climbing mutants, the retrotransposon has recombined to give an allele bearing only the long terminal repeat element, thus restoring a functional allele. In OF roses, seasonal regulation of the TFL1 homologue may explain the seasonal flowering, with low expression in spring to allow the first bloom. In woodland strawberry, Fragaria vesca, a 2-bp deletion in the coding region of the TFL1 homologue introduces a frame shift and is responsible for CF behaviour. A diversity analysis has revealed that this deletion is always associated with the CF phenotype. Our results demonstrate a new role of TFL1 in perennial plants in maintaining vegetative growth and modifying flowering seasonality.

  15. Vaccination against Bm86 Homologues in Rabbits Does Not Impair Ixodes ricinus Feeding or Oviposition

    Science.gov (United States)

    Coumou, Jeroen; Wagemakers, Alex; Trentelman, Jos J.; Nijhof, Ard M.; Hovius, Joppe W.

    2015-01-01

    Human tick-borne diseases that are transmitted by Ixodes ricinus, such as Lyme borreliosis and tick borne encephalitis, are on the rise in Europe. Diminishing I. ricinus populations in nature can reduce tick exposure to humans, and one way to do so is by developing an anti-vector vaccine against tick antigens. Currently, there is only one anti-vector vaccine available against ticks, which is a veterinary vaccine based on the tick antigen Bm86 in the gut of Rhipicephalus microplus. Bm86 vaccine formulations cause a reduction in the number of Rhipicephalus microplus ticks that successfully feed, i.e. lower engorgement weights and a decrease in the number of oviposited eggs. Furthermore, Bm86 vaccines reduce transmission of bovine Babesia spp. Previously two conserved Bm86 homologues in I. ricinus ticks, designated as Ir86-1 and Ir86-2, were described. Here we investigated the effect of a vaccine against recombinant Ir86-1, Ir86-2 or a combination of both on Ixodes ricinus feeding. Recombinant Ixodes ricinus Bm86 homologues were expressed in a Drosophila expression system and rabbits were immunized with rIr86-1, rIr86-2, a combination of both or ovalbumin as a control. Each animal was infested with 50 female adults and 50 male adults Ixodes ricinus and tick mortality, engorgement weights and egg mass were analyzed. Although serum IgG titers against rIr86 proteins were elicited, no effect was found on tick feeding between the rIr86 vaccinated animals and ovalbumin vaccinated animals. We conclude that vaccination against Bm86 homologues in Ixodes ricinus is not an effective approach to control Ixodes ricinus populations, despite the clear effects of Bm86 vaccination against Rhipicephalus microplus. PMID:25919587

  16. Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster.

    Science.gov (United States)

    M'Angale, P G; Staveley, B E

    2016-12-19

    Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. We used bioinformatic analyses to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy.

  17. First report of a thioredoxin homologue in jellyfish: molecular cloning, expression and antioxidant activity of CcTrx1 from Cyanea capillata.

    Directory of Open Access Journals (Sweden)

    Zengliang Ruan

    Full Text Available Thioredoxins (Trx proteins are a family of small, highly-conserved and ubiquitous proteins that play significant roles in the resistance of oxidative damage. In this study, a homologue of Trx was identified from the cDNA library of tentacle of the jellyfish Cyanea capillata and named CcTrx1. The full-length cDNA of CcTrx1 was 479 bp with a 312 bp open reading frame encoding 104 amino acids. Bioinformatics analysis revealed that the putative CcTrx1 protein harbored the evolutionarily-conserved Trx active site 31CGPC34 and shared a high similarity with Trx1 proteins from other organisms analyzed, indicating that CcTrx1 is a new member of Trx1 sub-family. CcTrx1 mRNA was found to be constitutively expressed in tentacle, umbrella, oral arm and gonad, indicating a general role of CcTrx1 protein in various physiological processes. The recombinant CcTrx1 (rCcTrx1 protein was expressed in Escherichia coli BL21 (DE3, and then purified by affinity chromatography. The rCcTrx1 protein was demonstrated to possess the expected redox activity in enzymatic analysis and protection against oxidative damage of supercoiled DNA. These results indicate that CcTrx1 may function as an important antioxidant in C. capillata. To our knowledge, this is the first Trx protein characterized from jellyfish species.

  18. Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

    Science.gov (United States)

    Goncharov, I; Palfi, Z; Bindereif, A; Michaeli, S

    1999-04-30

    Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

  19. First report of a thioredoxin homologue in jellyfish: molecular cloning, expression and antioxidant activity of CcTrx1 from Cyanea capillata.

    Science.gov (United States)

    Ruan, Zengliang; Liu, Guoyan; Guo, Yufeng; Zhou, Yonghong; Wang, Qianqian; Chang, Yinlong; Wang, Beilei; Zheng, Jiemin; Zhang, Liming

    2014-01-01

    Thioredoxins (Trx proteins) are a family of small, highly-conserved and ubiquitous proteins that play significant roles in the resistance of oxidative damage. In this study, a homologue of Trx was identified from the cDNA library of tentacle of the jellyfish Cyanea capillata and named CcTrx1. The full-length cDNA of CcTrx1 was 479 bp with a 312 bp open reading frame encoding 104 amino acids. Bioinformatics analysis revealed that the putative CcTrx1 protein harbored the evolutionarily-conserved Trx active site 31CGPC34 and shared a high similarity with Trx1 proteins from other organisms analyzed, indicating that CcTrx1 is a new member of Trx1 sub-family. CcTrx1 mRNA was found to be constitutively expressed in tentacle, umbrella, oral arm and gonad, indicating a general role of CcTrx1 protein in various physiological processes. The recombinant CcTrx1 (rCcTrx1) protein was expressed in Escherichia coli BL21 (DE3), and then purified by affinity chromatography. The rCcTrx1 protein was demonstrated to possess the expected redox activity in enzymatic analysis and protection against oxidative damage of supercoiled DNA. These results indicate that CcTrx1 may function as an important antioxidant in C. capillata. To our knowledge, this is the first Trx protein characterized from jellyfish species.

  20. Molecular Cloning of a Novel Bovine Homologue of the Drosophila Tumor Suppressor Gene, Lats

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Pervious studies demonstrate that lats, also known as warts, is a tumor suppressor gene in Drosophila[1,2]. Mutations of lats lead to an increase in cell number and organ size in Drosophila, indicating lats may be involved in organ size control. Furthermore, the high conservation of sequence and tumor suppression function of lats between Drosophila and human suggests that it may be also involved in organ size control of higher animals[3]. So here we isolated the bovine homologue of Drosophila lats. Sequence analysis indicates the bovine LATS1 to be very similar to other lats proteins.

  1. The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom.

    Science.gov (United States)

    Krizaj, I; Bieber, A L; Ritonja, A; Gubensek, F

    1991-12-18

    A new myotoxic phospholipase A2 homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipera ammodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier-reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle-cell culture from neonatal rats. The muscle-cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells.

  2. Partial functional complementation between human and mouse cytomegalovirus chemokine receptor homologues

    DEFF Research Database (Denmark)

    Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

    2011-01-01

    The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33......-mediated G protein-coupled signaling is critical for the salivary gland phenotype. In this report, we demonstrate that US28 and (to a lesser degree) UL33 restore reactivation from tissue explants and partially restore replication in salivary glands (compared to a signaling-deficient M33 mutant...

  3. Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue.

    Science.gov (United States)

    Cordery, Damien V; Kishore, Uday; Kyes, Sue; Shafi, Mohammed J; Watkins, Katherine R; Williams, Thomas N; Marsh, Kevin; Urban, Britta C

    2007-03-15

    Macrophage-migration inhibitory factor (MIF), one of the first cytokines described, has a broad range of proinflammatory properties. The genome sequencing project of Plasmodium falciparum identified a parasite homologue of MIF. The protein is expressed during the asexual blood stages of the parasite life cycle that cause malarial disease. The identification of a parasite homologue of MIF raised the question of whether it affects monocyte function in a manner similar to its human counterpart. Recombinant P. falciparum MIF (PfMIF) was generated and used in vitro to assess its influence on monocyte function. Antibodies generated against PfMIF were used to determine the expression profile and localization of the protein in blood-stage parasites. Antibody responses to PfMIF were determined in Kenyan children with acute malaria and in control subjects. PfMIF protein was expressed in asexual blood-stage parasites, localized to the Maurer's cleft. In vitro treatment of monocytes with PfMIF inhibited random migration and reduced the surface expression of Toll-like receptor (TLR) 2, TLR4, and CD86. These results indicate that PfMIF is released during blood-stage malaria and potentially modulates the function of monocytes during acute P. falciparum infection.

  4. Pescadillo homologue 1 and Peter Pan function during Xenopus laevis pronephros development.

    Science.gov (United States)

    Tecza, Aleksandra; Bugner, Verena; Kühl, Michael; Kühl, Susanne J

    2011-10-01

    pes1 (pescadillo homologue 1) and ppan (Peter Pan) are multifunctional proteins involved in ribosome biogenesis, cell proliferation, apoptosis, cell migration and regulation of gene expression. Both proteins are required for early neural development in Xenopus laevis, as previously demonstrated. We show that the expression of both genes in the developing pronephros depends on wnt4 and fzd3 (frizzled homologue 3) function. Loss of pes1 or ppan by MO (morpholino oligonucleotide)-based knockdown approaches resulted in strong malformations during pronephric tubule formation. Defects were already notable during specification of pronephric progenitor cells, as shown by lhx1 expression. Moreover, we demonstrated that Xenopus pes1 and ppan interact physically and functionally and that pes1 and ppan can cross-rescue the loss of function phenotype of one another. Interference with rRNA synthesis, however, did not result in a similar early pronephros phenotype. These results demonstrate that pes1 and ppan are required for Xenopus pronephros development and indicate that their function in the pronephros is independent of their role in ribosome biosynthesis.

  5. Protease inhibitor homologues from mamba venoms: facilitation of acetylcholine release and interactions with prejunctional blocking toxins.

    Science.gov (United States)

    Harvey, A L; Karlsson, E

    1982-09-01

    1 Five polypeptides, which were isolated from elapid snake venoms and which are structurally related to protease inhibitors, were tested for action on isolated biventer cervicis nerve-muscle preparations of the chick. 2 Dendrotoxin from the Eastern green mamba (Dendroaspis angusticeps) and toxins K and I from the black mamba (Dendroaspis polylepis polylepis) increased to indirect stimulation without affecting responses to exogenous acetylcholine, carbachol of KCl. 3 The two other protease inhibitor homologues, HHV-II from Ringhals cobra (Hemachatus haemachatus) and NNV-II from Cape cobra (Naja nivea) did not increase responses to nerve stimulation. Trypsin inhibitor from bovine pancreas also had no facilitatory effects on neuromuscular transmission. 4 The facilitatory toxins from mamba venoms interacted with the prejunctional blocking toxins, beta-bungarotoxin, crotoxin and notexin, but not with taipoxin. The blocking effects of beta-bungarotoxin were reduced by pretreatment with the mamba toxins, whereas the blocking actions of crotoxin and notexin were enhanced. 5 The results indicate that protease inhibitor homologues from mamba venoms form a new class of neurotoxin, which acts to increase the release of acetylcholine in response to motor nerve stimulation. 6 From the interaction studies it is concluded that the facilitatory toxins bind to motor nerve terminals at sites related to those occupied by the prejunctional blocking toxins. However, differences in interactions with individual toxins suggest that there must be several related binding sites on the nerve terminals.

  6. Heavy Atom Effect on the First Hyperpolarizabilities of Squaric Acid Homologues Studied by Ab Initio and DFT Methods

    Institute of Scientific and Technical Information of China (English)

    FU Wei-Wei; ZHOU Li-Xin; WAN Hua-Ping

    2004-01-01

    We have calculated the first hyperpolarizabilities of four squaric acid homologue molecules: 3,4-dithiohydroxy-3-cyclobutene-1,2-dione (OSSQ), 3,4-dithiohydroxy-3-cyclobutene-1, 2-dithione (SSSQ), 3,4-dithiohydroxy-3-cyclobutene-1,2-diselenone (SeSSQ) and 3,4-dithiohydroxy- 3-cyclobutene-1,2-ditellurone (TeSSQ). The correlation effect was investigated at the second-order Mφller-Plesset (MP2) perturbation and density functional theory (DFT) levels. The frequency disper- sion and solvent effect were considered to compare the theoretical values with the experimental observations. Based on all of these studies, it is worthy to point out that the heavy atom effect dis- covered for furan homologues is an influence on the first hyperpolarizabilities of squaric acid homologues.

  7. PAB-1, a Caenorhabditis elegans poly(A-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Directory of Open Access Journals (Sweden)

    Sunhee Ko

    Full Text Available Poly(A-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  8. PAB-1, a Caenorhabditis elegans poly(A)-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Science.gov (United States)

    Ko, Sunhee; Kawasaki, Ichiro; Shim, Yhong-Hee

    2013-01-01

    Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  9. Association of SelS mRNA expression in omental adipose tissue with Homa-IR and serum amyloid A in patients with type 2 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    DU Jian-ling; SUN Chang-kai; L(U) Bo; MEN Li-li; YAO Jun-jie; AN Li-jia; SONG Gui-rong

    2008-01-01

    Background Tanis was repoded as a putative receptor for serum amyloid A(SAA)involving glucose regulated protein in insulin regulated resistance.It was found to be dysregulated in diabetic rats(Psammomys obesus,Israeli sand rat)and its homologue for humans is SelS/AD-015.The present study analyzed mRNA expression of SelS in omental adipose tissue biopsies from patients with type 2 diabetes mellitus (T2DM),and age-and weight-matched nondiabetic patients,the relationship of SelS mRNA with Homa-IR and serum SAA level.Methods Human omental adipose tissues from ten cases of type 2 diabetic patients and twelve cases of nondiabetic individuals were analyzed for the expression level of SelS mRNA by semiquantitative polymerase chain reaction(PCR),Homa-IR estimated by standard formula and SM level by enzyme-linked immunosorbent assay(ELISA).Results SelS mRNA expression.Homa-IR and serum SAA were higher in T2DM sufferers than in nondiabeUc control group.SelS mRNA level was positively correlated with Homa-IR and SAA level in each group.Conclusions SelS protein may be involved in insulin resistarice;in Chinese with T2DM by acting as the SAA receptor,thus playing an important role in the development of T2DM and atherosclerosis.

  10. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Xenopus BTBD6 and its Drosophila homologue lute are required for neuronal development.

    Science.gov (United States)

    Bury, Frédéric J; Moers, Virginie; Yan, Jiekun; Souopgui, Jacob; Quan, Xiao-Jiang; De Geest, Natalie; Kricha, Sadia; Hassan, Bassem A; Bellefroid, Eric J

    2008-11-01

    BBP proteins constitute a subclass of CUL3 interacting BTB proteins whose in vivo function remains unknown. Here, we show that the Xenopus BBP gene BTBD6 and the single Drosophila homologue of mammalian BBP genes lute are strongly expressed in the developing nervous system. In Xenopus, BTBD6 expression responds positively to proneural and negatively to neurogenic gene overexpression. Knockdown of BTBD6 in Xenopus or loss of Drosophila lute result in embryos with strong defects in late neuronal markers and strongly reduced and disorganized axons while early neural development is unaffected. XBTBD6 knockdown in Xenopus also affects muscle development. Together, these data indicate that BTBD6/lute is required for proper embryogenesis and plays an essential evolutionary conserved role during neuronal development.

  12. Synthesis and Characterization of a New Five and Six Membered Selenoheterocyclic Compounds Homologues of Ebselen

    Directory of Open Access Journals (Sweden)

    Mouslim Messali

    2011-01-01

    Full Text Available The discovery of the antioxidant activity of selenoenzyme glutathione peroxidase (GPx has attracted growing attention in the biochemistry of selenium. Among molecules which mimic the structure of the active site of the enzyme, N-phenyl-1,2-benzisoselenazolin-3-one 1, Ebselen, exhibited useful anti-inflammatory properties. It has been extensively investigated and has undergone clinical trials as an anti-inflammatory agent. Unfortunately, Ebselen exhibits relatively poor catalytic activity, prompting attempts to design more efficacious GPx mimetics that would retain his low toxicity while manifesting improved catalytic properties. In this context, novel 1,2-benzoselenazine and 1,2-benzoselenazols, which are five and six membered homologues of Ebselen were synthesized and characterized. One structure has been proven by single crystal X-ray crystallography.

  13. Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae).

    Science.gov (United States)

    Finkler, Carla; Giacomet, Carolina; Muschner, Valéria C; Salzano, Francisco M; Freitas, Loreta B

    2005-07-01

    The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora, DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.

  14. Homologue Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves

    Energy Technology Data Exchange (ETDEWEB)

    Y Chen; L Hu; M Punta; R Bruni; B Hillerich; B Kloss; B Rost; J Love; S Siegelbaum; W Hendrickson

    2011-12-31

    The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

  15. A Salmonella Typhi homologue of bacteriophage muramidases controls typhoid toxin secretion.

    Science.gov (United States)

    Hodak, Hélène; Galán, Jorge E

    2013-01-01

    Unlike other Salmonella, which can infect a broad range of hosts causing self-limiting infection, Salmonella Typhi is an exclusively human pathogen that causes typhoid fever, a life-threatening systemic disease. Typhoid toxin is a unique virulence factor of Salmonella Typhi, which is expressed when the bacteria are within mammalian cells. Here, we report that an N-acetyl-β-D-muramidase similar to phage endolysins encoded within the same pathogenicity islet as the toxin is required for typhoid toxin secretion. Genetic and functional analysis of TtsA revealed unique amino acids at its predicted peptidoglycan-binding domain that are essential for protein secretion and that distinguishes this protein from other homologues. We propose that TtsA defines a new protein secretion mechanism recently evolved from the machine that mediates phage release.

  16. Iron uptake by melanoma cells from the soluble form of the transferrin homologue, melanotransferrin.

    Science.gov (United States)

    Food, Michael R; Des Richardson, R

    2002-01-01

    Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that can also exist in a soluble form (sMTf). Considering the high homology of MTf to Tf, it is possible to suggest that sMTf could bind to the high affinity transferrin receptor 1 (TfR1) or lower affinity TfR2. We have used sMTf labelled with 59Fe to examine its ability to donate Fe to cells. Our experiments demonstrate that sMTf is far less effective than Tf at donating Fe to cells and this does not occur via specific receptors. Indeed, the uptake of sMTf by cells occurred via a non-specific process (e.g. adsorptive pinocytosis).

  17. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  18. The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics.

    Directory of Open Access Journals (Sweden)

    Marcel Sluijter

    Full Text Available The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn and RuvA(Mge, respectively were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn and RuvA(Mge (68.8% identity, substantial differences were found between these proteins in their activities. First, RuvA(Mge was found to preferentially bind to HJs, whereas RuvA(Mpn displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn is able to form two distinct complexes with HJs, RuvA(Mge only produced a single HJ complex. Third, RuvA(Mge stimulated the DNA helicase and ATPase activities of RuvB(Mge, whereas RuvA(Mpn did not augment RuvB activity. Finally, while both RuvA(Mge and RecU(Mge efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge.

  19. The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics.

    Science.gov (United States)

    Sluijter, Marcel; Estevão, Silvia; Hoogenboezem, Theo; Hartwig, Nico G; van Rossum, Annemarie M C; Vink, Cornelis

    2012-01-01

    The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).

  20. Isolation and characterization of AtMLH1, a MutL homologue from Arabidopsis thaliana.

    Science.gov (United States)

    Jean, M; Pelletier, J; Hilpert, M; Belzile, F; Kunze, R

    1999-12-01

    DNA mismatch repair systems play an essential role in the maintenance of genetic information in living organisms and are also implicated in genetic recombination and genome stability. Using degenerate primers, we have cloned the first plant homologue of the E. coli MutL gene, which we have called AtMLH1 for Arabidopsis thaliana MutL-homologue 1. AtMLH1 is present as a single-copy gene in the Arabidopsis genome and is located on the top arm of chromosome 4. Sequence analysis revealed that the product of this gene shows extensive sequence homology with other eukaryotic MLH1 proteins. As mlh1-deficient lines would be useful for studying the biological function of this gene, several populations that had been mutagenized using T-DNA and transposon insertions were screened to identify such mutants. One line that carries a T-DNA insertion in the promoter region of the AtMLH1 gene was isolated. Surprisingly, although the insertion occurred only approximately 80 bp upstream of the putative transcription start site, Northern analyses revealed very low but similar amounts of AtMLH1 transcript in both the wild type and the T-DNA insertion lines. RT-PCR analyses suggest, however, that transcription is initiated further upstream in the insertion line and that the T-DNA may supply this novel initiation site. Finally, no increase in microsatellite instability - a phenotype often associated with mutations in mismatch repair genes - was observed in plants homozygous for this insertion.

  1. Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis of its expression pattern in eye and brain.

    NARCIS (Netherlands)

    Hollander, A.I. den; Ghiani, M.; Kok, Y.J.M. de; Wijnholds, J.; Ballabio, A.; Cremers, F.P.M.; Broccoli, V.

    2002-01-01

    Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies. CRB1 is homologous to Drosophila Crumbs, a protein essential for establishing and maintaining epithelial polarity. We have isolated the mouse orthologue, Crb1, and analyzed its expression pattern in embryonic and

  2. Predominant recognition of species-specific determinants of the GroES homologues from Mycobacterium leprae and M. tuberculosis

    NARCIS (Netherlands)

    Chua-Intra, B.; Ivanyi, J.; Hills, A.; Thole, J.; Moreno, C.; Vordermeier, H.M.

    1998-01-01

    The Mycobacterium leprae and M. tuberculosis 10000 MW heat-shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequenc

  3. GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin.

    NARCIS (Netherlands)

    Läppchen, T.; Hartog, A.F.; Pinas, V.; Koomen, G.J.; den Blaauwen, T.

    2005-01-01

    The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubul

  4. 4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase : Synthesis and evaluation of 2-fluoro substrate analogues

    NARCIS (Netherlands)

    Johnson, William H; Wang, Susan C; Stanley, Thanuja M; Czerwinski, Robert M; Almrud, Jeffrey J; Poelarends, Gerrit J; Murzin, Alexey G; Whitman, Christian P

    2004-01-01

    A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in B

  5. Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses

    Directory of Open Access Journals (Sweden)

    Sharp Tyler M

    2012-09-01

    Full Text Available Abstract Background Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. Findings All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. Conclusions These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.

  6. Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses.

    Science.gov (United States)

    Sharp, Tyler M; Crawford, Sue E; Ajami, Nadim J; Neill, Frederick H; Atmar, Robert L; Katayama, Kazuhiko; Utama, Budi; Estes, Mary K

    2012-09-03

    Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES) motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.

  7. 4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase : Synthesis and evaluation of 2-fluoro substrate analogues

    NARCIS (Netherlands)

    Johnson, William H; Wang, Susan C; Stanley, Thanuja M; Czerwinski, Robert M; Almrud, Jeffrey J; Poelarends, Gerrit J; Murzin, Alexey G; Whitman, Christian P

    2004-01-01

    A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in B

  8. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  9. Gene regulation by mRNA editing

    Energy Technology Data Exchange (ETDEWEB)

    Ashkenas, J. [Univ. of Washington, Seattle, WA (United States)

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  10. Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori.

    Science.gov (United States)

    Hong, Sun Mee; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2014-04-01

    The silkworm genome encodes three iron storage proteins or ferritins, Fer1HCH, Fer2LCH, and Fer3HCH. Probing our EST library constructed from 1-day-old silkworm eggs revealed only Fer2LCH mRNA, which encoded for a protein with a predicted putative N-glycosylation site. Developmental and tissue expression analyses during embryogenesis revealed that Fer2LCH mRNA was abundant from 6 h to 6 days after oviposition. Transcriptional expression of Fer2LCH during the postembryonic stage is also high in the larval fat body and mid-gut, and then is upregulated in all pupal tissues tested. We found that Fer2LCH mRNA contains an iron-responsive element, suggesting this ferritin subunit is subject to translational control. Although ferritin expression has been shown to increase following immune challenge in other insects, the levels of Fer2LCH mRNA were not significantly induced following viral or bacterial infection of Bombyx mori. Using a baculovirus expression system we expressed recombinant BmFer2LCH protein, which was detectable in the cytoplasmic fraction, likely in a compartment of the secretory pathway, and was shown to undergo posttranslational modifications including N-glycosylation. In particular, rBmFer2LCH carbohydrate chains were composed of mannose and GlcNAc. We suggest that Fer2LCH is important for iron homeostasis and maintaining normal organ function in silkworms.

  11. Studies of the Cataluminescence of Benzene Homologues onNanosized γ–Al2O3/Eu2O3 and the Development of a Gas Sensorfor Benzene Homologue Vapors

    Directory of Open Access Journals (Sweden)

    Cuiqin Wu

    2006-12-01

    Full Text Available The cataluminescence (CTL of benzene and the benzene homologues tolueneand xylene on nanosized γ–Al2O3 doped with Eu2O3 (γ–Al2O3/Eu2O3 was studied and asensor of determining these gases was designed. The proposed sensor showed highsensitivity and selectivity at an optimal temperature of 432 ºC, a wavelength of 425 nm anda flow rate of 400 mL/min. Quantitative analysis was performed at the optimal conditions.The linear ranges of CTL intensity versus concentration of the benzene homologues were asfollows: benzene 2.4~5000 mL/m3, toluene 4.0~5000 mL/m3 and xylene 6.8~5000 mL/m3,with detection limits (3σ of 1.8 mL/m3, 3.0 mL/m3 and 3.4 mL/m3 for each one,respectively. The response time of this system was less than 3 s. The coexistence of othergases, such as SO2, CO and NH3, caused interference at levels around 11.7%, 5.8% and8.9% respectively. The technique is a convenient and fast way of determining the vapors ofbenzene homologues in air.

  12. A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue.

    Science.gov (United States)

    Li, Wei; Feng, Zhaoyang; Sternberg, Paul W; Xu, X Z Shawn

    2006-03-30

    The nematode Caenorhabditis elegans is commonly used as a genetic model organism for dissecting integration of the sensory and motor systems. Despite extensive genetic and behavioural analyses that have led to the identification of many genes and neural circuits involved in regulating C. elegans locomotion behaviour, it remains unclear whether and how somatosensory feedback modulates motor output during locomotion. In particular, no stretch receptors have been identified in C. elegans, raising the issue of whether stretch-receptor-mediated proprioception is used by C. elegans to regulate its locomotion behaviour. Here we have characterized TRP-4, the C. elegans homologue of the mechanosensitive TRPN channel. We show that trp-4 mutant worms bend their body abnormally, exhibiting a body posture distinct from that of wild-type worms during locomotion, suggesting that TRP-4 is involved in stretch-receptor-mediated proprioception. We show that TRP-4 acts in a single neuron, DVA, to mediate its function in proprioception, and that the activity of DVA can be stimulated by body stretch. DVA both positively and negatively modulates locomotion, providing a unique mechanism whereby a single neuron can fine-tune motor activity. Thus, DVA represents a stretch receptor neuron that regulates sensory-motor integration during C. elegans locomotion.

  13. The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

    Directory of Open Access Journals (Sweden)

    Alessia Soldano

    Full Text Available Wnt Planar Cell Polarity (PCP signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs are intensely investigated because of their link to Alzheimer's disease (AD. APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh by Abelson kinase (Abl. Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

  14. A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells.

    Science.gov (United States)

    Lindskog, Eva; Svensson, Ingrid; Häggström, Lena

    2006-07-01

    Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

  15. GET_HOMOLOGUES, a versatile software package for scalable and robust microbial pangenome analysis.

    Science.gov (United States)

    Contreras-Moreira, Bruno; Vinuesa, Pablo

    2013-12-01

    GET_HOMOLOGUES is an open-source software package that builds on popular orthology-calling approaches making highly customizable and detailed pangenome analyses of microorganisms accessible to nonbioinformaticians. It can cluster homologous gene families using the bidirectional best-hit, COGtriangles, or OrthoMCL clustering algorithms. Clustering stringency can be adjusted by scanning the domain composition of proteins using the HMMER3 package, by imposing desired pairwise alignment coverage cutoffs, or by selecting only syntenic genes. The resulting homologous gene families can be made even more robust by computing consensus clusters from those generated by any combination of the clustering algorithms and filtering criteria. Auxiliary scripts make the construction, interrogation, and graphical display of core genome and pangenome sets easy to perform. Exponential and binomial mixture models can be fitted to the data to estimate theoretical core genome and pangenome sizes, and high-quality graphics can be generated. Furthermore, pangenome trees can be easily computed and basic comparative genomics performed to identify lineage-specific genes or gene family expansions. The software is designed to take advantage of modern multiprocessor personal computers as well as computer clusters to parallelize time-consuming tasks. To demonstrate some of these capabilities, we survey a set of 50 Streptococcus genomes annotated in the Orthologous Matrix (OMA) browser as a benchmark case. The package can be downloaded at http://www.eead.csic.es/compbio/soft/gethoms.php and http://maya.ccg.unam.mx/soft/gethoms.php.

  16. The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.

    Science.gov (United States)

    Cecchini, Marco; Changeux, Jean-Pierre

    2015-09-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger - a neurotransmitter - into an ion flux through the postsynaptic membrane. Here, we present an overview of the most recent advances on the signal transduction mechanism boosted by X-ray crystallography of both prokaryotic and eukaryotic homologues of the nicotinic acetylcholine receptor (nAChR) in conjunction with time-resolved analyses based on single-channel electrophysiology and Molecular Dynamics simulations. The available data consistently point to a global mechanism of gating that involves a large reorganization of the receptor mediated by two distinct quaternary transitions: a global twisting and a radial expansion/contraction of the extracellular domain. These transitions profoundly modify the organization of the interface between subunits, which host several sites for orthosteric and allosteric modulatory ligands. The same mechanism may thus mediate both positive and negative allosteric modulations of pLGICs ligand binding at topographically distinct sites. The emerging picture of signal transduction is expected to pave the way to new pharmacological strategies for the development of allosteric modulators of nAChR and pLGICs in general. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

    Science.gov (United States)

    Petkowski, Janusz J; Bonsignore, Lindsay A; Tooley, John G; Wilkey, Daniel W; Merchant, Michael L; Macara, Ian G; Schaner Tooley, Christine E

    2013-12-15

    NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2). We show in the present paper for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and MS experiments indicate that they differ in their specific catalytic functions. Although NRMT1 is a distributive methyltransferase that can mono-, di- and tri-methylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation.

  18. A caseian point for the evolution of a diaphragm homologue among the earliest synapsids.

    Science.gov (United States)

    Lambertz, Markus; Shelton, Christen D; Spindler, Frederik; Perry, Steven F

    2016-12-01

    The origin of the diaphragm remains a poorly understood yet crucial step in the evolution of terrestrial vertebrates, as this unique structure serves as the main respiratory motor for mammals. Here, we analyze the paleobiology and the respiratory apparatus of one of the oldest lineages of mammal-like reptiles: the Caseidae. Combining quantitative bone histology and functional morphological and physiological modeling approaches, we deduce a scenario in which an auxiliary ventilatory structure was present in these early synapsids. Crucial to this hypothesis are indications that at least the phylogenetically advanced caseids might not have been primarily terrestrial but rather were bound to a predominantly aquatic life. Such a lifestyle would have resulted in severe constraints on their ventilatory system, which consequently would have had to cope with diving-related problems. Our modeling of breathing parameters revealed that these caseids were capable of only limited costal breathing and, if aquatic, must have employed some auxiliary ventilatory mechanism to quickly meet their oxygen demand upon surfacing. Given caseids' phylogenetic position at the base of Synapsida and under this aquatic scenario, it would be most parsimonious to assume that a homologue of the mammalian diaphragm had already evolved about 50 Ma earlier than previously assumed.

  19. The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

    Science.gov (United States)

    Soldano, Alessia; Okray, Zeynep; Janovska, Pavlina; Tmejová, Kateřina; Reynaud, Elodie; Claeys, Annelies; Yan, Jiekun; Atak, Zeynep Kalender; De Strooper, Bart; Dura, Jean-Maurice; Bryja, Vítězslav; Hassan, Bassem A

    2013-01-01

    Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

  20. DNA Damage Induced MutS Homologue hMSH4 Acetylation

    Directory of Open Access Journals (Sweden)

    Chengtao Her

    2013-10-01

    Full Text Available Acetylation of non-histone proteins is increasingly recognized as an important post-translational modification for controlling the actions of various cellular processes including DNA repair and damage response. Here, we report that the human MutS homologue hMSH4 undergoes acetylation following DNA damage induced by ionizing radiation (IR. To determine which acetyltransferases are responsible for hMSH4 acetylation in response to DNA damage, potential interactions of hMSH4 with hTip60, hGCN5, and hMof were analyzed. The results of these experiments indicate that only hMof interacts with hMSH4 in a DNA damage-dependent manner. Intriguingly, the interplay between hMSH4 and hMof manipulates the outcomes of nonhomologous end joining (NHEJ-mediated DNA double strand break (DSB repair and thereby controls cell survival in response to IR. This study also shows that hMSH4 interacts with HDAC3, by which HDAC3 negatively regulates the levels of hMSH4 acetylation. Interestingly, elevated levels of HDAC3 correlate with increased NHEJ-mediated DSB repair, suggesting that hMSH4 acetylation per se may not directly affect the role of hMSH4 in DSB repair.

  1. The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK.

    Science.gov (United States)

    Singh, Rajinder; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ong-Abdullah, Meilina; Ting, Ngoot-Chin; Nagappan, Jayanthi; Nookiah, Rajanaidu; Amiruddin, Mohd Din; Rosli, Rozana; Manaf, Mohamad Arif Abdul; Chan, Kuang-Lim; Halim, Mohd Amin; Azizi, Norazah; Lakey, Nathan; Smith, Steven W; Budiman, Muhammad A; Hogan, Michael; Bacher, Blaire; Van Brunt, Andrew; Wang, Chunyan; Ordway, Jared M; Sambanthamurthi, Ravigadevi; Martienssen, Robert A

    2013-08-15

    A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation.

  2. Novel Type Ⅱ Peroxiredoxin Gene Homologue from Chinese Wildrye Enhancing Salt Stress Tolerance of Transgenic Yeast

    Institute of Scientific and Technical Information of China (English)

    YU Ying; LIU Xiang-guo; LU Yang; DOU Yao; WANG Hu-yi; HAN Si-ping; FENG Shu-dan; HAO Dong-yun

    2011-01-01

    Peroxiredoxins(Prxs)are a large family of antioxidant enzymes of various types that take part in signal transduction via decomposing reactive oxygen species(ROS).Although extensive efforts have been made over the last decades in understanding the structures and functions of Prxs,type Ⅱ Prxs in monocots are hardly studied.In this work,a monocot type Ⅱ Prx gene homologue from Chinese wildrye(Leymus Chinensis),designated as LcTpxⅡ,was isolated and characterized.LcTpxⅡ encoding a 162-amino acid protein contains a thioredoxin domain and a cysteine residue at position 51,suggesting that it is a member of the Prxs family.The LcTpxⅡ is capable of decomposing H2O2 and protecting plasmid DNA from damage caused by ROS.The expression of LcTpxⅡ in Chinese wildrye was induced by 400 mmol/L NaCl and 100 mmol/L Na2CO3 in the experiment.The overexpression of LcTpxⅡ enhances the tolerance of transgenic yeast to 1.6 mol/L NaCl and 10 mmol/L Na2CO3.

  3. Positive selection in AvrP4 avirulence gene homologues across the genus Melampsora.

    Science.gov (United States)

    Van der Merwe, Marlien M; Kinnear, Mark W; Barrett, Luke G; Dodds, Peter N; Ericson, Lars; Thrall, Peter H; Burdon, Jeremy J

    2009-08-22

    Pathogen genes involved in interactions with their plant hosts are expected to evolve under positive Darwinian selection or balancing selection. In this study a single copy avirulence gene, AvrP4, in the plant pathogen Melampsora lini, was used to investigate the evolution of such a gene across species. Partial translation elongation factor 1-alpha sequences were obtained to establish phylogenetic relationships among the Melampsora species. We amplified AvrP4 homologues from species pathogenic on hosts from different plant families and orders, across the inferred phylogeny. Translations of the AvrP4 sequences revealed a predicted signal peptide and towards the C-terminus of the protein, six identically spaced cysteines were identified in all sequences. Maximum likelihood analysis of synonymous versus non-synonymous substitution rates indicated that positive selection played a role in the evolution of the gene during the diversification of the genus. Fourteen codons under significant positive selection reside in the C-terminal 28 amino acid region, suggesting that this region interacts with host molecules in most sequenced accessions. Selection pressures on the gene may be either due to the pathogenicity or avirulence function of the gene or both.

  4. TRAUCO, a Trithorax-group gene homologue, is required for early embryogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Aquea, Felipe; Johnston, Amal J; Cañon, Paola; Grossniklaus, Ueli; Arce-Johnson, Patricio

    2010-02-01

    Embryogenesis is a critical stage during the plant life cycle in which a unicellular zygote develops into a multicellular organism. Co-ordinated gene expression is thus necessary for proper embryo development. Polycomb and Trithorax group genes are members of evolutionarily conserved machinery that maintains the correct expression patterns of key developmental regulators by repressing and activating gene transcription. TRAUCO (TRO), a gene homologous to the Trithorax group of genes that can functionally complement a BRE2P yeast mutant, has been identified in Arabidopsis thaliana. It is demonstrated that TRO is a nuclear gene product expressed during embryogenesis, and loss of TRO function leads to impaired early embryo development. Embryos that arrested at the globular stage in the tro-1 mutant allele were fully rescued by a TRO expression clone, a demonstration that the tro-1 mutation is a true loss-of-function in TRO. Our data have established that TRO is the first trithorax-group gene homologue in plants that is required for early embryogenesis.

  5. TRBP and eIF6 homologue in Marsupenaeus japonicus play crucial roles in antiviral response.

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    Shuai Wang

    Full Text Available Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC. Trans-activation response RNA-binding protein (TRBP, consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6. In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi pathway of shrimp. The double-stranded RNA binding domains (dsRBDs B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV. These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp.

  6. Crystal Structure of the Heterotrimer Core of Saccharomyces cerevisiae AMPK Homologue SNF1

    Energy Technology Data Exchange (ETDEWEB)

    Amodeo,G.; Rudolph, M.; Tong, L.

    2007-01-01

    AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals and is an attractive target for drug discovery against diabetes, obesity and other diseases. The AMPK homologue in Saccharomyces cerevisiae, known as SNF1, is essential for responses to glucose starvation as well as for other cellular processes, although SNF1 seems to be activated by a ligand other than AMP. Here we report the crystal structure at 2.6 resolution of the heterotrimer core of SNF1. The ligand-binding site in the {gamma}-subunit (Snf4) has clear structural differences from that of the Schizosaccharomyces pombe enzyme, although our crystallographic data indicate that AMP can also bind to Snf4. The glycogen-binding domain in the {beta}-subunit (Sip2) interacts with Snf4 in the heterotrimer but should still be able to bind carbohydrates. Our structure is supported by a large body of biochemical and genetic data on this complex. Most significantly, the structure reveals that part of the regulatory sequence in the {alpha}-subunit (Snf1) is sequestered by Snf4, demonstrating a direct interaction between the {alpha}- and {gamma}-subunits and indicating that our structure may represent the heterotrimer core of SNF1 in its activated state.

  7. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells.

    Science.gov (United States)

    Lavial, Fabrice; Acloque, Hervé; Bertocchini, Federica; Macleod, David J; Boast, Sharon; Bachelard, Elodie; Montillet, Guillaume; Thenot, Sandrine; Sang, Helen M; Stern, Claudio D; Samarut, Jacques; Pain, Bertrand

    2007-10-01

    Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

  8. Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

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    Kurz, M. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); Iturbe-Ormaetxe, I. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Jarrott, R. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); O’Neill, S. L. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Byriel, K. A.; Martin, J. L., E-mail: j.martin@imb.uq.edu.au; Heras, B., E-mail: j.martin@imb.uq.edu.au [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia)

    2008-02-01

    The first crystallization of a W. pipientis protein, α-DsbA1, was achieved using hanging-drop and sitting-drop vapour diffusion. α-DsbA1 is one of two DsbA homologues encoded by the Gram-negative α-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The α-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet α-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, b = 49.5, c = 69.3 Å, β = 107.0° and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 Å using a copper anode and data from SeMet α-DsbA1 crystals were recorded to 2.45 Å resolution using a chromium anode.

