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  1. Messenger RNA transcripts

    Treesearch

    Dan Cullen

    2004-01-01

    In contrast to DNA, messenger RNA (mRNA) in complex substrata is rarely analyzed, in large part because labile RNA molecules are difficult to purify. Nucleic acid extractions from fungi that colonize soil are particularly difficult and plagued by humic substances that interfere with Taq polymerase (Tebbe and Vahjen 1993 and references therein). Magnetic capture...

  2. Who discovered messenger RNA?

    PubMed

    Cobb, Matthew

    2015-06-29

    The announcement of the discovery of messenger RNA (mRNA) and the cracking of the genetic code took place within weeks of each other in a climax of scientific excitement during the summer of 1961. Although mRNA is of decisive importance to our understanding of gene function, no Nobel Prize was awarded for its discovery. The large number of people involved, the complex nature of the results, and the tortuous path that was taken over half a century ago, all show that simple claims of priority may not reflect how science works.

  3. Messenger RNA Decay.

    PubMed

    Kushner, Sidney R

    2007-04-01

    This chapter discusses several topics relating to the mechanisms of mRNA decay. These topics include the following: important physical properties of mRNA molecules that can alter their stability; methods for determining mRNA half-lives; the genetics and biochemistry of proteins and enzymes involved in mRNA decay; posttranscriptional modification of mRNAs; the cellular location of the mRNA decay apparatus; regulation of mRNA decay; the relationships among mRNA decay, tRNA maturation, and ribosomal RNA processing; and biochemical models for mRNA decay. Escherichia coli has multiple pathways for ensuring the effective decay of mRNAs and mRNA decay is closely linked to the cell's overall RNA metabolism. Finally, the chapter highlights important unanswered questions regarding both the mechanism and importance of mRNA decay.

  4. Bifunctional transfer-messenger RNA

    PubMed Central

    Ramadoss, Nitya S.

    2011-01-01

    Transfer-messenger RNA (tmRNA) is a bifunctional RNA that has properties of a tRNA and an mRNA. tmRNA uses these two functions to release ribosomes stalled during translation and target the nascent polypeptides for degradation. This concerted reaction, known as trans-translation, contributes to translational quality control and regulation of gene expression in bacteria. tmRNA is conserved throughout bacteria, and is one of the most abundant RNAs in the cell, suggesting that trans-translation is of fundamental importance for bacterial fitness. Mutants lacking tmRNA activity typically have severe phenotypes, including defects in viability, virulence, and responses to environmental stresses. PMID:21664408

  5. Nuclear Export of Messenger RNA

    PubMed Central

    Katahira, Jun

    2015-01-01

    Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

  6. Messenger RNA Methylation Regulates Glioblastoma Tumorigenesis.

    PubMed

    Dixit, Deobrat; Xie, Qi; Rich, Jeremy N; Zhao, Jing Crystal

    2017-04-10

    Messenger RNA (mRNA) modification provides an additional layer of gene regulation in cells. In this issue of Cancer Cell, Zhang et al. report that ALKBH5, a demethylase of the mRNA modification N(6)-methyladenosine, regulates proliferation and self-renewal of glioblastoma stem-like cells by modulating pre-mRNA stability and expression of the FOXM1 gene.

  7. [Posttranscriptional messenger RNA modifications in eukaryotes].

    PubMed

    Laptev, I G; Golovina, A Ya; Sergiev, P V; Dontsova, O A

    2015-01-01

    Genomewide mapping of posttranscriptional modification in eukaryotic RNA allowed to reveal tens of thousands modification sites. Among modified nucleotides of eukaryotic RNA 6-methyladenosine, 5-methylcytidine, pseudouridine, inosine, and others. Many modification sites are conserved, many are regulated. Function is known for a small subset of modified nucleotides, while the role of majority of them is still obscure. Global character of mRNA modifications allowed scientists to coin a new term, RNA epigenetics. The review is about posttranscriptional messenger RNA modifications in eukaryotes. Main modifications, their role in cell, their mapping techniques and proteins, that are responsible for such RNA modifications are observed.

  8. Messenger RNA modifications: Form, distribution, and function.

    PubMed

    Gilbert, Wendy V; Bell, Tristan A; Schaening, Cassandra

    2016-06-17

    RNA contains more than 100 distinct modifications that promote the functions of stable noncoding RNAs in translation and splicing. Recent technical advances have revealed widespread and sparse modification of messenger RNAs with N(6)-methyladenosine (m(6)A), 5-methylcytosine (m(5)C), and pseudouridine (Ψ). Here we discuss the rapidly evolving understanding of the location, regulation, and function of these dynamic mRNA marks, collectively termed the epitranscriptome. We highlight differences among modifications and between species that could instruct ongoing efforts to understand how specific mRNA target sites are selected and how their modification is regulated. Diverse molecular consequences of individual m(6)A modifications are beginning to be revealed, but the effects of m(5)C and Ψ remain largely unknown. Future work linking molecular effects to organismal phenotypes will broaden our understanding of mRNA modifications as cell and developmental regulators. Copyright © 2016, American Association for the Advancement of Science.