  9. A new MOF-5 homologue for selective separation of methane from C2 hydrocarbons at room temperature

    Directory of Open Access Journals (Sweden)

    Yabing He

    2014-12-01

    Full Text Available A new MOF-5 homologue compound UTSA-10 has been obtained under solvothermal conditions from a mixture of Zn(NO32⋅6H2O and commercially available linker, 2-methylfumaric acid, in N,N-dimethylformamide. The moderate surface area and suitable pore sizes enable the activated UTSA-10a to separate methane from C2 hydrocarbons at room temperature.

  10. Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi.

    Science.gov (United States)

    Lacaze, Isabelle; Lalucque, Hervé; Siegmund, Ulrike; Silar, Philippe; Brun, Sylvain

    2015-03-01

    NADPH oxidases (Nox) are membrane complexes that produce O2(-). Researches in mammals, plants and fungi highlight the involvement of Nox-generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91(phox)/Nox2 is associated with p22(phox) forming the flavocytochrome b558 ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22(phox) gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22(phox) homologue as being mutated in the Podospora anserina mutant IDC(509). Functional studies show that the fungal p22(phox), PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91(phox) homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co-localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system.

  11. Species-specific flight styles of flies are reflected in the response dynamics of a homologue motion sensitive neuron

    Directory of Open Access Journals (Sweden)

    Bart eGeurten

    2012-03-01

    Full Text Available Hoverflies and blowflies have distinctly different flight styles. Yet, both species have been shown to structure their flight behaviour in a way that facilitates extraction of 3D information from the image flow on the retina (optic flow. Neuronal candidates to analyse the optic flow are the tangential cells in the third optical ganglion – the lobula complex. These neurons are directionally selective and integrate the optic flow over large parts of the visual field. Homologue tangential cells in hoverflies and blowflies have a similar morphology. Because blowflies and hoverflies have similar neuronal layout but distinctly different flight behaviours, they are an ideal substrate to pinpoint potential neuronal adaptations to the different flight styles.In this article we describe the relationship between locomotion behaviour and motion vision on three different levels:1.We compare the different flight styles based on the categorisation of flight behaviour into prototypical movements.2.We measure the species specific dynamics of the optic flow under naturalistic flight conditions. We found the translational optic flow of both species to be very different.3.We describe possible adaptations of a homologue motion sensitive neuron. We stimulate this cell in blowflies (Calliphora and hoverflies (Eristalis with naturalistic optic flow generated by both species during free flight. The characterized hoverfly tangential cell responds faster to transient changes in the optic flow than its blowfly homologue. It is discussed whether and how the different dynamical response properties aid optic flow analysis.

  12. Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p.

    Science.gov (United States)

    Maceluch, J; Kmieciak, M; Szweykowska-Kulińska, Z; Jarmołowski, A

    2001-01-01

    Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

  13. The OxyR homologue in Tannerella forsythia regulates expression of oxidative stress responses and biofilm formation.

    Science.gov (United States)

    Honma, Kiyonobu; Mishima, Elina; Inagaki, Satoru; Sharma, Ashu

    2009-06-01

    Tannerella forsythia is an anaerobic periodontal pathogen that encounters constant oxidative stress in the human oral cavity due to exposure to air and reactive oxidative species from coexisting dental plaque bacteria as well as leukocytes. In this study, we sought to characterize a T. forsythia ORF with close similarity to bacterial oxidative stress response sensor protein OxyR. To analyse the role of this OxyR homologue, a gene deletion mutant was constructed and characterized. Aerotolerance, survival after hydrogen peroxide challenge and transcription levels of known bacterial antioxidant genes were then determined. Since an association between oxidative stress and biofilm formation has been observed in bacterial systems, we also investigated the role of the OxyR protein in biofilm development by T. forsythia. Our results showed that aerotolerance, sensitivity to peroxide challenge and the expression of oxidative stress response genes were significantly reduced in the mutant as compared with the wild-type strain. Moreover, the results of biofilm analyses showed that, as compared with the wild-type strain, the oxyR mutant showed significantly less autoaggregation and a reduced ability to form mixed biofilms with Fusobacterium nucleatum. In conclusion, a gene annotated in the T. forsythia genome as an oxyR homologue was characterized. Our studies showed that the oxyR homologue in T. forsythia constitutively activates antioxidant genes involved in resistance to peroxides as well as oxygen stress (aerotolerance). In addition, the oxyR deletion attenuates biofilm formation in T. forsythia.

  14. Strawberry homologue of terminal flower1 integrates photoperiod and temperature signals to inhibit flowering.

    Science.gov (United States)

    Rantanen, Marja; Kurokura, Takeshi; Jiang, Panpan; Mouhu, Katriina; Hytönen, Timo

    2015-04-01

    Photoperiod and temperature are major environmental signals affecting flowering in plants. Although molecular pathways mediating these signals have been well characterized in the annual model plant Arabidopsis, much less information is known in perennials. Many perennials including the woodland strawberry (Fragaria vesca L.) are induced to flower in response to decreasing photoperiod and temperature in autumn and they flower following spring. We showed earlier that, in contrast with Arabidopsis, the photoperiodic induction of flowering in strawberry occurs in short days (SD) when the decrease in FvFT1 (flowering locus T) and FvSOC1 (suppressor of the overexpression of constans1) expression leads to lower mRNA levels of the floral repressor, FvTFL1 (terminal flower1). By using transgenic lines and gene expression analyses, we show evidence that the temperature-mediated changes in the FvTFL1 mRNA expression set critical temperature limits for the photoperiodic flowering in strawberry. At temperatures below 13 °C, low expression level of FvTFL1 in both SD and long days (LD) allows flower induction to occur independently of the photoperiod. Rising temperature gradually increases FvTFL1 mRNA levels under LD, and at temperatures above 13 °C, SD is required for the flower induction that depends on the deactivation of FvSOC1 and FvTFL1. However, an unknown transcriptional activator, which functions independently of FvSOC1, enhances the expression of FvTFL1 at 23 °C preventing photoperiodic flowering. We suggest that the observed effect of the photoperiod × temperature interaction on FvTFL1 mRNA expression may allow strawberry to induce flowers in correct time in different climates.

  15. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  16. A novel salting-out assisted extraction coupled with HPLC- fluorescence detection for trace determination of vitamin K homologues in human plasma.

    Science.gov (United States)

    Ahmed, Sameh; Mahmoud, Ashraf M

    2015-11-01

    Recently, new physiological roles of vitamin K homologues have been established in the treatment of rheumatoid arthritis, osteoporosis, hepatocellular carcinoma and leukemia. However, relatively high plasma protein binding, low plasma concentrations and occurrences of interfering lipids make accurate determination of vitamin K homologues a challenging task. Therefore, a sensitive and reliable salting-out assisted liquid/liquid extraction (SALLE) method coupled with HPLC-Fluorescence detection was designed for efficient extraction and quantification of trace levels of vitamin K homologues in human plasma. The investigated vitamin K homologues were phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4) and menaquinone-7 (MK-7). The method employed a new efficient fluorescence derivatization reaction using ethanolic solution of stannous chloride in acidic solution to generate highly fluorescent naphthohydroquinone derivatives. Correlation coefficients were more than 0.998 in the concentration ranges of 0.3-100 ng mL(-1) with detection limits of 0.1-0.17 ng mL(-1) in human plasma. The developed HPLC-FL system was successfully applied for sensitive determination of vitamin K homologues in plasma of healthy volunteers. The developed method may provide a valuable tool in the pharmacoinformatic studies concerning the roles of vitamin K homologues.

  17. Characterization of the promoter and extended C-terminal domain of Arabidopsis WRKY33 and functional analysis of tomato WRKY33 homologues in plant stress responses.

    Science.gov (United States)

    Zhou, Jie; Wang, Jian; Zheng, Zuyu; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2015-08-01

    Arabidopsis AtWRKY33 plays a critical role in broad plant stress responses. Whether there are evolutionarily conserved homologues of AtWRKY33 in other plants and what make AtWRKY33 such an important protein in plant stress responses are largely unknown. We compared AtWRKY33 with its close homologues to identify AtWRKY33-specific regulatory and structural elements, which were then functionally analysed through complementation. We also performed phylogenetic analysis to identify structural AtWRKY33 homologues in other plants and functionally analysed two tomato homologues through complementation and gene silencing. AtWRKY33 has an extended C-terminal domain (CTD) absent in its close homologue AtWRKY25. Both its CTD and the strong pathogen/stress-responsive expression of AtWRKY33 are necessary to complement the critical phenotypes of atwrky33. Structural AtWRKY33 homologues were identified in both dicot and monocot plants including two (SlWRKY33A and SlWRKY33B) in tomato. Molecular complementation and gene silencing confirmed that the two tomato WRKY genes play a critical role similar to that of AtWRKY33 in plant stress responses. Thus, WRKY33 proteins are evolutionarily conserved with a critical role in broad plant stress responses. Both its CTD and promoter are critical for the uniquely important roles of WRKY33 in plant stress responses.

  18. Regulatory elements and transcriptional control of chicken vasa homologue (CVH) promoter in chicken primordial germ cells.

    Science.gov (United States)

    Jin, So Dam; Lee, Bo Ram; Hwang, Young Sun; Lee, Hong Jo; Rim, Jong Seop; Han, Jae Yong

    2017-01-01

    Primordial germ cells (PGCs), the precursors of functional gametes, have distinct characteristics and exhibit several unique molecular mechanisms to maintain pluripotency and germness in comparison to somatic cells. They express germ cell-specific RNA binding proteins (RBPs) by modulating tissue-specific cis- and trans-regulatory elements. Studies on gene structures of chicken vasa homologue (CVH), a chicken RNA binding protein, involved in temporal and spatial regulation are thus important not only for understanding the molecular mechanisms that regulate germ cell fate, but also for practical applications of primordial germ cells. However, very limited studies are available on regulatory elements that control germ cell-specific expression in chicken. Therefore, we investigated the intricate regulatory mechanism(s) that governs transcriptional control of CVH. We constructed green fluorescence protein (GFP) or luciferase reporter vectors containing the various 5' flanking regions of CVH gene. From the 5' deletion and fragmented assays in chicken PGCs, we have identified a CVH promoter that locates at -316 to +275 base pair fragment with the highest luciferase activity. Additionally, we confirmed for the first time that the 5' untranslated region (UTR) containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs. Furthermore, using a transcription factor binding prediction, transcriptome analysis and siRNA-mediated knockdown, we have identified that a set of transcription factors play a role in the PGC-specific CVH gene expression. These results demonstrate that cis-elements and transcription factors localizing in the 5' flanking region including the 5' UTR and an intron are important for transcriptional regulation of the CVH gene in chicken PGCs. Finally, this information will contribute to research studies in areas of reproductive biology, constructing of germ cell-specific synthetic promoter for tracing primordial germ cells as well as

  19. Engineering the glutamate transporter homologue GltPh using protein semisynthesis.

    Science.gov (United States)

    Focke, Paul J; Annen, Alvin W; Valiyaveetil, Francis I

    2015-03-03

    Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homologue of glutamate transporters. Semisynthesis allows the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site, with the unnatural analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of our results, we propose that Arg397 is involved in a Na+-dependent remodeling of the substrate binding site required for high-affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.

  20. The Mode of Hedgehog Binding to Ihog Homologues is Not Conserved Across Different Phyla

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, J.; Zheng, X; Hauk, G; Ghirlando, R; Beachy, P; Leahy, D

    2008-01-01

    Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses1, 2. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer3, 4, 5, 6, 7. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle8, 9, 10. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner12, 13. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO12, 14. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.

  1. Characterization of two 20kDa-cement protein (cp20k homologues in Amphibalanus amphitrite.

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    Li-Sheng He

    Full Text Available The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT, suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing.

  2. The Drosophila Arf1 homologue Arf79F is essential for lamellipodium formation

    Science.gov (United States)

    Humphreys, Daniel; Liu, Tao; Davidson, Anthony C.; Hume, Peter J.; Koronakis, Vassilis

    2012-01-01

    Summary The WAVE regulatory complex (WRC) drives the polymerisation of actin filaments located beneath the plasma membrane to generate lamellipodia that are pivotal to cell architecture and movement. By reconstituting WRC-dependent actin assembly at the membrane, we recently discovered that several classes of Arf family GTPases directly recruit and activate WRC in cell extracts, and that Arf cooperates with Rac1 to trigger actin polymerisation. Here, we demonstrate that the Class 1 Arf1 homologue Arf79F colocalises with the WRC at dynamic lamellipodia. We report that Arf79F is required for lamellipodium formation in Drosophila S2R+ cells, which only express one Arf isoform for each class. Impeding Arf function either by dominant-negative Arf expression or by Arf double-stranded RNA interference (dsRNAi)-mediated knockdown uncovered that Arf-dependent lamellipodium formation was specific to Arf79F, establishing that Class 1 Arfs, but not Class 2 or Class 3 Arfs, are crucial for lamellipodia. Lamellipodium formation in Arf79F-silenced cells was restored by expressing mammalian Arf1, but not by constitutively active Rac1, showing that Arf79F does not act via Rac1. Abolition of lamellipodium formation in Arf79F-silenced cells was not due to Golgi disruption. Blocking Arf79F activation with guanine nucleotide exchange factor inhibitors impaired WRC localisation to the plasma membrane and concomitant generation of lamellipodia. Our data indicate that the Class I Arf GTPase is a central component in WRC-driven lamellipodium formation. PMID:22992458

  3. SQL-1, homologue of the Golgi protein GMAP210, modulates intraflagellar transport in C. elegans.

    Science.gov (United States)

    Broekhuis, Joost R; Rademakers, Suzanne; Burghoorn, Jan; Jansen, Gert

    2013-04-15

    Primary cilia are microtubule-based organelles that have important sensory functions. For their function, cilia rely on the delivery of specific proteins, both by intracellular trafficking and intraflagellar transport (IFT). In the cilia of Caenorhabditis elegans, anterograde IFT is mediated by kinesin-II and OSM-3. Previously, we have shown that expression of a dominant active G protein α subunit (GPA-3QL) in amphid channel neurons affects the coordination of kinesin-II and OSM-3 and also affects cilia length, suggesting that environmental signals can modulate these processes. Here, we show that loss-of-function of sql-1 (suppressor of gpa-3QL 1), which encodes the homologue of the mammalian Golgi protein GMAP210, suppresses the gpa-3QL cilia length phenotype. SQL-1 localizes to the Golgi apparatus, where it contributes to maintaining Golgi organization. Loss of sql-1 by itself does not affect cilia length, whereas overexpression of sql-1 results in longer cilia. Using live imaging of fluorescently tagged IFT proteins, we show that in sql-1 mutants OSM-3 moves faster, kinesin-II moves slower and that some complex A and B proteins move at an intermediate velocity, while others move at the same velocity as OSM-3. This indicates that mutation of sql-1 destabilizes the IFT complex. Finally, we show that simultaneous inactivation of sql-1 and activation of gpa-3QL affects the velocity of OSM-3. In summary, we show that in C. elegans the Golgin protein SQL-1 plays an important role in maintaining the stability of the IFT complex.

  4. Diversity in domain architectures of Ser/Thr kinases and their homologues in prokaryotes

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    Krupa A

    2005-09-01

    Full Text Available Abstract Background Ser/Thr/Tyr kinases (STYKs commonly found in eukaryotes have been recently reported in many bacterial species. Recent studies elucidating their cellular functions have established their roles in bacterial growth and development. However functions of a large number of bacterial STYKs still remain elusive. The organisation of domains in a large dataset of bacterial STYKs has been investigated here in order to recognise variety in domain combinations which determine functions of bacterial STYKs. Results Using sensitive sequence and profile search methods, domain organisation of over 600 STYKs from 125 prokaryotic genomes have been examined. Kinase catalytic domains of STYKs tethered to a wide range of enzymatic domains such as phosphatases, HSP70, peptidyl prolyl isomerases, pectin esterases and glycoproteases have been identified. Such distinct preferences for domain combinations are not known to be present in either the Histidine kinase or the eukaryotic STYK families. Domain organisation of STYKs specific to certain groups of bacteria has also been noted in the current anlaysis. For example, Hydrophobin like domains in Mycobacterial STYK and penicillin binding domains in few STYKs of Gram-positive organisms and FHA domains in cyanobacterial STYKs. Homologues of characterised substrates of prokaryotic STYKs have also been identified. Conclusion The domains and domain architectures of most of the bacterial STYKs identified are very different from the known domain organisation in STYKs of eukaryotes. This observation highlights distinct biological roles of bacterial STYKs compared to eukaryotic STYKs. Bacterial STYKs reveal high diversity in domain organisation. Some of the modular organisations conserved across diverse bacterial species suggests their central role in bacterial physiology. Unique domain architectures of few other groups of STYKs reveal recruitment of functions specific to the species.

  5. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.

    Science.gov (United States)

    Wyroba, E; Surmacz, L; Osinska, M; Wiejak, J

    2007-01-01

    Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  6. Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus.

    Science.gov (United States)

    Zhang, Chuanshan; Wang, Limin; Wang, Hui; Pu, Hongwei; Yang, Le; Li, Jing; Wang, Junhua; Lü, Guodong; Lu, Xiaomei; Zhang, Wenbao; Vuitton, Dominique A; Wen, Hao; Lin, Renyong

    2014-10-01

    Smad family proteins are essential cellular mediators of the transforming growth factor-β superfamily. In the present study, we identified two members of the Smad proteins, Smad8 and Smad4 homologues (termed as EgSmadE and EgSmadD, respectively), from Echinococcus granulosus, the causative agent of cystic echinococcosis (CE). Phylogenetic analysis placed EgSmadE in the Smad1, 5, and 8 subgroup of the R-Smad sub-family and EgSmadD in the Co-Smad family. Furthermore, EgSmadE and EgSmadD attained a high homology to EmSmadE and EmSmadD of E. multilocularis, respectively. Both EgSmadE and EgSmadD were co-expressed in the larval stages and exhibited the highest transcript levels in activated protoscoleces, and their encoded proteins were co-localized in the sub-tegumental and tegumental layer of the parasite. As shown by yeast two-hybrid and pull-down analysis, EgSmadE displayed a positive binding interaction with EgSmadD. In addition, EgSmadE localized in the nuclei of Mv1Lu cells (mink lung epithelial cells) upon treatment with human TGF-β1 or human BMP2, indicating that EgSmadE is capable of being translocated into nucleus, in vitro. Our study suggests that EgSmadE and EgSmadD may take part in critical biological processes, including echinococcal growth, development, and parasite-host interaction.

  7. Development and mapping of SSR markers linked to resistance-gene homologue clusters in common bean

    Institute of Scientific and Technical Information of China (English)

    Luz; Nayibe; Garzon; Matthew; Wohlgemuth; Blair

    2014-01-01

    Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.

  8. Synthesis, Electrochemistry and Electrogenerated Chemiluminesce of two BODIPY-Appended Bipyridine Homologues

    Science.gov (United States)

    Qi, Honglan; Teesdale, Justin J.; Pupillo, Rachel C.

    2014-01-01

    Two new 2,2’-bipyridine (bpy) derivatives containing ancillary BODIPY chromophores attached at the 5- and 5’-positions (BB3) or 6- and 6’-positions (BB4) were prepared and characterized. In this work, the basic photophysics, electrochemistry and electrogenerated chemiluminescence (ECL) of BB3 and BB4 are compared with those previously reported for a related bpy-BODIPY derivative (BB2) (J. Phys. Chem. C 2011, 115, 17993–18001). Cyclic voltammetry revealed that BB3 and BB4 display reversible 2e− oxidation and reduction waves, which consist of two closely spaced (50 – 70 mV) 1e− events. This redox behavior is consistent with the frontier molecular orbitals calculated for BB3 and BB4 and indicates that the 2,2’-bipyridine spacer of each bpy- BODIPY homologue does not facilitate efficient electronic communication between the tethered indacene units. In the presence of a coreactant such as tri-n-propylamine (TPA) or benzoyl peroxide (BPO), BB3 and BB4 exhibit strong ECL and produce spectra that are very similar to their corresponding photoluminescence profiles. The ECL signal obtained under annihilation conditions, however, is significantly different and is characterized by two distinct bands. One of these bands is centered at ~570 nm and is attributed to emission via an S- or T-route. The second band, occurs at longer wavelengths and is centered around ~740 nm. The shape and concentration dependence of this long-wavelength ECL signal is not indicative of emission from an excimer or aggregate, but rather is suggests that a new emissive species is formed from the bpy-BODIPY luminophores during the annihilation process. PMID:23980850

  9. Ligand Binding in the Extracellular Vestibule of the Neurotransmitter Transporter Homologue LeuT.

    Science.gov (United States)

    Grouleff, Julie; Koldsø, Heidi; Miao, Yinglong; Schiøtt, Birgit

    2017-03-15

    The human monoamine transporters (MATs) facilitate the reuptake of monoamine neurotransmitters from the synaptic cleft. MATs are linked to a number of neurological diseases and are the targets of both therapeutic and illicit drugs. Until recently, no high-resolution structures of the human MATs existed, and therefore, studies of this transporter family have relied on investigations of the homologues bacterial transporters such as the leucine transporter LeuT, which has been crystallized in several conformational states. A two-substrate transport mechanism has been suggested for this transporter family, which entails that high-affinity binding of a second substrate in an extracellular site is necessary for the substrate in the central binding site to be transported. Compelling evidence for this mechanism has been presented, however, a number of equally compelling accounts suggest that the transporters function through a mechanism involving only a single substrate and a single high-affinity site. To shed light on this apparent contradiction, we have performed extensive molecular dynamics simulations of LeuT in the outward-occluded conformation with either one or two substrates bound to the transporter. We have also calculated the substrate binding affinity in each of the two proposed binding sites through rigorous free energy simulations. Results show that substrate binding is unstable in the extracellular vestibule and the substrate binding affinity within the suggested extracellular site is very low (0.2 and 3.3 M for the two dominant binding modes) compared to the central substrate binding site (14 nM). This suggests that for LeuT in the outward-occluded conformation only a single high-affinity substrate binding site exists.

  10. Neurophysiological Evidence That Musical Training Influences the Recruitment of Right Hemispheric Homologues for Speech Perception

    Directory of Open Access Journals (Sweden)

    McNeel Gordon Jantzen

    2014-03-01

    Full Text Available Musicians have a more accurate temporal and tonal representation of auditory stimuli than their non-musician counterparts (Kraus & Chandrasekaran, 2010; Parbery-Clark, Skoe, & Kraus, 2009; Zendel & Alain, 2008; Musacchia, Sams, Skoe, & Kraus, 2007. Musicians who are adept at the production and perception of music are also more sensitive to key acoustic features of speech such as voice onset timing and pitch. Together, these data suggest that musical training may enhance the processing of acoustic information for speech sounds. In the current study, we sought to provide neural evidence that musicians process speech and music in a similar way. We hypothesized that for musicians, right hemisphere areas traditionally associated with music are also engaged for the processing of speech sounds. In contrast we predicted that in non-musicians processing of speech sounds would be localized to traditional left hemisphere language areas. Speech stimuli differing in voice onset time was presented using a dichotic listening paradigm. Subjects either indicated aural location for a specified speech sound or identified a specific speech sound from a directed aural location. Musical training effects and organization of acoustic features were reflected by activity in source generators of the P50. This included greater activation of right middle temporal gyrus (MTG and superior temporal gyrus (STG in musicians. The findings demonstrate recruitment of right hemisphere in musicians for discriminating speech sounds and a putative broadening of their language network. Musicians appear to have an increased sensitivity to acoustic features and enhanced selective attention to temporal features of speech that is facilitated by musical training and supported, in part, by right hemisphere homologues of established speech processing regions of the brain.

  11. Phenotypic characterization of OmpX, an Ail homologue of Yersinia pestis KIM.

    Science.gov (United States)

    Kolodziejek, Anna M; Sinclair, Dylan J; Seo, Keun S; Schnider, Darren R; Deobald, Claudia F; Rohde, Harold N; Viall, Austin K; Minnich, Scott S; Hovde, Carolyn J; Minnich, Scott A; Bohach, Gregory A

    2007-09-01

    The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6(+) parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 degrees C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.

  12. Determination of homologues of quaternary ammonium surfactants by capillary electrophoresis using indirect UV detection.

    Science.gov (United States)

    Liu, Hsueh-Ying; Ding, Wang-Hsien

    2004-02-06

    This investigation describes the simultaneous separation of two major non-chromophoric quaternary ammonium surfactants, alkyltrimethyl- and dialkyldimethylammonium compounds (ATMACs and DADMACs, respectively), by capillary electrophoresis (CE) using indirect UV detection. The most effective separation conditions was 10 mM phosphate buffer with 57.5% tetrahydrofuran and 3 mM sodium dodecyl sulfate (SDS) at pH 4.3, and the sample hydrodynamic injection of up to 20 s at 1 psi (approximately 60 nl), and an applied voltage of 25 kV (1 psi = 6.9 kPa). Specially, the selection of an appropriate chromophore and an internal standard (I.S.) to improve the peak identification and quantitation was systematically investigated. Decylbenzyldimethyl ammonium chloride (C10-BDMA+C-) as a chromophore with 3 mM sodium dodecyl sulfate provided the best detectability for all homologues. The reproducibility of the migration time and quantitative analysis can be improved by using tetraoctyl ammonium ion as an internal standard, giving the relative standard deviation (R.S.D.) less than 0.8% for the relative migration times, and 2.5-5.5% for the relative peak areas. A good linearity of CE analysis was obtained in the range of 1.0-20 microg/ml with r2 values of above 0.999. The analysis of cationic surfactants in commercial products of hair conditioners and fabric softeners was also performed. Electrospray mass spectrometric method was applied to evaluate the CE method, and the compatible results were obtained.

  13. Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides.

    Science.gov (United States)

    Thynne, Elisha; Saur, Isabel M L; Simbaqueba, Jaime; Ogilvie, Huw A; Gonzalez-Cendales, Yvonne; Mead, Oliver; Taranto, Adam; Catanzariti, Ann-Maree; McDonald, Megan C; Schwessinger, Benjamin; Jones, David A; Rathjen, John P; Solomon, Peter S

    2016-06-13

    In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.

  14. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues

    Directory of Open Access Journals (Sweden)

    E Wyroba

    2009-08-01

    Full Text Available Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein and LAMP-2 (lysosomal membrane protein 2 as well as a7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2 in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100 000 x g of equal load were quantified by immunoblotting. LAMP-2 crossreacting polypeptide of ~106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The a7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILPrelated polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  15. A transglutaminase homologue as a condensation catalyst in antibiotic assembly lines.

    Science.gov (United States)

    Fortin, Pascal D; Walsh, Christopher T; Magarvey, Nathan A

    2007-08-16

    The unrelenting emergence of antibiotic-resistant bacterial pathogens demands the investigation of antibiotics with new modes of action. The pseudopeptide antibiotic andrimid is a nanomolar inhibitor of the bacterial acetyl-CoA carboxylase that catalyses the first committed step in prokaryotic fatty acid biosynthesis. Recently, the andrimid (adm) biosynthetic gene cluster was isolated and heterologously expressed in Escherichia coli. This establishes a heterologous biological host in which to rapidly probe features of andrimid formation and to use biosynthetic engineering to make unnatural variants of this important and promising new class of antibiotics. Bioinformatic analysis of the adm cluster revealed a dissociated biosynthetic assembly system lacking canonical amide synthases between the first three carrier protein domains. Here we report that AdmF, a transglutaminase (TGase) homologue, catalyses the formation of the first amide bond, an N-acyl-beta-peptide link, in andrimid biosynthesis. Hence, AdmF is a newly discovered biosynthetic enzyme that acts as a stand-alone amide synthase between protein-bound, thiotemplated substrates in an antibiotic enzymatic assembly line. TGases (enzyme class (EC) 2.3.2.13) normally catalyse the cross-linking of (poly)peptides by creating isopeptidic bonds between the gamma-carboxamide group of a glutamine side chain of one protein and various amine donors, including lysine side chains. To the best of our knowledge, the present study constitutes the first report of a TGase-like enzyme recruited for the assembly of an antibiotic. Moreover, genome mining using the AdmF sequence yielded additional TGases in unassigned natural product biosynthetic pathways. With many more microbial genomes being sequenced, such a strategy could potentially unearth biosynthetic pathways producing new classes of antibiotics.

  16. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli

    Science.gov (United States)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert E.; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9× dilution sample was 55, suggesting that ˜20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  17. The Drosophila Kctd-family homologue Kctd12-like modulates male aggression and mating behaviour.

    Science.gov (United States)

    Williams, Michael J; Goergen, Philip; Phad, Ganesh; Fredriksson, Robert; Schiöth, Helgi B

    2014-08-01

    In Drosophila, serotonin (5-HT) regulates aggression, mating behaviour and sleep/wake behaviour through different receptors. Currently, how these various receptors are themselves regulated is still not completely understood. The KCTD12-family of proteins, which have been shown to modify G-protein-coupled receptor (GPCR) signalling in mammals, are one possibility of auxiliary proteins modulating 5-HT receptor signalling. The KCTD12-family was found to be remarkably conserved and present in species from C. elegans to humans. The Drosophila KCTD12 homologue Kctd12-like (Ktl) was highly expressed in both the larval and adult CNS. By performing behavioural assays in male Drosophila, we now reveal that Ktl is required for proper male aggression and mating behaviour. Previously, it was shown that Ktl is in a complex with the Drosophila 5-HT receptor 5-HT7, and we observed that both Ktl and the 5-HT1A receptor are required in insulin-producing cells (IPCs) for proper adult male behaviour, as well as for hyperaggressive activity induced by the mammalian 5-HT1A receptor agonist 8-hydroxy-2-dipropylaminotetralin-hydrobromide. Finally, we show that Ktl expression in the IPCs is necessary to regulate locomotion and normal sleep/wake patterns in Drosophila, but not the 5-HT1A receptor. Similar to what was observed with mammalian KCTD12-family members that interact physically with a GPCR receptor to regulate desensitization, in Drosophila Ktl may function in GPCR 5-HT receptor pathways to regulate their signalling, which is required for proper adult male behaviour. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  18. Bursaphelenchus xylophilus is killed by homologues of 2-(1-undecyloxy)-1-ethanol

    Science.gov (United States)

    Kim, Junheon; Lee, Sang-Myeong; Park, Chung Gyoo

    2016-07-01

    2-(1-Undecyloxy)-1-ethanol, monochamol, is a male-produced aggregation pheromone of the Monochamus species, which are efficient vectors of the pine wood nematode (PWN), Bursaphelenchus xylophilus, which cause devastating damage to pines worldwide. The nematicidal activity of synthetic monochamol and its homologues (ROEtOH: R = C7-C13) were investigated to find potential alternatives to the currently used PWN control agents abamectin and emamectin. Compounds with C7-C13 chain length alkyl groups exhibited 100% nematicidal activity at a concentration of 1000 mg/L. At a concentration of 100 mg/L, 2-(1-nonyloxy)-1-ethanol (C9OEtOH), 2-(1-decyloxy)-1-ethanol (C10OEtOH), 2-(1-undecyloxy)-1-ethanol (C11OEtOH), and 2-(1-dodecyloxy)-1-ethanol (C12OEtOH) showed 100% nematicidal activity, but the others showed weaker activities. C11OEtOH showed similar nematicidal activity to abamectin in terms of LD90 values, which were 13.30 and 12.53 mg/L, respectively. However, C9OEtOH, C10OEtOH, and C12OEtOH (LC90 values: 53.63, 38.18, and 46.68 mg/L, respectively) were less effective than C11OEtOH and abamectin. These results indicate that monochamol could be an effective alternative agent against PWN. The relationship of insecticidal and nematicidal activity to different carbon chain lengths in compounds is discussed.

  19. Characterization of Two 20kDa-Cement Protein (cp20k) Homologues in Amphibalanus amphitrite

    KAUST Repository

    He, Li-Sheng

    2013-05-22

    The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing. © 2013 He et al.

  20. The two PPX-GppA homologues from Mycobacterium tuberculosis have distinct biochemical activities.

    Directory of Open Access Journals (Sweden)

    Mei Y Choi

    Full Text Available Inorganic polyphosphate (poly-P, guanosine pentaphosphate (pppGpp and guanosine tetraphosphate (ppGpp are ubiquitous in bacteria. These molecules play a variety of important physiological roles associated with stress resistance, persistence, and virulence. In the bacterial pathogen Mycobacterium tuberculosis, the identities of the proteins responsible for the metabolism of polyphosphate and (pppGpp remain to be fully established. M. tuberculosis encodes two PPX-GppA homologues, Rv0496 (MTB-PPX1 and Rv1026, which share significant sequence similarity with bacterial exopolyphosphatase (PPX and guanosine pentaphosphate 5'-phosphohydrolase (GPP proteins. Here we delineate the respective biochemical activities of the Rv0496 and Rv1026 proteins and benchmark these against the activities of the PPX and GPP proteins from Escherichia coli. We demonstrate that Rv0496 functions as an exopolyphosphatase, showing a distinct preference for relatively short-chain poly-P substrates. In contrast, Rv1026 has no detectable exopolyphosphatase activities. Analogous to the E. coli PPX and GPP enzymes, the exopolyphosphatase activities of Rv0496 are inhibited by pppGpp and, to a lesser extent, by ppGpp alarmones, which are produced during the bacterial stringent response. However, neither Rv0496 nor Rv1026 have the ability to hydrolyze pppGpp to ppGpp; a reaction catalyzed by E. coli PPX and GPP. Both the Rv0496 and Rv1026 proteins have modest ATPase and to a lesser extent ADPase activities. pppGpp alarmones inhibit the ATPase activities of Rv1026 and, to a lesser extent, the ATPase activities of Rv0496. We conclude that PPX-GppA family proteins may not possess all the catalytic activities implied by their name and may play distinct biochemical roles involved in polyphosphate and (pppGpp metabolic pathways.

  1. Recent innovations in mRNA vaccines.

    Science.gov (United States)

    Ulmer, Jeffrey B; Geall, Andrew J

    2016-08-01

    Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have yet been licensed for human use. Recently, mRNA based vaccines have emerged as an alternative approach. They promise the flexibility of plasmid DNA vaccines, without the need for electroporation, but with enhanced immunogenicity and safety. In addition, they avoid the limitations of anti-vector immunity seen with viral vectors, and can be dosed repeatedly. This review highlights the key papers published over the past few years and summarizes prospects for the near future.

  2. Alternative polyadenylation of mRNA precursors

    Science.gov (United States)

    Tian, Bin; Manley, James L.

    2017-01-01

    Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. PMID:27677860

  3. Identification, localization, and functional analysis of the homologues of mouse CABS1 protein in porcine testis

    OpenAIRE

    Shawki, Hossam H.; Kigoshi, Takumi; Katoh, Yuki; Matsuda, Manabu; Ugboma, Chioma M.; Takahashi, Satoru; Oishi, Hisashi; KAWASHIMA Akihiro

    2016-01-01

    Previously, we have identified a calcium-binding protein that is specifically expressed in spermatids and localized to the flagella of the mature sperm in mouse, so-called mCABS1. However, the physiological roles of CABS1 in the male reproductive system have not been fully elucidated yet. In the current study, we aimed to localize and clarify the role of CABS1 in porcine (pCABS1). We determined for the first time the full nucleotides sequence of pCABS1 mRNA. pCABS1 protein was detected on SDS...

  4. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA-binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    regulating mRNA turnover in eukaryotes. However, bacterial Hfq proteins are homohexameric, whereas eukaryotic Sm/Lsm proteins are heteroheptameric. Recently, Hfq proteins with poor sequence conservation were identified in archaea and cyanobacteria. In this article, we describe crystal structures of the Hfq...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  5. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    regulating mRNA turnover in eukaryotes. However, bacterial Hfq proteins are homohexameric, whereas eukaryotic Sm/Lsm proteins are heteroheptameric. Recently, Hfq proteins with poor sequence conservation were identified in archaea and cyanobacteria. In this article, we describe crystal structures of the Hfq...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  6. The opiorphin gene (ProL1) and its homologues function in erectile physiology.

    Science.gov (United States)

    Tong, Yuehong; Tar, Moses; Melman, Arnold; Davies, Kelvin

    2008-09-01

    To determine if ProL1, a member of the opiorphin family of genes, can modulate erectile physiology, as it encodes a peptide which acts as a neutral endopeptidase inhibitor, other examples of which (Vcsa1, hSMR3A) modulate erectile physiology. We cloned members of the opiorphin family of genes into the same mammalian expression backbone (pVAX); 100 microg of these plasmids (pVAX-Vcsa1, -hSMR3A, -hSMR3B and -ProL1) were injected intracorporally into retired breeder rats and the affect on erectile physiology assessed visually, by histology and by measuring the intracavernous pressure (ICP) and blood pressure (BP). As a positive control, rats were treated with pVAX-hSlo (expressing the MaxiK potassium channel) and as a negative control the empty backbone plasmid was injected (pVAX). We also compared the level of expression of ProL1 in corporal tissue of patients not reporting erectile dysfunction (ED), ED associated with diabetes and ED not caused by diabetes. Gene transfer of plasmids expressing all members of the opiorphin family had a similar and significant effect on erectile physiology. At the concentration used in these experiments (100 microg) they resulted in higher resting ICP, and histological and visual analysis showed evidence of a priapic-like condition. After electrostimulation of the cavernous nerve, rats had significantly better ICP/BP than the negative control (pVAX). Gene transfer of pVAX-hSlo increased the ICP/BP ratio to a similar extent to the opiorphin homologues, but with no evidence for a priapic-like condition. Corpora cavernosa tissue samples obtained from men with ED, regardless of underlying causes, had significant down-regulation of both hSMR3A and ProL1. All members of the human opiorphin family of genes can potentially modulate erectile physiology. Both hSMR3 and ProL1 are down-regulated in the corpora of men with ED, and therefore both genes can potentially act as markers of ED.

  7. Structure, Mechanism, and Substrate Profile for Sco3058: The Closest Bacterial Homologue to Human Renal Dipeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Cummings, J.; Nguyen, T; Fedorov, A; Kolb, P; Xu, C; Fedorov, E; Shoichet, B; Barondeau, D; Almo, S; Raushel, F

    2010-01-01

    Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of {beta}-lactams, is similar in sequence to a cluster of 400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of L-Xaa-L-Xaa, L-Xaa-D-Xaa, and D-Xaa-L-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an l-amino acid at the N-terminus and a d-amino acid at the C-terminus. The best substrate identified was L-Arg-D-Asp (k{sub cat}/K{sub m} = 7.6 x 10{sup 5} M{sup -1} s{sup -1}). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of L-Ala-D-Asp. The enzyme folds as a ({beta}/{alpha}){sub 8} barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D{sub 2}O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for k{sub cat} and k{sub cat}/K{sub m} indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of L/D-Ala-L/D-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.

  8. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli.

    Science.gov (United States)

    Paproski, Robert J; Li, Yan; Barber, Quinn; Lewis, John D; Campbell, Robert E; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9×dilution sample was 55, suggesting that ∼20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene

  9. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue.

    Science.gov (United States)

    Zhao, Yongfang; Terry, Daniel S; Shi, Lei; Quick, Matthias; Weinstein, Harel; Blanchard, Scott C; Javitch, Jonathan A

    2011-06-02

    Neurotransmitter/Na(+) symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na(+) electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na(+) are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan, and the Na(+)-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na(+)-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively

  10. Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats

    Directory of Open Access Journals (Sweden)

    Roussel Anne M

    2007-01-01

    Full Text Available Abstract Background Tristetraprolin (TTP/ZFP36 family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has anti-inflammatory properties but the molecular mechanisms have not been completely elucidated. We hypothesized that TTP and/or its homologues might contribute to the beneficial effects of tea as an anti-inflammatory product. Methods Quantitative real-time PCR was used to investigate the effects of green tea (0, 1, and 2 g solid extract/kg diet on the expression of Ttp family genes (Ttp/Tis11/Zfp36, Zfp36l1/Tis11b, Zfp36l2/Tis11d, Zfp36l3, pro-inflammatory genes (Tnf, Csf2/Gm-csf, Ptgs2/Cox2, and Elavl1/Hua/Hur and Vegf genes in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance, oxidative stress, inflammation, and TNF-alpha levels. Results Ttp and Zfp36l1 mRNAs were the major forms in both liver and skeletal muscle. Ttp, Zfp36l1, and Zfp36l2 mRNA levels were more abundant in the liver than those in the muscle. Csf2/Gm-csf and Zfp36l3 mRNAs were undetectable in both tissues. Tea (1 g solid extract/kg diet increased Ttp mRNA levels by 50–140% but Tnf mRNA levels decreased by 30% in both tissues, and Ptgs2/Cox2 mRNA levels decreased by 40% in the muscle. Tea (2 g solid extract/kg diet increased Elavl1/Hua/Hur mRNA levels by 40% in the liver but did not affect any of the other mRNA levels in liver or muscle. Conclusion These results show that tea can modulate Ttp mRNA levels in animals and suggest that a post-transcriptional mechanism through TTP could partially account for tea's anti-inflammatory properties. The results also suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.

  11. Improved detection of remote homologues using cascade PSI-BLAST: influence of neighbouring protein families on sequence coverage.

    Directory of Open Access Journals (Sweden)

    Swati Kaushik

    Full Text Available BACKGROUND: Development of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST. METHODOLOGY/PRINCIPAL FINDINGS: We have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of ~100% and Mathew's correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families. CONCLUSIONS/SIGNIFICANCE: Our study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for

  12. Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

    Science.gov (United States)

    Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

    2015-07-01

    Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

  13. A C-type lectin collaborates with a CD45 phosphatase homologue to facilitate West Nile virus infection of mosquitoes

    OpenAIRE

    Cheng, Gong; Cox, Jonathan; Wang, Penghua; Krishnan, Manoj N; Dai, Jianfeng; Qian, Feng; Anderson, John F.; Fikrig, Erol

    2010-01-01

    West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homologue of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells, and to enhance viral entry. ...

  14. Prenylated Indolediketopiperazine Peroxides and Related Homologues from the Marine Sediment-Derived Fungus Penicillium brefeldianum SD-273

    Directory of Open Access Journals (Sweden)

    Chun-Yan An

    2014-01-01

    Full Text Available Three new indolediketopiperazine peroxides, namely, 24-hydroxyverruculogen (1, 26-hydroxyverruculogen (2, and 13-O-prenyl-26-hydroxyverruculogen (3, along with four known homologues (4–7, were isolated and identified from the culture extract of the marine sediment-derived fungus Penicillium brefeldianum SD-273. Their structures were determined based on the extensive spectroscopic analysis and compound 1 was confirmed by X-ray crystallographic analysis. The absolute configuration of compounds 1–3 was determined using chiral HPLC analysis of their acidic hydrolysates. Each of the isolated compounds was evaluated for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina lethality.

  15. Crystal structure of myotoxin-II: a myotoxic phospholipase A{sub 2} - homologue from Bothrops moojeni venom

    Energy Technology Data Exchange (ETDEWEB)

    Azevedo, W.F.; Ward, R.J.; Lombardi, F.R.; Arni, R.K. [UNESP, Sao Jose do Rio Preto, SP (Brazil). Inst. de Biociencias, Letras e Ciencias Exatas; Soares, A.M.; Giglio, J.R. [Sao Paulo Univ., Ribeirao Preto, SP (Brazil). Escola de Medicina; Fontes, M.R.M. [UNESP, Botucatu, SP (Brazil). Inst. Biofisica

    1997-12-31

    Full text. Phospho lipases A2 (PLA{sub 2}; E C 3.1.1.4, phosphatides s n-2 acyl hydrolases) hydrolysis the s n-2 ester bond of phospholipids showing enhanced activity at lamellar or membrane surfaces. Intracellular PLA{sub 2} s are involved at phospholipid metabolism and signal transduction, whereas extracellular PLA{sub 2} s are found in mammalian pancreatic juices, the venoms of snakes, lizards and insects. Based on their high primary sequence similarity, extracellular PLA{sub 2} s are separated into Classes I, II and III. Class II PLA{sub 2} s are found in snake venoms of Crotalidae an Viperidae species, and include the sub-family of Lys PLA{sub 2} s homologue. he coordination of the Ca{sup 2+} ion in the PLA{sub 2} calcium-binding loop includes and aspartate at position 49. In the catalytically active PLA{sub 2} s, this calcium ion plays a critical role in the stabilization of the tetrahedral transition state intermediate in the catalytic mechanism. The conservative substitution Asp49-Lys results in a decreased calcium affinity with a concomitant loss of catalytic activity, and naturally occurring PLA{sub 2} s-homologues showing the same substitution are catalytically inactive. However, the Lys PLA{sub 2} s possess cytolytic and myotoxic activities and furthermore retain the ability to disrupt the integrity of both plasma membranes and model lipid layers by a ca{sup 2+}-independent mechanism for which there is no evidence of lipid hydrolysis. Lys 49 PLA{sub 2} homologues have been isolated from several Bothrops spp. venoms including B. moojeni. Therefore, in order to improve our understanding of the molecular basis of the myotoxic and Ca{sup 2+} independent membrane damaging activities we have determined the crystal structure of MjTX-II, a Lys 49 homologue from the venom of B. moojeni. The model presented has been determined at 2.0 A resolution and refined to a crystallographic residual of 19.7% (R{sub f}ree=28.1%). (author)

  16. An Arabidopsis ctpA homologue is involved in the repair of photosystem Ⅱ under high light

    Institute of Scientific and Technical Information of China (English)

    YIN ShuMing; SUN XuWu; ZHANG LiXin

    2008-01-01

    A T-DNA insertion mutant AtctpA 1 was identified to study the physiological roles of a carboxyl-terminal processing protease (CtpA) homologue in Arabidopsis. Under normal growth conditions, disruption of AtctpA1 did not result in any apparent alterations in growth rate and thylakoid membrane protein components. However the mutant plants exhibited increased sensitivity to high irradiance. Degradation of PSII reaction center protein D1 was accelerated in the mutant during photoinhibition. These results demostrated that AtctpA1 was required for efficient repair of PSII in Arabidopsis under high irradiance.

  17. A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae.

    Science.gov (United States)

    Kwon, T H; Kim, M S; Choi, H W; Joo, C H; Cho, M Y; Lee, B L

    2000-10-01

    Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S. , Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615-621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-k

  18. Reversed-phase ion-pair high-performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues.

    Science.gov (United States)

    Kozlov, Vyacheslav V; Danilov, Leonid L

    2016-02-01

    A reversed-phase ion-pair high-performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion-pair reagent, and the dependence of the separation quality on pH of ion-pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Mouse Homologue of the Schizophrenia Susceptibility Gene ZNF804A as a Target of Hoxc8

    Directory of Open Access Journals (Sweden)

    Hyun Joo Chung

    2010-01-01

    Full Text Available Using a ChIP-cloning technique, we identified a Zinc finger protein 804a (Zfp804a as one of the putative Hoxc8 downstream target genes. We confirmed binding of Hoxc8 to an intronic region of Zfp804a by ChIP-PCR in F9 cells as well as in mouse embryos. Hoxc8 upregulated Zfp804a mRNA levels and augmented minimal promoter activity in vitro. In E11.5 mouse embryos, Zfp804a and Hoxc8 were coexpressed. Recent genome-wide studies identified Zfp804a (or ZNF804A in humans as a plausible marker for schizophrenia, leading us to hypothesize that this embryogenic regulatory control might also exert influence in development of complex traits such as psychosis.

  20. Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus.

    Science.gov (United States)

    Hayata, Koutarou; Asada, Seiya; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Sawayama, Shigeki

    2014-11-01

    The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

  1. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknot...

  2. Functional Integration of mRNA Translational Control Programs

    Directory of Open Access Journals (Sweden)

    Melanie C. MacNicol

    2015-07-01

    Full Text Available Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  3. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Sugahara, Mitsuaki [Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    2007-01-01

    An archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue from P. horikoshii OT3 was overexpressed as native and selenomethionine-substituted protein, purified and crystallized. The native and selenomethionine-derivative crystals are isomorphous and diffract X-rays to 2.1 and 2.9 Å resolution, respectively. 6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V{sub M} = 2.24 Å{sup 3} Da{sup −1}). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å.

  4. Biodegradation of diesel fuel by a microbial consortium in the presence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues

    DEFF Research Database (Denmark)

    Chrzanowski, L; Stasiewicz, M; Owsianiak, Mikolaj

    2009-01-01

    Fast development of ionic liquids as gaining more and more attention valuable chemicals will undoubtedly lead to environmental pollution. New formulations and application of ionic liquids may result in contamination in the presence of hydrophobic compounds, such as petroleum mixtures. We hypothes......Fast development of ionic liquids as gaining more and more attention valuable chemicals will undoubtedly lead to environmental pollution. New formulations and application of ionic liquids may result in contamination in the presence of hydrophobic compounds, such as petroleum mixtures. We...... hypothesize that in the presence of diesel fuel low-water-soluble ionic liquids may become more toxic to hydrocarbon-degrading microorganisms. In this study the influence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues (side-chain length from C-3 to C-18) on biodegradation of diesel fuel...... by a bacterial consortium was investigated. Whereas test performed for the consortium cultivated on disodium succinate showed that toxicity of the investigated ionic liquids decreased with increase in side-chain length, only higher homologues (C-8-C-18) caused a decrease in diesel fuel biodegradation...

  5. A 1.4-Mb interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues.

    Science.gov (United States)

    Lee, Eun-Joon; Raudsepp, Terje; Kata, Srinivas R; Adelson, David; Womack, James E; Skow, Loren C; Chowdhary, Bhanu P

    2004-02-01

    Comparative genomics has served as a backbone for the rapid development of gene maps in domesticated animals. The integration of this approach with radiation hybrid (RH) analysis provides one of the most direct ways to obtain physically ordered comparative maps across evolutionarily diverged species. We herein report the development of a detailed RH and comparative map for horse chromosome 17 (ECA17). With markers distributed at an average interval of every 1.4 Mb, the map is currently the most informative among the equine chromosomes. It comprises 75 markers (56 genes and 19 microsatellites), of which 50 gene specific and 5 microsatellite markers were generated in this study and typed to our 5000-rad horse x hamster whole genome RH panel. The markers are dispersed over six RH linkage groups and span 825 cR(5000). The map is among the most comprehensive whole chromosome comparative maps currently available for domesticated animals. It finely aligns ECA17 to human and mouse homologues (HSA13 and MMU1, 3, 5, 8, and 14, respectively) and homologues in other domesticated animals. Comparisons provide insight into their relative organization and help to identify evolutionarily conserved segments. The new ECA17 map will serve as a template for the development of clusters of BAC contigs in regions containing genes of interest. Sequencing of these regions will help to initiate studies aimed at understanding the molecular mechanisms for various diseases and inherited disorders in horse as well as human.

  6. Determination of the fate of alcohol ethoxylate homologues in a laboratory continuous activated-sludge unit study.

    Science.gov (United States)

    Wind, T; Stephenson, R J; Eadsforth, C V; Sherren, A; Toy, R

    2006-05-01

    Environmental monitoring indicates that the distribution of alcohol ethoxylate (AE) homologues in wastewater treatment plant (WWTP) effluents differs from the distribution in commercial AE products, with a relative higher proportion of fatty alcohol (AOH, which is AE with zero ethoxylation). To determine the contribution of AE-derived AOH to the total concentration of AE and AOH in WWTP effluents, we conducted a laboratory continuous activated-sludge study (CAS). This consisted of a test unit fed with AE-amended synthetic sewage and a control unit fed with only synthetic sewage to avoid AE contamination from the feed. The removal efficiencies of some 114 AE homologues were determined by the application of a specific and sensitive analytical method. The extent of the removal of AE ranged from 99.70% for C18 compounds to > 99.98% for C12-16. Relatively high-AOH concentrations were observed in the effluents from blank and test units. By building the concentration difference from the test minus the control unit, the AE in the CAS effluent originating from AE in the influent was determined. Thus, it could be shown that AOH represented only 19% of the total AE (EO0-18) in the CAS, while monitoring in 29 WWTP effluents (European, Canadian, and US) revealed in total a mean AOH fraction of 55% (5-82%) of the total AE (EO0-18). This shows that only a small fraction of AOH in WWTP effluents originates from AE entering the WWTP.

  7. Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8.

    Directory of Open Access Journals (Sweden)

    Rachel Senturia

    Full Text Available Human DiGeorge Critical Region 8 (DGCR8 is an essential microRNA (miRNA processing factor that is activated via direct interaction with Fe(III heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD. We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star also binds heme. The extinction coefficients (ε of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.

  8. The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death.

    Science.gov (United States)

    Mills, Kathryn; Daish, Tasman; Harvey, Kieran F; Pfleger, Cathie M; Hariharan, Iswar K; Kumar, Sharad

    2006-03-13

    The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.

  9. A WD40-repeat gene from Malus x domestica is a functional homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1.

    Science.gov (United States)

    Brueggemann, Julian; Weisshaar, Bernd; Sagasser, Martin

    2010-03-01

    The WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) is involved in a multitude of developmental and biochemical reactions in Arabidopsis thaliana such as the production of seed coat colour and mucilage, pigmentation by anthocyanins as well as the formation of trichomes and root hairs. In this study, a putative TTG1 homologue was isolated from apple (Malus x domestica Borkh.) showing 80.2% identity to A. thaliana TTG1 on nucleotide and 90.7% similarity on amino acid level. The MdTTG1 candidate was able to activate the AtBAN promoter in cooperation with the A. thaliana transcription factors TT2 and TT8 in A. thaliana protoplasts. This indicates that the encoded protein can be integrated into the complex that activates BAN in A. thaliana, and that a similar complex might also be present in apple. When transformed into ttg1 mutants of A. thaliana, the apple sequence was able to restore trichome growth, anthocyanin production in young seedlings as well as proanthocyanidin production in seeds. Additionally, roots of complemented mutant plants showed root hair formation resembling wild type. These results show that the studied apple WD40 gene is a functional homologue of AtTTG1 and we refer to this gene as MdTTG1.

  10. Probing dimensionality beyond the linear sequence of mRNA.

    Science.gov (United States)

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions.

  11. UPF1, a conserved nonsense-mediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Yi-Hsiu Chen

    Full Text Available The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

  12. In situ hydrothermal growth of metal-organic framework 199 films on stainless steel fibers for solid-phase microextraction of gaseous benzene homologues.

    Science.gov (United States)

    Cui, Xiao-Yan; Gu, Zhi-Yuan; Jiang, Dong-Qing; Li, Yan; Wang, He-Fang; Yan, Xiu-Ping

    2009-12-01

    Metal-organic frameworks (MOFs) have received great attention due to their fascinating structures and intriguing potential applications in various fields. Herein, we report the first example of the utilization of MOFs for solid-phase microextraction (SPME). MOF-199 with unique pores and open metal sites (Lewis acid sites) was employed as the coating for SPME fiber to extract volatile and harmful benzene homologues. The SPME fiber was fabricated by in situ hydrothermal growth of thin MOF-199 films on etched stainless steel wire. The MOF-199-coated fiber not only offered large enhancement factors from 19,613 (benzene) to 110,860 (p-xylene), but also exhibited wide linearity with 3 orders of magnitude for the tested benzene homologues. The limits of detection for the benzene homologues were 8.3-23.3 ng L(-1). The relative standard deviation (RSD) for six replicate extractions using one SPME fiber ranged from 2.0% to 7.7%. The fiber-to-fiber reproducibility for three parallel prepared fibers was 3.5%-9.4% (RSD). Indoor air samples were analyzed for the benzene homologues using the SPME with the MOF-199-coated fiber in combination with gas chromatography-flame ionization detection. The recoveries for the spiked benzene homologues in the collected indoor air samples were in the range of 87%-106%. The high affinity of the MOF-199-coated fiber to benzene homologues resulted from the combined effects of the large surface area and the unique porous structure of the MOF-199, the pi-pi interactions of the aromatic rings of the analytes with the framework 1,3,5-benzenetricarboxylic acid molecules, and the pi-complexation of the electron-rich analytes to the Lewis acid sites in the pores of MOF-199.

  13. Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability

    Directory of Open Access Journals (Sweden)

    Gorospe Myriam

    2005-05-01

    Full Text Available Abstract Background Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Results In order to investigate the relative contribution of gene transcription and changes in mRNA stability regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell and nuclear run-on (newly transcribed RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes. Conclusion We propose that regulation of mRNA stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.

  14. Effects of DNA replication on mRNA noise.

    Science.gov (United States)

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories.

  15. Identification, localization, and functional analysis of the homologues of mouse CABS1 protein in porcine testis.

    Science.gov (United States)

    Shawki, Hossam H; Kigoshi, Takumi; Katoh, Yuki; Matsuda, Manabu; Ugboma, Chioma M; Takahashi, Satoru; Oishi, Hisashi; Kawashima, Akihiro

    2016-07-29

    Previously, we have identified a calcium-binding protein that is specifically expressed in spermatids and localized to the flagella of the mature sperm in mouse, so-called mCABS1. However, the physiological roles of CABS1 in the male reproductive system have not been fully elucidated yet. In the current study, we aimed to localize and clarify the role of CABS1 in porcine (pCABS1). We determined for the first time the full nucleotides sequence of pCABS1 mRNA. pCABS1 protein was detected on SDS-PAGE gel as two bands at 75 kDa and 70 kDa in adult porcine testis, whereas one band at 70 kDa in epididymal sperm. pCABS1 immunoreactivity in seminiferous tubules was detected in the elongated spermatids, and that in the epididymal sperm was found in the acrosome as well as flagellum. The immunoreactivity of pCABS1 in the acrosomai region disappeared during acrosome reaction. We also identified that pCABS1 has a transmembrane domain using computational prediction of the amino acids sequence. The treatment of porcine capacitated sperm with anti-pCABS1 antiserum significantly decreased acrosome reactions. These results suggest that pCABS1 plays an important role in controlling calcium ion signaling during the acrosome reaction.

  16. Nutritional and hormonal factors control the gene expression of FoxOs, the mammalian homologues of DAF-16.

    Science.gov (United States)

    Imae, M; Fu, Z; Yoshida, A; Noguchi, T; Kato, H

    2003-04-01

    Transcription factors of the FoxO family in mammals are orthologues of the Caenorhabditis elegans forkhead factor DAF-16, which has been characterized as a target of insulin-like signalling. Three members of this family have been identified in rodents: FoxO1, FoxO3 and FoxO4, originally termed FKHR, FKHRL1 and AFX respectively. A number of in vitro studies have revealed that FoxOs are regulated through phosphorylation in response to insulin and related growth factors, resulting in their nuclear exclusion and inactivation. To clarify the mechanisms involved in the regulation of these factors in vivo, we investigated in the present study whether or not, and if so how, their mRNA levels in rat liver respond to the stimuli of several nutritional and hormonal factors. Imposed fasting for 48 h significantly elevated mRNA levels of FoxO1 (1.5-fold), FoxO3 (1.4-fold), and FoxO4 (1.6-fold). Refeeding for 3 h recovered the induced mRNA levels of FoxO1 and FoxO3 to the control levels, but did not affect that of FoxO4. FoxO1 and FoxO4 mRNA levels were proved to be highly reflective of their protein levels measured by Western immunoblotting. Of the three FoxO genes, FoxO4 only showed altered levels of mRNA (a 1.5-fold increase) in response to a protein-free diet. Streptozotocin-induced diabetes for 28 days decreased hepatic mRNA levels of FoxO1 and FoxO3 and increased the level of FoxO4 mRNA, but short-term (7 days) diabetes had fewer effects on the expression of these genes. Insulin replacement partially restored the FoxO1 and FoxO4 mRNA levels, but had no effect on the FoxO3 mRNA level. Daily administration for 1 week of dexamethasone, a synthetic glucocorticoid, increased the mRNA levels of FoxO1 (1.8-fold) and FoxO3 (2.4-fold). These results show that the FoxO genes respond differently to nutritional and hormonal factors, suggesting a new mechanism for the regulation of FoxO-dependent gene expression by these factors. Moreover, changes of FoxO1 and FoxO4 in the nucleus in

  17. Genetic analysis of the two zebrafish patched homologues identifies novel roles for the hedgehog signaling pathway

    Directory of Open Access Journals (Sweden)

    Groot Evelyn

    2008-02-01

    Full Text Available Abstract Background Aberrant activation of the Hedgehog (Hh signaling pathway in different organisms has shown the importance of this family of morphogens during development. Genetic screens in zebrafish have assigned specific roles for Hh in proliferation, differentiation and patterning, but mainly as a result of a loss of its activity. We attempted to fully activate the Hh pathway by removing both receptors for the Hh proteins, called Patched1 and 2, which are functioning as negative regulators in this pathway. Results Here we describe a splice-donor mutation in Ptc1, called ptc1hu1602, which in a homozygous state results in a subtle eye and somite phenotype. Since we recently positionally cloned a ptc2 mutant, a ptc1;ptc2 double mutant was generated, showing severely increased levels of ptc1, gli1 and nkx2.2a, confirming an aberrant activation of Hh signaling. As a consequence, a number of phenotypes were observed that have not been reported previously using Shh mRNA overexpression. Somites of ptc1;ptc2 double mutants do not express anteroposterior polarity markers, however initial segmentation of the somites itself is not affected. This is the first evidence that segmentation and anterior/posterior (A/P patterning of the somites are genetically uncoupled processes. Furthermore, a novel negative function of Hh signaling is observed in the induction of the fin field, acting well before any of the previously reported function of Shh in fin formation and in a way that is different from the proposed early role of Gli3 in limb/fin bud patterning. Conclusion The generation and characterization of the ptc1;ptc2 double mutant assigned novel and unexpected functions to the Hh signaling pathway. Additionally, these mutants will provide a useful system to further investigate the consequences of constitutively activated Hh signaling during vertebrate development.

  18. Cloning and expression of the rat homologue of the Huntington disease gene

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, I.; Epplen, J.T.; Riess, O. [Ruhr-Univ. Bochum (Germany)] [and others

    1994-09-01

    Huntington`s disease (HD) is an autosomal dominant neurodegenerative disorder which is manifested usually in adult life. The age of onset is variable and leads to progressive symptoms including involuntary choreatic movements and various cognitive and psychiatric disturbances. Recently, a gene (IT15) was cloned containing a (CAG){sub n} repeat which is elongated and unstable in HD patients. IT15 is widely expressed in human tissues but unrelated to any known deduced protein sequence. To further investigate the HD gene, 15 rat cDNA libraries were screened. 24 clones have been identified covering the Huntingtin gene. Comparison of the Huntingtin gene between human and rat revealed homologies between 80% and 87% at the DNA level and about 90% at the protein level. These analyses will help to define biologically important sequence regions, e.g., via evolutionary conservation. One clone contains the (CAG){sub n} repeat which consists of eight triplets compared to seven triplets in the mouse and a median of 17 in human. As in humans there are two transcripts arising from differential 3{prime}-polyadenylation. In the 3{prime}UTR a stretch of about 280 bp is exchanged for a 250 bp fragment with no homology in rodents and man. The cDNA clones are currently used to study Huntingtin gene expression during development in rodent tissues. RNA in situ hybridization of embryonic sections shows predominant signals in all neuronal tissues. In contrast to previously published data Huntingtin mRNA expression in testis is increased in spermatocytes vs. spermatogonia.

  19. Investigation of evaporation characteristics of polonium and its lighter homologues selenium and tellurium from liquid Pb-Bi-eutecticum

    CERN Document Server

    Neuhausen, J; Eichler, B

    2004-01-01

    The evaporation behaviour of polonium and its lighter homologues selenium and tellurium dissolved in liquid Pb-Bi-eutecticum (LBE) has been studied at various temperatures in the range from 482 K up to 1330 K under Ar/H2 and Ar/H2O-atmospheres using γ-ray spectroscopy. Polonium release in the temperature range of interest for technical applications is slow. Within short term (1h) experiments measurable amounts of polonium are evaporated only at temperatures above 973 K. Long term experiments reveal that a slow evaporation of polonium occurs at temperatures around 873 K resulting in a fractional polonium loss of the melt around 1% per day. Evaporation rates of selenium and tellurium are smaller than those of polonium. The presence of H2O does not enhance the evaporation within the error limits of our experiments. The thermodynamics and possible reaction pathways involved in polonium release from LBE are discussed.

  20. The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

    DEFF Research Database (Denmark)

    Xiao, Ruoyu; Wilkinson, Bonney; Solovyov, Anton

    2004-01-01

    In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae......, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI...... homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p...

  1. In mouse oocytes the mitochondrion-originated germinal body-like structures accumulate mouse Vasa homologue (MVH) protein.

    Science.gov (United States)

    Reunov, Arkadiy A; Reunova, Yulia A

    2015-08-01

    Mouse Vasa homologue (MVH) antibodies were applied to mouse Graafian oocytes to clarify if mitochondrion-originated germinal body-like structures, described previously by conventional electron microscopy, were associated with the germ plasm. It was found that both the mitochondrion-like structures with cristae and the germinal body-like structures that lacked any signs of cristae were labelled specifically by the anti-MVH antibody. Moreover, some granules were MVH-positive ultrastructural hybrids of the mitochondria and germinal body-like structures, the presence of which clearly supported the idea of a mitochondrial origin for the germinal body-like structures. This finding is the first evidence that mitochondrion-originated germinal body-like granules represent mouse germ plasm.

  2. mRNA trafficking and local translation: the Yin and Yang of regulating mRNA localization in neurons

    Institute of Scientific and Technical Information of China (English)

    John R. Sinnamon; Kevin Czaplinski

    2011-01-01

    Localized translation and the requisite trafficking of the mRNA template play significant roles in the nervous system including the establishment of dendrites and axons,axon path-finding,and synaptic plasticity.We provide a brief review on the regulation of localizing mRNA in mammalian neurons through critical posttranslational modifications of the factors involved.These examples highlight the relationship between mRNA trafficking and the translational regulation of trafficked mRNAs and provide insight into how extracellular signals target these events during signal transduction.

  3. Identification and characterisation of the angiotensin converting enzyme-3 (ACE3 gene: a novel mammalian homologue of ACE

    Directory of Open Access Journals (Sweden)

    Phelan Anne

    2007-06-01

    Full Text Available Abstract Background Mammalian angiotensin converting enzyme (ACE plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals. Results Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple sequence alignment and molecular modelling have been employed to characterise the predicted ACE3 protein. In mouse, rat, cow and dog, the predicted protein has mutations in some of the critical residues involved in catalysis, including the catalytic Glu in the HEXXH zinc binding motif which is Gln, and ESTs or reverse-transcription PCR indicate that the gene is expressed. In humans, the predicted ACE3 protein has an intact HEXXH motif, but there are other deletions and insertions in the gene and no ESTs have been identified. Conclusion In the genomes of several mammalian species there is a gene that encodes a novel, single domain ACE-like protein, ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, no evidence was found that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that ACE3 is a pseudogene.

  4. Homologue of Protein Kinase Mζ Maintains Context Aversive Memory and Underlying Long-Term Facilitation in Terrestrial Snail Helix.

    Directory of Open Access Journals (Sweden)

    Pavel M. Balaban

    2015-06-01

    Full Text Available It has been shown that a variety of long-term memories in different regions of the brain and in different species are quickly erased by local inhibition of PKMζ. Using antibodies to mammalian PKMζ, we describe in the present study the localization of immunoreactive molecules in the nervous system of the terrestrial snail Helix lucorum. Presence of a homologue of PKMζ was confirmed with transcriptomics. We have demonstrated in behavioral experiments that contextual fear memory disappeared under a blockade of PKMζ with a selective peptide blocker of PKMζ (ZIP, but not with scrambled ZIP. If ZIP was combined with a reminder (20 min in noxious context, no impairment of the long-term contextual memory was observed. In electrophysiological experiments we investigated whether PKMζ takes part in the maintenance of long-term facilitation (LTF in the neural circuit mediating tentacle withdrawal. LTF of excitatory synaptic inputs to premotor interneurons was induced by high-frequency nerve stimulation combined with serotonin bath applications and lasted at least four hours. We found that bath application of 2x10-6 M ZIP at the 90th min after the tetanization reduced the EPSP amplitude to the non-tetanized EPSP values. Applications of the scrambled ZIP peptide at a similar time and concentration didn't affect the EPSP amplitudes. In order to test whether effects of ZIP are specific to the synapses, we performed experiments with LTF of somatic membrane responses to local glutamate applications. It was shown earlier that serotonin application in such an artificial synapse condition elicits LTF of responses to glutamate. It was found that ZIP had no effect on LTF in these conditions, which may be explained by the very low concentration of PKMζ molecules in somata of these identified neurons, as evidenced by immunochemistry. Obtained results suggest that the Helix homologue of PKMζ might be involved in post-induction maintenance of long-term changes in

  5. Plasmodium falciparum Bloom homologue, a nucleocytoplasmic protein, translocates in 3' to 5' direction and is essential for parasite growth.

    Science.gov (United States)

    Rahman, Farhana; Tarique, Mohammed; Tuteja, Renu

    2016-05-01

    Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein.

  6. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  7. Allele mining in the gene pool of wild Solanum species for homologues of late blight resistance gene RB/Rpi-blb1

    Science.gov (United States)

    Solanum bulbocastanum comprising a CC-NBS-LRR gene RB/Rpi-blb1 confers broad-spectrum resistance to Phytophthora infestans and is currently employed in potato breeding for durable late blight (LB) resistance. Genomes of several Solanum species were reported to contain RB homologues with confirmed b...

  8. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results.

  9. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results. PMID:28120903

  10. The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Ajore Ram

    2012-03-01

    Full Text Available Abstract Background MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis. Results 5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA- and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16

  11. Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype

    Directory of Open Access Journals (Sweden)

    Dato Laura

    2010-01-01

    Full Text Available Abstract Background Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. Results The GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Δgas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Δgas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1β secretion disruption had no relevant effect. Conclusions The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as

  12. Intestinal expression of the anti-inflammatory interleukin-1 homologue IL-37 in pediatric inflammatory bowel disease.

    Science.gov (United States)

    Weidlich, Simon; Bulau, Ana-Maria; Schwerd, Tobias; Althans, Johanna; Kappler, Roland; Koletzko, Sibylle; Mayr, Doris; Bufler, Philip

    2014-08-01

    The function of interleukin (IL)-37 has not been resolved. We recently showed that IL-37 suppresses colonic inflammation in mice. To gain more insight into its relevance in human disease, we investigated the expression of IL-37 in the intestine of pediatric patients with chronic inflammatory bowel disease (IBD). Intestinal biopsies were obtained from children with IBD (18 Crohn disease [CD], 14 ulcerative colitis [UC] and 11 controls) during endoscopy and analyzed for IL-37 expression by immunohistochemistry and real-time polymerase chain reaction. Results were correlated with immunostaining for IL-18 and IL-17, messenger RNA (mRNA) levels of pro- and anti-inflammatory cytokines, and clinical parameters. IL-37 protein was detected in epithelial cells and submucosal lymphoid cells of patients with CD and UC as well as healthy controls. IL-37 protein expression tended to be higher with submucosal lymphoid cell infiltration of patients with CD and UC and correlated with histological severity score of inflammation. IL-18 showed a staining pattern similar to that of IL-37, whereas staining for IL-17 revealed distinct positive cells scattered in the submucosal layer. mRNA expression of IL-8, IL-17, and IL-10 was upregulated in patients with CD and UC. mRNA levels of IL-18 and IL-37 were not significantly elevated compared with controls. Levels of IL-37 and IL-18 mRNA showed a positive correlation in the CD group. IL-37 protein is expressed in healthy and diseased bowel tissue. IL-37 and IL-18 show a similar expression pattern and correlate at mRNA levels. Future studies are warranted to delineate the specific contribution of IL-37 to modulate chronic bowel inflammation in humans.

  13. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

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    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  14. Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization.

    Science.gov (United States)

    Noegel, A A; Rivero, F; Albrecht, R; Janssen, K P; Köhler, J; Parent, C A; Schleicher, M

    1999-10-01

    The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

  15. The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson's disease.

    Science.gov (United States)

    Merzetti, Eric M; Dolomount, Lindsay A; Staveley, Brian E

    2017-01-01

    Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson's disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. We show that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.

  16. Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Sameh [Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Kishikawa, Naoya [Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Nakashima, Kenichiro [Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Kuroda, Naotaka [Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan)]. E-mail: n-kuro@nagasaki-u.ac.jp

    2007-05-22

    A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.

  17. GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin.

    Science.gov (United States)

    Läppchen, Tilman; Hartog, Aloysius F; Pinas, Victorine A; Koomen, Gerrit-Jan; den Blaauwen, Tanneke

    2005-05-31

    The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.

  18. The Drosophila HP1 homologue Rhino is required for transposon silencing and piRNA production by dual strand clusters

    Science.gov (United States)

    Klattenhoff, Carla; Xi, Hualin; Li, Chengjian; Lee, Soohyun; Xu, Jia; Khurana, Jaspreet S.; Zhang, Fan; Schultz, Nadine; Koppetsch, Birgit S.; Nowosielska, Anetta; Seitz, Herve; Zamore, Phillip D.; Weng, Zhiping; Theurkauf, William E.

    2009-01-01

    Summary piRNAs silence transposons and maintain genome integrity during germ-line development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. We show that the HP1 homologue Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but do not block production of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster, but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle. PMID:19732946

  19. Modeling meiotic chromosomes indicates a size dependent contribution of telomere clustering and chromosome rigidity to homologue juxtaposition.

    Directory of Open Access Journals (Sweden)

    Christopher A Penfold

    Full Text Available Meiosis is the cell division that halves the genetic component of diploid cells to form gametes or spores. To achieve this, meiotic cells undergo a radical spatial reorganisation of chromosomes. This reorganisation is a prerequisite for the pairing of parental homologous chromosomes and the reductional division, which halves the number of chromosomes in daughter cells. Of particular note is the change from a centromere clustered layout (Rabl configuration to a telomere clustered conformation (bouquet stage. The contribution of the bouquet structure to homologous chromosome pairing is uncertain. We have developed a new in silico model to represent the chromosomes of Saccharomyces cerevisiae in space, based on a worm-like chain model constrained by attachment to the nuclear envelope and clustering forces. We have asked how these constraints could influence chromosome layout, with particular regard to the juxtaposition of homologous chromosomes and potential nonallelic, ectopic, interactions. The data support the view that the bouquet may be sufficient to bring short chromosomes together, but the contribution to long chromosomes is less. We also find that persistence length is critical to how much influence the bouquet structure could have, both on pairing of homologues and avoiding contacts with heterologues. This work represents an important development in computer modeling of chromosomes, and suggests new explanations for why elucidating the functional significance of the bouquet by genetics has been so difficult.

  20. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 A resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 A. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V(M) = 2.24 A3 Da(-1)). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 A.

  1. Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development

    Indian Academy of Sciences (India)

    Srividhya V. Iyer; Mukund Ramakrishnan; Durgadas P. Kasbekar

    2009-04-01

    The Neurospora crassa fmf-1 mutation exerts an unusual ‘perithecium-dominant’ developmental arrest; fmf-1 × fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste 11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 × fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.

  2. Anion exchange behaviour of Zr, Hf, Nb, Ta and Pa as homologues of RF and Db in fluoride medium

    Energy Technology Data Exchange (ETDEWEB)

    Monroy G, F. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico); Trubert, D.; Brillard, L.; Hussonnois, M.; Constantinescu, O.; Le Naour, C., E-mail: fabiola.monroy@inin.gob.m [Institut de Physique Nucleaire, F-91406 Orsay, France (France)

    2010-07-01

    Studies of the chemical property of trans actinide elements are very difficult due to their short half-lives and extremely small production yields. However it is still possible to obtain considerable information about their chemical properties, such as the most stable oxidation states in aqueous solution, complexing ability, etc., comparing their behaviour with their lighter homologous in the periodic table. In order to obtain a better knowledge of the behaviour of rutherfordium, RF (element 104), dub nium, Db (element 105) in HF medium, the sorption properties of Zr, Hf, Nb, Ta an Pa, homologues of RF and Db, were studied in NH{sub 4}F/HClO{sub 4} medium in this work. Stability constants of the fluoride complexes of these elements were experimentally obtained from K{sub d} obtained at different F{sup -} and H{sup +} concentrations. The anionic complexes: [Zr(Hf)F{sub 5}]{sup -}, [Zr(Hf)F{sub 6}]{sup 2-}, [Zr(Hf)F{sub 7}]{sup 3-}, [Ta(Pa)F{sub 6}]{sup -}, [Ta(Pa)F{sub 7}]{sup 2-}, [Ta(Pa)F{sub 8}]{sup 3-}, [NbOF{sub 4}]{sup -} and [NbOF{sub 5}]{sup 2-} are present as predominant species in the HF range over investigation. (Author)

  3. Ustilago maydis Rho1 and 14-3-3 homologues participate in pathways controlling cell separation and cell polarity.

    Science.gov (United States)

    Pham, Cau D; Yu, Zhanyang; Sandrock, Björn; Bölker, Michael; Gold, Scott E; Perlin, Michael H

    2009-07-01

    Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3epsilon and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.

  4. Deletion of the fission yeast homologue of human insulinase reveals a TORC1-dependent pathway mediating resistance to proteotoxic stress.

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    Clémentine Beuzelin

    Full Text Available Insulin Degrading Enzyme (IDE is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1 modulates cellular sensitivity to endoplasmic reticulum (ER stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1. Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1(+ promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.

  5. The ClpP protease homologue is required for the transmission traits and cell division of the pathogen Legionella pneumophila

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    Zhang Qin-fen

    2010-02-01

    Full Text Available Abstract Background Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality. Results In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii. Conclusions The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.

  6. Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway.

    Science.gov (United States)

    Shimamura, Mai; Kyotani, Akane; Azuma, Yumiko; Yoshida, Hideki; Binh Nguyen, Thanh; Mizuta, Ikuko; Yoshida, Tomokatsu; Mizuno, Toshiki; Nakagawa, Masanori; Tokuda, Takahiko; Yamaguchi, Masamitsu

    2014-08-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.

  7. The Caenorhabditis elegans p120 catenin homologue, JAC-1, modulates cadherin-catenin function during epidermal morphogenesis.

    Science.gov (United States)

    Pettitt, Jonathan; Cox, Elisabeth A; Broadbent, Ian D; Flett, Aileen; Hardin, Jeff

    2003-07-07

    The cadherin-catenin complex is essential for tissue morphogenesis during animal development. In cultured mammalian cells, p120 catenin (p120ctn) is an important regulator of cadherin-catenin complex function. However, information on the role of p120ctn family members in cadherin-dependent events in vivo is limited. We have examined the role of the single Caenorhabditis elegans p120ctn homologue JAC-1 (juxtamembrane domain [JMD]-associated catenin) during epidermal morphogenesis. Similar to other p120ctn family members, JAC-1 binds the JMD of the classical cadherin HMR-1, and GFP-tagged JAC-1 localizes to adherens junctions in an HMR-1-dependent manner. Surprisingly, depleting JAC-1 expression using RNA interference (RNAi) does not result in any obvious defects in embryonic or postembryonic development. However, jac-1(RNAi) does increase the severity and penetrance of morphogenetic defects caused by a hypomorphic mutation in the hmp-1/alpha-catenin gene. In these hmp-1 mutants, jac-1 depletion causes failure of the embryo to elongate into a worm-like shape, a process that involves contraction of the epidermis. Associated with failed elongation is the detachment of actin bundles from epidermal adherens junctions and failure to maintain cadherin in adherens junctions. These results suggest that JAC-1 acts as a positive modulator of cadherin function in C. elegans.