  9. ACTH Action on Messenger RNA Stability Mechanisms

    PubMed Central

    Desroches-Castan, Agnès; Feige, Jean-Jacques; Cherradi, Nadia

    2017-01-01

    The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3′-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology. PMID:28163695

  10. Messenger RNA processing in Methanocaldococcus (Methanococcus) jannaschii

    PubMed Central

    Zhang, Jian; Olsen, Gary J.

    2009-01-01

    Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12–16 nucleotides upstream of the corresponding translation start site. Analyses with 3′-RACE (rapid amplification of cDNA ends) and 5′-RACE indicate that the processing is endonucleolytic. Analyses of the sequences surrounding the processing sites for functional sites, sequence motifs, or potential RNA secondary structure elements did not reveal any recurring features except for an AUG translation start codon and (in most cases) a ribosome binding site. These properties differ from those of all previously described mRNA processing systems. Our data suggest that the processing alters the representation of various genes in the RNA pool and therefore, may play a significant role in defining the balance of proteins in the cell. PMID:19717546

  11. Processivity and Coupling in Messenger RNA Transcription

    PubMed Central

    Aitken, Stuart; Robert, Marie-Cécile; Alexander, Ross D.; Goryanin, Igor; Bertrand, Edouard; Beggs, Jean D.

    2010-01-01

    Background The complexity of messenger RNA processing is now being uncovered by experimental techniques that are capable of detecting individual copies of mRNA in cells, and by quantitative real-time observations that reveal the kinetics. This processing is commonly modelled by permitting mRNA to be transcribed only when the promoter is in the on state. In this simple on/off model, the many processes involved in active transcription are represented by a single reaction. These processes include elongation, which has a minimum time for completion and processing that is not captured in the model. Methodology In this paper, we explore the impact on the mRNA distribution of representing the elongation process in more detail. Consideration of the mechanisms of elongation leads to two alternative models of the coupling between the elongating polymerase and the state of the promoter: Processivity allows polymerases to complete elongation irrespective of the promoter state, whereas coupling requires the promoter to be active to produce a full-length transcript. We demonstrate that these alternatives have a significant impact on the predicted distributions. Models are simulated by the Gillespie algorithm, and the third and fourth moments of the resulting distribution are computed in order to characterise the length of the tail, and sharpness of the peak. By this methodology, we show that the moments provide a concise summary of the distribution, showing statistically-significant differences across much of the feasible parameter range. Conclusions We conclude that processivity is not fully consistent with the on/off model unless the probability of successfully completing elongation is low—as has been observed. The results also suggest that some form of coupling between the promoter and a rate-limiting step in transcription may explain the cell's inability to maintain high mRNA levels at low noise—a prediction of the on/off model that has no supporting evidence. PMID

  12. Release, Identification, and Isolation of Messenger RNA from Mammalian Ribosomes

    PubMed Central

    Blobel, Günter

    1971-01-01

    The puromycin-induced dissociation, at high ionic strength, of active ribosomes from reticulocytes resulted in the release of protein-free, apparently undegraded, messenger RNA for globin, which was identified by centrifugation on a sucrose density gradient. This procedure should make messenger RNA from various cells available for isolation on a large scale, and has several advantages over procedures that use detergents or magnesium chelators. PMID:5279524

  13. Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system

    PubMed Central

    2010-01-01

    Background A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink). Results Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc) and of the enhanced green fluorescent protein (eGFP). Peptide tags specific to the endogenous E. coli AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured. Conclusions The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in new research areas such as

  14. Chemical and structural effects of base modifications in messenger RNA

    NASA Astrophysics Data System (ADS)

    Harcourt, Emily M.; Kietrys, Anna M.; Kool, Eric T.

    2017-01-01

    A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification -- the epitranscriptome -- is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.

  15. Messenger RNA Degradation in Bacterial Cells

    PubMed Central

    Hui, Monica P.; Foley, Patricia L.; Belasco, Joel G.

    2015-01-01

    mRNA degradation is an important mechanism for controlling gene expression in bacterial cells. This process involves the orderly action of a battery of cellular endonucleases and exonucleases, some universal and others present only in certain species. They function with the assistance of ancillary enzymes that covalently modify the 5’ or 3’ end of RNA or unwind base-paired regions. Triggered by initiating events at either the 5’ terminus or an internal site, mRNA decay occurs at diverse rates that are transcript-specific and governed by features such as RNA sequence and structure, translating ribosomes, and bound sRNAs or proteins. In response to environmental cues, bacteria are able to orchestrate widespread changes in mRNA lifetimes by modulating the concentration or specific activity of cellular ribonucleases or by unmasking the mRNA-degrading activity of cellular toxins. PMID:25292357

  16. Messenger RNA degradation in bacterial cells.

    PubMed

    Hui, Monica P; Foley, Patricia L; Belasco, Joel G

    2014-01-01

    mRNA degradation is an important mechanism for controlling gene expression in bacterial cells. This process involves the orderly action of a battery of cellular endonucleases and exonucleases, some universal and others present only in certain species. These ribonucleases function with the assistance of ancillary enzymes that covalently modify the 5' or 3' end of RNA or unwind base-paired regions. Triggered by initiating events at either the 5' terminus or an internal site, mRNA decay occurs at diverse rates that are transcript specific and governed by RNA sequence and structure, translating ribosomes, and bound sRNAs or proteins. In response to environmental cues, bacteria are able to orchestrate widespread changes in mRNA lifetimes by modulating the concentration or specific activity of cellular ribonucleases or by unmasking the mRNA-degrading activity of cellular toxins.