  8. Mapping of ionomic traits in Mimulus guttatus reveals Mo and Cd QTLs that colocalize with MOT1 homologues.

    Directory of Open Access Journals (Sweden)

    David B Lowry

    Full Text Available Natural variation in the regulation of the accumulation of mineral nutrients and trace elements in plant tissues is crucial to plant metabolism, development, and survival across different habitats. Studies of the genetic basis of natural variation in nutrient metabolism have been facilitated by the development of ionomics. Ionomics is a functional genomic approach for the identification of the genes and gene networks that regulate the elemental composition, or ionome, of an organism. In this study, we evaluated the genetic basis of divergence in elemental composition between an inland annual and a coastal perennial accession of Mimulus guttatus using a recombinant inbred line (RIL mapping population. Out of 20 elements evaluated, Mo and Cd were the most divergent in accumulation between the two accessions and were highly genetically correlated in the RILs across two replicated experiments. We discovered two major quantitative trait loci (QTL for Mo accumulation, the largest of which consistently colocalized with a QTL for Cd accumulation. Interestingly, both Mo QTLs also colocalized with the two M. guttatus homologues of MOT1, the only known plant transporter to be involved in natural variation in molybdate uptake.

  9. Biological characterization of a myotoxin phosphoplipase A2 homologue purified from the venom of the snake Bothrops moojeni

    Directory of Open Access Journals (Sweden)

    MR Queiroz

    2011-01-01

    Full Text Available A myotoxin phospholipase A2 homologue, BmooMtx, was isolated from the venom of Bothrops moojeni by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. SDS-PAGE showed the enzyme to be a monomer with a molecular weight of 16,500. BmooMtx induced release of creatine kinase and morphological analyses indicated that it provoked an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 hours after injection. Anti-BmooMTx antibodies partially neutralized the myotoxic activity of BmooMtx and crude B. moojeni venom, as judged by determination of plasma creatine kinase levels and histological evaluation of skeletal muscle in mice. Anti-BmooMTx antibodies were effective in reducing the plasma creatine kinase levels of crude B. alternatus and B. leucurus venoms, evidencing immunological cross-reactivity between BmooMTx and other bothropic venoms. Intraplantar (i.pl. injection of BmooMtx (1 to 15 μg/animal caused a dose- and time-dependent hyperalgesia and edematogenic responses. Dexamethasone (0.4 mg/kg, meloxicam (2 mg/kg and promethazine (5 mg/kg markedly reduced the hyperalgesia. Our data suggest that these drugs may likely serve as complementary therapies in cases of accidents with Bothrops moojeni, provided that such pharmacological treatments are administered immediately after the incident.

  10. Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development.

    Science.gov (United States)

    Iyer, Srividhya V; Ramakrishnan, Mukund; Kasbekar, Durgadas P

    2009-04-01

    The Neurospora crassa fmf-1 mutation exerts an unusual 'perithecium-dominant' developmental arrest; fmf-1 x fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 x fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.

  11. Transcriptomic analysis of common carp anterior kidney during Cyprinid herpesvirus 3 infection: Immunoglobulin repertoire and homologue functional divergence

    Science.gov (United States)

    Neave, Matthew J.; Sunarto, Agus; McColl, Kenneth A.

    2017-01-01

    Cyprinid herpesvirus 3 (CyHV-3) infects koi and common carp and causes widespread mortalities. While the virus is a significant concern for aquaculture operations in many countries, in Australia the virus may be a useful biocontrol agent for pest carp. However, carp immune responses to CyHV-3, and the molecular mechanisms underpinning resistance, are not well understood. Here we used RNA-Seq on carp during different phases of CyHV-3 infection to detect the gene expression dynamics of both host and virus simultaneously. During acute CyHV-3 infection, the carp host modified the expression of genes involved in various immune systems and detoxification pathways. Moreover, the activated pathways were skewed toward humoral immune responses, which may have been influenced by the virus itself. Many immune-related genes were duplicated in the carp genome, and often these were expressed differently across the infection phases. Of particular interest were two interleukin-10 homologues that were not expressed synchronously, suggesting neo- or sub-functionalization. The carp immunoglobulin repertoire significantly diversified during active CyHV-3 infection, which was followed by the selection of high-affinity B-cells. This is indicative of a developing adaptive immune response, and is the first attempt to use RNA-Seq to understand this process in fish during a viral infection. PMID:28148967

  12. Mammalian mitochondrial intermediate peptidase: Structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases

    Energy Technology Data Exchange (ETDEWEB)

    Isaya, G.; Sakati, W.R.; Rollins, R.A. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others

    1995-08-10

    Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1{Delta} mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X){sub 12}-G-(X){sub 5}-D-(X){sub 2}-E-X-P-S-(X){sub 3}-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments. 48 refs., 8 figs.

  13. Mammalian mitochondrial intermediate peptidase: structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases.

    Science.gov (United States)

    Isaya, G; Sakati, W R; Rollins, R A; Shen, G P; Hanson, L C; Ullrich, R C; Novotny, C P

    1995-08-10

    Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1 delta mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments.

  14. 2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins.

    Science.gov (United States)

    Jeckelmann, Jean-Marc; Harder, Daniel; Ucurum, Zöhre; Fotiadis, Dimitrios

    2014-10-01

    Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.

  15. Differential expression of metallothionein type-2 homologues in leaves and roots of Black pepper (Piper nigrum L

    Directory of Open Access Journals (Sweden)

    Susan M. Alex

    2008-01-01

    Full Text Available Black pepper (Piper nigrum L., member of the family Piperaceae is indigenous to India and is one of the most widely used spices in the world. In this paper we report the results of our attempts to identify a set of genes differentially expressed in the leaves of Piper nigrum, which could facilitate targeted engineering of this valuable crop. A PCR-based Suppression Subtractive Hybridization (SSH technique was used to generate a leaf-specific subtracted cDNA library of Piper nigrum. A tester population of leaf cDNA was subtracted with a root derived driver cDNA. The efficiency of subtraction was confirmed by PCR analysis using the housekeeping gene actin. On sequence analysis, almost 30% of the clones showed homology to metallothionein type-2 gene. The predominance of metallothionein transcripts in the leaf was further confirmed using Real-Time PCR analyses and Northern blot. The possible role of metallothionein type-2 homologues in the leaf is discussed along with the feasibility of using SSH technique for identification of more number of tissue-specific genes from Piper nigrum.

  16. Transcriptomic analysis of common carp anterior kidney during Cyprinid herpesvirus 3 infection: Immunoglobulin repertoire and homologue functional divergence.

    Science.gov (United States)

    Neave, Matthew J; Sunarto, Agus; McColl, Kenneth A

    2017-02-02

    Cyprinid herpesvirus 3 (CyHV-3) infects koi and common carp and causes widespread mortalities. While the virus is a significant concern for aquaculture operations in many countries, in Australia the virus may be a useful biocontrol agent for pest carp. However, carp immune responses to CyHV-3, and the molecular mechanisms underpinning resistance, are not well understood. Here we used RNA-Seq on carp during different phases of CyHV-3 infection to detect the gene expression dynamics of both host and virus simultaneously. During acute CyHV-3 infection, the carp host modified the expression of genes involved in various immune systems and detoxification pathways. Moreover, the activated pathways were skewed toward humoral immune responses, which may have been influenced by the virus itself. Many immune-related genes were duplicated in the carp genome, and often these were expressed differently across the infection phases. Of particular interest were two interleukin-10 homologues that were not expressed synchronously, suggesting neo- or sub-functionalization. The carp immunoglobulin repertoire significantly diversified during active CyHV-3 infection, which was followed by the selection of high-affinity B-cells. This is indicative of a developing adaptive immune response, and is the first attempt to use RNA-Seq to understand this process in fish during a viral infection.

  17. Su(dx) E3 ubiquitin ligase-dependent and -independent functions of polychaetoid, the Drosophila ZO-1 homologue.

    Science.gov (United States)

    Djiane, Alexandre; Shimizu, Hideyuki; Wilkin, Marian; Mazleyrat, Sabine; Jennings, Martin D; Avis, Johanna; Bray, Sarah; Baron, Martin

    2011-01-10

    Zona occludens (ZO) proteins are molecular scaffolds localized to cell junctions, which regulate epithelial integrity in mammals. Using newly generated null alleles, we demonstrate that polychaetoid (pyd), the unique Drosophila melanogaster ZO homologue, regulates accumulation of adherens junction-localized receptors, such as Notch, although it is dispensable for epithelial polarization. Pyd positively regulates Notch signaling during sensory organ development but acts negatively on Notch to restrict the ovary germline stem cell niche. In both contexts, we identify a core antagonistic interaction between Pyd and the WW domain E3 ubiquitin ligase Su(dx). Pyd binds Su(dx) directly, in part through a noncanonical WW-binding motif. Pyd also restricts epithelial wing cell numbers to control adult wing shape, a function associated with the FERM protein Expanded and independent of Su(dx). As both Su(dx) and Expanded regulate trafficking, we propose that a conserved role of ZO proteins is to coordinate receptor trafficking and signaling with junctional organization.

  18. PP-O and PP-V, Monascus pigment homologues, production, and phylogenetic analysis in Penicillium purpurogenum.

    Science.gov (United States)

    Arai, Teppei; Kojima, Ryo; Motegi, Yoshiki; Kato, Jun; Kasumi, Takafumi; Ogihara, Jun

    2015-12-01

    The production of pigments as secondary metabolites by microbes is known to vary by species and by physiological conditions within a single strain. The fungus strain Penicillium purpurogenum IAM15392 has been found to produce violet pigment (PP-V) and orange pigment (PP-O),Monascus azaphilone pigment homologues, when grown under specific culture conditions. In this study, we analysed PP-V and PP-O production capability in seven strains of P. purpurogenum in addition to strain IAM15392 under specific culture conditions. The pigment production pattern of five strains cultivated in PP-V production medium was similar to that of strain IAM15392, and all violet pigments produced by these five strains were confirmed to be PP-V. Strains that did not produce pigment were also identified. In addition, two strains cultivated in PP-O production medium produced a violet pigment identified as PP-V. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region sequences from the eight P. purpurogenum strains were sequenced and used to construct a neighbor-joining phylogenetic tree. PP-O and PP-V production of P. purpurogenum was shown to be related to phylogenetic placement based on rDNA ITS sequence. Based on these results, two hypotheses for the alteration of pigment production of P. purpurogenum in evolution were proposed.

  19. Characterization of Fetal Antigen 1/Delta-Like 1 Homologue Expressing Cells in the Rat Nigrostriatal System

    DEFF Research Database (Denmark)

    Liechti, Rémy; Ducray, Angélique D; Jensen, Pia;

    2015-01-01

    Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been su...... adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co......-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata...... as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc...

  20. Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate.

    Science.gov (United States)

    Lavial, Fabrice; Acloque, Hervé; Bachelard, Elodie; Nieto, M Angela; Samarut, Jacques; Pain, Bertrand

    2009-06-01

    When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.

  1. An alpha-catulin homologue controls neuromuscular function through localization of the dystrophin complex and BK channels in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Linu S Abraham

    2010-08-01

    Full Text Available The large conductance, voltage- and calcium-dependent potassium (BK channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 Mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.

  2. The C. elegans CBFbeta homologue BRO-1 interacts with the Runx factor, RNT-1, to promote stem cell proliferation and self-renewal.

    Science.gov (United States)

    Kagoshima, Hiroshi; Nimmo, Rachael; Saad, Nicole; Tanaka, Junko; Miwa, Yoshihiro; Mitani, Shohei; Kohara, Yuji; Woollard, Alison

    2007-11-01

    In this report, we investigate the C. elegans CBFbeta homologue, BRO-1. bro-1 mutants have a similar male-specific sensory ray loss phenotype to rnt-1 (the C. elegans homologue of the mammalian CBFbeta-interacting Runx factors), caused by failed cell divisions in the seam lineages. Our studies indicate that BRO-1 and RNT-1 form a cell proliferation-promoting complex, and that BRO-1 increases both the affinity and specificity of RNT-1-DNA interactions. Overexpression of bro-1, like rnt-1, leads to an expansion of seam cell number and co-overexpression of bro-1 and rnt-1 results in massive seam cell hyperplasia. Finally, we find that BRO-1 appears to act independently of RNT-1 in certain situations. These studies provide new insights into the function and regulation of this important cancer-associated DNA-binding complex in stem cells and support the view that Runx/CBFbeta factors have oncogenic potential.

  3. Snake venom toxins. The amino-acid sequence of a short-neurotoxin homologue from Dendroaspis polylepis polylepis (black mamba) venom.

    Science.gov (United States)

    Strydom, D J

    1977-06-01

    The third most abundant component of black mamba venom, named FS2, was sequenced with the aid of sequenator studies and peptides derived by tryptic and chymotryptic digestion. Cyanogen bromide digests provided extra information to support the proposed structure. This protein is a homologue of the short neurotoxins of snake venom, but is much less toxic. Its structure is quite different from both neurotoxins and the other mamba proteins, called angusticeps types (neurotoxin homologues). Comparison of the known angusticeps-type toxins from mamba venom with mamba neurotoxins and each other leads to proposals that these proteins of low toxicity are inventions of the group of mambas and that three different, as yet unknown, functions will be associated with the three subgroups that are discernable.

  4. Ionization potentials and electron affinities of the superheavy elements 115-117 and their sixth-row homologues Bi, Po, and At

    Science.gov (United States)

    Borschevsky, A.; Pašteka, L. F.; Pershina, V.; Eliav, E.; Kaldor, U.

    2015-02-01

    Calculations of the first and second ionization potentials and electron affinities of superheavy elements 115-117 are presented. The calculations are performed in the framework of the Dirac-Coulomb Hamiltonian, and the results are corrected for the Breit and QED contributions. Correlation is treated by the relativistic coupled cluster approach with single, double, and perturbative triple excitations [CCSD(T)]. The same approach is used to calculate the ionization potentials and electron affinities of the lighter homologues, Bi, Po, and At. Comparison of the available experimental values for these atoms, namely, the first ionization potentials (IPs) of Bi, Po, and At and the second IP and EA of Bi, with our results shows excellent agreement, within a few hundredths of an eV, lending credence to our predictions for their superheavy homologues. High-accuracy predictions are also made for the second ionization potentials and electron affinities of Po and At, where no experiment is available.

  5. Characterization of synthetic routes to 'Bromo-DragonFLY' and benzodifuranyl isopropylamine homologues utilizing ketone intermediates. Part 1: synthesis of ketone precursors.

    Science.gov (United States)

    O'Connor, Richard E; Keating, John J

    2014-01-01

    Bromo-DragonFLY (BDF) and many of its analogues are misused as recreational drugs due to their potency as psychoactive substances. To date, none of the published routes to these designer amphetamines have exploited a ketone intermediate. It is well known that benzyl methyl ketone (BMK) can be employed as a precursor in the synthesis of amphetamine. Similarly, it is reasonable to assume that ketone precursors may potentially be utilized in the clandestine synthesis of BDF and its homologues. This paper describes the multifaceted synthesis of novel precursor ketones structurally related to BDF, namely benzodifuranyl propanone 16, its tetrahydrobenzodifuranyl homologue 8, and their brominated analogues 12 and 20. Their characterization by Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy ((1) H-NMR), carbon nuclear magnetic resonance spectroscopy ((13) C-NMR), high performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS) is also described.

  6. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)

    2011-01-01

    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in

  7. Influenza virus mRNA trafficking through host nuclear speckles.

    Science.gov (United States)

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W; García-Sastre, Adolfo; Fontoura, Beatriz M A

    2016-05-27

    Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

  8. Thermodynamics of molecular recognition of mRNA 5' cap by yeast eukaryotic initiation factor 4E.

    Science.gov (United States)

    Kiraga-Motoszko, Katarzyna; Niedzwiecka, Anna; Modrak-Wojcik, Anna; Stepinski, Janusz; Darzynkiewicz, Edward; Stolarski, Ryszard

    2011-07-14

    Molecular mechanisms underlying the recognition of the mRNA 5' terminal structure called "cap" by the eukaryotic initiation factor 4E (eIF4E) are crucial for cap-dependent translation. To gain a deeper insight into how the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van't Hoff analysis based on intrinsic protein fluorescence quenching upon titration with a series of chemical cap analogs were performed, providing a consistent thermodynamic description of the binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed similarities and differences between yeast and mammalian eIF4Es. The yeast eIF4E complex formation was enthalpy-driven and entropy-opposed for each cap analog at 293 K. A nontrivial isothermal enthalpy–entropy compensation was found, described by a compensation temperature, T(c) = 411 ± 18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected, ΔC(p)° = +5.2 ± 1.3 kJ·mol(-1)·K(-1). The charge-related interactions involving the 5′-5′ triphosphate bridge of the cap and basic amino acid side chains at the yeast eIF4E cap-binding site were significantly weaker (by ΔΔH°(vH) of about +10 kJ·mol(-1)) than those for the mammalian homologues, suggesting their optimization during the evolution. © 2011 American Chemical Society

  9. Cloning of the Rhesus Lymphocryptovirus Viral Capsid Antigen and Epstein-Barr Virus-Encoded Small RNA Homologues and Use in Diagnosis of Acute and Persistent Infections

    OpenAIRE

    Rao, Pasupuleti; Jiang, Hua; Wang, Fred

    2000-01-01

    Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is associated with the development of several human malignancies. A closely related herpesvirus in the same lymphocryptovirus (LCV) genera as EBV naturally infects rhesus monkeys and provides an important animal model for studying EBV pathogenesis. We cloned the small viral capsid antigen (sVCA) homologue from the rhesus LCV and developed a peptide enzyme-linked immunosorbent assay (ELISA) to determine whether e...

  10. Structure and Function of CC-Chemokine Receptor 5 Homologues Derived from Representative Primate Species and Subspecies of the Taxonomic Suborders Prosimii and Anthropoidea

    OpenAIRE

    2003-01-01

    A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family L...

  11. Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

    Directory of Open Access Journals (Sweden)

    Kah Yan How

    2015-08-01

    Full Text Available Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3. The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity.

  12. Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4.

    Science.gov (United States)

    How, Kah Yan; Hong, Kar Wai; Chan, Kok-Gan

    2015-01-01

    Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity.

  13. Metal-Organic Framework with Aromatic Rings Tentacles: High Sulfur Storage in Li-S Batteries and Efficient Benzene Homologues Distinction.

    Science.gov (United States)

    Li, Meng-Ting; Sun, Yu; Zhao, Kai-Sen; Wang, Zhao; Wang, Xin-Long; Su, Zhong-Min; Xie, Hai-Ming

    2016-12-07

    We designed and fabricated a fluorophore-containing tetradentate carboxylate ligand-based metal-organic framework (MOF) material with open and semiopen channels, which acted as the host for sulfur trapped in Li-S batteries and sensor of benzene homologues. These channels efficiently provide a π-π* conjugated matrix for the charge transfer and guest molecule trapping. The open channel ensured a much higher loading quantitative of sulfur (S content-active material, 72 wt %; electrode, 50.4 wt %) than most of the MOF/sulfur composites, while the semiopen channel possessing aromatic rings tentacles guaranteed an outstanding specific discharge capacity (1092 mA h g(-1) at 0.1 C) accompanied by good cycling stability. To our surprise, benefiting from special π-π* conjugated conditions, compound 1 could be a chemical sensor for benzene homologues, especially for 1,2,4-trimethylbenzene (1,2,4-TMB). This is the first example of MOFs materials serving as a sensor of 1,2,4-TMB among benzene homologues. Our works may be worthy of use for references in other porous materials systems to manufacture more long-acting Li-S batteries and sensitive chemical sensors.

  14. Fragile X mental retardation protein control of neuronal mRNA metabolism: Insights into mRNA stability.

    Science.gov (United States)

    De Rubeis, Silvia; Bagni, Claudia

    2010-01-01

    The fragile X mental retardation protein (FMRP) is an RNA binding protein that has an essential role in neurons. From the soma to the synapse, FMRP is associated with a specific subset of messenger RNAs and controls their posttranscriptional fates, i.e., dendritic localization and local translation. Because FMRP target mRNAs encode important neuronal proteins, the deregulation of their expression in the absence of FMRP leads to a strong impairment of synaptic function. Here, we review emerging evidence indicating a critical role for FMRP in the control of mRNA stability. To date, two mRNAs have been identified as being regulated in this manner: PSD-95 mRNA, encoding a scaffolding protein, and Nxf1 mRNA, encoding a general export factor. Moreover, expression studies suggest that the turnover of other neuronal mRNAs, including those encoding for the GABA(A) receptors subunits, could be affected by the loss of FMRP. According to the specific target and/or cellular context, FMRP could influence mRNA stability in the brain. Copyright 2009 Elsevier Inc. All rights reserved.

  15. Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans.

    Science.gov (United States)

    Hajeri, Vinita A; Stewart, Anil M; Moore, Landon L; Padilla, Pamela A

    2008-02-04

    The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable san-1(ok1580) deletion mutant. The san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.

  16. Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Moore Landon L

    2008-02-01

    Full Text Available Abstract Background The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi to identify genes synthetic lethal with the viable san-1(ok1580 deletion mutant. Results The san-1(ok1580 animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580 embryos is significantly reduced when HCP-1 (CENP-F homologue, MDF-1 (MAD-1 homologue, MDF-2 (MAD-2 homologue or BUB-3 (predicted BUB-3 homologue are reduced by RNAi. Interestingly, the viability of san-1(ok1580 embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580;hcp-1(RNAi embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging. Several of the san-1(ok1580;hcp-1(RNAi animals displayed abnormal kinetochore (detected by MPM-2 and microtubule structure. The survival of mdf-2(RNAi;hcp-1(RNAi embryos but not bub-3(RNAi;hcp-1(RNAi embryos was also compromised. Finally, we found that san-1(ok1580 and bub-3(RNAi, but not hcp-1(RNAi embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. Conclusion Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580;hcp-1(RNAi animals had a severe viability defect whereas in the san-1(ok1580;hcp-2(RNAi and san-1(ok1580;hcp-2(ok1757 animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.

  17. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Daugaard, Tina Fuglsang; Holm, Ida E;

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapa is the most predominant isoform. The Gfapd isoform is expressed in proliferating......RNA localization patterns were dependent on the different 39-exon sequences included in Gfapd and Gfapa mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential...

  18. A spinach O-acetylserine(thiollyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms

    Directory of Open Access Journals (Sweden)

    Miki Noda

    2016-06-01

    Full Text Available An enzyme, O-acetylserine(thiollyase (OASTL, also known as O-acetylserine sulfhydrylase or cysteine synthase (CSase, catalyses the incorporation of sulfide into O-acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from Spinacia oleracea, (SoCSaseLP, but a recombinant SoCSaseLP produced in Escherichia coli did not show OASTL activity. The exon-intron structure of the SoCSaseLP gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a Beta vulgaris homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the SoCSaseLP gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione S-transferase (GST-SoCSaseLP fusion protein was expressed in E. coli together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.

  19. Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shimamura, Mai; Kyotani, Akane [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Azuma, Yumiko [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Yoshida, Hideki; Binh Nguyen, Thanh [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Mizuta, Ikuko; Yoshida, Tomokatsu; Mizuno, Toshiki [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Nakagawa, Masanori [North Medical Center, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 (Japan); Tokuda, Takahiko, E-mail: ttokuda@koto.kpu-m.ac.jp [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan)

    2014-08-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.

  20. Strboh A homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers.

    Science.gov (United States)

    Kumar, G N Mohan; Iyer, Suresh; Knowles, N Richard

    2007-12-01

    During 30-months of storage at 4 degrees C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18-24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca(+2) declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25-30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.

  1. Gibberellins regulate the transcription of the continuous flowering regulator, RoKSN, a rose TFL1 homologue.

    Science.gov (United States)

    Randoux, Marie; Jeauffre, Julien; Thouroude, Tatiana; Vasseur, François; Hamama, Latifa; Juchaux, Marjorie; Sakr, Soulaiman; Foucher, Fabrice

    2012-11-01

    The role of gibberellins (GAs) during floral induction has been widely studied in the annual plant Arabidopsis thaliana. Less is known about this control in perennials. It is thought that GA is a major regulator of flowering in rose. In spring, low GA content may be necessary for floral initiation. GA inhibited flowering in once-flowering roses, whereas GA did not block blooming in continuous-flowering roses. Recently, RoKSN, a homologue of TFL1, was shown to control continuous flowering. The loss of RoKSN function led to continuous flowering behaviour. The objective of this study was to understand the molecular control of flowering by GA and the involvement of RoKSN in this inhibition. In once-flowering rose, the exogenous application of GA(3) in spring inhibited floral initiation. Application of GA(3) during a short period of 1 month, corresponding to the floral transition, was sufficient to inhibit flowering. At the molecular level, RoKSN transcripts were accumulated after GA(3) treatment. In spring, this accumulation is correlated with floral inhibition. Other floral genes such as RoFT, RoSOC1, and RoAP1 were repressed in a RoKSN-dependent pathway, whereas RoLFY and RoFD repression was RoKSN independent. The RoKSN promoter contained GA-responsive cis-elements, whose deletion suppressed the response to GA in a heterologous system. In summer, once-flowering roses did not flower even after exogenous application of a GA synthesis inhibitor that failed to repress RoKSN. A model is presented for the GA inhibition of flowering in spring mediated by the induction of RoKSN. In summer, factors other than GA may control RoKSN.

  2. Atlantic cod (Gadus morhua L.) possesses three homologues of ISG15 with different expression kinetics and conjugation properties.

    Science.gov (United States)

    Furnes, Clemens; Kileng, Øyvind; Rinaldo, Christine Hanssen; Seppola, Marit; Jensen, Ingvill; Robertsen, Børre

    2009-12-01

    Two new interferon stimulated gene 15 (ISG15) family members were identified in a subtractive cDNA library constructed from a mixture of head kidney and spleen of Atlantic cod (Gadus morhua) stimulated with polyinosinic:polycytidylic acid (poly I:C). Two full-length Atlantic cod (Ac) ISG15-2 and AcISG15-3 cDNAs were cloned with rapid amplification of cDNA ends (RACE). The cDNA sequence of AcISG15-2 encodes a 16.9kDa protein and AcISG15-3 encodes a 18.4kDa protein, both of which possess the characteristic structural features of two tandem ubiquitin-like domains and the LRGG motif necessary for conjugation. Furthermore, the AcISG15-3 protein is expressed with a C-terminal extension in common with the human ISG15 protein. Gene expression analysis using quantitative reverse transcriptase PCR (RT-qPCR) showed that AcISG15-1, AcISG15-2, and AcISG15-3 transcripts were up-regulated in head kidney after poly I:C stimulation, suggesting that these proteins may be involved in the cod immune response. However, transient expression of myc-tagged AcISG15 proteins revealed differences in their abilities to form conjugates in vitro. We show that AcISG15-2 forms covalent conjugates to a range of cellular protein as a response to poly I:C, recombinant Atlantic salmon IFNa1 (rSasaIFNa1) and infectious pancreatic necrosis virus (IPNV), whereas conjugation was absent for AcISG15-1 and AcISG15-3. Thus, these results suggest there are three ISG15 homologues in Atlantic cod and that the three proteins may play different roles in innate immunity.

  3. Cloning, identification and functional analysis of a β-catenin homologue from Pacific white shrimp, Litopenaeus vannamei.

    Science.gov (United States)

    Zhang, Shuang; Shi, Lili; L, Kai; Li, Haoyang; Wang, Sheng; He, Jianguo; Li, Chaozheng

    2016-07-01

    Wnt signaling is known to control multiple of cellular processes such as cell differentiation, communication, apoptosis and proliferation, and is also reported to play a role during microbial infection. β-catenin is a key regulator of the Wnt signaling cascade. In the present study, we cloned and identified a β-catenin homologue from Litopenaeus vannamei termed Lvβ-catenin. The full-length of Lvβ-catenin transcript was 2797 bp in length within a 2451 bp open reading frame (ORF) that encoded a protein of 816 amino acids. Lvβ-catenin protein was comprised of several characteristic domains such as an N-terminal region of GSK-β consensus phosphorylation site and Coed coil section, a central region of 12 continuous Armadillo/β-Catenin-like repeat (ARM) domains and a C-terminal region. Real-time PCR showed Lvβ-catenin expression was responsive to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infection. Dual-reporter analysis showed that over-expression of Lvβ-catenin could induce activation of the promoter activities of several antimicrobial peptides (AMPs) such as shrimp PEN4, suggesting that Lvβ-catenin could play a role in regulating the production of AMPs. Knockdown of Lvβ-catenin enhanced the sensitivity of shrimps to V. parahaemolyticus and WSSV challenge, suggesting Lvβ-catenin could play a positive role against bacterial and viral pathogens. In summary, the results presented in this study provided some insights into the function of Wnt/β-catenin of shrimp in regulating AMPs and the host defense against invading pathogens.

  4. Health risk equations and risk assessment of airborne benzene homologues exposure to drivers and passengers in taxi cabins.

    Science.gov (United States)

    Chen, Xiaokai; Feng, Lili; Luo, Huilong; Cheng, Heming

    2016-03-01

    Interior air environment and health problems of vehicles have attracted increasing attention, and benzene homologues (BHs) including benzene, toluene, ethylbenzene, xylenes, and styrene are primary hazardous gases in vehicular cabins. The BHs impact on the health of passengers and drivers in 38 taxis is assessed, and health risk equations of in-car BHs to different drivers and passengers are induced. The health risk of in-car BHs for male drivers is the highest among all different receptors and is 1.04, 6.67, and 6.94 times more than ones for female drivers, male passengers, and female passengers, respectively. In-car BHs could not lead to the non-cancer health risk to all passengers and drivers as for the maximal value of non-cancer indices is 0.41 and is less than the unacceptable value (1.00) of non-cancer health risk from USEPA. However, in-car BHs lead to cancer health risk to drivers as for the average value of cancer indices is 1.21E-04 which is 1.21 times more than the unacceptable value (1.00E-04) of cancer health risk from USEPA. Finally, for in-car airborne benzene concentration (X, μg/m(3)) to male drivers, female drivers, male passengers, and female passengers, the cancer health risk equations are Y = 1.48E-06X, Y = 1.42E-06X, Y = 2.22E-07X, and Y = 2.13E-07X, respectively, and the non-cancer health risk equations are Y = 1.70E-03X, Y = 1.63E-03X, Y = 2.55E-04X, and Y = 2.45E-04X, respectively.

  5. Sex-dependent modulation of longevity by two Drosophila homologues of human Apolipoprotein D, GLaz and NLaz.

    Science.gov (United States)

    Ruiz, Mario; Sanchez, Diego; Canal, Inmaculada; Acebes, Angel; Ganfornina, Maria D

    2011-07-01

    Apolipoprotein D (ApoD), a member of the Lipocalin family, is the gene most up-regulated with age in the mammalian brain. Its expression strongly correlates with aging-associated neurodegenerative and metabolic diseases. Two homologues of ApoD expressed in the Drosophila brain, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz), are known to alter longevity in male flies. However, sex differences in the aging process have not been explored so far for these genes. Here we demonstrate that NLaz alters lifespan in both sexes, but unexpectedly the lack of GLaz influences longevity in a sex-specific way, reducing longevity in males but not in females. While NLaz has metabolic functions similar to ApoD, the regulation of GLaz expression upon aging is the closest to ApoD in the aging brain. A multivariate analysis of physiological parameters relevant to lifespan modulation uncovers both common and specialized functions for the two Lipocalins, and reveals that changes in protein homeostasis account for the observed sex-specific patterns of longevity. The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. We also demonstrate that food composition is an important environmental parameter influencing stress resistance and reproductive phenotypes of both Lipocalin mutants. Since ApoD shares many properties with the common ancestor of invertebrate Lipocalins, we must benefit from this global comparison with both GLaz and NLaz to understand the complex functions of ApoD in mammalian aging and neurodegeneration.

  6. MutS homologue hMSH5: recombinational DSB repair and non-synonymous polymorphic variants.

    Science.gov (United States)

    Wu, Xiling; Xu, Yang; Feng, Katey; Tompkins, Joshua D; Her, Chengtao

    2013-01-01

    Double-strand breaks (DSBs) constitute the most deleterious form of DNA lesions that can lead to genome alterations and cell death, and the vast majority of DSBs arise pathologically in response to DNA damaging agents such as ionizing radiation (IR) and chemotherapeutic agents. Recent studies have implicated a role for the human MutS homologue hMSH5 in homologous recombination (HR)-mediated DSB repair and the DNA damage response. In the present study, we show that hMSH5 promotes HR-based DSB repair, and this property resides in the carboxyl-terminal portion of the protein. Our results demonstrate that DSB-triggered hMSH5 chromatin association peaks at the proximal regions of the DSB and decreases gradually with increased distance from the break. Furthermore, the DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal regions of the DSB. Lastly, the potential effects of hMSH5 non-synonymous variants (L85F, Y202C, V206F, R351G, L377F, and P786S) on HR and cell survival in response to DSB-inducing anticancer agents have been analyzed. These experiments show that the expression of hMSH5 variants elicits different survival responses to anticancer drugs cisplatin, bleomycin, doxorubicin and camptothecin. However, the effects of hMSH5 variants on survival responses to DSB-inducing agents are not directly correlated to their effects exerted on HR-mediated DSB repair, suggesting that the roles of hMSH5 variants in the processes of DNA damage response and repair are multifaceted.

  7. The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.

    Directory of Open Access Journals (Sweden)

    John C Pascall

    Full Text Available The GIMAPs (GTPases of the immunity-associated proteins are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s. Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

  8. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    Science.gov (United States)

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  9. Translation with frameshifting of ribosome along mRNA transcript

    CERN Document Server

    Li, Jingwei

    2015-01-01

    Translation is an important process for prokaryotic and eukaryotic cells to produce necessary proteins for cell growth. Numerious experiments have been performed to explore the translational properties. Diverse models have also been developed to determine the biochemical mechanism of translation. However, to simplify the majority of the existing models, the frameshifting of ribosome along the mRNA transcript is neglected, which actually occurs in real cells and has been extensively experimentally studied. The frameshifting of ribosome evidently influences the efficiency and speed of translation, considering that the peptide chains synthesized by shifted ribosomes will not fold into functional proteins and will degrade rapidly. In this study, a theoretical model is presented to describe the translational process based on the model for totally asymmetric simple exclusion process. In this model, the frameshifting of the ribosome along the mRNA transcript and the attachment/detachment of the ribosome to/from the ...

  10. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, C.G.; Paludan, Søren Riis;

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...

  11. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

    Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B;

    2009-01-01

    BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane....... In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to beta-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients. RESULTS: The alpha1(IV......). The level of alpha 6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p collagen was visualized by immunohistochemical staining. CONCLUSION: Our results suggest that the down-regulation of alpha 6(IV) mRNA coincides...

  12. High lib mRNA expression in breast carcinomas.

    Science.gov (United States)

    Satoh, Kazuki; Hata, Mitsumi; Yokota, Hiroshi

    2004-06-30

    Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.

  13. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  14. Membrane-Coupled mRNA Trafficking in Fungi.

    Science.gov (United States)

    Haag, Carl; Steuten, Benedikt; Feldbrügge, Michael

    2015-01-01

    Intracellular logistics are essential for delivery of newly synthesized material during polar growth of fungal hyphae. Proteins and lipids are actively transported throughout the cell by motor-dependent movement of small vesicles or larger units such as endosomes and the endoplasmic reticulum. A remarkably tight link is emerging between active membrane trafficking and mRNA transport, a process that determines the precise subcellular localization of translation products within the cell. Here, we report on recent insights into the mechanism and biological role of these intricate cotransport processes in fungal models such as Saccharomyces cerevisiae, Candida albicans, and Ustilago maydis. In the latter, we focus on the new finding of endosomal mRNA transport and its implications for protein targeting, complex assembly, and septin biology.

  15. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, C.G.; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  16. Vibrational force alters mRNA expression in osteoblasts

    Science.gov (United States)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  17. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting pro...... nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome. (C) 1996 Wiley-Liss, Inc....

  18. Analysis of specific mRNA destabilization during Dictyostelium development.

    Science.gov (United States)

    Mangiarotti, G; Bulfone, S; Giorda, R; Morandini, P; Ceccarelli, A; Hames, B D

    1989-07-01

    A number of specific mRNAs are destabilized upon disaggregation of developing Dictyostelium discoideum cells. Analysis of a family of cloned genes indicates that only prespore-enriched mRNAs are affected; constitutive mRNAs that are expressed throughout development and mRNAs that accumulate preferentially in prestalk cells are stable under these conditions. The decay of sensitive prespore mRNAs can be halted by allowing the cells to reaggregate, indicating that destabilization occurs by the progressive selection of individual molecules rather than on all members of an mRNA subpopulation at the time of disaggregation. Individual molecules of the sensitive mRNA species remain engaged in protein synthesis in the disaggregated cells until selected. Destabilization of sensitive mRNAs is induced by cell dissociation even in the presence of concentrations of nogalamycin that inhibit RNA synthesis. The reported prevention of disaggregation-induced mRNA decay by actinomycin D and daunomycin is therefore probably a secondary effect unrelated to the inhibition of transcription.

  19. mRNA and DNA PCR tests in cutaneous tuberculosis

    Directory of Open Access Journals (Sweden)

    Chandanmal Suthar

    2013-01-01

    Full Text Available Background: The microbiologic diagnosis of cutaneous tuberculosis is difficult because most lesions harbor only a small number of mycobacteria that cannot usually be detected by staining for the organism or by culture. Nucleic acid amplification tests based on the polymerase chain reaction (PCR are potentially useful in this situation. Aims: To evaluate the utility of mRNA PCR and DNA PCR in the diagnosis of cutaneous tuberculosis. Methods: Biopsies from 28 cases of cutaneous tuberculosis and 19 controls with other diseases were subjected to microbiologic tests including direct smears for mycobacteria, culture and both mRNA PCR and DNA PCR. The laboratory was blinded to the clinical diagnosis. Results: None of the patients or controls showed a positive reaction on mRNA PCR test. Seven of 28 cases and 5 out of 19 controls showed a positive result on DNA PCR test yielding a sensitivity of 25% and a specificity of 73.7%. Conclusion: The results of PCR tests in cutaneous tuberculosis should be interpreted in the light of clinical and histopathological findings.

  20. Midbrain expression of Delta-like 1 homologue is regulated by GDNF and is associated with dopaminergic differentiation

    DEFF Research Database (Denmark)

    Christophersen, Nicolai S.; Gronborg, Mette; Petersen, Thomas Nordahl;

    2007-01-01

    Affymetrix GeneChip technology and quantitative real-time PCR (Q-PCR) were used to examine changes in gene expression in the adult murine substantia nigra pars compacta (SNc) following lentiviral glial cell line-derived neurotrophic factor (GDNF) delivery in adult striatum. We identified several...... upregulation with increased positive staining of cell bodies in the SNc and fibers in the striatum. Analysis of the developmental regulation of Dlk1 in the murine ventral midbrain showed that the upregulation of Dlk1 mRNA correlated with the generation of tyrosine hydroxylase (TH)-positive neurons. Furthermore...

  1. Endoplasmic reticulum-directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Kol, Stefan; Andersen, Mikael Rørdam

    2016-01-01

    for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding...... the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER-directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER-directed mRNA, with no cytoplasmic translation of ER-directed recombinant proteins observed....... These observations indicate that the recombinant mRNA, encoding ER-directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also...