  17. A new modification for mammalian messenger RNA.

    PubMed

    Liu, Fange; He, Chuan

    2017-09-01

    The discovery of multiple RNA modifications in the past few years has broadened our views of the structures and potential functions of RNA species, but deciphering which modifications are made where and how remains a challenge. A new study by Xu et al. applies a combination of mass spectrometry, biochemistry, genetics, and cellular biology tools to reveal the two mammalian methyltransferases that are responsible for m(3)C installation in tRNA and a third that mediates the previously unknown installation of m(3)C in mammalian mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Estrogen Regulation of Messenger RNA Stability

    DTIC Science & Technology

    1990-08-17

    ribonuclease inhibitor, inhibits activity of RNase A-type enzymes. RNP-CS- ribonucleoprotein consensus sequence (K/R)G(F/Y)(G/A)FVX(F/Y) rRNA - ribosomal...CJ r i ɡ a 5S ^ S i C9 3 3 *» • - M 19 > • h- O C9 ^ h- 5 C9 > l - « < • - U f t - o CJ k u a u Q. < 2 C9 S C9 3 3 "ai t- 41 (9...mRNA molecules will need to be examined. Which of these factors degrade mRNAs? Which factors degrade other types of RNA molecules such as rRNA and

  19. Ovalbumin Messenger RNA: Evidence That the Initial Product of Transcription Is the Same Size as Polysomal Ovalbumin Messenger

    PubMed Central

    McKnight, G. Stanley; Schimke, Robert T.

    1974-01-01

    The messenger RNA for ovalbumin, the major secretory protein of the chick oviduct, appears not to be made as a high-molecular-weight precursor when artifacts due to aggregation are eliminated. No ovalbumin messenger RNA sequences that will hybridize to complementary DNA made against ovalbumin mRNA are found in concentrated samples of hen oviduct RNA larger than 28 S. The sensitivity of the hybridization assay is sufficient to detect less than one molecule of ovalbumin mRNA precursor per tubular gland cell. Newly synthesized ovalbumin messenger RNA isolated from immature chicks stimulated briefly by estrogen is the same size as that found in hen polyribosomes. We conclude that ovalbumin messenger RNA does not undergo any significant change in molecular weight from its initial transcription to its incorporation into polyribosomes. PMID:4530986

  20. Mapping the messenger RNA within the elongating ribosome

    NASA Astrophysics Data System (ADS)

    Jünemann, R.; Wadzack, J.; Burkhardt, N.; Schmitt, M.; Zhao, J.; Stuhrmann, H. B.; Nierhaus, K. H.

    1997-02-01

    The method of proton-spin contrast-variation was applied for determining the position of the messenger RNA within the elongating ribosome. Using an artificial mRNA fragment the mass center of the mRNA sequence covered by the ribosome could be localized for the pre- and the post-translocational elongation states. The mass center moves about 12 ± 5 Å upon translocation. The radius of gyration was 12 ± e Å. The data give an independent contribution for refining a structural model including the RNA ligands of the elongating ribosome.

  1. Chemical and structural effects of base modifications in messenger RNA

    PubMed Central

    Harcourt, Emily M.; Kietrys, Anna M.; Kool, Eric T.

    2017-01-01

    A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification — the epitranscriptome — is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles. PMID:28102265

  2. Turnover of messenger RNA: Polysome statistics beyond the steady state

    NASA Astrophysics Data System (ADS)

    Valleriani, A.; Ignatova, Z.; Nagar, A.; Lipowsky, R.

    2010-03-01

    The interplay between turnover or degradation and ribosome loading of messenger RNA (mRNA) is studied theoretically using a stochastic model that is motivated by recent experimental results. Random mRNA degradation affects the statistics of polysomes, i.e., the statistics of the number of ribosomes per mRNA as extracted from cells. Since ribosome loading of newly created mRNA chains requires some time to reach steady state, a fraction of the extracted mRNA/ribosome complexes does not represent steady state conditions. As a consequence, the mean ribosome density obtained from the extracted complexes is found to be inversely proportional to the mRNA length. On the other hand, the ribosome density profile shows an exponential decrease along the mRNA for prokaryotes and becomes uniform in eukaryotic cells.

  3. Messenger RNA processing sites in Trypanosoma brucei.

    PubMed

    Benz, Corinna; Nilsson, Daniel; Andersson, Björn; Clayton, Christine; Guilbride, D Lys

    2005-10-01

    In Kinetoplastids, protein-coding genes are transcribed polycistronically by RNA polymerase II. Individual mature mRNAs are generated from polycistronic precursors by 5' trans splicing of a 39-nt capped leader RNA and 3' polyadenylation. It was previously known that trans splicing generally occurs at an AG dinucleotide downstream of a polypyrimidine tract, and that polyadenylation is coupled to downstream trans splicing. The few polyadenylation sites that had been examined were 100-400 nt upstream of the polypyrimidine tract which marked the adjacent trans splice site. We wished to define the sequence requirements for trypanosome mRNA processing more tightly and to generate a predictive algorithm. By scanning all available Trypanosoma brucei cDNAs for splicing and polyadenylation sites, we found that trans splicing generally occurs at the first AG following a polypyrimidine tract of 8-25 nt, giving rise to 5'-UTRs of a median length of 68 nt. We also found that in general, polyadenylation occurs at a position with one or more A residues located between 80 and 140 nt from the downstream polypyrimidine tract. These data were used to calibrate free parameters in a grammar model with distance constraints, enabling prediction of polyadenylation and trans splice sites for most protein-coding genes in the trypanosome genome. The data from the genome analysis and the program are available from: .

  4. Messenger RNA-based vaccines: progress, challenges, applications.

    PubMed

    Kramps, Thomas; Probst, Jochen

    2013-01-01

    Twenty years after the demonstration that messenger RNA (mRNA) was expressed and immunogenic upon direct injection in mice, the first successful proof-of-concept of specific protection against viral infection in small and large animals was reported. These data indicate wider applicability to infectious disease and should encourage continued translation of mRNA-based prophylactic vaccines into human clinical trials. At the conceptual level, mRNA-based vaccines-more than other genetic vectors-combine the simplicity, safety, and focused immunogenicity of subunit vaccines with favorable immunological properties of live viral vaccines: (1) mRNA vaccines are molecularly defined and carry no excess information. In the environment and upon physical contact, RNA is rapidly degraded by ubiquitous RNases and cannot persist. These characteristics also guarantee tight control over their immunogenic profile (including avoidance of vector-specific immune responses that could interfere with repeated administration), pharmacokinetics, and dosing. (2) mRNA vaccines are synthetically produced by an enzymatic process, just requiring information about the nucleic acid sequence of the desired antigen. This greatly reduces general complications associated with biological vaccine production, such as handling of infectious agents, genetic variability, environmental risks, or restrictions to vaccine distribution. (3) RNA can be tailored to provide potent adjuvant stimuli to the innate immune system by direct activation of RNA-specific receptors; this may reduce the need for additional adjuvants. The formation of native antigen in situ affords great versatility, including intracellular localization, membrane association, posttranslational modification, supra-molecular assembly, or targeted structural optimization of delivered antigen. Messenger RNA vaccines induce balanced immune responses including B cells, helper T cells, and cytotoxic T lymphocytes, rendering them an extremely adaptable

  5. The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

    PubMed

    Dominissini, Dan; Nachtergaele, Sigrid; Moshitch-Moshkovitz, Sharon; Peer, Eyal; Kol, Nitzan; Ben-Haim, Moshe Shay; Dai, Qing; Di Segni, Ayelet; Salmon-Divon, Mali; Clark, Wesley C; Zheng, Guanqun; Pan, Tao; Solomon, Oz; Eyal, Eran; Hershkovitz, Vera; Han, Dali; Doré, Louis C; Amariglio, Ninette; Rechavi, Gideon; He, Chuan

    2016-02-25

    Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

  6. The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA

    PubMed Central

    Dominissini, Dan; Nachtergaele, Sigrid; Moshitch-Moshkovitz, Sharon; Peer, Eyal; Kol, Nitzan; Ben-Haim, Moshe Shay; Dai, Qing; Di Segni, Ayelet; Salmon-Divon, Mali; Clark, Wesley C.; Zheng, Guanqun; Pan, Tao; Solomon, Oz; Eyal, Eran; Hershkovitz, Vera; Han, Dali; Doré, Louis C.; Amariglio, Ninette; Rechavi, Gideon; He, Chuan

    2016-01-01

    Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N6-methyladenosine (m6A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N1-methyladenosine (m1A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m1A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m1A in promoting translation of methylated mRNA. PMID:26863196

  7. Length-dependent translation of messenger RNA by ribosomes

    NASA Astrophysics Data System (ADS)

    Valleriani, Angelo; Zhang, Gong; Nagar, Apoorva; Ignatova, Zoya; Lipowsky, Reinhard

    2011-04-01

    A simple measure for the efficiency of protein synthesis by ribosomes is provided by the steady state amount of protein per messenger RNA (mRNA), the so-called translational ratio, which is proportional to the translation rate. Taking the degradation of mRNA into account, we show theoretically that both the translation rate and the translational ratio decrease with increasing mRNA length, in agreement with available experimental data for the prokaryote Escherichia coli. We also show that, compared to prokaryotes, mRNA degradation in eukaryotes leads to a less rapid decrease of the translational ratio. This finding is consistent with the fact that, compared to prokaryotes, eukaryotes tend to have longer proteins.

  8. Nuclear networking fashions pre-messenger RNA and primary microRNA transcripts for function

    PubMed Central

    Pawlicki, Jan M.; Steitz, Joan A.