  2. A homologue of the defender against the apoptotic death gene (dad1) in UV-exposed Chlamydomonas cells is downregulated with the onset of programmed cell death

    Indian Academy of Sciences (India)

    Swati Moharikar; Jacinta S D’souza; Basuthkar J Rao

    2007-03-01

    We report here the isolation of a homologue of the potential anti-apoptotic gene, defender against apoptotic death (dad1) from Chlamydomonas reinhardtii cells. Using polymerase chain reaction (PCR), we investigated its expression in the execution process of programmed cell death (PCD) in UV-C exposed dying C. reinhardtii cells. Reverse-transcriptase (RT)-PCR showed that C. reinhardtii dad1 amplification was drastically reduced in UV-C exposed dying C. reinhardtii cells. We connect the downregulation of dad1 with the upregulation of apoptosis protease activating factor-1 (APAF-1) and the physiological changes that occur in C. reinhardtii cells upon exposure to 12 J/m2 UV-C in order to show a reciprocal relationship between proapoptotic and inhibitor of apoptosis factors. The temporal changes indicate a correlation between the onset of cell death and dad1 downregulation. The sequence of the PCR product of the cDNA encoding the dad1 homologue was aligned with the annotated dad1 (C_20215) from the Chlamydomonas database (http://genome.jgi-psf.org:8080/annotator/servlet/jgi.annotation.Annotation?pDb=chlre2); Annotation?pDb=chlre2); this sequence was found to show 100% identity, both at the nucleotide and amino acid level. The 327 bp transcript showed an open reading frame of 87 amino acid residues. The deduced amino acid sequence of the putative C. reinhardtii DAD1 homologue showed 54% identity with Oryza sativa, 56% identity with Drosophila melanogaster, 66% identity with Xenopus laevis, and 64% identity with Homo sapiens, Sus scrofa, Gallus gallus, Rattus norvegicus and Mus musculus.

  3. Cloning of the rhesus lymphocryptovirus viral capsid antigen and Epstein-Barr virus-encoded small RNA homologues and use in diagnosis of acute and persistent infections.

    Science.gov (United States)

    Rao, P; Jiang, H; Wang, F

    2000-09-01

    Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is associated with the development of several human malignancies. A closely related herpesvirus in the same lymphocryptovirus (LCV) genera as EBV naturally infects rhesus monkeys and provides an important animal model for studying EBV pathogenesis. We cloned the small viral capsid antigen (sVCA) homologue from the rhesus LCV and developed a peptide enzyme-linked immunosorbent assay (ELISA) to determine whether epitopes in the rhesus LCV sVCA are a reliable indicator of rhesus LCV infection. In order to define a "gold standard" for rhesus LCV infection, we also cloned the EBV-encoded small RNA 1 (EBER1) and EBER2 homologues from rhesus LCV and developed a reverse transcription (RT)-PCR assay to detect persistent LCV infection in rhesus monkey peripheral blood lymphocytes. Animals from a conventional and a hand-reared colony were studied to compare the prevalence of rhesus LCV infection in the two groups. There was a 100% correlation between the peptide ELISA and EBER RT-PCR results for rhesus LCV infection. In addition, specificity for LCV infection and exclusion of potential cross-reactivity to the rhesus rhadinovirus sVCA homologue could be demonstrated using sera from experimentally infected animals. These studies establish two novel assays for reliable diagnosis of acute and persistent rhesus LCV infections. The rhesus LCV sVCA peptide ELISA provides a sensitive and reliable assay for routine screening, and these studies of the hand-reared colony confirm the feasibility of raising rhesus LCV-naive animals.

  4. Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa.

    Science.gov (United States)

    Aoshima, Ryota; Hiraoka, Rieko; Shimada, Nao; Kawata, Takefumi

    2006-01-01

    A Dd-STATa-null mutant, which is defective in expression of a Dictyostelium homologue of the metazoan STAT (signal transducers and activators of transcription) proteins, fails to culminate and this phenotype correlates with the loss of expression of various prestalk (pst) genes. An EST clone, SSK395, encodes a close homologue of the adducin amino-terminal head domain and harbors a putative actin-binding domain. We fused promoter fragments of the cognate gene, ahhA (adducin head homologue A), to a lacZ reporter and determined their expression pattern. The proximal promoter region is necessary for the expression of ahhA at an early (pre-aggregative) stage of development and this expression is Dd-STATa independent. The distal promoter region is necessary for expression at later stages of development in pstA cells, of the slug and in upper cup and pstAB cells during culmination. The distal region is partly Dd-STATa-dependent. The ahhA-null mutant develops almost normally until culmination, but it forms slanting culminants that tend to collapse on to the substratum. The mutant also occasionally forms fruiting bodies with swollen papillae and with constrictions in the prestalk region. The AhhA protein localizes to the stalk tube entrance and also to the upper cup cells and in cells at or near to the constricted region where an F-actin ring is localized. These findings suggest that Dd-STATa regulates culmination and may be necessary for straight downward elongation of the stalk, via the putative actin-binding protein AhhA.

  5. Mechanisms of mRNA translation of interferon stimulated genes.

    Science.gov (United States)

    Joshi, Sonali; Kaur, Surinder; Kroczynska, Barbara; Platanias, Leonidas C

    2010-01-01

    Over the last two decades, a lot of research work has been focused on the interferon (IFN)-regulated JAK-STAT pathway and understanding the mechanisms governing the transcription of interferon stimulated genes (ISGs). Evidence suggests that the JAK-STAT pathway alone does not account in its entirety for mediating cellular responses to IFNs. There is emerging evidence that non-Stat pathways play important roles in mediating signals for the generation of IFN-responses. Various studies have underscored the importance of mitogen activated protein kinases (MAPKs), especially p38 and ERK1/2, as well as the PI 3'K/AKT pathway in transmitting signals that are of critical importance for the biological effects of IFNs. Besides regulating the transcription of ISGs in some cases, engagement of these signaling pathways by the IFN-receptor (IFNR) associated complexes also plays an important role in mediating the translation of ISGs. The mechanisms regulating mRNA translation of ISGs is an area of ongoing active research and a lot more efforts will be required to complete our understanding of the various cellular elements involved in this process. In this review we highlight the mechanisms regulating translation of ISGs. We focus on the proteins regulated by the PI 3'K/AKT pathway, their role in mediating mRNA translation of ISGs and the functional consequences of this regulation. In addition, MAPKs are known to regulate the phosphorylation of various eukaryotic initiation factors and we summarize the roles of eIF4B and eIF4E phosphorylations on the translation of ISGs. The emerging roles of microRNAs in mRNA translation of ISGs are also discussed.

  6. Crystal Structure of a NifS Homologue CsdB from Escherichia coli (MOLECULAR BIOLOGY AND INFORMATION-Biopolymer Structure)

    OpenAIRE

    Fujii, Tomomi; Hata, Yasuo

    2001-01-01

    Escherichia coli CsdB is a dimeric NifS-homologue belonging to the fold-type I family of PLPdependent enzymes, and catalyzes the decomposition of L-selenocysteine into selenium and L-alanine with specificity higher than that for a substrate of cysteine. The structure of the enzyme has been determined at 2.8 A resolution by an X-ray crystallographic method. The subunit of CsdB comprises a large domain, a small domain, and an N-terminal segment. A remarkable structural feature of CsdB is that a...

  7. Snake venom toxins. The amino acid sequence of toxin Vi2, a homologue of pancreatic trypsin inhibitor, from Dendroaspis polylepis polylepis (black mamba) venom.

    Science.gov (United States)

    Strydom, D J

    1977-04-25

    The amino acid sequence of venom component Vi2, a protein of low toxicity from Dendroaspis polylepis polylepis venom was determined by automatic sequence analysis in combination with sequence studies on tryptic peptides. This protein, the most retarded fraction of this venom on a cation-exchange resin, is a homologue of bovine pancreatic trypsin inhibitor consisting of a single chain of 57 amino acid residues containing six half-cystine residues. The active site lysyl residue of bovine trypsin inhibitor is conserved in Vi2 although large differences are found in the rest of the molecule.

  8. IL-2R{gamma} gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease

    Energy Technology Data Exchange (ETDEWEB)

    Henthorn, P.S.; Fimiani, V.M.; Patterson, D.F. [Univ. of Pennsylvania School of Veterinary Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    X-linked severe combined immunodeficiency (SCID) is characterized by profound defects in cellular and humoral immunity and, in humans, is associated with mutations in the gene for the {gamma} chain of the IL-2 receptor (IL-2R{gamma}). We have examined this gene in a colony of dogs established from a single X-linked SCID carrier female. Affected dogs have a 4-bp deletion in the first exon of the IL-2R{gamma} gene, which precludes the production of a functional protein, demonstrating that the canine disease is a true homologue of human X-linked SCID. 37 refs., 3 figs.

  9. Identification of common mechanisms by which human and mouse cytomegalovirus seven-transmembrane receptor homologues contribute to in vivo phenotypes in a mouse model

    DEFF Research Database (Denmark)

    Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

    2013-01-01

    The mouse cytomegalovirus chemokine receptor homologue (CKR) M33 is required for salivary gland tropism and efficient reactivation from latency, phenotypes partially rescued by the human cytomegalovirus CKR US28. Herein, we demonstrate that complementation of salivary gland tropism is mediated...... predominantly by G protein-dependent signaling conserved with that of M33; in contrast, both G protein-dependent and -independent pathways contribute to the latency phenotypes. A novel M33-dependent replication phenotype in cultured bone marrow macrophages is also described....

  10. mRNA analysis of single living cells

    Directory of Open Access Journals (Sweden)

    Ikai Atsushi

    2003-02-01

    Full Text Available Abstract Analysis of specific gene expression in single living cells may become an important technique for cell biology. So far, no method has been available to detect mRNA in living cells without killing or destroying them. We have developed here a novel method to examine gene expression of living cells using an atomic force microscope (AFM. AFM tip was inserted into living cells to extract mRNAs. The obtained mRNAs were analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enabled us to examine time-dependent gene expression of single living cells without serious damage to the cells.

  11. Control of Klebsiella pneumoniae nif mRNA synthesis.

    OpenAIRE

    1985-01-01

    Four probes, each specific for a single nif transcript, were used for an analysis of the regulation of nif mRNA synthesis. Transcription of the nifLA operon was repressed by NH4+ but not by amino acids, O2, or temperatures above 37 degrees C. The nifA gene product was required for the activation of transcription of the other nif operons but not nifLA. Synthesis of the other nif transcripts was rapidly turned off by the addition of O2, NH4+, serine, or glutamine. These regulatory effects requi...

  12. Transcriptional activators enhance polyadenylation of mRNA precursors

    OpenAIRE

    Nagaike, Takashi; Manley, James L.

    2011-01-01

    3′ processing of mRNA precursors is frequently coupled to transcription by RNA polymerase II (RNAP II). This coupling is well known to involve the C-terminal domain of the RNAP II largest subunit, but a variety of other transcription-associated factors have also been suggested to mediate coupling. Our recent studies have provided direct evidence that transcriptional activators can enhance the efficiency of transcription-coupled 3′ processing. In this point-of-view, we discuss the mechanisms t...

  13. Microinjection and Fluorescence In Situ Hybridization Assay for Studying mRNA Export in Mammalian Cells.

    Science.gov (United States)

    Wang, Ke; Shi, Min; Cheng, Hong

    2017-01-01

    Microinjection and Fluorescence in situ Hybridization (FISH) assay is a useful method for mRNA export studies, which can overcome the problems of traditional transfection in cells. Here, we describe the method of microinjection and FISH assay applied in investigation of mRNA export. By this method we can estimate the mRNA export kinetics, examining mRNA export in cells with low transfection efficiencies, and observing nuclear export of aberrant RNAs.

  14. [Age-dependent changes in mRNA transport (nucleus-cytoplasm)].

    Science.gov (United States)

    Müller, W E; Agutter, P S; Prochnow, D J; Fasold, H; Sève, A P; Tsiapalis, C M; Schröder, H C

    1993-01-01

    Transport of mRNA from nucleus to cytoplasm is an ATP-dependent process which occurs strictly vectorially. Because the mRNA is structurally bound during transport, mRNA transport is a "solid-state" process consisting of i) mRNA release from the nuclear matrix, ii) mRNA translocation through the nuclear pore, and iii) cytoskeletal binding. We identified and purified the following components involved in the translocation step: i) the nuclear envelope (NE) nucleoside triphosphatase (NTPase) which is stimulated by the 3'poly(A) tail of mRNA, ii) the poly(A)-recognizing mRNA carrier, iii) the NE protein kinase, and iv) the NE phosphatase. In addition, we found that an RNA helicase activity is present in NE, which also may be involved in RNA transport. Our results show that, besides poly(A), also double-stranded RNA structures may modulate RNA export. The amount of mRNA released from nuclei markedly decreases with age. Evidence is presented that this age-dependent change is caused by an impairment of polyadenylation of mRNA, hnRNA processing, release of mRNA from nuclear matrix, and translocations of mRNA from nuclear to cytoplasmic compartment (decrease in activities of NE NTPase, protein kinase, and phosphatase; decrease in poly(A)-binding affinity of mRNA carrier).

  15. Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN.

    Science.gov (United States)

    Shima, Yoko; Kitagawa, Mamiko; Fujisawa, Masaki; Nakano, Toshitsugu; Kato, Hiroki; Kimbara, Junji; Kasumi, Takafumi; Ito, Yasuhiro

    2013-07-01

    The tomato MADS-box transcription factor RIN acts as a master regulator of fruit ripening. Here, we identified MADS-box proteins that interact with RIN; we also provide evidence that these proteins act in the regulation of fruit ripening. We conducted a yeast two-hybrid screen of a cDNA library from ripening fruit, for genes encoding proteins that bind to RIN. The screen identified two MADS-box genes, FUL1 and FUL2 (previously called TDR4 and SlMBP7), both of which have high sequence similarity to Arabidopsis FRUITFULL. Expression analyses revealed that the FUL1 mRNA and FUL1 protein accumulate in a ripening-specific manner in tomato fruits and FUL2 mRNA and protein accumulate at the pre-ripening stage and throughout ripening. Biochemical analyses confirmed that FUL1 and FUL2 form heterodimers with RIN; this interaction required the FUL1 and FUL2 C-terminal domains. Also, the heterodimers bind to a typical target DNA motif for MADS-box proteins. Chromatin immunoprecipitation assays revealed that FUL1 and FUL2 bind to genomic sites that were previously identified as RIN-target sites, such as the promoter regions of ACS2, ACS4 and RIN. These findings suggest that RIN forms complexes with FUL1 and FUL2 and these complexes regulate expression of ripening-related genes. In addition to the functional redundancy between FUL1 and FUL2, we also found they have potentially divergent roles in transcriptional regulation, including a difference in genomic target sites.

  16. mRNA quality control pathways in Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    Satarupa Das; Biswadip Das

    2013-09-01

    Efficient production of translation-competent mRNAs involves processing and modification events both in the nucleus and cytoplasm which require a number of complex machineries at both co-transcriptional and post-transcriptional levels. Mutations in the genomic sequence sometimes result in the formation of mutant non-functional defective messages. In addition, the enormous amounts of complexities involved in the biogenesis of mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional counterpart. Subsequent translation of these mutant and defective populations of messenger RNAs could possibly result in the unfaithful transmission of genetic information and thus is considered a threat to the survival of the cell. To prevent this possibility, mRNA quality control systems have evolved both in the nucleus and cytoplasm in eukaryotes to scrutinize various stages of mRNP biogenesis and translation. In this review, we will focus on the physiological role of some of these mRNA quality control systems in the simplest model eukaryote Saccharomyces cerevisiae.

  17. Drosha mediates destabilization of Lin28 mRNA targets.

    Science.gov (United States)

    Qiao, Chong; Ma, Jing; Xu, Jie; Xie, Mingyi; Ma, Wei; Huang, Yingqun

    2012-10-01

    Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.

  18. Tracking single mRNA molecules in live cells

    Science.gov (United States)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  19. Imaging mRNA and protein interactions within neurons

    Science.gov (United States)

    Eliscovich, Carolina; Shenoy, Shailesh M.

    2017-01-01

    RNA–protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions. PMID:28223507

  20. Myc Regulation of mRNA Cap Methylation

    Science.gov (United States)

    Cowling, Victoria H.; Cole, Michael D.

    2010-01-01

    The c-myc proto-oncogene regulates the expression of 15% to 20% of all genes, depending on the cell type, and the regulation is usually modest (1.5- to 2.0-fold). The authors discovered that in addition to regulating mRNA abundance, c-Myc regulates the formation of the 7-methylguanosine cap on many mRNAs, including transcriptional target genes and others not transcriptionally activated. Because the 7-methylguanosine cap is required for effective translation, enhanced methyl cap formation leads to increased protein production from Myc-responsive genes that exceeds the transcriptional induction. Increased cap methylation is linked to Myc-dependent enhanced activity of 2 critical kinases, TFIIH and p-TEFb, which phosphorylate the RNA polymerase II carboxy-terminal domain (CTD). Phosphorylation of the CTD recruits RNGTT and RNMT, the enzymes involved in mRNA capping, to the nascent transcript. Evidence is accumulating that enhanced cap methylation makes a significant contribution to Myc-dependent gene regulation and protein production. PMID:21170289

  1. Cytokine mRNA quantification by real-time PCR.

    Science.gov (United States)

    Stordeur, Patrick; Poulin, Lionel F; Craciun, Ligia; Zhou, Ling; Schandené, Liliane; de Lavareille, Aurore; Goriely, Stanislas; Goldman, Michel

    2002-01-01

    Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

  2. Molecular Cloning and mRNA Profile of Insulin-like Growth Factor Type 1 Receptor in Orange-spotted Grouper, Epinephelus coioides

    Institute of Scientific and Technical Information of China (English)

    Yue-Mei KUANG; Wen-Sheng LI; Hao-Ran LIN

    2005-01-01

    The insulin-like growth factor type 1 receptor (IGF-IR) belongs to the tyrosine kinase (TK)receptor family. Besides being mitogenic, IGF-IR plays a crucial role in cell survival, transformation and maintenance of the malignant phenotype. In this study, we cloned the cDNA from the hypothalamus of the orange-spotted grouper (Epinephelus coioides) using reverse transcription PCR (RT-PCR) and the rapid amplification of cDNA ends (RACE) method. The deduced amino acid sequence showed that the receptor comprises 1413 amino acid residues. It contains cysteine-rich domains in its α-subunit, and a conserved transmembrane domain and TK domains in its β-subunit. Comparison of the amino acid sequence with those of other species showed that the grouper IGF-IR shares 90.2%, 89.6%, 71.9% and 72% similarity with the IGF-IR of the Japanese flounder, turbot, zebrafish-a and zebrafish-b, respectively. When compared with its mammalian homologue, grouper IGF-IR contains a large insertion at its C-terminus. Phylogenetic analysis has revealed that the grouper IGF-IR belongs to the b-type IGF-IRs and has a higher similarity with flounder and turbot IGF-IR, and a lower similarity (<70%) with human, mouse and avian IGF-IR. Grouper IGF-IR transcripts were detected in the brain, peripheral tissues, embryos and early development larvae by semi-quantitative RT-PCR assay. It was observed that IGF-IR mRNA expression was greater in the brain than in peripheral tissues. The level of IGF-IR mRNA expression was much higher in retina, gonad, skeletal muscle and gill tissues than in liver, heart and thymus tissues. The expression of IGF-IR can be visualized as a ubiquitous signalin unfertilized eggs, embryos and early development larvae. The distribution pattern of IGF-IR mRNA in grouper development suggests that IGF-IR plays an important role in the embryo and early larval development stages.

  3. Equine herpesvirus 1 gene 12, the functional homologue of herpes simplex virus VP16, transactivates via octamer sequences in the equine herpesvirus IE gene promoter.

    Science.gov (United States)

    Elliott, G; O'Hare, P

    1995-10-20

    The HSV-1 transactivator of immediate-early (IE) gene expression, VP16, has several functional homologues among the alphaherpesviruses which have not yet been extensively studied in relation to their modes of action. To date, nothing is known of the exact sites or mechanism of interaction of the equine herpesvirus type 1 (EHV-1) homologue, the gene 12 protein, with the EHV-1 IE promoter. We show that the gene 12 protein utilises the promoter proximal region of the IE gene to induce activation and identify four potential octamer DNA binding sites within that region. Although there was divergence from its consensus, Oct-1 bound to each of these sites in an in vitro complex formation assay, and in the presence of the gene 12 product a second complex of slower migration, which was also dependent on Oct-1, was detected. When each site was inserted into a basal promoter, two conferred activation by gene 12 with a resulting increase in expression of up to 50-fold compared to basal levels. These results show that, despite the differences between the two proteins, the mechanism of interaction of the gene 12 protein with its target is analogous to that of VP16.

  4. Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus.

    Science.gov (United States)

    Purves, Joanne; Cockayne, Alan; Moody, Peter C E; Morrissey, Julie A

    2010-12-01

    The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.

  5. Mycosphaerella graminicola LysM effector-mediated stealth pathogenesis subverts recognition through both CERK1 and CEBiP homologues in wheat.

    Science.gov (United States)

    Lee, Wing-Sham; Rudd, Jason J; Hammond-Kosack, Kim E; Kanyuka, Kostya

    2014-03-01

    Fungal cell-wall chitin is a well-recognized pathogen-associated molecular pattern. Recognition of chitin in plants by pattern recognition receptors activates pathogen-triggered immunity (PTI). In Arabidopsis, this process is mediated by a plasma membrane receptor kinase, CERK1, whereas in rice, a receptor-like protein, CEBiP, in addition to CERK1 is required. Secreted chitin-binding lysin motif (LysM) containing fungal effector proteins, such as Ecp6 from the biotrophic fungus Cladosporium fulvum, have been reported to interfere with PTI. Here, we identified wheat homologues of CERK1 and CEBiP and investigated their role in the interaction with the nonbiotrophic pathogen of wheat Mycosphaerella graminicola (synonym Zymoseptoria tritici). We show that silencing of either CERK1 or CEBiP in wheat, using Barley stripe mosaic virus-mediated virus-induced gene silencing, is sufficient in allowing leaf colonization by the normally nonpathogenic M. graminicola Mg3LysM (homologue of Ecp6) deletion mutant, while the Mg1LysM deletion mutant was fully pathogenic toward both silenced and wild-type wheat leaves. These data indicate that Mg3LysM is important for fungal evasion of PTI in wheat leaf tissue and that both CERK1 and CEBiP are required for activation of chitin-induced defenses, a feature conserved between rice and wheat, and perhaps, also in other cereal species.

  6. Muscle cell migrations of C. elegans are mediated by the alpha-integrin INA-1, Eph receptor VAB-1, and a novel peptidase homologue MNP-1.

    Science.gov (United States)

    Tucker, Morgan; Han, Min

    2008-06-15

    Cell migration is a fundamental process occurring during embryonic development and tissue morphogenesis. In the nematode Caenorhabditis elegans, morphogenesis of the body-wall musculature involves short-range migrations of 81 embryonic muscle cells from the lateral surface of the embryo towards the dorsal and ventral midlines. This study shows that mutations in ina-1 (alpha-integrin), as well as vab-1 (Eph receptor), and vab-2 (ephrin), display defects in embryonic muscle cell migration. Furthermore, an RNAi-based enhancer screen in an ina-1 weak loss-of-function background identified mnp-1 (matrix non-peptidase homologue-1) as a previously uncharacterized gene required for promoting proper migration of the embryonic muscle cells. mnp-1 encodes a membrane associated metalloproteinase homologue that is predicted to be catalytically inactive. Our data suggest that MNP-1 is expressed in migrating muscle cells and localizes to the plasma membrane with the non-peptidase domain exposed to the extra-cellular environment. Double-mutant analysis between mnp-1(RNAi), ina-1, and vab-1 mutations; as well as tissue specific rescue experiments; indicated that each of these gene products function predominantly independent of each other and from different cell types to affect muscle cell migration. Together these results suggest complex interactions between the adjacent epidermal, neuronal, and muscle cells are required to promote proper muscle cell migration during embryogenesis.

  7. DUF538 protein super family is predicted to be the potential homologue of bactericidal/permeability-increasing protein in plant system.

    Science.gov (United States)

    Gholizadeh, Ashraf; Kohnehrouz, Samira Baghban

    2013-03-01

    DUF538 protein super family includes a number of plant proteins that their role is not yet clear. These proteins have been frequently reported to be expressed in plants under various stressful stimuli such as bacteria and elicitors. In order to further understand about this protein family we utilized bioinformatics tools to analyze its structure in details. As a result, plants DUF538 was predicted to be the partial structural homologue of BPI (bactericidal/permeability increasing) proteins in mammalian innate immune system that provides the first line of defense against different pathogens including bacteria, fungi, viruses and parasites. Moreover, on the base of the experimental data, it was identified that exogenously applied purified fused product of Celosia DUF538 affects the bacterial growth more possibly similar to BPI through the binding to the bacterial membranes. In conclusion, as the first ever time report, we nominated DUF538 protein family as the potential structural and functional homologue of BPI protein in plants, providing a basis to study the novel functions of this protein family in the biological systems in the future.

  8. Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity zinc uptake system.

    Directory of Open Access Journals (Sweden)

    Cheryl M Vahling-Armstrong

    Full Text Available The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria.

  9. Comparison of the Regulation, Metabolic Functions, and Roles in Virulence of the Glyceraldehyde-3-Phosphate Dehydrogenase Homologues gapA and gapB in Staphylococcus aureus▿

    Science.gov (United States)

    Purves, Joanne; Cockayne, Alan; Moody, Peter C. E.; Morrissey, Julie A.

    2010-01-01

    The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence. PMID:20876289

  10. Analysis of linear alkylbenzene sulfonate homologues in environmental water samples by mixed admicelle-based extraction and liquid chromatography/mass spectrometry.

    Science.gov (United States)

    Lunar, Loreto; Rubio, Soledad; Pérez-Bendito, Dolores

    2006-07-01

    Hemimicelles and admicelles of cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC), adsorbed onto silica, were tested as sorbents for the solid phase extraction (SPE) of linear alkylbenzene sulfonate (LAS) homologues from environmental water samples. LASs were quantitatively retained on both surfactants due to high hydrophobic and ionic interactions, which led to the formation of analyte-extractant mixed aggregates. Parameters affecting the SPE of LASs were optimised. Recoveries of analytes from wastewater influent and effluent and river water samples ranged between 86 and 110%. Combination of SPE with liquid chromatography/mass spectrometry provided detection limits for the different LAS homologues of about 4 ng L(-1). The precision of the method, expressed as relative standard deviation, ranged from 5 to 9%. The method was applied to the analysis of LASs in wastewater and river samples using sample volumes between 10 and 25 mL. The LAS concentrations found ranged from 9 to 503 microg L(-1). No cleaning step was required to get accurate results.

  11. Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

    DEFF Research Database (Denmark)

    Leick, Lotte; Lindegaard, Birgitte; Stensvold, Dorthe

    2007-01-01

    OBJECTIVES: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin......: AT IL-18 mRNA content and plasma IL-18 concentration were higher (p insulin resistance. While acute exercise did not affect IL-18 mRNA expression...... at the studied time-points, exercise training reduced AT IL-18 mRNA content by 20% in both sexes. DISCUSSION: Because obesity and insulin resistance were associated with elevated AT IL-18 mRNA and plasma IL-18 levels, the training-induced lowering of AT IL-18 mRNA content may contribute to the beneficial effects...

  12. Lipidoid mRNA Nanoparticles for Myocardial Delivery in Rodents.

    Science.gov (United States)

    Turnbull, Irene C; Eltoukhy, Ahmed A; Anderson, Daniel G; Costa, Kevin D

    2017-01-01

    An area of active research in the field of cardiac gene therapy aims to achieve high transfection efficiency without eliciting immune or inflammatory reactions. Nanomedicine offers an attractive alternative to traditional viral delivery vehicles because nanoparticle technology can enable safer and more controlled delivery of therapeutic agents. Here we describe the use of lipidoid nanoparticles for delivery of modified mRNA (modRNA) to the myocardium in vivo, with a focus on rodent models that represent a first step toward preclinical studies. Three major procedures are discussed in this chapter: (1) preparation of lipid modRNA nanoparticles, (2) intramyocardial delivery of the lipid modRNA nanoparticles by direct injection with an open chest technique in rats, and (3) intracoronary delivery of the lipid modRNA nanoparticles with open chest and temporary aortic cross clamping in rats.

  13. Selenoprotein P mRNA expression in human hepatic tissues

    Institute of Scientific and Technical Information of China (English)

    Chun-Li Li; Ke-Jun Nan; Tao Tian; Chen-Guang Sui; Yan-Fang Liu

    2007-01-01

    AIM: To investigate the expression of Selenoprotein P mRNA (SePmRNA) in tissues of normal liver, liver cirrhosis and hepatocellular carcinoma (HCC), and its relationship with HCC occurrence and development.METHODS: The expression of SePmRNA in tissues of normal liver, liver cirrhosis and HCC were detected by in situ hybridization using a cDNA probe.RESULTS: The enzyme digesting products of pBluescript-Human Selenoprotein P were evaluated by electrophoresis.The positive expression of SePmRNA was found in the tissues of normal liver,liver cirrhosis and HCC.The expression of SeP mRNA was found in hepatic interstitial substance,especially in endothelial cells and lymphocytes of vasculature.The positive rate of SePmRNA in normal liver tissue was 84.6% (11/13) and the positive signals appeared in the nucleus and cytoplasm,mostly in the nucleolus,and the staining granules were larger in the nucleolus and around the nucleus.The positive rate of SePmRNA in liver cirrhosis tissue was 45.O% (9/20) and the positive signals were mainly in the nucleolus and cytoplasm,being less around the nucleus and inner nucleus than that in normal liver tissue. The positive rate of SePmRNA in HCC tissue was 30.0% (9/30) and the positive signals were in the cytoplasm, but less in the nucleus.CONCLUSION: SePmRNA expression in the tissues of normal liver and HCC is significantly different (84.6% vs 30.0%, P = 0.003), suggesting that SeP might play a role in the occurrence and development of HCC.

  14. A study of anaerobic biodegradation of pure homologue and isomers of linear alkylbenzene sulphonate (LAS) in batch digesters; Estudio de la biodegradacion anaerobia de homologos e isomeros puros de alquibenceno sulfonatos lineales (LAS) en digestores discontinuos

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, M. T.; Campos, E.; Illan, P.; Dalman, M.; Sanchez-Leal, J.

    2005-07-01

    In the present work the effect of different structural parameters on the anaerobic degradation of the LAS molecule has been studied. so, the effect both of the alkyl chain length and the phenyl position on the anaerobic degradation of the LAS molecule have been investigated. The behaviour of the individual homologues and isomers was studied applying discontinuous anaerobic tests (ECETOC-28). Sludge from the anaerobic digester of the waste water treatment plant of Manresa (Barcelona) was used as bacterial inoculum source. All the LAS homologues showed a poor primary biodegradation. In addition, no effect of the LAS isomer type was found on the anaerobic degradation kinetics. (Author) 6 refs.

  15. Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

    Energy Technology Data Exchange (ETDEWEB)

    Sampathkumar, Parthasarathy; Kim, Seung Joong; Manglicmot, Danalyn; Bain, Kevin T.; Gilmore, Jeremiah; Gheyi, Tarun; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Matsui, Tsutomu; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sali, Andrej; Sauder, J. Michael; Almo, Steven C.; Burley, Stephen K. (Einstein); (SLAC); (Rockefeller); (UCSF); (Lilly)

    2012-10-23

    The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of {approx}456 polypeptide chains contributed by {approx}30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal 'FG' repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 {angstrom} resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.

  16. qPCR based mRNA quality score show intact mRNA after heat stabilization

    Directory of Open Access Journals (Sweden)

    Oskar Karlsson

    2016-03-01

    Full Text Available Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

  17. Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1.

    Science.gov (United States)

    Mittag, D; Vieths, S; Vogel, L; Wagner-Loew, D; Starke, A; Hunziker, P; Becker, W-M; Ballmer-Weber, B K

    2005-08-01

    Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1. Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS). All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress. Food allergy to mungbean

  18. Highest trkB mRNA expression in the entorhinal cortex among hippocampal subregions in the adult rat: contrasting pattern with BDNF mRNA expression.

    Science.gov (United States)

    Tokuyama, W; Hashimoto, T; Li, Y X; Okuno, H; Miyashita, Y

    1998-11-20

    Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, regulate synaptic functions in the hippocampus of the adult rodent. In previous studies, in situ hybridization methods have been used to evaluate regional differences in BDNF and trkB mRNA expression levels in hippocampal subregions. However, these studies have failed to reach consensus regarding the regional differences in the mRNA expression levels. In the present study, we quantitated mRNA expression levels using two different methods, ribonuclease protection assays and a quantitative reverse-transcription polymerase chain reaction technique, in four hippocampal subregions: the entorhinal cortex, dentate gyrus (DG), CA3 and CA1. These two methods yielded the same results. We found that BDNF and trkB mRNA expression levels did not covary in the four subregions. BDNF and full length trkB (trkB FL) mRNA in the entorhinal cortex and the DG show contrasting expression patterns. The expression level of BDNF mRNA was highest in the DG among the hippocampal subregions and low in the entorhinal cortex and the CA1, whereas the trkB FL mRNA expression level was highest in the entorhinal cortex, low in the DG and lowest in the CA3. These results suggest regional differences in BDNF/TrkB signaling for maintenance and modifiability of neuronal connections in the hippocampal formation.

  19. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry

    2011-01-01

    concentrations of AMH, inhibin-B, progesterone and estradiol. Androgen Receptor mRNA expression in granulosa cells, and the FF content of androgens, both showed a highly significant positive association with to the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly...... with the expression of AMHR2, but did not correlate with any of the hormones in the FF. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the FF levels of androgen and FSHR expression...

  20. A FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA and its application

    Institute of Scientific and Technical Information of China (English)

    QING HUI ZHU; QING LU; GU SHENG ZHANG

    2006-01-01

    The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response (FQ-RT-PCR) method for the quantitative determination of IL-2 mRNA and IL-4mRNA in Th cells, with which the Th cells status of the patients with gynaecological tumors and chronic renal failure (CRF) can be analyzed. IL-2 cDNA and IL-4 cDNA were prepared, and the plasmid pMD18 carrying IL-2 cDNA or IL-4 cDNA fragment was constructed and cloned as the template for quantitative determination. The primers and probes labelled with 6-carboxy-fluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) were prepared, and the experimental conditions were optimized to set up the FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA. Th cells enriched from peripheral blood mononuclear cells (PBMCs) of 20 healthy volunteers (HVs), 16 gynaecological benign (GB) cases, 18 gynaecological malignant (GM) tumor cases and 16 chronic renal failure (CRF) patients were tested for IL-2 mRNA and IL-4 mRNA by FQ-RT-PCR. The house-keeping gene 3-actin was used as the internal control gene of the experiment. The standard curve for log concentration of series of quantitative templates vs threshold cycle (CT) was established by linear regression, and the linear range was 102-107 copies/μl. The imprecision test showed the CV of inter-assay and intra-assay of a high content sample by FQ-RT-PCR were 7.8% and 12.5%, respectively. The CV of inter-assay and intra-assay of a low content sample were 10.8% and 19.5%, respectively. The IL-2 mRNA expressions in Th of the patients with gynaecological malignant tumor (compared with the HVs and the patients with gynaecological benign disease) and in Th of the CRF patients (compared with the HVs) were declined significantly and at the same time the IL-4 mRNA expression increased significantly ( P < 0. 001 ). A simple, sensitive and accurate FQ-RT-PCR method for the quantitative detection of IL-2 mRNA and IL-4 mRNA has

  1. Repeated stress increases catalytic TrkB mRNA in rat hippocampus.

    Science.gov (United States)

    Nibuya, M; Takahashi, M; Russell, D S; Duman, R S

    1999-05-28

    Northern blot analysis was utilized to distinguish between catalytic and truncated TrkB mRNA on the basis of transcript size. Repeated (10 days), but not acute, immobilization stress significantly increased levels of catalytic TrkB mRNA, but did not influence expression of truncated TrkB transcripts in rat hippocampus. Exposure to another paradigm, a combination of different, unpredictable stressors, also increased levels of catalytic, but not truncated, TrkB mRNA. In situ hybridization analysis demonstrated that chronic stress up-regulated TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cells layers of hippocampus. As previously reported, both acute and chronic immobilization stress decreased expression of BDNF mRNA, suggesting that up-regulation of catalytic TrkB mRNA may be a compensatory adaptation to repeated stress.

  2. Studies on the Physicochemical Properties, Structure and Antitumor Activity of an Oligosaccharide Homologue SnS-2 from the Root of Scrophularia ningpoensis Hemsl

    Institute of Scientific and Technical Information of China (English)

    DENG,Jun-E(邓军娥); ZHANG,Jian(张健); CHEN,Xiao-Ming(陈晓明); KE,Wei(柯炜); TIAN,Geng-Yuan(田庚元)

    2004-01-01

    An oligosaccharide homologue named SnS-2 was isolated from the root of Scrophularia ningpoensis Hemsl.SnS-2 was purified by means of gel-permeation chromatography and ion-exchange chromatography.Its physicochemical properties,including carbohydrate content and molecular weight were determined.The structure of SnS-2was elucidated by chemical methods along with 1H and 13C NMR spectroscopy,including two-dimensional DQCOSY and H-detected 1H,13C HMQC experiments.These results show that SnS-2 possesses a backbone consisting of terminal α-Galp-(1→,α-Galp-(1→6),α-Glcp-(1→6) and nonreducing end fβ-Fruf-(2→.The bioactive assay showed that it could inhibit the growth of Lewis pulmonary carcinoma implanted in mice.

  3. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

    2013-11-29

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

  4. Tsp66E, the Drosophila KAI1 homologue, and Tsp74F function to regulate ovarian follicle cell and wing development by stabilizing integrin localization.

    Science.gov (United States)

    Han, Seung Yeop; Lee, Minjung; Hong, Yoon Ki; Hwang, Soojin; Choi, Gahee; Suh, Yoon Seok; Park, Seung Hwan; Lee, Soojin; Lee, Sang-Hee; Chung, Jongkyeong; Baek, Sung Hee; Cho, Kyoung Sang

    2012-11-16

    The metastasis suppressor KAI1/CD82 has been implicated in various cellular processes; however, its function in development is not fully understood. Here, we generated and characterized mutants of Tsp66E and Tsp74F, which are Drosophila homologues of KAI1/CD82 and Tspan11, respectively. These mutants exhibited egg elongation defects along with disturbed integrin localization and actin polarity. Moreover, the defects were enhanced by mutation of inflated, an αPS2 integrin gene. Mutant ovaries had elevated αPS2 integrin levels and reduced endocytic trafficking. These results suggest that Drosophila KAI1/CD82 affects the polarized localization and the level of integrin, which may contribute to epithelial cell polarity.

  5. Rye inhibitors of animal alpha-amylases show different specificities, aggregative properties and IgE-binding capacities than their homologues from wheat and barley.

    Science.gov (United States)

    García-Casado, G; Sánchez-Monge, R; López-Otín, C; Salcedo, G

    1994-09-01

    Three new members of the alpha-amylase/trypsin-inhibitor family of cereal endosperm have been isolated from rye. N-terminal amino acid sequence comparison revealed that two of the purified proteins were the rye homologues of the barley monomeric inhibitor (BMAI-1) previously described, while the other rye protein corresponded to one of the subunits of the barley and wheat heterotetrameric inhibitors. However, the inhibitory specificities (active against human salivary alpha-amylase), aggregative behaviours (mainly as dimeric forms) and IgE-binding capacities (not recognized by sera from allergic patients) of the rye inhibitors were clearly different from those of their wheat and barley counterparts. These results indicate that homologous inhibitors may have distinctive properties in different cereal species.

  6. Six3, a medaka homologue of the Drosophila homeobox gene sine oculis is expressed in the anterior embryonic shield and the developing eye.

    Science.gov (United States)

    Loosli, F; Köster, R W; Carl, M; Krone, A; Wittbrodt, J

    1998-06-01

    homologue Six3 (Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045-4055). sine oculis (so) is essential for the development of the larval and adult visual system (Cheyette, B.N.R., Green, P.J., Martin, K., Garren, H., Hartenstein, V., Zipursky, S.L., 1994. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron l2, 977-996). Six3 is expressed in the anterior neural plate and optic vesicles, lens, olfactory placodes and ventral forebrain (Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045-4055). Overexpression of mouse Six3 gene in medaka fish embryos (Orvzias latipes) results in the formation of an ectopic lens, indicating that Six3 activity can trigger the genetic pathway leading to lens formation (Oliver, G., Loosli, F., Koster, R., Wittbrodt, J., Gruss, P., 1996. Ectopic lens induction in fish in response to the murine homeobox gene Six3. Mech. Dev. 60, 233-239). We isolated the medaka Six3 homologue and analyzed its expression pattern in the medaka embryo. It is expressed initially in the anterior embryonic shield and later in the developing eye and prosencephalon. The early localized expression of Six3 suggests a role in the regionalization of the rostral head.

  7. Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.

  8. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    Science.gov (United States)

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.

  9. Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.

    OpenAIRE

    Bondurant, M C; Koury, M J

    1986-01-01

    Regulation of the production of erythropoietin occurs in the kidney and liver largely through control of accumulation of erythropoietin mRNA. Erythropoietin mRNA was first detected in kidneys at 1.5 h postanemia and reached a plateau value at least 200-fold above the control value by 4 to 8 h. A 20-base sequence immediately upstream from the reported erythropoietin mRNA initiation site is complementary to a hypervariable sequence in 18S rRNA.

  10. Dynamics of translation by single ribosomes through mRNA secondary structures

    OpenAIRE

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L.; Reiche, Michael; Farrell, Ian; Cooperman, Barry S.; Goldman, Yale E.

    2013-01-01

    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNA 2° structures. Downstream ...

  11. Mapping of a macular drusen susceptibility locus in rhesus macaques to the homologue of human chromosome 6q14-15.

    Science.gov (United States)

    Singh, Krishna K; Ristau, Steven; Dawson, William W; Krawczak, Michael; Schmidtke, Jörg

    2005-10-01

    Rhesus macaques (Macaca mulatta) are a natural model for retinal drusen formation. The present study aimed at clarifying whether chromosomal regions homologous to candidate genes for drusen formation and progression in humans are also associated with a drusen phenotype in rhesus macaques. Some 42 genetic markers from seven chromosomal regions implicated in macular degeneration syndromes in humans were tested for whether they identified homologous, polymorphic sequences in rhesus DNA. This was found to be the case for seven markers, all of which were subsequently screened for the presence of potentially disease-predisposing alleles in 52 randomly chosen adult animals from the Cayo Santiago population of rhesus macaques (Caribbean Primate Research Center, PR, USA). The high drusen prevalence expected in the Cayo Santiago colony was confirmed in our sample in that 38 animals were found to have drusen (73%). Logistic regression analysis revealed that some alleles of the rhesus homologue of anonymous human marker D6S1036 were consistently over-represented among affected animals. Of two candidate genes located in the respective region, allelic variation in one (IMPG1) showed strong association with drusen formation. We conclude that one or more genes located at the rhesus homologue of human 6q14-15 are likely to play a role in retinal drusen formation, a finding that represents a first step towards the identification of genetic factors implicated in macular drusen formation in rhesus macaques. This is an important tool for the separation of genetic and environmental factors which must occur before satisfactory management methods can be developed.

  12. Rabbit neutrophil chemotactic protein (NCP) activates both CXCR1 and CXCR2 and is the functional homologue for human CXCL6.

    Science.gov (United States)

    Catusse, Julie; Struyf, Sofie; Wuyts, Anja; Weyler, Myke; Loos, Tamara; Gijsbers, Klara; Gouwy, Mieke; Proost, Paul; Van Damme, Jo

    2004-11-15

    Neutrophil chemotactic protein (NCP) is a rabbit CXC chemokine with activating and chemotactic properties on neutrophilic granulocytes. Although its selective activity on neutrophils is demonstrated, its interactions with specific chemokine receptors are not defined. For further functional characterization, NCP was chemically synthesized and was found to be equipotent as natural NCP in neutrophil chemotaxis. To identify its human homologue, we separately expressed two potential rabbit NCP receptors (CXCR1 and CXCR2) in Jurkat cells. Pure synthetic NCP was equally efficient to promote chemotaxis through either rabbit CXCR1 or CXCR2. Moreover, chemotaxis assays on rabbit CXCR1 and CXCR2 transfectants showed that NCP uses the same receptors as interleukin-8 (IL-8), a major rabbit CXC chemokine, but not rabbit GROalpha, which only recognized CXCR2. In addition, specific inhibitors for CXCR1 or CXCR2 reduced rabbit neutrophil chemotaxis induced by NCP and rabbit IL-8. Furthermore, NCP and the structurally related human CXCR1/CXCR2 agonist CXCL6/GCP-2 (granulocyte chemotactic protein-2) cross-desensitized each other in intracellular calcium release assays on human neutrophils, further indicating that both chemokines share the same receptors. The inflammatory role of NCP was also evidenced by its potent granulocytosis inducing capacity in rabbits upon systemic administration. This study provides in vitro and in vivo evidences that NCP is the functional rabbit homologue for human CXCL6/GCP-2 rather than the most related CXCR2 agonist CXCL5/ENA-78 (epithelial cell-derived neutrophil activating peptide-78). It is concluded that the rabbit is a better model to study human neutrophil activation compared to mice, which lack CXCL8/IL-8.

  13. New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for Saccharomyces cerevisiae Regulated by 2,4-Diacetylphloroglucinol and Other Ligands.

    Science.gov (United States)

    Ikushima, Shigehito; Boeke, Jef D

    2017-03-17

    Here we describe the development of tightly regulated expression switches in yeast, by engineering distant homologues of Escherichia coli TetR, including the transcriptional regulator PhlF from Pseudomonas and others. Previous studies demonstrated that the PhlF protein bound its operator sequence (phlO) in the absence of 2,4-diacetylphloroglucinol (DAPG) but dissociated from phlO in the presence of DAPG. Thus, we developed a DAPG-Off system in which expression of a gene preceded by the phlO-embedded promoter was activated by a fusion of PhlF to a multimerized viral activator protein (VP16) domain in a DAPG-free environment but repressed when DAPG was added to growth medium. In addition, we constructed a DAPG-On system with the opposite behavior of the DAPG-Off system; i.e., DAPG triggers the expression of a reporter gene. Exposure of DAPG to yeast cells did not cause any serious deleterious effect on yeast physiology in terms of growth. Efforts to engineer additional Tet repressor homologues were partially successful and a known mammalian switch, the p-cumate switch based on CymR from Pseudomonas, was found to function in yeast. Orthogonality between the TetR (doxycycline), CamR (d-camphor), PhlF (DAPG), and CymR (p-cumate)-based Off switches was demonstrated by evaluating all 4 ligands against suitably engineered yeast strains. This study expands the toolbox of "On" and "Off" switches for yeast biotechnology.

  14. The Ca{sup 2+} channel TRPML3 specifically interacts with the mammalian ATG8 homologue GATE16 to regulate autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Suzy; Kim, Hyun Jin, E-mail: kimhyunjin@skku.edu

    2014-01-03

    Highlights: •Split-ubiquitin MY2H screen identified GATE16 as an interacting protein of TRPML3. •TRPML3 specifically binds to a mammalian ATG8 homologue GATE16, not to LC3B. •The interaction of TRPML3 with GATE16 facilitates autophagosome formation. •GATE16 is expressed in both autophagosome and extra-autophagosomal compartments. -- Abstract: TRPML3 is a Ca{sup 2+} permeable cation channel expressed in multiple intracellular compartments. Although TRPML3 is implicated in autophagy, how TRPML3 can regulate autophagy is not understood. To search interacting proteins with TRPML3 in autophagy, we performed split-ubiquitin membrane yeast two-hybrid (MY2H) screening with TRPML3-loop as a bait and identified GATE16, a mammalian ATG8 homologue. GST pull-down assay revealed that TRPML3 and TRPML3-loop specifically bind to GATE16, not to LC3B. Co-immunoprecipitation (co-IP) experiments showed that TRPML3 and TRPML3-loop pull down only the lipidated form of GATE16, indicating that the interaction occurs exclusively at the organellar membrane. The interaction of TRPML3 with GATE16 and GATE16-positive vesicle formation were increased in starvation induced autophagy, suggesting that the interaction facilitates the function of GATE16 in autophagosome formation. However, GATE16 was not required for TRPML3 trafficking to autophagosomes. Experiments using dominant-negative (DN) TRPML3(D458K) showed that GATE16 is localized not only in autophagosomes but also in extra-autophagosomal compartments, by contrast with LC3B. Since GATE16 acts at a later stage of the autophagosome biogenesis, our results suggest that TRPML3 plays a role in autophagosome maturation through the interaction with GATE16, by providing Ca{sup 2+} in the fusion process.

  15. In situ hybridization analysis of the expression of futsch, tau, and MESK2 homologues in the brain of the European honeybee (Apis mellifera L..

    Directory of Open Access Journals (Sweden)

    Kumi Kaneko

    Full Text Available BACKGROUND: The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs. METHODOLOGY/PRINCIPAL FINDINGS: Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2, which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste. CONCLUSIONS: Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau or ventral medulla-preferential manner (AmMESK2 in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral

  16. Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation.

    Science.gov (United States)

    Pynyaha, Yuriy V; Boretsky, Yuriy R; Fedorovych, Daria V; Fayura, Lubov R; Levkiv, Andriy I; Ubiyvovk, Vira M; Protchenko, Olha V; Philpott, Caroline C; Sibirny, Andriy A

    2009-12-01

    Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B2) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-L-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3-3.5 times higher as compared to the parental strain. It produced 50-70 times more riboflavin in iron sufficient synthetic media relative to the parental wildtype strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.

  17. Ischemia and reperfusion induce differential expression of calpastatin and its homologue high molecular weight calmodulin-binding protein in murine cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Sreejit Parameswaran

    Full Text Available In the heart, calpastatin (Calp and its homologue high molecular weight calmodulin-binding protein (HMWCaMBP regulate calpains (Calpn by inhibition. A rise in intracellular myocardial Ca2+ during cardiac ischemia activates Calpn thereby causing damage to myocardial proteins, which leads to myocyte death and consequently to loss of myocardial structure and function. The present study aims to elucidate expression of Calp and HMWCaMBP with respect to Calpn during induced ischemia and reperfusion in primary murine cardiomyocyte cultures. Ischemia and subsequently reperfusion was induced in ∼ 80% confluent cultures of neonatal murine cardiomyocytes (NMCC. Flow cytometric analysis (FACS has been used for analyzing protein expression concurrently with viability. Confocal fluorescent microscopy was used to observe protein localization. We observed that ischemia induces increased expression of Calp, HMWCaMBP and Calpn. Calpn expressing NMCC on co-expressing Calp survived ischemic induction compared to NMCC co-expressing HMWCaMBP. Similarly, living cells expressed Calp in contrast to dead cells which expressed HMWCaMBP following reperfusion. A significant difference in the expression of Calp and its homologue HMWCaMBP was observed in localization studies during ischemia. The current study adds to the existing knowledge that HMWCaMBP could be a putative isoform of Calp. NMCC on co-expressing Calp and Calpn-1 survived ischemic and reperfusion inductions compared to NMCC co-expressing HMWCaMBP and Calpn-1. A significant difference in expression of Calp and HMWCaMBP was observed in localization studies during ischemia.

  18. A gonococcal homologue of meningococcal γ-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent

    Directory of Open Access Journals (Sweden)

    Watanabe Haruo

    2005-10-01

    Full Text Available Abstract Background It has been speculated that the γ-glutamyl transpeptidase (ggt gene is present only in Neisseria meningitidis and not among related species such as Neisseria gonorrhoeae and Neisseria lactamica, because N. meningitidis is the only bacterium with GGT activity. However, nucleotide sequences highly homologous to the meningococcal ggt gene were found in the genomes of N. gonorrhoeae isolates. Results The gonococcal homologue (ggt gonococcal homologue; ggh was analyzed. The nucleotide sequence of the ggh gene was approximately 95 % identical to that of the meningococcal ggt gene. An open reading frame in the ggh gene was disrupted by an ochre mutation and frameshift mutations induced by a 7-base deletion, but the amino acid sequences deduced from the artificially corrected ggh nucleotide sequences were approximately 97 % identical to that of the meningococcal ggt gene. The analyses of the sequences flanking the ggt and ggh genes revealed that both genes were localized in a common DNA region containing the fbp-ggt (or ggh-glyA-opcA-dedA-abcZ gene cluster. The expression of the ggh RNA could be detected by dot blot, RT-PCR and primer extension analyses. Moreover, the truncated form of ggh-translational product was also found in some of the gonococcal isolates. Conclusion This study has shown that the gonococcal ggh gene is a pseudogene of the meningococcal ggt gene, which can also be designated as Ψggt. The gonococcal ggh (Ψggt gene is the first identified bacterial pseudogene that is transcriptionally active but phenotypically silent.

  19. Characterization of the Distal-less gene homologue, NlDll, in the brown planthopper, Nilaparvata lugens (Stål).

    Science.gov (United States)

    Lin, Xinda; Yao, Yun; Jin, Minna; Li, Qilin

    2014-02-10

    The brown planthopper, Nilaparvata lugens (Stål), is a globally devastating insect pest of rice, particularly in eastern Asia. Distal-less or Dll is a highly conserved and well studied transcription factor required for limb formation in invertebrates and vertebrates. We have identified a homologue of this gene, NlDll, and demonstrated that it is expressed in all life stages of N. lugens, particularly in adult brachypterous females. When we compared between specific adult tissues it was expressed most strongly in wings. Using RNAi techniques we demonstrated that downregulation of NlDll in the 3rd instar larvae led to the disrupted development of the leg, while downregulation of NlDll in the 5th instar larvae led to abnormal wing formation. Ectopic over-expression of NlDll in Drosophila melanogaster using the GAL4-UAS system led to fatal or visible phenotypic changes such as the loss of normal wing structure and disrupted haltere structure. Our work suggests that NlDll is a conserved homologue of Distal-less and is required for both leg development and wing structure. Since researches have shown that Dll is required for wing morphogenesis, understanding the role of NlDll during the wing development will further provide a basis for revealing the molecular mechanism of the wing dimorphism in brown planthopper. In the future, NlDll could be used as a target gene for brown planthopper pest management in the field. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  20. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-08-25

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  1. mRNA related to insulin family in human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-03-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  2. Nuclear imprisonment: viral strategies to arrest host mRNA nuclear export.

    Science.gov (United States)

    Kuss, Sharon K; Mata, Miguel A; Zhang, Liang; Fontoura, Beatriz M A

    2013-07-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  3. Correlation of mRNA and protein in complex biological samples.

    Science.gov (United States)

    Maier, Tobias; Güell, Marc; Serrano, Luis

    2009-12-17

    The correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

  4. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Science.gov (United States)

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  5. Elicitor induction of mRNA activity. Rapid effects of elicitor on phenylalanine ammonia-lyase and chalcone synthase mRNA activities in bean cells.

    Science.gov (United States)

    Lawton, M A; Dixon, R A; Hahlbrock, K; Lamb, C J

    1983-01-17

    Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total

  6. BUB1 mRNA is significantly co-expressed with AURKA and AURKB mRNA in advanced-stage ovarian serous carcinoma.

    Science.gov (United States)

    Davidson, Ben; Nymoen, Dag Andre; Elgaaen, Bente Vilming; Staff, Anne Cathrine; Tropé, Claes G; Kærn, Janne; Reich, Reuven; Falkenthal, Thea E Hetland

    2014-06-01

    The objective of this study was to investigate the expression and clinical role of the spindle checkpoint kinase budding uninhibited by benzimidazole 1 (Bub1) in primary and metastatic advanced-stage ovarian serous carcinoma. BUB1 mRNA expression was analyzed in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction (PCR). Bub1 protein expression by Western blotting was studied in 63 carcinomas (30 effusions and 33 solid lesions). BUB1 mRNA expression at different anatomic sites was studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. BUB1 mRNA was universally expressed in serous carcinomas, irrespective of anatomic site. BUB1 mRNA levels were uniformly low in six ovarian surface epithelium specimens analyzed for comparative purposes. Bub1 protein was expressed in 22/30 effusions and 28/33 solid lesions. BUB1 mRNA expression was significantly higher in chemo-naïve primary carcinomas and solid metastases compared to specimens obtained following neoadjuvant chemotherapy (p cancer. BUB1 mRNA levels are lower following chemotherapy exposure in solid lesions, though its presence is unrelated to clinical behavior including response to chemotherapy and survival. BUB1 is co-expressed with AURKA and AURKB suggesting biological relationship between these spindle cell components.

  7. Effect of Exercise on the Expression of Adiponectin mRNA and GLUT4 mRNA in Type 2 Diabetic Rats

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the impact of exercise on the expression of adiponectin and GLUT4 mR NA in type 2 diabetic rats, type 2 diabetic rat model was made. The diabetic rats were treated with swimming training for 8 weeks. The expression of adiponectin mRNA in perirenal fat and GLUT4mRNA in skeletal muscles were assessed by reverse transcription polymerase chain reaction (RT PCR) and the levels of blood glucose, serum insulin, and blood lipid were measured. Our results showed that the expression of adiponectin mRNA and GLUT4 mRNA in diabetic model group was decreased by 45 % (P<0.01), 43 % (P<0.01) respectively. The gene expression of adiponectin and GLUT4 was increased significantly in swimming group (P<0.05 and P<0.01, respectively).Compared with the model group, fasting insulin, TG, TC and FFA were decreased significantly in the training group (P<0.05 or P<0.01) as compared with model group. It is concluded that exercise can promote the expression of adiponectin mRNA and GLUT4 mRNA in type 2 diabetic rats,which may be one of the mechanisms responsible for the amelioration of insulin resistance in the rats.

  8. Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

    Institute of Scientific and Technical Information of China (English)

    朱长虹; 田红

    2001-01-01

    In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT-PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

  9. 阴道毛滴虫Rac1蛋白的cDNA克隆和序列分析%Molecular Cloning and Characterization of a Rac1 Homologue cDNA from Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    傅玉才; 章家新; 郑晓虹; 刘红

    2004-01-01

    Objective To clone and characterize a Racl homologue from Trichomonas vaginalis for studying cell cycle of the organism. Methods A cDNA library derived from T. vaginalis mRNA was constructed into λ TriplEx2 phage vector. An expression sequence tag program was launched. Sequences of cDNA clones were analyzed using NCBI BLAST algorithms, and ClustalW and Treeview programs. Results A cDNA clone with a length of 714 base pairs was isolated. The sequence analysis showed that the cDNA clone has an open reading frame with 600 bp. The deduced amino acid sequence from the open reading frame contains 200 residuals and is most homologous to Rac1 subfamily of Rho GTPases with > 60% identity. The conserved sequence elements of Rho GTPases, such as GTP-binding sites, GTPase-activating protein (GAP) interaction motifs, GTP-dissociation inhibitors (GDI) interaction motifs, guanine nucleotide exchange factor (GEF) interaction elements, etc, were detected in the amino acid sequence. The phylogenetic analysis showed that the cDNA clone is grouped in the Rac subfamily and is more closely related to Rac1 proteins of protozoa. Conclusion The cDNA clone isolated belongs to Rac subfamily of Rho GTPases and is probably a Rac1 protein of T. vaginalis.%目的获得阴道毛滴虫Rac1蛋白的cDNA克隆,研究其在细胞周期中的调解作用.方法提取阴道毛滴虫总RNA,构建cDNA表达文库,随机分离cDNA克隆并测序.用在线生物分析软件NCBI BLAST、ClustalW以及Treeview等程序进行序列分析.结果获得一株有714 bp的cDNA克隆.序列分析表明,该克隆开放阅读框具600 bp,推测肽链具200个氨基酸.该肽链与Rho家族中Rac1鸟苷三磷酸(GTP)酶同源性最高(>60%),并具多种Rho GTP酶的保守基序,如GTP结合部位、GTP酶激活蛋白作用基序、GTP分离抑制因子作用基序、鸟嘌呤核苷酸交换因子作用基序等.进化树分析显示该克隆属于Rac亚家族GTP酶,与原虫Rac1蛋白最接近.结论该克隆

  10. Elevated neurofilament light chain (NFL) mRNA levels in prediabetic peripheral neuropathy.

    Science.gov (United States)

    Celikbilek, Asuman; Tanik, Nermin; Sabah, Seda; Borekci, Elif; Akyol, Lutfi; Ak, Hakan; Adam, Mehmet; Suher, Murat; Yilmaz, Neziha

    2014-06-01

    Evidence suggests that peripheral nerve injury occurs during the early stages of disease with mild glycemic dysregulation. Two proteins, neuron-specific enolase (NSE) and neurofilament light chain (NFL), have been examined previously as possible markers of neuronal damage in the pathophysiology of neuropathies. Herein, we aimed to determine the potential value of circulatory NSE and NFL mRNA levels in prediabetic patients and in those with peripheral neuropathy. This prospective clinical study included 45 prediabetic patients and 30 age- and sex-matched controls. All prediabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy and nephropathy. mRNA levels of NSE and NFL were determined in the blood by real-time polymerase chain reaction. NSE mRNA levels were similar between prediabetic and control groups (p > 0.05), whereas NFL mRNA levels were significantly higher in prediabetics than in controls (p 0.05), while NFL mRNA levels were significantly higher in prediabetics with peripheral neuropathy than in those without (p = 0.038). According to correlation analysis, NFL mRNA levels were positively correlated with the Douleur Neuropathique 4 questionnaire score in prediabetic patients (r = 0.302, p = 0.044). This is the first study to suggest blood NFL mRNA as a surrogate marker for early prediction of prediabetic peripheral neuropathy, while NSE mRNA levels may be of no diagnostic value in prediabetic patients.

  11. Cotranscriptional assembly of mRNP complexes that determine the cytoplasmic fate of mRNA

    OpenAIRE

    Forget, Amélie; Chartrand, Pascal

    2011-01-01

    Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence has revealed that the full picture is more complex and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA.

  12. Impaired expression of glycogen synthase mRNA in skeletal muscle of NIDDM patients

    DEFF Research Database (Denmark)

    Vestergaard, H; Bjørbaek, C; Andersen, P H

    1991-01-01

    with NIDDM were accompanied by a 39% reduction (P less than 0.02) in the steady state level of GS mRNA per microgram DNA of muscle. In both diabetic and control subjects, the mRNA expression of GS was unaffected after euglycemic-hyperinsulinemic clamp for 4 h. With single-stranded conformation polymorphism...

  13. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Directory of Open Access Journals (Sweden)

    Cristian Del Campo

    2015-10-01

    Full Text Available Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  14. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Science.gov (United States)

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  15. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    NARCIS (Netherlands)

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

    1990-01-01

    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in

  16. Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

    Science.gov (United States)

    Michaud, Morgane; Ubrig, Elodie; Filleur, Sophie; Erhardt, Mathieu; Ephritikhine, Geneviève; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

    2014-06-17

    Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

  17. Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

    KAUST Repository

    Arae, Toshihiro

    2017-04-18

    Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

  18. Distribution of the mRNA for protein phosphatase T in rat brain

    NARCIS (Netherlands)

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG

    1996-01-01

    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  19. Impaired expression of glycogen synthase mRNA in skeletal muscle of NIDDM patients

    DEFF Research Database (Denmark)

    Vestergaard, H; Bjørbaek, C; Andersen, P H

    1991-01-01

    with NIDDM were accompanied by a 39% reduction (P less than 0.02) in the steady state level of GS mRNA per microgram DNA of muscle. In both diabetic and control subjects, the mRNA expression of GS was unaffected after euglycemic-hyperinsulinemic clamp for 4 h. With single-stranded conformation polymorphism...

  20. Neurotrophin-3 mRNA expression in rat intrafusal muscle fibres after denervation and reinnervation

    NARCIS (Netherlands)

    Copray, JCVM; Brouwer, N

    1997-01-01

    We have studied the regulation of the expression of neurotrophin-3 (NT-3) mRNA in neonatal and adult rat muscle spindles after denervation and after denervation followed by reinnervation. Denervation of the intrafusal fibres did not result in an upregulation of the NT-3 mRNA expression but decreased

  1. The intragraft cytokine mRNA pattern reflects the efficacy of steroid anti rejection therapy

    NARCIS (Netherlands)

    Baan, C C; Niesters, H G; Balk, A H; Mochtar, B; Zondervan, P E; Weimar, W

    1996-01-01

    BACKGROUND: We studied the effect of antirejection therapy on intragraft cytokine mRNA expression. METHODS: Therapy consisted of three doses of 1 gm of intravenous methylprednisolone. We determined its effect on intragraft mRNA expression of immunoregulatory (interleukin-2, interleukin-4) and inflam

  2. Distribution of the mRNA for protein phosphatase T in rat brain

    NARCIS (Netherlands)

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG

    1996-01-01

    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  3. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    NARCIS (Netherlands)

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

    1990-01-01

    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in ter

  4. Simultaneous isolation of mRNA and native protein from minute samples of cells

    DEFF Research Database (Denmark)

    Petersen, Tonny Studsgaard; Andersen, Claus Yding

    2014-01-01

    Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native pr...

  5. Relative workload determines exercise-induced increases in PGC-1alpha mRNA

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai Baastrup; Lundby, Carsten; Leick, Lotte;

    2010-01-01

    trial. No change in HIF1alpha, PFK, CS, LDH-A or LDH-B mRNA expression was detected after any of the exercise trials. CONCLUSION:: The relative intensity of brief intermittent exercise is of major importance for the exercise induced increase of several mRNA's, including PGC-1alpha....

  6. Correlation between indoleamine 2,3 dioxygenase mRNA and CDKN2A/p16 mRNA: a combined strategy to cervical cancer diagnosis.

    Science.gov (United States)

    Saffi Junior, Mario Cezar; Duarte, Ivone da Silva; Brito, Rodrigo Barbosa de Oliveira; Prado, Giovana Garcia; Makabe, Sergio; Dellê, Humberto; Camacho, Cleber P

    2016-11-01

    Cervical cancer (CC) is one of the most common cancers among women worldwide. The relation of the human papillomavirus (HPV) with CC and its precursor lesions was first suspected for over 40 years. The indoleamine 2,3 dioxygenase (IDO) is an immune modulator enzyme responsible for the immune system tissue protection mechanism, which may be the key to the tumoural persistence. HPV oncoprotein E7 promotes the increase in cyclin-dependent kinase inhibitor p16 (CDKN2A/p16). The isolated and combined analysis of CDKN2A/p16 mRNA to CC diagnosis was done with promising results. The aim of this study is to evaluate the correlation between IDO mRNA and CDKN2A/p16 mRNA. We will explore the potential of both as diagnostic tools. RNA was extracted from tissue samples. cDNA was generated with High Capacity RNA-to-cDNA kit. The real-time PCR results were analysed using nonlinear curve estimation, ROC curve, Chi-squared test, the proportion of variance explained and Galen and Gambino formulas. From 270 patients attended, colposcopy examination was performed in 110 and the biopsy in 75 patients. We found a positive correlation in patients older than 28 years old with low-risk lesions, but the correlation is lost in high-risk lesions. Although cytology, IDO mRNA and CDKN2A/p16 mRNA could not differentiate the risk groups, IDO combined with CDKN2A/p16 mRNA results could (p = 0.028). The best diagnostic result was achieved by IDO coupled with CDKN2A/p16 mRNA, which may considerably increase the sensitivity of screening for CC.

  7. mRNA expression of adipocytokines and glucocorticoid-related genes are associated with downregulation of E-cadherin mRNA in colorectal adenocarcinomas.

    Science.gov (United States)

    Størkson, Ragnhild H; Aamodt, Rolf; Vetvik, Katja Kannisto; Pietilainen, Kirsi; Bukholm, Geir; Jonsdottir, Kristin; Vollan, Hilde S; Sonerud, Tonje; Lüders, Torben; Jacobsen, Morten B; Bukholm, Ida R K

    2012-08-01

    There is a consistently reported relationship between the incidence of colon cancer and obesity. It is thought that adipose tissue, particularly visceral fat, which secretes systemic factors that alter immunological, metabolic and endocrine milieu and promotes insulin resistance by producing adipocytokines, is important in cancer progression. Systemic high concentrations of adipocytokines, such as tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and glucocorticoid metabolism-related genes have been associated with gastrointestinal cancer. However, limited information exists about the expression of these cytokines within tumour tissue. mRNA expression of TNF-α, IL-6,IL-8, IL-10, IL-1RN, glucocorticoid receptor alpha (GR-α), 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), plasminogen activator inhibitor-1 (PAI-1), Slug, vimentin, Snail and E-cadherin was analysed in paired samples of tumour tissue and normal mucosa in 60 surgical patients for Dukes B and C colorectal adenocarcinomas using quantitative reverse transcription PCR and microarray technology. The mRNA expression level of analysed genes was compared between tumour tissue and normal mucosa from the same patients, and a correlation to mRNA expression of E-cadherin in the same tissue samples was also performed. A highly significant difference in mRNA expression level of several of the analysed genes was observed between tumour tissue and the normal intestinal mucosa. Inverse correlation between mRNA expression of 11βHSD1, IL-6, GR-α and PAI-1 on one hand and mRNA expression of E-cadherin on the other hand was observed. Results show that the adipocytokines and glucocorticoid metabolism-related genes are overexpressed in colorectal adenocarcinomas, and expression of these genes is associated with the downregulation of E-cadherin mRNA, connecting these genes to carcinogenesis and progression of colorectal cancer.

  8. Relationship Between Heart Damages and HSPs mRNA in Persistent Heat Stressed Broilers

    Institute of Scientific and Technical Information of China (English)

    SUN Pei-ming; LIU Yu-tian; ZHAO Yong-gang; BAO En-dong; WANG Zhi-liang

    2007-01-01

    The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activities of creatine kinase (CK) and glutamic-pyruvic transaninase (GPT) were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC70) mRNA was changeable. The transcription of heat shock protein 70 (HSP70) mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.

  9. The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民

    2003-01-01

    Objective To evaluate the potential role of insulin-like growth factor-1 receptor mRNA(IGF-IR mRNA) in the onset and development of retinopathy in diabetic rats.Methods A diabetic model was duplicated in Wistar rats. The early changes in the retina were examined using light and transmission electron microscopy. Expression of IGF-IR mRNA was analyzed using in situ hybridization.Results Weak expression of IGF-IR mRNA(5%) was found in retinas of normal rats, but was significantly increased (15% and 18%) in the retinas of diabetic rats after 3 and 6 months of diabetes (P<0.01). In situ hybridization and morphological study demonstrated that there was a positive correlation between IGF-IR mRNA expression and retinal changes at various stages.Conclusion Increased IGF-IR mRNA might play an important role in the onset and development of diabetic retinopathy.

  10. Expression of hippocampal adrenergic receptor mRNA in a rat model of depression

    Institute of Scientific and Technical Information of China (English)

    Jianbin Zhang; Lingling Wang; Xinjun Wang; Jingfeng Jiang; Xiaoren Xiang; Tianjun Wang

    2011-01-01

    Adrenergic receptor dysfunction is suggested as a potential cause of hippocampal vulnerability to stress-related pathology. We examined mRNA expression of adrenergic receptor (AR) subtypes α1-AR, α2-AR, and β1-AR in hippocampal subregions (CA1, CA3, dentate gyrus) using in situ hybridization in a depression model induced by chronic unpredictable mild stress and social isolation. α1-AR mRNA expression was significantly increased in the CA3 and dentate gyrus, β1-AR mRNA was significantly increased in the CA1, and α2-AR mRNA remained unchanged in all regions of depression rats compared with controls. Thus, different AR subtypes exhibit a differing pattern of mRNA expression in various hippocampal subregions following depression.

  11. Quantification of mRNA Levels Using Real-Time Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Li, Yiyi; Wang, Kai; Chen, Longhua; Zhu, Xiaoxia; Zhou, Jie

    2016-01-01

    Real-time quantitative reverse transcription PCR technique has advanced greatly over the past 20 years. Messenger RNA (mRNA) levels in cells or tissues can be quantified by this approach. It is well known that changes in mRNA expression in disease, and correlation of mRNA expression profiles with clinical parameters, serve as clinically relevant biomarkers. Hence, accurate determination of the mRNA levels is critically important in describing the biological, pathological, and clinical roles of genes in health and disease. This chapter describes a real-time PCR approach to detect and quantify mRNA expression levels, which can be used for both laboratorial and clinical studies in breast cancer research.

  12. Challenges and advances towards the rational design of mRNA vaccines.

    Science.gov (United States)

    Pollard, Charlotte; De Koker, Stefaan; Saelens, Xavier; Vanham, Guido; Grooten, Johan

    2013-12-01

    In recent years, mRNA vaccines have emerged as a safe and potent approach for the induction of cellular immune responses. Whereas initial studies were limited to the ex vivo loading of dendritic cells (DCs) with antigen-encoding mRNA, recent progress has led to the development of improved mRNA vaccines that enable direct in vivo targeting of DCs. Although preclinical studies demonstrated their potency in inducing antitumor immunity, several bottlenecks hinder the broader application of mRNA vaccines. In this review, we discuss the challenges associated with mRNA-based vaccination strategies, the technological advances that have been made to overcome these limitations, and the hurdles that remain to be tackled for the development of an optimal mRNA vaccine.

  13. Glucocorticoids modulate BDNF mRNA expression in the rat hippocampus after traumatic brain injury.

    Science.gov (United States)

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2000-10-20

    Brain-derived neurotrophic factor (BDNF) expression in rat hippocampus is increased after experimental traumatic brain injury (TBI) and may be neuroprotective. Glucocorticoids are important regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) on the expression of BDNF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury (FPI) and in situ hybridization to evaluate the expression of BDNF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomized rats (with or without corticosterone replacement). FPI and ADX independently increased expression of BDNF mRNA. In animals undergoing FPI, prior ADX caused further elevation of BDNF mRNA and this upregulation was prevented by corticosterone replacement in ADX rats. These findings suggest that glucocorticoids are involved in the modulation of the BDNF mRNA response to TBI.

  14. Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury.

    Science.gov (United States)

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2001-02-23

    Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.

  15. Ontogeny of pituitary growth hormone and growth hormone mRNA in the chicken.

    Science.gov (United States)

    McCann-Levorse, L M; Radecki, S V; Donoghue, D J; Malamed, S; Foster, D N; Scanes, C G

    1993-01-01

    The changes in pituitary growth hormone (GH) mRNA levels have been determined by Northern blot analysis and laser densitometry during embryonic development and posthatch growth of white Leghorn cockerels. Pituitary GH mRNA levels were observed to progressively increase between 18 days of embryonic development to a maximum at 4 weeks of age (posthatch). Subsequently, pituitary GH mRNA levels declined between 4 and 8 weeks of age, and between 12 weeks of age and adulthood. Pituitary GH contents showed increases during embryonic development and posthatch growth that paralleled the rise in GH mRNA. The decline in pituitary GH mRNA levels between 4 weeks of age and adulthood occurs when GH secretion has been observed previously to decline.

  16. Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin M1 resistance.

    Science.gov (United States)

    Kawachi, R; Akashi, T; Kamitani, Y; Sy, A; Wangchaisoonthorn, U; Nihira, T; Yamada, Y

    2000-04-01

    Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a gamma-butyrolactone autoregulator and the corresponding receptor protein of the Streptomyces species, and has been shown to regulate the production of antibiotic virginiamycin (VM) in S. virginiae. A divergently transcribed barX gene is situated 259 bp upstream of the barA gene, and the BarX protein has been shown to be highly homologous (39.8% identity, 74. 6% similarity) to S. griseus AfsA. Although AfsA is thought to be a biosynthetic enzyme for A-factor, another member of the family of gamma-butyrolactone autoregulators, the in vivo function of S. virginiae BarX was investigated in this study by phenotypic and transcriptional comparison between wild-type S. virginiae and a barX deletion mutant. With the same growth rate as wild-type S. virginiae on both solid and liquid media, the barX mutant showed no apparent changes in its morphological behaviour, indicating that barX does not participate in morphological control in S. virginiae. However, the barX mutant became more sensitive to virginiamycin M1 than did the wild-type strain (minimum inhibitory concentration, 50 microgram ml-1 compared with > 200 microgram ml-1) and exhibited reduced VB and VM production. The VM production was not restored by exogenous addition of VB, suggesting that BarX per se is not a biosynthetic enzyme of VBs but a pleiotropic regulatory protein controlling VB biosynthesis. DNA sequencing of a 5.6 kbp downstream region of barX revealed the presence of five open reading frames (ORFs): barZ, encoding a BarB-like regulatory protein; orf2, encoding a Streptomyces coelicolor RedD-like pathway specific regulator; varM, encoding a homologue of ATP-dependent transporters for macrolide antibiotics; orf4, encoding a homologue of beta-ketoacyl ACP/CoA reductase; and orf5, encoding a homologue of dNDP-glucose dehydratase. Reverse transcription polymerase chain reaction (RT-PCR) analyses of the

  17. The impact of different intensities of green light on the bacteriochlorophyll homologue composition of the Chlorobiaceae Prosthecochloris aestuarii and Chlorobium phaeobacteroides.

    Science.gov (United States)

    Massé, Astrid; Airs, Ruth L; Keely, Brendan J; de Wit, Rutger

    2004-08-01

    Members of the Chlorobiaceae and Chloroflexaceae are unique among the phototrophic micro-organisms in having a remarkably rich chlorophyll pigment diversity. The physiological regulation of this diversity and its ecological implications are still enigmatic. The bacteriochlorophyll composition of the chlorobiaceae Prosthecochloris aestuarii strain CE 2404 and Chlorobium phaeobacteroides strain UdG 6030 was therefore studied by both HPLC with photodiode array (PDA) detection and liquid chromatography-mass spectrometry (LC-MS). These strains were grown in liquid cultures under green light (480-615 nm) at different light intensities (0.2-55.7 micromol photons m(-2) s(-1)), simulating the irradiance regime at different depths of the water column of deep lakes. The specific growth rates of Ptc. aestuarii under green light achieved a maximum of 0.06 h(-1) at light intensities exceeding 6 micromol photons m(-2) s(-1), lower than the maximum observed under white light (approx. 0.1 h(-1)). The maximal growth rates of Chl. phaeobacteroides under green light were slightly higher (0.07 h(-1)) than observed for Ptc. aestuarii and were achieved at 3.5 and 4.3 micromol photons m(-2) s(-1). LC-MS/MS analysis of pigment extracts revealed most (>90 %) BChl c homologues of Ptc. aestuarii to be esterified with farnesol. The homologues differed in mass by multiples of 14 Da, reflecting different alkyl subsituents at positions C-8 and C-12 on the tetrapyrrole macrocycle. The relative proportions of the individual homologues varied only slightly among different light intensities. The specific content of BChl c was maximal at 3-5 micromol photons m(-2) s(-1) [400+/-150 nmol BChl c (mg protein)(-1)]. In the case of Chl. phaeobacteroides, the specific content of BChl e was maximal at 4.3 micromol photons m(-2) s(-1) [115 nmol BChl e (mg protein)(-1)], and this species was characterized by high carotenoid (isorenieratene) contents. The major BChl e forms were esterified with a range of

  18. Cellular stress increases RGS2 mRNA and decreases RGS4 mRNA levels in SH-SY5Y cells.

    Science.gov (United States)

    Song, Ling; Jope, Richard S

    2006-07-24

    Modulation of the expression of regulator of G-protein signaling (RGS) proteins is a major mechanism used to modulate their actions. Besides control by second messengers, the expression of RGS proteins, particularly RGS2, can be regulated by cell stress. Because RGS2 and RGS4 expression can be regulated by the cell cycle, we examined if cell cycle signals are involved in their regulation following stress. Treatment of SH-SY5Y cells with camptothecin increased RGS2 mRNA and decreased RGS4 mRNA levels. This effect on RGS2 mRNA was blocked by the cyclin-dependent kinase-2 (cdk2) inhibitors roscovitine and purvalanol. Cell cycle arrest was further implicated in regulating RGS mRNA levels because geldanamycin, which causes cell cycle arrest by inhibiting the actions of heat shock protein 90, caused changes in the mRNA levels of RGS2 and RGS4 similar to, and additive with, the effects of camptothecin. Overall, these results indicate that cell cycle arrest regulates the expression of RGS2 and RGS4, and that the expression of these two RGS family members is oppositely regulated by stress that causes cell cycle arrest.

  19. Structure, supramolecular organization and phase behavior of N-acyl-β-alanines: Structural homologues of mammalian brain constituents N-acylglycine and N-acyl-GABA.