    2010-01-01

    The expression of protein-coding genes is enhanced by the exquisite coupling of transcription by RNA polymerase II with pre-messenger RNA processing reactions, such as 5′-end capping, splicing and 3′-end formation. Integration between cotranscriptional processing events extends beyond the nucleus, as proteins that bind cotranscriptionally can affect the localization, translation and degradation of the mature messenger RNA. MicroRNAs are RNA polymerase II transcripts with crucial roles in the regulation of gene expression. Recent data demonstrate that processing of primary microRNA transcripts might be yet another cotranscriptional event that is woven into this elaborate nuclear network. This review discusses the extensive molecular interactions that couple the earliest steps in gene expression and therefore influence the final fate and function of the mature messenger RNA or microRNA produced. PMID:20004579

  9. Translation of globin messenger RNA by the mouse ovum

    PubMed Central

    Brinster, R. L.; Chen, H. Y.; Trumbauer, M. E.; Avarbock, M. R.

    2016-01-01

    It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell1. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species2–4. This system has proved useful in understanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte5,6. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell7,8. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system. PMID:7352032

  10. Messenger RNA (mRNA) nanoparticle tumour vaccination

    NASA Astrophysics Data System (ADS)

    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.

  11. Multifunctional triblock copolymers for intracellular messenger RNA delivery.

    PubMed

    Cheng, Connie; Convertine, Anthony J; Stayton, Patrick S; Bryers, James D

    2012-10-01

    Messenger RNA (mRNA) is a promising alternative to plasmid DNA (pDNA) for gene vaccination applications, but safe and effective delivery systems are rare. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize a series of triblock copolymers designed to enhance the intracellular delivery of mRNA. These materials are composed of a cationic dimethylaminoethyl methacrylate (DMAEMA) segment to mediate mRNA condensation, a hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) segment to enhance stability and biocompatibility, and a pH-responsive endosomolytic copolymer of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) designed to facilitate cytosolic entry. The blocking order and PEGMA segment length were systematically varied to investigate the effect of different polymer architectures on mRNA delivery efficacy. These polymers were monodisperse, exhibited pH-dependent hemolytic activity, and condensed mRNA into 86-216 nm particles. mRNA polyplexes formed from polymers with the PEGMA segment in the center of the polymer chain displayed the greatest stability to heparin displacement and were associated with the highest transfection efficiencies in two immune cell lines, RAW 264.7 macrophages (77%) and DC2.4 dendritic cells (50%). Transfected DC2.4 cells were shown to be capable of subsequently activating antigen-specific T cells, demonstrating the potential of these multifunctional triblock copolymers for mRNA-based vaccination strategies.

  12. BACTERIAL IDENTIFICATION USING SSRA ENCODING TRANSFER-MESSENGER RNA.

    PubMed

    Osawa, Kayo; Shigemura, Katsumi; Shirai, Hiroki; Kato, Ayaka; Okuya, Yuma; Jikimoto, Takumi; Arakawa, Soichi; Fujisawa, Masato; Shirakawa, Toshiro

    2015-07-01

    Abstract. Ribosomal DNA (rDNA) sequences are widely used for phylogenetic and bacterial identification. However, rDNA of different species often reveals similar or identical same sequences. This study employed the bacterial stable small RNA (ssrA) gene encoding transfer-messenger RNA (tmRNA) as a tool for identification of Staphylococcus aureus, Enterococcus spp, Pseudomonas spp and Enterobacteriaceae from clinical isolates as representative groups using PCR and species specific primers. The method correctly identified 11 standard strains and 99 clinical isolates. Quantitative PCR revealed a limit of detection of 10(-5) µg of DNA for S. aureus and Enterococcus spp, and 10(-6) µg for Pseudomonas spp and Enterobacteriaceae. Further studies with a greater number of bacteria especially from clinical samples will need to be undertaken before this bacterial molecular marker can be applied in a clinical setting.

  13. Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

    SciTech Connect

    Jbilo, O.; Barteles, C.F.; Chatonnet, A.; Toutant, J.P.; Lockridge, O.

    1994-12-31

    Tissue distribution of human acetyicholinesterase and butyryicholinesterase messenger RNA. 1 Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetyicholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

  14. Messenger RNA modifications – Form, distribution, and function

    PubMed Central

    Gilbert, Wendy V.; Bell, Tristan A.; Schaening, Cassandra

    2016-01-01

    RNA contains more than 100 distinct modifications that promote the functions of stable non-coding RNAs in translation and splicing. Recent technical advances have revealed widespread and sparse modification of messenger RNAs with N6-methyladenosine (m6A), 5-methylcytosine (m5C) and pseudouridine (Ψ). Here we discuss the rapidly evolving understanding of the location, regulation and function of these dynamic mRNA marks, collectively termed the epitranscriptome. We highlight differences among modifications and between species that could instruct ongoing efforts to understand how specific mRNAs target sites are selected and how their modification is regulated. Diverse molecular consequences of individual m6A modifications are beginning to be revealed but the effects of m5C and Ψ remain largely unknown. Future work linking molecular effects to organismal phenotypes will broaden our understanding of mRNA modifications as cell and developmental regulators. PMID:27313037

  15. Pseudo–Messenger RNA: Phantoms of the Transcriptome

    PubMed Central

    Frith, Martin C; Wilming, Laurens G; Forrest, Alistair; Kawaji, Hideya; Tan, Sin Lam; Wahlestedt, Claes; Bajic, Vladimir B; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Bailey, Timothy L; Huminiecki, Lukasz

    2006-01-01

    The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo–messenger RNA to be RNA molecules that resemble protein-coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo–messenger RNAs (approximately half of which are transposon-associated) among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein-coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense-mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non-standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level. PMID:16683022

  16. m6A-dependent regulation of messenger RNA stability

    PubMed Central

    Wang, Xiao; Lu, Zhike; Gomez, Adrian; Hon, Gary C.; Yue, Yanan; Han, Dali; Fu, Ye; Parisien, Marc; Dai, Qing; Jia, Guifang; Ren, Bing; Pan, Tao; He, Chuan

    2013-01-01

    N6-methyladenosine (m6A) is the most prevalent internal (non-cap) modification present in the messenger RNA (mRNA) of all higher eukaryotes1,2. Although essential to cell viability and development3–5, the exact role of m6A modification remains to be determined. The recent discovery of two m6A demethylases in mammalian cells highlighted the importance of m6A in basic biological functions and disease6–8. Here we show that m6A is selectively recognized by the human YTH domain family 2 (YTHDF2) protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m6A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies9. The C-terminal domain of YTHDF2 selectively binds to m6A-containing mRNA whereas the N-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m6A modification is recognized by selective-binding proteins to affect the translation status and lifetime of mRNA. PMID:24284625

  17. [Deregulation of pre-messenger RNA splicing and rare diseases].

    PubMed

    de la Grange, Pierre

    2016-12-01

    Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein.

  18. Human Bocavirus Capsid Messenger RNA Detection in Children With Pneumonia.

    PubMed

    Schlaberg, Robert; Ampofo, Krow; Tardif, Keith D; Stockmann, Chris; Simmon, Keith E; Hymas, Weston; Flygare, Steven; Kennedy, Brett; Blaschke, Anne; Eilbeck, Karen; Yandell, Mark; McCullers, Jon A; Williams, Derek J; Edwards, Kathryn; Arnold, Sandra R; Bramley, Anna; Jain, Seema; Pavia, Andrew T

    2017-09-15

    The role of human bocavirus (HBoV) in respiratory illness is uncertain. HBoV genomic DNA is frequently detected in both ill and healthy children. We hypothesized that spliced viral capsid messenger RNA (mRNA) produced during active replication might be a better marker for acute infection. As part of the Etiology of Pneumonia in the Community (EPIC) study, children aged <18 years who were hospitalized with community-acquired pneumonia (CAP) and children asymptomatic at the time of elective outpatient surgery (controls) were enrolled. Nasopharyngeal/oropharyngeal specimens were tested for HBoV mRNA and genomic DNA by quantitative polymerase chain reaction. HBoV DNA was detected in 10.4% of 1295 patients with CAP and 7.5% of 721 controls (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.0-2.0]); HBoV mRNA was detected in 2.1% and 0.4%, respectively (OR, 5.1 [95% CI, 1.6-26]). When adjusted for age, enrollment month, and detection of other respiratory viruses, HBoV mRNA detection (adjusted OR, 7.6 [95% CI, 1.5-38.4]) but not DNA (adjusted OR, 1.2 [95% CI, .6-2.4]) was associated with CAP. Among children with no other pathogens detected, HBoV mRNA (OR, 9.6 [95% CI, 1.9-82]) was strongly associated with CAP. Detection of HBoV mRNA but not DNA was associated with CAP, supporting a pathogenic role for HBoV in CAP. HBoV mRNA could be a useful target for diagnostic testing.

  19. A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA

    PubMed Central

    Martick, Monika; Horan, Lucas H.; Noller, Harry F.; Scott, William G.

    2008-01-01

    Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5′ UTR1. This discovery has raised the question of whether gene-regulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes2,3 are present in the 3′ UTRs of rodent C-type lectin type II (Clec2) genes4–7. Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads8–10, occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the poly-adenylation signals within the 3′ UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of post-transcriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals. PMID:18615019

  20. Protein secondary structural types are differentially coded on messenger RNA.

    PubMed Central

    Thanaraj, T. A.; Argos, P.