    Science.gov (United States)

    Sivaramakrishna, D; Swamy, Musti J

    2016-12-01

    N-Acyl-β-alanines (NABAs) are structural homologues of N-acylglycines (NAGs) and N-acyl-γ-aminobutyric acids (NAGABAs), and achiral isomers of N-acylalanines, which are all present in mammalian brain and other tissues and modulate activity of biological receptors with various functions. In the present study, we synthesized and characterized a homologous series of NABAs bearing saturated acyl chains (n=8-20) and investigated their supramolecular organization and thermotropic phase behavior. In differential scanning calorimetric (DSC) studies, most of the NABAs gave one or two minor transitions before the main chain-melting phase transition in the dry state as well as upon hydration with water, but gave only a single transition when hydrated with buffer (pH7.6). Transition enthalpies (ΔHt) and entropies (ΔSt), obtained from the DSC studies showed linear dependence on the chain length in the dry state and upon hydration with buffer, whereas odd-even alteration was observed when hydrated with water. The crystal structures of N-lauroyl-β-alanine (NLBA) and N-myristoyl-β-alanine (NMBA) were solved in monoclinic system in the P21/c space group. Both NLBA and NMBA were packed in tilted bilayers with head-to-head (and tail-to-tail) arrangement with tilt angles of 33.28° and 34.42°, respectively. Strong hydrogen bonding interactions between COOH groups of the molecules from opposite leaflets as well as NH⋯O hydrogen bonds between the amide groups from adjacent molecules in the same leaflet as well as dispersion interactions between the acyl chains stabilize the bilayer structure. The d-spacings calculated from powder X-ray diffraction studies showed odd-even alteration with odd-chain length compounds exhibiting higher values as compared to the even-chain length ones and the tilt angles calculated from the PXRD data are higher for the even chain NABAs. These observations are relevant to developing structure-activity relationships for these amphiphiles and understand

  20. Structure of a NifS homologue: X-ray structure analysis of CsdB, an Escherichia coli counterpart of mammalian selenocysteine lyase.

    Science.gov (United States)

    Fujii, T; Maeda, M; Mihara, H; Kurihara, T; Esaki, N; Hata, Y

    2000-02-15

    Escherichia coli CsdB, a NifS homologue with a high specificity for L-selenocysteine, is a pyridoxal 5'-phosphate (PLP)-dependent dimeric enzyme that belongs to aminotransferases class V in fold-type I of PLP enzymes and catalyzes the decomposition of L-selenocysteine into selenium and L-alanine. The crystal structure of the enzyme has been determined by the X-ray crystallographic method of multiple isomorphous replacement and refined to an R-factor of 18.7% at 2.8 A resolution. The subunit structure consists of three parts: a large domain of an alpha/beta-fold containing a seven-stranded beta-sheet flanked by seven helices, a small domain containing a four-stranded antiparallel beta-sheet flanked by three alpha-helices, and an N-terminal segment containing two alpha-helices. The overall fold of the subunit is similar to those of the enzymes belonging to the fold-type I family represented by aspartate aminotransferase. However, CsdB has several structural features that are not observed in other families of the enzymes. A remarkable feature is that an alpha-helix in the lobe extending from the small domain to the large domain in one subunit of the dimer interacts with a beta-hairpin loop protruding from the large domain of the other subunit. The extended lobe and the protruded beta-hairpin loop form one side of a limb of each active site in the enzyme. The most striking structural feature of CsdB lies in the location of a putative catalytic residue; the side chain of Cys364 on the extended lobe of one subunit is close enough to interact with the gamma-atom of a modeled substrate in the active site of the subunit. Moreover, His55 from the other subunit is positioned so that it interacts with the gamma- or beta-atom of the substrate and may be involved in the catalytic reaction. This is the first report on three-dimensional structures of NifS homologues.

  1. Bonding analysis using localized relativistic orbitals: water, the ultrarelativistic case and the heavy homologues H2X (X = Te, Po, eka-Po).

    Science.gov (United States)

    Dubillard, S; Rota, J-B; Saue, T; Faegri, K

    2006-04-21

    We report the implementation of Pipek-Mezey [J. Chem. Phys. 90, 4916 (1989)] localization of molecular orbitals in the framework of a four-component relativistic molecular electronic structure theory. We have used an exponential parametrization of orbital rotations which allows the use of unconstrained optimization techniques. We demonstrate the strong basis set dependence of the Pipek-Mezey localization criterion and how it can be eliminated. We have employed localization in conjunction with projection analysis to study the bonding in the water molecule and its heavy homologues. We demonstrate that in localized orbitals the repulsion between hydrogens in the water molecule is dominated by electrostatic rather than exchange interactions and that freezing the oxygen 2s orbital blocks polarization of this orbital rather than hybridization. We also point out that the bond angle of the water molecule cannot be rationalized from the potential energy alone due to the force term of the molecular virial theorem that comes into play at nonequilibrium geometries and which turns out to be crucial in order to correctly reproduce the minimum of the total energy surface. In order to rapidly assess the possible relativistic effects we have carried out the geometry optimizations of the water molecule at various reduced speed of light with and without spin-orbit interaction. At intermediate speeds, the bond angle is reduced to around 90 degrees , as is known experimentally for H(2)S and heavier homologues, although our model of ultrarelativistic water by construction does not allow any contribution from d orbitals to bonding. At low speeds of light the water molecule becomes linear which is in apparent agreement with the valence shell electron pair repulsion (VSEPR) model since the oxygen 2s12 and 2p12 orbitals both become chemically inert. However, we show that linearity is brought about by the relativistic stabilization of the (n + 1)s orbital, the same mechanism that leads to an

  2. Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation.

    Science.gov (United States)

    Srivastava, Sudhakar; Sangwan, Rajender Singh; Tripathi, Sandhya; Mishra, Bhawana; Narnoliya, L K; Misra, L N; Sangwan, Neelam S

    2015-11-01

    Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control

  3. Short hairpin RNA targeting NP mRNA inhibiting Newcastle disease virus production and other viral structural mRNA transcription.

    Science.gov (United States)

    Yue, Hua; Deng, Shu; Yang, Fa-Long; Li, Ding-Fei; Fu, An-Jing; Yang, Fan; Tang, Cheng

    2009-02-01

    Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiological agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry. Better understanding of the molecular basis of viral structural genes involved with production should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here we show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding mRNA but also P, HN, F, M gene mRNA. The findings reveal that newly synthesized NP mRNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

  4. Heat shock response in yeast involves changes in both transcription rates and mRNA stabilities.

    Directory of Open Access Journals (Sweden)

    Laia Castells-Roca

    Full Text Available We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25 °C to 37 °C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins.

  5. Circulating hTERT mRNA as a tumor marker in cholangiocarcinoma patients

    Institute of Scientific and Technical Information of China (English)

    Kawin Leelawat; Surang Leelawat; Thawee Ratanachu-Ek; Somboon Trubwongchareon; Jerasak Wannaprasert; Saad Tripongkaruna; Suchart Chantawibul; Panadda Tepaksorn

    2006-01-01

    AIM: To investigate human telomerase reverse transcriptase (hTERT) mRNA in the serum of cholangiocarcinoma patients.METHODS: The serum of thirty three cholangiocarcinoma patients, forty one benign biliary tract disease patients and ten healthy volunteers were collected and analyzed for the expression of hTERT mRNA by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).We then examined the correlation between values of serum hTERT mRNA and the pathological staging of cholangiocarcinoma.RESULTS: hTERT mRNA was detected in 28 of 33(84.85%) of serum obtained from cholangiocarcinoma patients and 9 of 41 (21.9%) of serum obtained from benign biliary tract disease patients. hTERT mRNA was not detected in any serum obtained from healthy volunteers. on the other hand the common tumor marker, CA19-9 was detected in 20 of 33 (60.6%) of serum obtained from cholangiocarcinoma patients and 8 of 41 (19.5%) of serum obtained from benign biliary tract disease patients. However, no correlation was found between the present of serum hTERT mRNA and tumor staging.CONCLUSION: These results indicate that the detection of circulating hTERT mRNA was identified in almost all cholangiocarcinoma patients. It offers anovel tumor marker, which can be used as a complementary study for diagnosis of cholangiocarcinoma.

  6. Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation.

    Science.gov (United States)

    Mezquita, Pau; Parghi, Sean S; Brandvold, Kimberly A; Ruddell, Alanna

    2005-01-27

    Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

  7. Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

    Science.gov (United States)

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Fleischer, Jennifer G; Woster, Patrick M; Casero, Robert A

    2005-03-15

    The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

  8. Myxovirus Resistance Protein A mRNA Expression Kinetics in Multiple Sclerosis Patients Treated with IFNβ

    Science.gov (United States)

    Libertinova, Jana; Meluzinova, Eva; Tomek, Ales; Horakova, Dana; Kovarova, Ivana; Matoska, Vaclav; Kumstyrova, Simona; Zajac, Miroslav; Hyncicova, Eva; Liskova, Petra; Houzvickova, Eva; Martinkovic, Lukas; Bojar, Martin; Havrdova, Eva; Marusic, Petr

    2017-01-01

    Introduction Interferon-β (IFNß) is the first-line treatment for relapsing-remitting multiple sclerosis. Myxovirus resistance protein A (MxA) is a marker of IFNß bioactivity, which may be reduced by neutralizing antibodies (NAbs) against IFNß. The aim of the study was to analyze the kinetics of MxA mRNA expression during long-term IFNβ treatment and assess its predictive value. Methods A prospective, observational, open-label, non-randomized study was designed in multiple sclerosis patients starting IFNß treatment. MxA mRNA was assessed prior to initiation of IFNß therapy and every three months subsequently. NAbs were assessed every six months. Assessment of relapses was scheduled every three months during 24 months of follow up. The disease activity was correlated to the pretreatment baseline MxA mRNA value. In NAb negative patients, clinical status was correlated to MxA mRNA values. Results 119 patients were consecutively enrolled and 107 were included in the final analysis. There was no correlation of MxA mRNA expression levels between baseline and month three. Using survival analysis, none of the selected baseline MxA mRNA cut off points allowed prediction of time to first relapse on the treatment. In NAb negative patients, mean MxA mRNA levels did not significantly differ in patients irrespective of relapse status. Conclusion Baseline MxA mRNA does not predict the response to IFNß treatment or the clinical status of the disease and the level of MxA mRNA does not correlate with disease activity in NAb negative patients. PMID:28081207

  9. Isolation and amplification of mRNA within a simple microfluidic lab on a chip.

    Science.gov (United States)

    Reinholt, Sarah J; Behrent, Arne; Greene, Cassandra; Kalfe, Ayten; Baeumner, Antje J

    2014-01-07

    The major modules for realizing molecular biological assays in a micro-total analysis system (μTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 10(15) fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.

  10. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

    2016-08-05

    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

  11. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    Directory of Open Access Journals (Sweden)

    Sayaka Aizawa

    Full Text Available The pars tuberalis (PT is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD, such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2 mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

  12. COX-2 mRNA expression in esophageal squamous cell carcinoma (ESCC) and effect by NSAID.

    Science.gov (United States)

    Liu, X; Li, P; Zhang, S-T; You, H; Jia, J-D; Yu, Z-L

    2008-01-01

    To investigate cyclooxygenase-2 (COX-2) mRNA expression in human esophageal squamous cell carcinoma and the effect of a non-steroidal anti-inflammatory drug (NSAID) on it, in order to explore the mechanism of COX-2 in esophageal squamous cell carcinoma (ESCC) carcinogenesis and the ability of NSAID to prevent or treat ESCC. Frozen specimens of human ESCC and adjacent normal esophageal squamous epithelium pairs (n = 22) were examined for COX-2 mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). After incubation with aspirin (a non-selective COX inhibitor) or Nimesulide (a selective COX-2 inhibitor), the proliferation status of two human esophageal squamous cancer cell lines, EC-9706 and EC-109, was quantified by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide assay. The expression of COX-2 mRNA in these cells was detected by RT-PCR. COX-2 mRNA was expressed in 12 of 22 (54.5%) ESCC tissue samples, but it was undetectable in all the specimens of adjacent normal esophageal squamous epithelium COX-2 mRNA expression. Both aspirin (5-20 mmol/L) and Nimesulide (0.1-0.8 mmol/L) inhibited EC-9706 cell line proliferation and suppressed its COX-2 mRNA expression dose-dependently. However, only aspirin (5-20 mmol/L) could inhibit proliferation in the EC-109 cell line and suppress COX-2 mRNA expression. Nimesulide (0.1-0.8 mmol/L) could neither inhibit EC-109 cell growth nor suppress COX-2 mRNA expression. COX-2 mRNA expression is a frequent phenomenon in human ESCC tissue samples and plays an important role in the carcinogenesis of ESCC. NSAID may be useful in the chemoprevention and therapy of human ESCC and its effects are likely to be mediated by modulating COX-2 activity.

  13. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

    DEFF Research Database (Denmark)

    Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina

    2003-01-01

    between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed...

  14. Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

    Science.gov (United States)

    Sartor, Francesca; Anderson, Jihan; McCaig, Colin; Miedzybrodzka, Zosia; Müller, Berndt

    2015-12-01

    Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention.

  15. An Experimental Study on the Expression of Heme Oxygenase-2 mRNA in Hirschsprung's Disease

    Institute of Scientific and Technical Information of China (English)

    朱珉; 魏明发; 刘芳

    2002-01-01

    Summary: In order to investigate the relationship between the expression of heme oxygenase-2 (HO-2) mRNA and the pathogenesis of Hirschsprung's disease (HD), total ribonucleic acid (RNA) was extracted in the aganglionic and ganglionic segments of colon respectively from 15 cases of HD. The single-stranded cDNA of HO-2 was synthesized and further amplified by reverse transcription-poly merase chain reaction (RT-PCR). The expression of HO-2 mRNA was normal in ganglionic seg ments, but absent in aganglionic segments. It is concluded that the absence of HO-2 mRNA expres sion may be an important mechanism responsible for HD.

  16. Assessing mRNA nuclear export in mammalian cells by microinjection.

    Science.gov (United States)

    Lee, Eliza S; Palazzo, Alexander F

    2017-08-15

    The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Overexpression of protease nexin-1 mRNA and protein in oral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Gao, Shan; Krogdahl, Annelise; Sørensen, Jens Ahm;

    2007-01-01

    -1 has been almost totally neglected. We have now compared the level of PN-1 mRNA in 20 cases of oral squamous cell carcinomas and in matched samples of the corresponding normal oral tissues. We found that the average PN-1 mRNA level in tumours and normal tissues was significantly different, being...... increased up to 13 fold in tumour samples compared with the average level in normal tissues. The PN-1 mRNA level was significantly higher in tumours from patients with lymph node metastasis than in tumours from patients without. We could conclude that PN-1 is frequently overexpressed in oral squamous cell...

  18. Stable RNA hairpins in 88 coding regions of human mRNA

    Institute of Scientific and Technical Information of China (English)

    PAN Min; WANG Chuanming; LIU Ciquan

    2004-01-01

    RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R=G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

  19. The topological configuration and conformational analysis of mRNA in translation

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The theoretical model construction of mRNA hairpin structure and single-stranded structure as well as the simulation studies on RNA structure determined by the X-ray crystal diffraction and nuclear magnetic resonance revealed that in translation, after mRNA being unfolded into single-stranded structure, its topological configuration was closely correlative with the original hairpin structure. The conformational features of single-stranded mRNA appeared as helical regions alternating with curly regions to different extents, which might exert the influence on the folding of nascent polypeptide by various regulating effects including different translational rates.

  20. The wild boar (Sus scrofa Lymphocyte function-associated antigen-1 (CD11a/CD18 receptor: cDNA sequencing, structure analysis and comparison with homologues

    Directory of Open Access Journals (Sweden)

    Bergh Philippe

    2007-10-01

    Full Text Available Abstract Background The most predominant beta2-integrin lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2, expressed on all leukocytes, is essential for many adhesive functions of the immune system. Interestingly, RTX toxin-producing bacteria specifically target this leukocyte beta2-integrin which exacerbates lesions and disease development. Results This study reports the sequencing of the wild boar beta2-integrin CD11a and CD18 cDNAs. Predicted CD11a and CD18 subunits share all the main structural characteristics of their mammalian homologues, with a larger interspecies conservation for the CD18 than the CD11a. Besides these strong overall similarities, wild boar and domestic pig LFA-1 differ by 2 (CD18 and 1 or 3 (CD11a substitutions, of which one is located in the crucial I-domain (CD11a, E168D. Conclusion As most wild boars are seropositive to the RTX toxin-producing bacterium Actinobacillus pleuropneumoniae and because they have sustained continuous natural selection, future studies addressing the functional impact of these polymorphisms could bring interesting new information on the physiopathology of Actinobacillus pleuropneumoniae-associated pneumonia in domestic pigs.

  1. Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

    Science.gov (United States)

    Eklund, D Magnus; Thelander, Mattias; Landberg, Katarina; Ståldal, Veronika; Nilsson, Anders; Johansson, Monika; Valsecchi, Isabel; Pederson, Eric R A; Kowalczyk, Mariusz; Ljung, Karin; Ronne, Hans; Sundberg, Eva

    2010-04-01

    The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

  2. The shf gene of a Shigella flexneri homologue on the virulent plasmid pAA2 of enteroaggregative Escherichia coli 042 is required for firm biofilm formation.

    Science.gov (United States)

    Fujiyama, Rika; Nishi, Junichiro; Imuta, Naoko; Tokuda, Koichi; Manago, Kunihiro; Kawano, Yoshifumi

    2008-05-01

    Enteroaggregative Escherichia coli (EAEC) is an increasingly important cause of diarrhea in both developing and industrialized countries, and is characterized by strong biofilm formation on the intestinal mucosa. Sequencing of the virulent plasmid pAA2 of the prototype EAEC 042 revealed a cluster of three open reading frames (ORFs; shf, capU, and virK) ca. 93% identical to a similar cluster located in Shigella flexneri. The function of the first ORF Shf protein is not known, but the closest well-characterized homologue is the IcaB protein of Staphylococcus epidermidis, which plays a crucial role in exopolysaccharide modification in bacterial biofilm formation. To investigate the role of this cluster in the virulence of EAEC, we mutated three genes at this locus. All the mutants maintained the aggregative phenotype in the liquid phase. However, the insertional mutant of shf formed a less abundant biofilm in a microtiter plate assay than did the wild type, while the capU mutant and the virK mutant did not. The complementation of the shf mutant with this cluster restored the thick biofilm similar to that of the wild type. The shf transcriptional level decreased in the transcriptional regulator aggR mutant and was restored when the mutant was complemented with aggR. These results suggest that the shf gene is required for the firm biofilm formation of EAEC 042, and transcription of the shf gene is dependent on AggR.

  3. LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

    Science.gov (United States)

    Pazour, Gregory J.; Koutoulis, Anthony; Benashski, Sharon E.; Dickert, Bethany L.; Sheng, Hong; Patel-King, Ramila S.; King, Stephen M.; Witman, George B.

    1999-01-01

    Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility. PMID:10512883

  4. Co-precipitation of phosphate and iron limits mitochondrial phosphate availability in Saccharomyces cerevisiae lacking the yeast frataxin homologue (YFH1).

    Science.gov (United States)

    Seguin, Alexandra; Santos, Renata; Pain, Debkumar; Dancis, Andrew; Camadro, Jean-Michel; Lesuisse, Emmanuel

    2011-02-25

    Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.

  5. Initial characterization of a bolA homologue from Pseudomonas fluorescens indicates different roles for BolA-like proteins in P. fluorescens and Escherichia coli.

    Science.gov (United States)

    Koch, Birgit; Nybroe, Ole

    2006-09-01

    The RpoS-regulated bolA gene in Escherichia coli is important for the decrease in cell size during stationary phase or sudden carbon starvation. A Pseudomonas fluorescens strain mutated in a gene with homology to bolA reduced its cell size upon carbon starvation, and RpoS had little effect on bolA expression. The mutant grew slower than the wild-type strain in minimal medium with L-serine as the sole nitrogen source, while growth rates were similar on a mixture of L-serine and L-cysteine. Reverse transcriptase polymerase chain reaction analysis indicated that the bolA homologue is the second gene in an operon where the two next ORFs encode putative proteins with homology to sulphurtransferases and protein disulphide isomerases. Complementation of the mutant phenotypes was only obtained by plasmids encoding BolA as well as the above two proteins. Growth phenotypes and gene homologies suggest that BolA-like proteins have different functions in E. coli and Pseudomonas.

  6. SLAH1, a homologue of the slow type anion channel SLAC1, modulates shoot Cl − accumulation and salt tolerance in Arabidopsis thaliana

    KAUST Repository

    Qiu, Jiaen

    2016-06-23

    Salinity tolerance is correlated with shoot chloride (Cl–) exclusion in multiple crops, but the molecular mechanisms of long-distance Cl– transport are poorly defined. Here, we characterize the in planta role of AtSLAH1 (a homologue of the slow type anion channel-associated 1 (SLAC1)). This protein, localized to the plasma membrane of root stelar cells, has its expression reduced by salt or ABA, which are key predictions for a protein involved with loading Cl– into the root xylem. Artificial microRNA knockdown mutants of AtSLAH1 had significantly reduced shoot Cl− accumulation when grown under low Cl–, whereas shoot Cl– increased and the shoot nitrate/chloride ratio decreased following AtSLAH1 constitutive or stelar-specific overexpression when grown in high Cl–. In both sets of overexpression lines a significant reduction in shoot biomass over the null segregants was observed under high Cl– supply, but not low Cl– supply. Further in planta data showed AtSLAH3 overexpression increased the shoot nitrate/chloride ratio, consistent with AtSLAH3 favouring nitrate transport. Heterologous expression of AtSLAH1 in Xenopus laevis oocytes led to no detectible transport, suggesting the need for post-translational modifications for AtSLAH1 to be active. Our in planta data are consistent with AtSLAH1 having a role in controlling root-to-shoot Cl– transport.

  7. Re-fraction: a machine learning approach for deterministic identification of protein homologues and splice variants in large-scale MS-based proteomics.

    Science.gov (United States)

    Yang, Pengyi; Humphrey, Sean J; Fazakerley, Daniel J; Prior, Matthew J; Yang, Guang; James, David E; Yang, Jean Yee-Hwa

    2012-05-04

    A key step in the analysis of mass spectrometry (MS)-based proteomics data is the inference of proteins from identified peptide sequences. Here we describe Re-Fraction, a novel machine learning algorithm that enhances deterministic protein identification. Re-Fraction utilizes several protein physical properties to assign proteins to expected protein fractions that comprise large-scale MS-based proteomics data. This information is then used to appropriately assign peptides to specific proteins. This approach is sensitive, highly specific, and computationally efficient. We provide algorithms and source code for the current version of Re-Fraction, which accepts output tables from the MaxQuant environment. Nevertheless, the principles behind Re-Fraction can be applied to other protein identification pipelines where data are generated from samples fractionated at the protein level. We demonstrate the utility of this approach through reanalysis of data from a previously published study and generate lists of proteins deterministically identified by Re-Fraction that were previously only identified as members of a protein group. We find that this approach is particularly useful in resolving protein groups composed of splice variants and homologues, which are frequently expressed in a cell- or tissue-specific manner and may have important biological consequences.

  8. Structural studies on a non toxic homologue of type II RIPs from bitter gourd: Molecular basis of non toxicity, conformational selection and glycan structure

    Indian Academy of Sciences (India)

    Thyageshwar Chandran; Alok Sharma; M Vijayan

    2015-12-01

    The structures of nine independent crystals of bitter gourd seed lectin (BGSL), a non-toxic homologue of type II RIPS, and its sugar complexes have been determined. The four-chain, two-fold symmetric, protein is made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. The lectin chain is made up of two domains. Each domain carries a carbohydrate binding site in type II RIPS of known structure. BGSL has a sugar binding site only on one domain, thus impairing its interaction at the cell surface. The adenine binding site in the catalytic chain is defective. Thus, defects in sugar binding as well as adenine binding appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well defined conformations of a surface loop in the lectin chain. One of them is chosen in the sugar complexes, in a case of conformational selection, as the chosen conformation facilitates an additional interaction with the sugar, involving an arginyl residue in the loop. The -glycosylation of the lectin involves a plant-specific glycan while that in toxic type H RIPS of known structure involves a glycan which is animal as well as plant specific.

  9. CDX2 Inhibits Invasion and Migration of Gastric Cancer Cells by Phosphatase and Tensin Homologue Deleted from Chromosome 10/Akt Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Liu

    2015-01-01

    Full Text Available Background: Gastric cancer (GC is one of the most prevalent malignancies in the world today, with a high mortality rate. CDX2 is a Drosophila caudal-related homeobox transcription factor that plays an important role in GC. Phosphatase and tensin homologue deleted from chromosome 10 (PTEN is an important tumor suppressor which is widely expressed in normal human tissues. The aim of the study was to determine the relationship and mechanism between CDX2 and PTEN in invasion and migration of GC cells. Methods: pcDNA3-CDX2 plasmids were transfected into MGC-803 cells to up-regulate CDX2 protein, and small interfering RNA-CDX2 was transfected to down-regulate CDX2. The influence of CDX2 or PTEN on cell migration and invasion was measured by invasion, migration and wound healing assays. Western blotting assay and immunofluorescence were used to detect the expression of CDX2, PTEN, phosphorylation of Akt, E-cadherin and N-cadherin. Statistical significance was determined by one-way analysis of variance. Results: The results showed that CDX2 reduced the migration and invasion of GC cells (P < 0.05, and inhibited the activity of Akt through down-regulating PTEN expression (P < 0.05. CDX2 also restrained epithelial-mesenchymal transition of GC cells. Conclusions: CDX2 inhibited invasion and migration of GC cells by PTEN/Akt signaling pathway, and that may be used for potential therapeutic target.

  10. C-type lectins in immune defense against pathogens: the murine DC-SIGN homologue SIGNR3 confers early protection against Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Tanne, Antoine; Neyrolles, Olivier

    2010-01-01

    Host defense against pathogens involves various receptors expressed in cells of the immune system. Upon pathogen recognition, these proteins mediate a plethora of effector functions, such as the secretion of key protective cytokines and other immune mediators. These receptors include C-type lectins (CTLs), which are increasingly being recognized as major players in the host response to microbes. One particular CTL, DCSIGN/CD209, recognizes conserved sugar motifs in a number of viruses, parasites and bacteria. In particular, we and others have shown that DC-SIGN plays an important part in the recognition by dendritic cells and macrophages of Mycobacterium tuberculosis, the causal agent of tuberculosis in humans. Using the mouse as a model: host for M. tuberculosis, we recently showed that the DC-SIGN homologue SIGNR3 mediates protection against the tubercle bacillus, possibly through secretion of the key cytokines interleukin 6 and tumor necrosis factor. Here, we summarize and discuss these findings and their implications for the design of future studies aiming to improve our understanding of the role of DC-SIGN and other C-type lectins in immunity to mycobacteria and other pathogens.

  11. A novel C-terminal homologue of Aha1 co-chaperone binds to heat shock protein 90 and stimulates its ATPase activity in Entamoeba histolytica.

    Science.gov (United States)

    Singh, Meetali; Shah, Varun; Tatu, Utpal

    2014-04-17

    Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with Kd values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10(-4) min(-1) μM(-1). Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.

  12. Physicochemical studies on ion-pair amphiphiles: Solution and interfacial behaviour of systems derived from sodium dodecylsulfate and -alkyltrimethylammonium bromide homologues

    Indian Academy of Sciences (India)

    Kajari Maiti; Subhash C Bhattacharya; Satya P Moulik; Amiya K Panda

    2010-11-01

    Bulk and interfacial properties of ion-pair amphiphiles (IPA), formed between sodium dodecylsulfate (SDS) and -alkyltrimethylammonium bromide homologues (CTAB; = 10, 12, 14, 16, and 18), have been investigated. Different phases and aggregated states, formed in the ternary combinations of CTAB/SDS/H2O, have been identified and described. Equimolar mixture of IPAs in water yielded precipitates, in the form of coacervates. Aqueous solubility of isolated coacervates in presence and absence of additives like cholesterol and bile salts have been examined. The isolated coacervates have been characterized by 1H NMR, FTIR, X-ray diffraction (XRD), differential scanning calorimetry (DSC) and polarization microscopic measurements. The coacervates have appeared in the shape of needle and complex flower-like aggregates. Surface pressure ()-area (A) isotherm of the coacervates at the air/water interface have been constructed and compared with the lipid 1,2-dipalmitoyl--glycero-3-phosphocholine (DPPC). Morphologies of the IPA monolayers at different surface pressures have been also examined by epifluorescence microscopy. The compressed interfacial monolayers have produced spherical (both regular and irregular) and fern-leaf like domains.

  13. Isolation of new genes in distal Xq28: transcriptional map and identification of a human homologue of the ARD1 N-acetyl transferase of Saccharomyces cerevisiae.

    Science.gov (United States)

    Tribioli, C; Mancini, M; Plassart, E; Bione, S; Rivella, S; Sala, C; Torri, G; Toniolo, D

    1994-07-01

    In this paper, we describe the physical and transcriptional organization of a region of 140 kb in Xq28, 5' to the L1CAM gene. By isolation and mapping of CpG islands to the physical map of the region, isolation of cDNAs, determination of partial nucleotide sequences and study of the pattern of expression and of the orientation of the transcripts identified we have established a transcriptional map of this region. In this map, previously identified genes (L1CAM, V2R, HCF1 and RnBP) have been positioned as well as 3 new genes. All genes in the region are rather small, ranging in size from 2 to 30 kb, and very close to one another. With the exception of the V2R gene, they are housekeeping, have a CpG island at their 5' end and the same orientation of transcription. This kind of organization is consistent with the one previously described for the more distal portion of Xq28, between the Color Vision (CV) and the G6PD genes and indicates that genes with housekeeping and tissue specific pattern of expression are interspersed in the genome but they are probably found in different 'transcriptional domains'. Among the new genes, TE2 demonstrated 40% identity with the protein N-acetyl transferase ARD1 of S. cerevisiae: TE2 may be the human homologue of the S. cerevisiae gene.

  14. Identification of linear B-cell epitopes on myotoxin II, a Lys49 phospholipase A₂ homologue from Bothrops asper snake venom.

    Science.gov (United States)

    Lomonte, Bruno

    2012-10-01

    Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as "conventional" dimer.

  15. The PAM1 gene of petunia, required for intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi, encodes a homologue of VAPYRIN.

    Science.gov (United States)

    Feddermann, Nadja; Muni, Rajasekhara Reddy Duvvuru; Zeier, Tatyana; Stuurman, Jeroen; Ercolin, Flavia; Schorderet, Martine; Reinhardt, Didier

    2010-11-01

    Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.

  16. Robust identification of orthologues and paralogues for microbial pan-genomics using GET_HOMOLOGUES: a case study of pIncA/C plasmids.

    Science.gov (United States)

    Vinuesa, Pablo; Contreras-Moreira, Bruno

    2015-01-01

    GET_HOMOLOGUES is an open-source software package written in Perl and R to define robust core- and pan-genomes by computing consensus clusters of orthologous gene families from whole-genome sequences using the bidirectional best-hit, COGtriangles, and OrthoMCL clustering algorithms. The granularity of the clusters can be fine-tuned by a user-configurable filtering strategy based on a combination of blastp pairwise alignment parameters, hmmscan-based scanning of Pfam domain composition of the proteins in each cluster, and a partial synteny criterion. We present detailed protocols to fit exponential and binomial mixture models to estimate core- and pan-genome sizes, compute pan-genome trees from the pan-genome matrix using a parsimony criterion, analyze and graphically represent the pan-genome structure, and identify lineage-specific gene families for the 12 complete pIncA/C plasmids currently available in NCBI's RefSeq. The software package, license, and detailed user manual can be downloaded for free for academic use from two mirrors: http://www.eead.csic.es/compbio/soft/gethoms.php and http://maya.ccg.unam.mx/soft/gethoms.php.

  17. Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

    Science.gov (United States)

    Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

    2000-04-01

    The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

  18. The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy.

    Science.gov (United States)

    Patel, Prajal H; Costa-Mattioli, Mauro; Schulze, Karen L; Bellen, Hugo J

    2009-06-29

    Hypusination is a unique posttranslational modification by which lysine is transformed into the atypical amino acid hypusine. eIF5A (eukaryotic initiation factor 5A) is the only known protein to contain hypusine. In this study, we describe the identification and characterization of nero, the Drosophila melanogaster deoxyhypusine hydroxylase (DOHH) homologue. nero mutations affect cell and organ size, bromodeoxyuridine incorporation, and autophagy. Knockdown of the hypusination target eIF5A via RNA interference causes phenotypes similar to nero mutations. However, loss of nero appears to cause milder phenotypes than loss of eIF5A. This is partially explained through a potential compensatory mechanism by which nero mutant cells up-regulate eIF5A levels. The failure of eIF5A up-regulation to rescue nero mutant phenotypes suggests that hypusination is required for eIF5A function. Furthermore, expression of enzymatically impaired forms of DOHH fails to rescue nero clones, indicating that hypusination activity is important for nero function. Our data also indicate that nero and eIF5A are required for cell growth and affect autophagy and protein synthesis.

  19. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

    Energy Technology Data Exchange (ETDEWEB)

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)] [and others

    1996-07-01

    In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.

  20. Recombinant vacuolar iron transporter family homologue PfVIT from human malaria-causing Plasmodium falciparum is a Fe2+/H+exchanger

    Science.gov (United States)

    Labarbuta, Paola; Duckett, Katie; Botting, Catherine H.; Chahrour, Osama; Malone, John; Dalton, John P.; Law, Christopher J.

    2017-01-01

    Vacuolar iron transporters (VITs) are a poorly understood family of integral membrane proteins that can function in iron homeostasis via sequestration of labile Fe2+ into vacuolar compartments. Here we report on the heterologous overexpression and purification of PfVIT, a vacuolar iron transporter homologue from the human malaria-causing parasite Plasmodium falciparum. Use of synthetic, codon-optimised DNA enabled overexpression of functional PfVIT in the inner membrane of Escherichia coli which, in turn, conferred iron tolerance to the bacterial cells. Cells that expressed PfVIT had decreased levels of total cellular iron compared with cells that did not express the protein. Qualitative transport assays performed on inverted vesicles enriched with PfVIT revealed that the transporter catalysed Fe2+/H+ exchange driven by the proton electrochemical gradient. Furthermore, the PfVIT transport function in this system did not require the presence of any Plasmodium-specific factor such as post-translational phosphorylation. PfVIT purified as a monomer and, as measured by intrinsic protein fluorescence quenching, bound Fe2+ in detergent solution with low micromolar affinity. This study of PfVIT provides material for future detailed biochemical, biophysical and structural studies to advance understanding of the vacuolar iron transporter family of membrane proteins from important human pathogens. PMID:28198449

  1. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length.

    Science.gov (United States)

    Nakazawa, M; Yabe, N; Ichikawa, T; Yamamoto, Y Y; Yoshizumi, T; Hasunuma, K; Matsui, M

    2001-01-01

    A novel dominant mutant designated 'dwarf in light 1' (dfl1-D) was isolated from screening around 1200 Arabidopsis activation-tagged lines. dfl1-D has a shorter hypocotyl under blue, red and far-red light, but not in darkness. Inhibition of cell elongation in shoots caused an exaggerated dwarf phenotype in the adult plant. The lateral root growth of dfl1-D was inhibited without any reduction of primary root length. The genomic DNA adjacent to the right border of T-DNA was cloned by plasmid rescue. The rescued genomic DNA contained a gene encoding a GH3 homologue. The transcript was highly accumulated in the dfl1-D. The dfl1-D phenotype was confirmed by over-expression of the gene in the wild-type plant. The dfl1-D showed resistance to exogenous auxin treatment. Moreover, over-expression of antisense DFL1 resulted in larger shoots and an increase in the number of lateral roots. These results indicate that the gene product of DFL1 is involved in auxin signal transduction, and inhibits shoot and hypocotyl cell elongation and lateral root cell differentiation in light.

  2. Altered expression of GFLO, the Gesneriaceae homologue of FLORICAULA/LEAFY, is associated with the transition to bulbil formation in Titanotrichum oldhamii.

    Science.gov (United States)

    Wang, Chun-Neng; Möller, Michael; Cronk, Quentin C B

    2004-03-01

    Titanotrichum oldhamii inflorescences switch from flower to bulbil production at the end of the flowering season. The structure of the bulbiliferous shoots resembles the abnormal meristematic organization of the Antirrhinum mutant, floricaula. Gesneriaceae- FLORICAULA (GFLO) is thus a candidate gene in the regulation of bulbil formation. To investigate this hypothesis, part of the GFLO gene (between the second and third exon) was isolated using degenerate primers designed in regions conserved between Antirrhinum, Nicotiana and Arabidopsis, followed by genome walking to obtain the complete gene and flanking sequences. RT-PCR results showed that the GFLO homologue is strongly expressed in inflorescence apical meristems and young flowers. However, in meristems that had switched to bulbil formation, GFLO transcription was greatly reduced. The down-regulation of GFLO in bulbil primordia indicates that this gene is connected to, or part of, the bulbil-flower regulatory pathway. Phylogenetic analysis confirms the orthology of GFLO and FLO, and indicates that the gene may be useful for phylogenetic reconstruction at the genus or family level.

  3. Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC-homologue HgdD in protein secretion.

    Science.gov (United States)

    Hahn, Alexander; Stevanovic, Mara; Brouwer, Eva; Bublak, Daniela; Tripp, Joanna; Schorge, Tobias; Karas, Michael; Schleiff, Enrico

    2015-03-01

    Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC-homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst-forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss-of-function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild-type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin-tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD-mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD-dependent pathway.

  4. Prorenin Receptor Homologue VHA-20 is Critical for Intestinal pH Regulation, Ion and Water Management and Larval Development in C. elegans.

    Science.gov (United States)

    Zima, V; Šebková, K; Šimečková, K; Dvořák, T; Saudek, V; Kostrouchová, M

    2015-01-01

    The prorenin receptor (ATP6AP2) is a multifunctional transmembrane protein; it is a constituent of proton-translocating V-ATPase, a non-proteolytic activator of renin and an adaptor in the Wnt/β-catenin pathway. Here, we studied vha-20, one of the two prorenin receptor homologues that are identified by sequence similarity in the C. elegans genome. We show that vha-20 (R03E1.2) is prominently expressed in the intestine, in the excretory cell and in amphid neurons, tissues critical for regulation of ion and water management. The expression of vha-20 in the intestine is dependent on NHR-31, a nuclear receptor related to HNF4. VHA-20 is indispensable for normal larval development, acidification of the intestine, and is required for nutrient uptake. Inhibition of vha-20 by RNAi leads to complex deterioration of water and pH gradients at the level of the whole organism including distention of pseudocoelome cavity. This suggests new roles of prorenin receptor in the regulation of body ion and water management and in acidification of intestinal lumen in nematodes.

  5. Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrio anguillarum strain 775.

    Science.gov (United States)

    Di Lorenzo, Manuela; Stork, Michiel; Crosa, Jorge H

    2011-08-01

    Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

  6. Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism.

    Science.gov (United States)

    Tatebayashi, K; Tani, T; Ikeda, H

    2001-04-01

    We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.

  7. Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p.

    Science.gov (United States)

    Jaspersen, Sue L; Giddings, Thomas H; Winey, Mark

    2002-12-23

    Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.

  8. Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue.

    Science.gov (United States)

    Coddeville, Bernadette; Wu, Sz-Wei; Fabre, Emeline; Brassart, Colette; Rombouts, Yoann; Burguière, Adeline; Kremer, Laurent; Khoo, Kay-Hooi; Elass-Rochard, Elisabeth; Guérardel, Yann

    2012-10-22

    The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Wax crystal-sparse leaf2, a rice homologue of WAX2/GL1, is involved in synthesis of leaf cuticular wax.