    1996-01-01

    Tricodon regions on messenger RNAs corresponding to a set of proteins from Escherichia coli were scrutinized for their translation speed. The fractional frequency values of the individual codons as they occur in mRNAs of highly expressed genes from Escherichia coli were taken as an indicative measure of the translation speed. The tricodons were classified by the sum of the frequency values of the constituent codons. Examination of the conformation of the encoded amino acid residues in the corresponding protein tertiary structures revealed a correlation between codon usage in mRNA and topological features of the encoded proteins. Alpha helices on proteins tend to be preferentially coded by translationally fast mRNA regions while the slow segments often code for beta strands and coil regions. Fast regions correspondingly avoid coding for beta strands and coil regions while the slow regions similarly move away from encoding alpha helices. Structural and mechanistic aspects of the ribosome peptide channel support the relevance of sequence fragment translation and subsequent conformation. A discussion is presented relating the observation to the reported kinetic data on the formation and stabilization of protein secondary structural types during protein folding. The observed absence of such strong positive selection for codons in non-highly expressed genes is compatible with existing theories that mutation pressure may well dominate codon selection in non-highly expressed genes. PMID:8897597

  1. Messenger RNA patterns in rat liver nuclei before and after treat-ment with growth hormone.

    PubMed

    Drews, J; Brawerman, G

    1967-06-09

    Like cortisol, growth hormone enhances RNA synthesis in rat liver nuclei. However, DNA-RNA hybridization experiments show that the application of growth hormone does not stimulate the formation of new species of messenger RNA. The latter phenomenon was observed after treatment with cortisol.

  2. Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates.

    PubMed Central

    Damha, M J; Ganeshan, K; Hudson, R H; Zabarylo, S V

    1992-01-01

    The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described. The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA [(Xp)nA2' (pN)n3'(pN)n] related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA. The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis. Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo. Images PMID:1480476

  3. Control of Messenger RNA Fate by RNA-Binding Proteins: An Emphasis on Mammalian Spermatogenesis

    PubMed Central

    IDLER, R. KEEGAN; YAN, WEI

    2017-01-01

    Posttranscriptional status of messenger RNAs (mRNA) can be affected by many factors, most of which are RNA-binding proteins (RBP) that either bind mRNA in a nonspecific manner or through specific motifs, usually located in the 39 untranslated regions. RBPs can also be recruited by small noncoding RNAs (sncRNA), which have been shown to be involved in posttranscriptional regulations and transposon repression (eg, microRNAs or P-element–induced wimpy testis–interacting RNA) as components of the sncRNA effector complex. Non–sncRNA-binding RBPs have much more diverse effects on their target mRNAs. Some can cause degradation of their target transcripts and/or repression of translation, whereas others can stabilize and/or activate translation. The splicing and exportation of transcripts from the nucleus to the cytoplasm are often mediated by sequence-specific RBPs. The mechanisms by which RBPs regulate mRNA transcripts involve manipulating the 39 poly(A) tail, targeting the transcript to polysomes or to other ribonuclear protein particles, recruiting regulatory proteins, or competing with other RBPs. Here, we briefly review the known mechanisms of posttranscriptional regulation mediated by RBPs, with an emphasis on how these mechanisms might control spermatogenesis in general. PMID:21757510

  4. Radioautographic localization of prolactin messenger RNA on histological sections by in situ hybridization.

    PubMed

    Pochet, R; Brocas, H; Vassart, G; Toubeau, G; Seo, H; Refetoff, S; Dumont, J E; Pasteels, J L

    1981-05-04

    In situ hybridization of complementary [H3]DNA ([H3]cDNA) synthetized from purified rat prolactin messenger RNA (rPRL mRNA) was performed to specifically identify on histologic sections of rat hypophysis cells expressing the PRL gene. Radioautographic labelling occurred over weakly acidophilic cells, while other acidophils, with darker cytoplasm did not contain more silver grains than blood vessels.

  5. A Contemporary, Laboratory-Intensive Course on Messenger RNA Transcription and Processing

    ERIC Educational Resources Information Center

    Carson, Sue; Miller, Heather

    2012-01-01

    Messenger ribonucleic acid (mRNA) plays a pivotal role in the central dogma of molecular biology. Importantly, molecular events occurring during and after mRNA synthesis have the potential to create multiple proteins from one gene, leading to some of the remarkable protein diversity that genomes hold. The North Carolina State University…

  6. A Contemporary, Laboratory-Intensive Course on Messenger RNA Transcription and Processing

    ERIC Educational Resources Information Center

    Carson, Sue; Miller, Heather

    2012-01-01

    Messenger ribonucleic acid (mRNA) plays a pivotal role in the central dogma of molecular biology. Importantly, molecular events occurring during and after mRNA synthesis have the potential to create multiple proteins from one gene, leading to some of the remarkable protein diversity that genomes hold. The North Carolina State University…

  7. MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity.

    PubMed

    Deng, Youping; Ai, Junmei; Guan, Xin; Wang, Zhaohui; Yan, Bin; Zhang, Daqin; Liu, Chang; Wilbanks, Mitch S; Escalon, Barbara Lynn; Meyers, Sharon A; Yang, Mary Qu; Perkins, Edward J

    2014-01-01

    RDX is a well-known pollutant to induce neurotoxicity. MicroRNAs (miRNA) and messenger RNA (mRNA) profiles are useful tools for toxicogenomics studies. It is worthy to integrate MiRNA and mRNA expression data to understand RDX-induced neurotoxicity. Rats were treated with or without RDX for 48 h. Both miRNA and mRNA profiles were conducted using brain tissues. Nine miRNAs were significantly regulated by RDX. Of these, 6 and 3 miRNAs were up- and down-regulated respectively. The putative target genes of RDX-regulated miRNAs were highly nervous system function genes and pathways enriched. Fifteen differentially genes altered by RDX from mRNA profiles were the putative targets of regulated miRNAs. The induction of miR-71, miR-27ab, miR-98, and miR-135a expression by RDX, could reduce the expression of the genes POLE4, C5ORF13, SULF1 and ROCK2, and eventually induce neurotoxicity. Over-expression of miR-27ab, or reduction of the expression of unknown miRNAs by RDX, could up-regulate HMGCR expression and contribute to neurotoxicity. RDX regulated immune and inflammation response miRNAs and genes could contribute to RDX- induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure. Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity.