    Science.gov (United States)

    Mao, Bigang; Cheng, Zhijun; Lei, Cailin; Xu, Fenghua; Gao, Suwei; Ren, Yulong; Wang, Jiulin; Zhang, Xin; Wang, Jie; Wu, Fuqing; Guo, Xiuping; Liu, Xiaolu; Wu, Chuanyin; Wang, Haiyang; Wan, Jianmin

    2012-01-01

    Epicuticular wax in plants limits non-stomatal water loss, inhibits postgenital organ fusion, protects plants against damage from UV radiation and imposes a physical barrier against pathogen infection. Here, we give a detailed description of the genetic, physiological and morphological consequences of a mutation in the rice gene WSL2, based on a comparison between the wild-type and an EMS mutant. The mutant's leaf cuticle membrane is thicker and less organized than that of the wild type, and its total wax content is diminished by ~80%. The mutant is also more sensitive to drought stress. WSL2 was isolated by positional cloning, and was shown to encode a homologue of the Arabidopsis thaliana genes CER3/WAX2/YRE/FLP1 and the maize gene GL1. It is expressed throughout the plant, except in the root. A transient assay carried out in both A. thaliana and rice protoplasts showed that the gene product is deposited in the endoplasmic reticulum. An analysis of the overall composition of the wax revealed that the mutant produces a substantially reduced quantity of C22-C32 fatty acids, which suggests that the function of WSL2 is associated with the elongation of very long-chain fatty acids.

  10. Small cell lung cancer, an epithelial to mesenchymal transition (EMT)-like cancer: significance of inactive Notch signaling and expression of achaete-scute complex homologue 1.

    Science.gov (United States)

    Ito, Takaaki; Kudoh, Shinji; Ichimura, Takaya; Fujino, Kosuke; Hassan, Wael Ahmed Maher Abdo; Udaka, Naoko

    2017-01-01

    Small cell lung cancer (SCLC) is one of the most malignant neoplasms in common human cancers. The tumor is composed of small immature-looking cells with a round or fusiform shape, which possesses weak adhesion features among them, suggesting that SCLC shows the morphological characteristics of epithelial to mesenchymal transition (EMT). SCLC is characterized by high metastatic and recurrent rates, sensitivity to the initial chemotherapy, and easy acquirement of chemoresistance afterwards. These characters may be related to the EMT phenotype of SCLC. Notch signaling is an important signaling pathway, and could have roles in regulating neuroendocrine differentiation, proliferation, cell adhesion, EMT, and chemoresistance. Notch1 is usually absent in SCLC in vivo, but could appear after chemotherapy. Notch1 can enhance cell adhesion by induction of E-cadherin in SCLC, which indicates mesenchymal to epithelial transition. On the other hand, achaete-scute complex homologue 1 (ASCL1), negatively regulated by Notch signaling, is a lineage-specific gene of SCLC, and functions to promote neuroendocrine differentiation as well as EMT. ASCL1-transfected adenocarcinoma cell lines induced neuroendocrine phenotypes and lost epithelial cell features. SCLC is characterized by neuroendocrine differentiation and EMT-like features, which could be produced by inactive Notch signaling and ASCL1 expression. In addition, chemical and radiation treatments can activate Notch signaling, which suppress neuroendocrine differentiation and induces chemoradioresistance, accompanied by secession from EMT. Thus, the status of Notch signaling and ASCL1 expression may determine the cell behaviors of SCLC partly through modifying EMT phenotypes.

  11. CRP-ductin, the mouse homologue of gp-340/deleted in malignant brain tumors 1 (DMBT1), binds gram-positive and gram-negative bacteria and interacts with lung surfactant protein D

    DEFF Research Database (Denmark)

    Madsen, Jens; Tornøe, Ida; Nielsen, Ole

    2003-01-01

    CRP-ductin is a protein expressed mainly by mucosal epithelial cells in the mouse. Sequence homologies indicate that CRP-ductin is the mouse homologue of human gp-340, a glycoprotein that agglutinates microorganisms and binds the lung mucosal collectin surfactant protein-D (SP-D). Here we report......-ductin and SP-D expression by reverse transcription-PCR. The pancreas was the main site of synthesis of CRP-ductin, but transcripts were also readily amplified from salivary gland, the gastrointestinal tract, liver, testis, uterus and lung. Lung was the main site of synthesis of SP-D, but transcripts were also...... amplified from uterus, salivary gland, thymus, thyroid gland, pancreas and testis. We conclude that CRP-ductin is the mouse homologue of human gp-340 and that its capacity to bind SP-D as well as gram-negative and gram-positive bacteria suggests a role in mucosal immune defense....

  12. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    Science.gov (United States)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick

    2015-07-07

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    DEFF Research Database (Denmark)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent;

    2015-01-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation......-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor....

  14. Short-chain chlorinated paraffins in soil, paddy seeds (Oryza sativa) and snails (Ampullariidae) in an e-waste dismantling area in China: Homologue group pattern, spatial distribution and risk assessment.

    Science.gov (United States)

    Yuan, Bo; Fu, Jianjie; Wang, Yawei; Jiang, Guibin

    2017-01-01

    Short-chain chlorinated paraffins (SCCPs) in multi-environmental matrices are studied in Taizhou, Zhejiang Province, China, which is a notorious e-waste dismantling area. The investigated matrices consist of paddy field soil, paddy seeds (Oryza sativa, separated into hulls and rice unpolished) and apple snails (Ampullariidae, inhabiting the paddy fields). The sampling area covered a 65-km radius around the contamination center. C10 and C11 are the two predominant homologue groups in the area, accounting for about 35.7% and 33.0% of total SCCPs, respectively. SCCPs in snails and hulls are generally higher than in soil samples (30.4-530 ng/g dw), and SCCPs in hulls are approximate five times higher than in corresponding rice samples (4.90-55.1 ng/g dw). Homologue pattern analysis indicates that paddy seeds (both hull and rice) tend to accumulate relatively high volatile SCCP homologues, especially the ones with shorter carbon chain length, while snails tend to accumulate relatively high lipophilic homologues, especially the ones with more substituted chlorines. SCCPs in both paddy seeds and snails are linearly related to those in the soil. The e-waste dismantling area, which covers a radius of approximate 20 km, shows higher pollution levels for SCCPs according to their spatial distribution in four matrices. The preliminary assessment indicates that SCCP levels in local soils pose no significant ecological risk for soil dwelling organisms, but higher risks from dietary exposure of SCCPs are suspected for people living in e-waste dismantling area.

  15. Single-Crystal X-Ray Diffraction Studies of Homologues in the Series nBa(Nb,Zr)O 3+3 mNbO with n=2, 3, 4, 5 and m=1

    Science.gov (United States)

    Nilsson, G.; Svensson, G.

    2001-01-01

    Single crystals of four homologues in the series nBa(Nb,Zr)O3+3mNbO, with n:m=2:1, 3:1, 4:1, and 5:1, were found in the reduced Ba-Nb-Zr-O system. Single-crystal X-ray diffraction data were collected for all the crystals. For all homologues the space group was found to be P4/mmm. The structures can be described as intergrowths of Ba(Nb,Zr)O3 perovskite and NbO slabs. The refined cell parameters and compositions of the 2:1, 3:1, and 4:1 homologues are a=4.1768(5) Å and c=12.269(2) Å for Ba2Nb4.5(1)Zr0.5(1)O9, a=4.1769(5) Å and c=16.493(3) Å for Ba3+δNb4.8(2)-δ Zr1.2(2)O12-δ (δ=0.098(4)), and a=4.1747(6) Å and c= 20.619(4) Å for Ba4+δNb5.1(4)-δZr1.9(4)O15-δ (δ=0.270(9)). The refined cell parameters of the 5:1 homologue are a=4.1727(3) Å and c=24.804(3) Å. Zr replaces Nb only in the NbO6 octahedra found in the perovskite slabs.

  16. Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

    Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR......-based poly(A) test and in vitro poly(A) assay. Taken together, our results suggest that hCPEB1 and hGLD-2 are antagonizing factors regulating p53 mRNA stability....

  17. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  18. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    Science.gov (United States)

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing

    2014-09-01

    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  19. c-myc mRNA in cytoskeletal-bound polysomes in fibroblasts.

    Science.gov (United States)

    Hesketh, J E; Campbell, G P; Whitelaw, P F

    1991-03-01

    3T3 fibroblasts were treated sequentially with 25 mM-KCl/0.05% Nonidet P40, 130 mM-KCl/0.05% Nonidet P40 and finally with 1% Nonidet P40/1% deoxycholate in order to release free, cytoskeletal-bound and membrane-bound polysomes respectively. The membrane-bound fraction was enriched in the mRNA for the membrane protein beta 2-microglobulin, whereas the cytoskeletal-bound polysomes were enriched in c-myc mRNA. Actin mRNA was present in both free and cytoskeletal-bound polysomes. The results suggest that cytoskeletal-bound polysomes are involved in the translation of specific mRNA species.

  20. Lipoprotein Lipase mRNA expression in different tissues of farm ...

    African Journals Online (AJOL)

    Lipoprotein Lipase mRNA expression in different tissues of farm animals. ... Lipoprotein lipase (LPL) controls triacylglycerol partitioning between adipose tissues and muscles, so it is important enzyme for ... Article Metrics. Metrics Loading .

  1. Control of mRNA stability during development of Dictyostelium discoideum.

    Science.gov (United States)

    Mangiarotti, G

    1989-01-01

    A large group of mRNA species (which are mainly pre-spore specific) accumulate only after the formation of multicellular aggregates. They are transcribed at a constant rate from the beginning of development and their accumulation is controlled by a 10-20-fold increase in their stability. This mRNA stabilization is dependent upon multicellularity. When aggregates are dispersed, the mRNAs are destabilized; if cells are allowed to reaggregate, the destabilization is reversed. Destabilization is not due to a selective exclusion of mRNA from polyribosomes, but is a primary control event. It does not require synthesis of new RNA or protein, but it may require an interaction between ribosome and the 5'-end of mRNA molecules.

  2. The potential lipolysis function of musclin and its mRNA expression ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-16

    Nov 16, 2009 ... Musclin mRNA levels in adipose, muscle tissues and primary adipocytes were examined by quantitative PCR. The ... reduced fat mass in mice. Here, we ..... increased, suggesting that musclin might be involved in adipocyte ...

  3. Mapping interactions between mRNA export factors in living cells.

    Directory of Open Access Journals (Sweden)

    I-Fang Teng

    Full Text Available The TREX complex couples nuclear mRNA processing events with subsequent export to the cytoplasm. TREX also acts as a binding platform for the mRNA export receptor Nxf1. The sites of mRNA transcription and processing within the nucleus have been studied extensively. However, little is known about where TREX assembly takes place and where Nxf1 is recruited to TREX to form the export competent mRNP. Here we have used sensitized emission Förster resonance energy transfer (FRET and fluorescence lifetime imaging (FLIM-FRET, to produce a spatial map in living cells of the sites for the interaction of two TREX subunits, Alyref and Chtop, with Nxf1. Prominent assembly sites for export factors are found in the vicinity of nuclear speckles in regions known to be involved in transcription, splicing and exon junction complex formation highlighting the close coupling of mRNA export with mRNP biogenesis.

  4. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Directory of Open Access Journals (Sweden)

    Beatriz M. A. Fontoura

    2013-07-01

    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  5. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    Science.gov (United States)

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P

    2016-05-25

    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol.

  6. Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

    Science.gov (United States)

    Bire, Solenne; Gosset, David; Jégot, Gwenhael; Midoux, Patrick; Pichon, Chantal; Rouleux-Bonnin, Florence

    2013-09-26

    Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration

  7. TGF beta 2 mRNA expression and pregnancy failure in mice.

    Science.gov (United States)

    Gorivodsky, M; Torchinsky, A; Zemliak, I; Savion, S; Fein, A; Toder, V

    1999-08-01

    We describe here a pattern of transforming growth factor (TGF) beta2 mRNA expression at the fetomaternal interface in mice with high rate of resorptions as well as its expression following maternal immunopotentiation. TGF beta 2 mRNA expression was evaluated in the uteroplacental units of mice with spontaneous (CBA/J x DBA/2J mouse combination) or cyclophosphamide (CP)-induced pregnancy loss. The effect of immunopotentiation on TGF beta 2 mRNA expression was determined in CP-treated females who underwent nonspecific immunostimulation with xenogeneic (rat) leukocytes. A quantitative analysis of TGF beta 2 mRNA level was performed using RNase protection assay. Distribution of TGF beta 2 mRNA transcripts at the fetomaternal interface was studied by in situ hybridization analysis. RNase protection analysis revealed four TGF beta 2 specific mRNA forms (330, 270, 230, and 170 bp) in the uteroplacental units of mice with either normal or decreased reproductive performance. A significant decrease (about 50%) in the level of TGF beta 2 mRNA was registered in the uteroplacental unit of mice with pregnancy loss as compared to the control mice. TGF beta 2 transcripts were abundant in the uterine epithelium and stroma. A specific hybridization signal was detected also in metrial gland cells and it was found to be substantially lower in CP-treated as compared to intact mice. In the resorbing uteroplacental unit, the expression of TGF beta 2 mRNA was completely lost in the uterine epithelium, and the number of TGF beta 2 mRNA-positive metrial gland cells was lower as compared to the control. Immunopotentiation decreased the resorption rate in mice with CP-induced pregnancy loss and caused a dramatic increase in TGF beta 2 mRNA expression: the level of TGF beta 2 mRNA was found to be higher by 2.0-3.2 fold in the uteroplacental unit of immunized as compared to nonimmunized CP-treated mice. These data suggest that distortion of TGF beta 2 expression at the fetomaternal interface

  8. OX40 mRNA in peripheral blood as a biomarker of acute renal allograft rejection

    Institute of Scientific and Technical Information of China (English)

    WANG Yu-liang; FU Ying-xin; ZHU Zhi-jun; WANG Hui; SHEN Zhong-yang

    2012-01-01

    Background Acute rejection remains an important cause of renal allograft dysfunction and the need for accurate diagnosis is essential to successfully treat transplant recipients.The purpose of this study was to determine the costimulatory molecules OX40 and OX40L messenger RNA (mRNA) levels in peripheral blood mononuclear cells (PBMCs) to predict acute renal transplant rejection.Methods The whole blood samples from 20 recipients with biopsy-confirmed acute rejection (rejection group),20 recipients with stable graft function and normal biopsy results (stable group) after kidney transplantation,and 20 healthy volunteers (control group) were collected.The mRNA levels of OX40 and OX40L were analyzed with TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR).The association of OX40 and OX40L mRNA levels with disease severity was investigated.Results There was no significant difference of OX40,OX40L mRNA levels in PBMCs between the stable group and control group (P>0.05).The levels of OX40 and OX40L mRNA were significantly higher in the rejection group than in the control group (P<0.01 and P<0.05,respectively).Non-significantly higher OX40L mRNA and significantly higher OX40 mRNA in PBMCs were observed in subjects in the rejection group compared with the stable group (P >0.05 and P <0.01,respectively).Receiver operating characteristic (ROC) curve analysis demonstrated that OX40 mRNA levels could discriminate recipients who subsequently suffered acute allograft rejection (area under the curve,0.908).OX40 and OX40L mRNA levels did not significantly correlate with serum creatinine levels in the rejection group (P >0.05).Levels of OX40 mRNA after anti-rejection therapy were lower than those at the time of protocol biopsy in the rejection group (P<0.05).Conclusion Our data suggest that measurement of OX40 mRNA levels after transplant might offer a noninvasive means for recognizing recipients at risk of acute renal allograft rejection.

  9. Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA.

    Science.gov (United States)

    Sharon, Ronit; Abramovitz, Rene; Miskin, Ruth

    2002-08-15

    Plasminogen (Plg), which can be converted to the active protease plasmin by plasminogen activators, has been previously implicated in brain plasticity and in toxicity inflicted in hippocampal pyramidal neurons by kainate. Here we have localized Plg. mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate injection (i.p.). The results indicated that Plg mRNA was undetectable in the normal brain, but after kainate injection it was induced in neuronal cells in multiple, but specific areas, including layers II-III of the neocortex; the olfactory bulb, anterior olfactory nucleus, and the piriform cortex; the caudate/putamen and accumbens nucleus shell; throughout the amygdaloid complex; and in the CAI/CA3 subfields of the hippocampus. Interestingly, this distribution pattern coincided with what we have recently described for the plasminogen activator inhibitor-2 (PAI-2) mRNA, however differing from that of the plasminogen activator inhibitor-1 (PAI-1) mRNA, as also shown here. These results suggest that enhanced Plg gene expression could be involved in events associated with olfactory, striatal, and limbic structures. Furthermore, because PAI-2 is thought to intracellularly counteract cytotoxic events, our results raise the possibility that PAI-2 can act in the brain as an intracellular neuroprotector against potential plasmin-mediated toxicity.

  10. Unique variability of tocopherol composition in various seed oils recovered from by-products of apple industry: rapid and simple determination of all four homologues (α, β, γ and δ) by RP-HPLC/FLD.

    Science.gov (United States)

    Górnaś, Paweł

    2015-04-01

    The tocochromanol profile was studied in seed oils recovered from by-products of fruit industry, five dessert and seven crab apple varieties grown in Eastern Europe (Latvia). The seed oils obtained from dessert apples were characterized by higher contents of tocopherols (191.05-379.08 mg/100g oil) when compared to seed oils recovered from crab apples (130.55-202.54 mg/100g oil). The predominant homologues of tocopherol in all the studied samples were α and β over γ and δ. However, seed oils recovered from the apple cultivars 'Antej' and 'Beforest' had a unique profile of four tocopherol homologues (α:β:γ:δ) 91.41:80.55:72.46:79.03 and 114.55:112.84:78.69:73.00 mg/100g oil, respectively. A single dilution of seed oils in 2-propanol facilitated the direct use samples in the DPPH assay as well as injection into the RP-HPLC system containing a PFP (pentafluorophenyl) column, which resulted in a rapid separation of all four tocopherol homologues with excellent repeatability and reproducibility. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Involvement of the p53 and HPV-16 early 3'UTRs in mRNA regulation

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald

    tilstede. hCPEB1 nedsætter i øvrigt marginelt halveringtiden på mRNA. hGLD-2 kan delvis fjerne den hCPEB1 inducerede repression. Andre proteiner regulerer også p53 mRNA stabiliteten så som Wig-1, der ogå induceres af p53, hvilket medfører en positiv feed-back loop i p53 reguleringen. Som konklusion lader...

  12. Methylation Inactivation Mechanism of Parkin Gene mRNA Expression in Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ni Haifeng; Jiang Bo; Zhou Zhen; Li Yong; Huang Guangwu

    2014-01-01

    Objective:To investigate the methylation inactivation and the clinical signiifcance of Parkin gene mRNA expression in nasopharyngeal carcinoma (NPC). Methods: The methylation-speciifc PCR (MSP) and semi-quantitative reverse transcription PCR (RT-PCR) were used to detect methylation and the mRNA expression level of Parkin gene in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues.The mRNA expression of Parkin gene in two NPC cell lines (CNE1 and CNE2) were detected before and after treatment with the methyltransferase inhibitor 5-aza-2-deoxycytidine so as to analyze the effects of Parkin gene methylation and demethylation on Parkin gene mRNA expression and the relationship between Parkin gene mRNA expression and clinical factors. Results: The methylation frequency of Parkin gene in human NPC tissues was 62.96% (34/54), but didn’t happen in any of 16 cases of NNE tissues. The mRNA expression level was (0.3430±0.4947) in 54 cases of NPC tissues and (1.0052±0.4911) in NNE tissues, showing that the mRNA expression level of NPC tissues was significantly down-regulated (P0.05), but was closely related to lymph node metastasis (P<0.05). Conclusion:Parkin gene mRNA expression, serving as a cancer suppressor gene in the occurrence and development of NPC, is inactivated and regulated by methylation, which also has a negative correlation with lymph node metastasis and could be considered as the judgment of predictive index of clinical prognosis of NPC.

  13. Rapid changes in ovarian mRNA induced by brief photostimulation in Siberian hamsters (Phodopus sungorus).

    Science.gov (United States)

    Shahed, Asha; McMichael, Carling F; Young, Kelly A

    2015-11-01

    This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2 (PT day-2), 4 (PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone β-subunit (FSHβ-subunit), luteinizing hormone β-subunit (LHβ-subunit), FSH and LH receptors, estrogen receptors α and β (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Müllerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHβ- and LHβ-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation.

  14. mRNA levels of TLR4 and TLR5 are independent of H pylori

    Institute of Scientific and Technical Information of China (English)

    Elvira Garza-González; Virgilio Bocanegra-García; Francisco Javier Bosques-Padilla; Juan Pablo Flores-Gutiérrez; Francisco Moreno; Guillermo Ignacio Perez-Perez

    2008-01-01

    AIM:To determine if the presence Hpylori or its virulence affect toll-like receptor 4 (TLR4) and TLR5 mRNA expression levels.METHODS:For the in vivo assays,gastric biopsies were obtained from 40 patients and H pylori status was determined.For the in vitro assays,human gastric adenocarcinoma mucosal cells (AGS) were cultured in the presence or absence of twelve selected H pylori strains.H pylori strains isolated from culture-positive patients and selected strains were genotyped for cagA and vacA.The cDNA was obtained from mRNA extracted from biopsies and from infected AGS cells.TLR4 and TLR5 mRNA levels were examined by real-time PCR.RESULTS:The presence of Hpylori did not affect the mRNA levels of TLR4 or TLR5 in gastric biopsies.The mRNA levels of both receptors were not influenced by the vacA status (P>0.05 for both receptors) and there were no differences in TLR4 or TLR5 mRNA levels among the different clinical presentations/histological findings (P>0.05).In the in vitro assay,the mRNA levels of TLR4 or TLR5 in AGS cells were not influenced by the vacAsl status or the clinical condition associated with the strains (P>0.05 for both TLR4 and TLR5).CONCLUSION:The results of this study show that the mRNA levels of TLR4 and TLR5 in gastric cells,both in vivo and in vitro,are independent of H pylori colonization and suggest that vacA may not be a significant player in the first step of innate immune recognition mediated by TLR4 or TLR5.

  15. Changes in mRNA for metabotropic glutamate receptors after transient cerebral ischaemia

    DEFF Research Database (Denmark)

    Rosdahl, D; Seitzberg, D A; Christensen, Thomas;

    1994-01-01

    Using a rat 4-vessel occlusion model of cerebral ischaemia we studied the changes in the mRNA level for the metabotropic receptor subtypes mGluR1 alpha, mGluR1 beta, mGluR2, mGluR3, mGluR4, and mGluR5 by means of in situ hybridization with oligonucleotides. After 24 hours of reperfusion the mRNA ...

  16. Expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patient

    Institute of Scientific and Technical Information of China (English)

    Li Zhang; Zhen Yang; Bao-Min Shi; Da-Peng Li; Chong-Yun Fang; Fa-Zu Qiu

    2003-01-01

    AIM: To investigate the expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patients.METHODS: The expression of local renin and angiotensinogen mRNA in the liver, splenic artery and vein of PH patients was detected by RT-PCR analysis.RESULTS: Expression of local renin mRNA in the liver of control group was (0.19±0.11), significantly lower than that in splenic artery(0.45±0.17)or splenic vein(0.39±0.12)respectively, (P<0.05). Expression of local angiotensinogen mRNA in the liver was (0.64±0.21), significantly higher than that in splenic artery(0.41±0.15) or in splenic vein (0.35±0.18)respectively, (P<0.05). Expression of local renin mRNA in the liver, splenic artery and vein of PH group was (0.78±0.28),(0.86±0.35) and (0.81±0.22) respectively, significantly higher than that in the control group, (P<0.05). Expression of local angiotensinogen mRNA in the liver, splenic artery and vein of PH group was (0.96±0.25), (0.83±0.18) and (0.79±0.23)respectively, significantly higher than that in the control group,(P<0.05). There was no significant difference between the liver, splenic artery and vein in the expression of local renin or local angiotensinogen mRNA in PH group, (P<0.05).CONCLUSION: In normal subjects the expression of local renin and angiotensinogen mRNA was organ specific, but with increase of the expression of LRAS, the organ-specificity became lost in cirrhotic patients. LRAS may contribute to increased resistance of portal vein with liver and formation of splanchnic vasculopathy.

  17. Maternally inherited npm2 mRNA is crucial for egg developmental competence in zebrafish.

    Science.gov (United States)

    Bouleau, Aurélien; Desvignes, Thomas; Traverso, Juan Martin; Nguyen, Thaovi; Chesnel, Franck; Fauvel, Christian; Bobe, Julien

    2014-08-01

    The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates.

  18. Carboxylesterase 1 gene duplication and mRNA expression in adipose tissue are linked to obesity and metabolic function

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Poulsen, Pernille; Wojtaszewski, Jørgen

    2013-01-01

    involved in the control of mRNA expression. Here, we investigated mRNA expression level in adipose tissue and its association with measures of adiposity and metabolic function in a population of elderly twins. Furthermore, the heritability of mRNA expression level in adipose tissue and the effect of gene...

  19. Chamber-dependent expression of brain natriuretic peptide and its mRNA in normal and diabetic pig heart

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Goetze, Jens P; Bartels, Emil D;

    2002-01-01

    -induced diabetes doubled the expression of BNP mRNA in porcine atrial myocardium (P=0.03), but was without effect on BNP mRNA in the ventricular myocardium. The data suggest that BNP mRNA processing and proBNP storage differ between the atrial and ventricular myocardium. The results also imply that diabetes...

  20. Prognostic relevance of circulating CK19 mRNA in advanced malignant biliary tract diseases

    Institute of Scientific and Technical Information of China (English)

    Kawin Leelawat; Siriluck Narong; Wandee Udomchaiprasertkul; Jerasak Wannaprasert; Sa-ard Treepongkaruna; Somboon Subwongcharoen; Tawee Ratanashu-ek

    2012-01-01

    AIM: To determine the role of circulating tumor cells (CTCs) in prediction of the overall survival of patients with advanced malignant biliary tract obstruction. METHODS: We investigated the prognostic value of CTCs by examining two markers, cytokeratin (CK) 19 and human telomerase reverse transcriptase (hTERT) mRNA, in 40 patients diagnosed with advanced malignant biliary tract diseases. Quantitative real-time reverse transcription polymerase chain reaction was used to detect CK19 and hTERT mRNA in the peripheral blood of these patients. Overall survival was analyzed using the Kaplan-Meier method and Cox regression modeling. RESULTS: Positive CK19 and hTERT mRNA expression was detected in 45% and 60%, respectively, of the 40 patients. Univariable analysis indicated that positive CK19 mRNA expression was significantly associated with worse overall survival (P = 0.009). Multivariable analysis determined that positive CK19 mRNA expression, patient's age and serum bilirubin were each independently associated with overall survival. CONCLUSION: CK19 mRNA expression levels in peripheral blood appear to provide a valuable marker to predict the overall survival of patients with advanced malignant biliary tract obstruction.

  1. Foxg1 mRNA overexpression in neonatal rats following hypoxic brain injury

    Institute of Scientific and Technical Information of China (English)

    Luquan Li; Yi Zheng; Guoliang Mo; Fang Li; Jialin Yu

    2011-01-01

    Forkhead box G1 (Foxg1) is expressed during the embryonic stage and in postnatal brain regions sensitive to hypoxia/ischemia injury,such as the hippocampus and cerebral cortex.To date,very little is known about Foxg 1 expression changes in the brain following hypoxia injury (HI).The present study measured Foxg 1 mRNA expression using reverse-transcription polymerase chain reaction on days 3,7,14,28,and 56 following HI to determine self-restorative features in the injured brain.In addition,mRNA expression of other related layer markers,such as Reelin,RORB,Foxp1,Foxp2,ER81,and Otx-1,was detected following HI.Results revealed significantly decreased Foxg1 mRNA expression at 3 days after HI,which significantly increased by 56 days.Reelin and Foxp2 mRNA expression were upregulated until 56 days after HI,but Foxp1 and ER81 mRNA expression decreased from day 14 to 56 following HI.In addition,Otx-1 and RORB mRNA expression decreased from day 3 to 28 after HI.These findings revealed Fxog1 mRNA overexpression and varying degrees of restoration in the neonatal rat brain following HI.

  2. mRNA transfection of mouse and human neural stem cell cultures.

    Directory of Open Access Journals (Sweden)

    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  3. Drosophila glutamate receptor mRNA expression and mRNP particles.

    Science.gov (United States)

    Ganesan, Subhashree; Karr, Julie E; Featherstone, David E

    2011-01-01

    The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.

  4. [Interrelationship between protein synthesis and mRNA metabolism in rat liver cells].

    Science.gov (United States)

    Arbuzov, V A

    1976-08-01

    It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.

  5. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

    Directory of Open Access Journals (Sweden)

    Steven J. Schnell

    2014-11-01

    Full Text Available The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE. Plenty of nuclear pore complexes (NPCs embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

  6. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  7. RNase III controls the degradation of corA mRNA in Escherichia coli.

    Science.gov (United States)

    Lim, Boram; Sim, Se-Hoon; Sim, Minji; Kim, Kyungsub; Jeon, Che Ok; Lee, Younghoon; Ha, Nam-Chul; Lee, Kangseok

    2012-05-01

    In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.

  8. Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA.

    Directory of Open Access Journals (Sweden)

    Thomas J Stoddard

    Full Text Available Plant genome engineering using sequence-specific nucleases (SSNs promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA encoding TAL effector nucleases (TALENs. mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were 10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5' and 3' untranslated regions (UTRs from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740 enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing.

  9. HDAC3 regulates stability of estrogen receptor α mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Murayama, Akiko; Yanagisawa, Junn, E-mail: junny@agbi.tsukuba.ac.jp

    2013-03-08

    Highlights: ► HDAC inhibitors decrease the stability of ERα mRNA in MCF-7 cells. ► HDAC3 is involved in maintaining ERα mRNA stability in MCF-7 cells. ► ERα mRNA instability by knockdown of HDAC3 reduces the estrogen-dependent proliferation of ERα-positive MCF-7 cells. ► HDAC3 specific inhibitor will be one of new drugs for ERα-positive breast cancers. -- Abstract: Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.

  10. Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

    Science.gov (United States)

    Giraldo-Vela, Juan P; Kang, Wonmo; McNaughton, Rebecca L; Zhang, Xuemei; Wile, Brian M; Tsourkas, Andrew; Bao, Gang; Espinosa, Horacio D

    2015-05-01

    New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

  11. Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

    Science.gov (United States)

    Soetanto, R; Hynes, C J; Patel, H R; Humphreys, D T; Evers, M; Duan, G; Parker, B J; Archer, S K; Clancy, J L; Graham, R M; Beilharz, T H; Smith, N J; Preiss, T

    2016-05-01

    miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Histone gene expression and histone mRNA 3' end structure in Caenorhabditis elegans

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    Pettitt Jonathan

    2007-06-01

    Full Text Available Abstract Background Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development. Results Sequence analysis of replication-dependent histone genes revealed the presence of several highly conserved sequence elements in the 3' untranslated region of histone pre-mRNAs, including an RNA hairpin element and a polyadenylation signal. To determine whether in C. elegans histone mRNA 3' end formation occurs at this polyadenylation signal and results in polyadenylated histone mRNA, we investigated the mRNA 3' end structure of histone mRNA. Using poly(A selection, RNAse protection and sequencing of histone mRNA ends, we determined that a majority of C. elegans histone mRNAs lack a poly(A tail and end three to six nucleotides after the hairpin structure, after an A or a U, and have a 3' OH group. RNAi knock down of CDL-1, the C. elegans HBP/SLBP, does not significantly affect histone mRNA levels but severely depletes histone protein levels. Histone gene expression varies during development and is reduced in L3 animals compared to L1 animals and adults. In adults, histone gene expression is restricted to the germ line, where cell division occurs. Conclusion Our findings indicate that the expression of C. elegans histone genes is subject to control mechanisms similar to the ones in other

  13. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

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    Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

    2013-08-30

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

  14. The Lon protease homologue LonA, not LonC, contributes to the stress tolerance and biofilm formation of Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Xie, Fang; Li, Gang; Zhang, Yanhe; Zhou, Long; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

    2016-04-01

    Lon proteases are a family of ATP-dependent proteases that are involved in the degradation of abnormal proteins in bacteria exposed to adverse environmental stress. An analysis of the genome sequence of Actinobacillus pleuropneumoniae revealed the unusual presence of two putative ATP-dependent Lon homologues, LonA and LonC. Sequence comparisons indicated that LonA has the classical domain organization of the LonA subfamily, which includes the N-terminal domain, central ATPase (AAA) domain, and C-terminal proteolytic (P) domain. LonC belongs to the recently classified LonC subfamily, which includes Lon proteases that contain neither the N-terminal domain of LonA nor the transmembrane region that is present only in LonB subfamily members. To investigate the roles of LonA and LonC in A. pleuropneumoniae, mutants with deletions in the lonA and lonC genes were constructed. The impaired growth of the △lonA mutant exposed to low and high temperatures and osmotic and oxidative stress conditions indicates that the LonA protease is required for the stress tolerance of A. pleuropneumoniae. Furthermore, the △lonA mutant exhibited significantly reduced biofilm formation compared to the wild-type strain. However, no significant differences in stress responses or biofilm formation were observed between the △lonC mutant and the wild-type strain. The △lonA mutant exhibited reduced colonization ability and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model compared to the wild-type strain. Disruption of lonC gene did not significantly influence the colonization and virulence of A. pleuropneumoniae. The data presented in this study illustrate that the LonA protease, but not the LonC protease, is required for the stress tolerance, biofilm formation and pathogenicity of A. pleuropneumoniae. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Shrimp Serine Proteinase Homologues PmMasSPH-1 and -2 Play a Role in the Activation of the Prophenoloxidase System

    Science.gov (United States)

    Jearaphunt, Miti; Amparyup, Piti; Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Senapin, Saengchan; Tassanakajon, Anchalee

    2015-01-01

    Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system. PMID:25803442

  16. Requirements of Multiple Domains of SLI-1, a Caenorhabditis elegans Homologue of c-Cbl, and an Inhibitory Tyrosine in LET-23 in Regulating Vulval Differentiation

    Science.gov (United States)

    Yoon, Charles H.; Chang, Chieh; Hopper, Neil A.; Lesa, Giovanni M.; Sternberg, Paul W.

    2000-01-01

    SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin–protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss of sli-1 but not of another negative regulator, ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin–ligase domain. PMID:11071924

  17. Characterization and mapping to human chromosome 8q24.3 of Ly-6-related gene 9804 encoding an apparent homologue of mouse TSA-1.

    Science.gov (United States)

    Shan, X; Bourdeau, A; Rhoton, A; Wells, D E; Cohen, E H; Landgraf, B E; Palfree, R G

    1998-01-01

    The 9804 gene, which encodes a human Ly-6 protein most similar to mouse differentiation Ag TSA-1/Sca-2, has also been called RIG-E. Like mouse TSA-1, it has a broad tissue distribution with varied expression levels in normal human tissues and tumor cell lines. Like some members of the murine Ly-6 family, the 9804 gene is responsive to IFNs, particularly IFN-alpha. Overlapping genomic fragments spanning the 9804 gene (5543 bp) have been isolated and characterized. The gene organization is analogous to that of known mouse Ly-6 genes. The first exon, 2296 bp upstream from exon II, is entirely untranslated. The three coding exons (II, III, and IV) are separated by short introns of 321 and 131 bp, respectively. Primers were developed for specific amplification of 9804 gene fragments. Screening of human-hamster somatic cell hybrids and yeast artificial chromosomes (YACs) indicated that the gene is distal to c-Myc, located in the q arm of human chromosome 8. No positives were detected from the Centre d'Etude du Polymorphisme Humain mega-YAC A or B panels, nor from bacterial artificial chromosome libraries; two positive cosmids (c101F1 and c157F6) were isolated from a human chromosome 8 cosmid library (LA08NC01). Fluorescence in situ hybridization of metaphase spreads of chromosome 8, containing hybrid cell line 706-B6 clone 17 (CL-17) with cosmid c101F1, placed the 9804 gene close to the telomere at 8q24.3. This mapping is significant, since the region shares a homology with a portion of mouse chromosome 15, which extends into band E where Ly-6 genes reside. Moreover, the gene encoding E48, the homologue of mouse Ly-6 molecule ThB, has also been mapped to 8q24.

  18. CDX2 Inhibits Invasion and Migration of Gastric Cancer Cells by Phosphatase and Tensin Homologue Deleted from Chromosome 10/Akt Signaling Pathway

    Institute of Scientific and Technical Information of China (English)

    Yong-Qiang Liu; Zhi-Gang Bai; Xue-Mei Ma; Zhong-Tao Zhang

    2015-01-01

    Background:Gastric cancer (GC) is one of the most prevalent malignancies in the world today,with a high mortality rate.CDX2 is a Drosophila caudal-related homeobox transcription factor that plays an important role in GC.Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) is an important tumor suppressor which is widely expressed in normal human tissues.The aim of the study was to determine the relationship and mechanism between CDX2 and PTEN in invasion and migration of GC cells.Methods:pcDNA3-CDX2 plasmids were transfected into MGC-803 cells to up-regulate CDX2 protein,and small interfering RNA-CDX2 was transfected to down-regulate CDX2.The influence of CDX2 or PTEN on cell migration and invasion was measured by invasion,migration and wound healing assays.Western blotting assay and immunofluorescence were used to detect the expression ofCDX2,PTEN,phosphorylation ofAkt,E-cadherin and N-cadherin.Statistical significance was determined by one-way analysis of variance.Results:The results showed that CDX2 reduced the migration and invasion of GC cells (P < 0.05),and inhibited the activity of Akt through down-regulating PTEN expression (P < 0.05).CDX2 also restrained epithelial-mesenchymal transition of GC cells.Conclusions:CDX2 inhibited invasion and migration of GC cells by PTEN/Akt signaling pathway,and that may be used for potential therapeutic target.

  19. Expression of Phosphatase and Tensin Homologue, phospho-Akt, and p53 in Acral Benign and Malignant Melanocytic Neoplasms (Benign Nevi, Dysplastic Nevi, and Acral Melanomas)

    Science.gov (United States)

    Lyu, So Min; Wu, Ju Yeon; Byun, Ji Yeon; Choi, Hae Young; Park, Sang Hee

    2016-01-01

    Background The role of the phosphatidylinositol-3 kinase signaling pathway in the development of acral melanoma has recently gained evidence. Phosphatase and tensin homologue (PTEN), one of the key molecules in the pathway, acts as a tumor suppressor through either an Akt-dependent or Akt-independent pathway. Akt accelerates degradation of p53. Objective We assessed the expression of PTEN, phospho-Akt (p-Akt), and p53 by immunohistochemistry in benign acral nevi, acral dysplastic nevi, and acral melanomas in the radial growth phase and with a vertical growth component. Methods Ten specimens in each group were included. Paraffin-embedded specimens were immunostained with antibodies for PTEN, p-Akt, and p53. We scored both the staining intensity and the proportion of positive cells. The final score was calculated by multiplying the intensity score by the proportion score. Results All specimens of benign acral nevi except one showed some degree of PTEN-negative cells. The numbers of p-Akt and p53-positive cells were higher in acral dysplastic nevi and melanoma than in benign nevi. P-Akt scores were 1.7, 1.8, 2.6, and 4.4, and p53 scores were 2.0, 2.1, 3.8, and 4.1 in each group. PTEN and p-Akt scores in advanced acral melanoma were higher than in the other neoplasms. Conclusion The expression of PTEN was decreased and the expression of p-Akt was increased in acral melanoma, especially in advanced cases. The PTEN-induced pathway appears to affect the late stage of melanomagenesis. Altered expression of p-Akt is thought to be due to secondary changes following the loss of PTEN. PMID:27746632

  20. Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting

    Science.gov (United States)

    Singer, Josef; Weichselbaumer, Marlene; Stockner, Thomas; Mechtcheriakova, Diana; Sobanov, Yury; Bajna, Erika; Wrba, Friedrich; Horvat, Reinhard; Thalhammer, Johann G.; Willmann, Michael; Jensen-Jarolim, Erika

    2012-01-01

    To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology. Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G0/G1 phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules. Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development. PMID:22424313