  8. MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity

    PubMed Central

    2014-01-01

    Background RDX is a well-known pollutant to induce neurotoxicity. MicroRNAs (miRNA) and messenger RNA (mRNA) profiles are useful tools for toxicogenomics studies. It is worthy to integrate MiRNA and mRNA expression data to understand RDX-induced neurotoxicity. Results Rats were treated with or without RDX for 48 h. Both miRNA and mRNA profiles were conducted using brain tissues. Nine miRNAs were significantly regulated by RDX. Of these, 6 and 3 miRNAs were up- and down-regulated respectively. The putative target genes of RDX-regulated miRNAs were highly nervous system function genes and pathways enriched. Fifteen differentially genes altered by RDX from mRNA profiles were the putative targets of regulated miRNAs. The induction of miR-71, miR-27ab, miR-98, and miR-135a expression by RDX, could reduce the expression of the genes POLE4, C5ORF13, SULF1 and ROCK2, and eventually induce neurotoxicity. Over-expression of miR-27ab, or reduction of the expression of unknown miRNAs by RDX, could up-regulate HMGCR expression and contribute to neurotoxicity. RDX regulated immune and inflammation response miRNAs and genes could contribute to RDX- induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure. Conclusions Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity. PMID:25559034

  9. Guardian of Genetic Messenger-RNA-Binding Proteins.

    PubMed

    Anji, Antje; Kumari, Meena

    2016-01-06

    RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

  10. Guardian of Genetic Messenger-RNA-Binding Proteins

    PubMed Central

    Anji, Antje; Kumari, Meena

    2016-01-01

    RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins. PMID:26751491

  11. Characterization of long noncoding RNA and messenger RNA signatures in melanoma tumorigenesis and metastasis

    PubMed Central

    Wang, Siqi; Fan, Wenliang; Wan, Bing; Tu, Mengqi; Jin, Feng; Liu, Fang; Xu, Haibo; Han, Ping

    2017-01-01

    The incidence of melanoma, the most aggressive and life-threatening form of skin cancer, has significantly risen over recent decades. Therefore, it is essential to identify the mechanisms that underlie melanoma tumorigenesis and metastasis and to explore novel and effective melanoma treatment strategies. Accumulating evidence s uggests that aberrantly expressed long noncoding RNAs (lncRNAs) have vital functions in multiple cancers. However, lncRNA functions in melanoma tumorigenesis and metastasis remain unclear. In this study, we investigated lncRNA and messenger RNA (mRNA) expression profiles in primary melanomas, metastatic melanomas and normal skin samples from the Gene Expression Omnibus database. We used GSE15605 as the training set (n = 74) and GSE7553 as the validation set (n = 58). In three comparisons (primary melanoma versus normal skin, metastatic melanoma versus normal skin, and metastatic melanoma versus primary melanoma), 178, 295 and 48 lncRNAs and 847, 1758, and 295 mRNAs were aberrantly expressed, respectively. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses to examine the differentially expressed mRNAs, and potential core lncRNAs were predicted by lncRNA-mRNA co-expression networks. Based on our results, 15 lncRNAs and 144 mRNAs were significantly associated with melanoma tumorigenesis and metastasis. A subsequent analysis suggested a critical role for a five-lncRNA signature during melanoma tumorigenesis and metastasis. Low expression of U47924.27 was significantly associated with decreased survival of patients with melanoma. To the best of our knowledge, this study is the first to explore the expression patterns of lncRNAs and mRNAs during melanoma tumorigenesis and metastasis by re-annotating microarray data from the Gene Expression Omnibus (GEO) microarray dataset. These findings reveal potential roles for lncRNAs during melanoma tumorigenesis and metastasis and provide a rich candidate reservoir for

  12. Characterization of long noncoding RNA and messenger RNA signatures in melanoma tumorigenesis and metastasis.

    PubMed

    Wang, Siqi; Fan, Wenliang; Wan, Bing; Tu, Mengqi; Jin, Feng; Liu, Fang; Xu, Haibo; Han, Ping

    2017-01-01

    The incidence of melanoma, the most aggressive and life-threatening form of skin cancer, has significantly risen over recent decades. Therefore, it is essential to identify the mechanisms that underlie melanoma tumorigenesis and metastasis and to explore novel and effective melanoma treatment strategies. Accumulating evidence s uggests that aberrantly expressed long noncoding RNAs (lncRNAs) have vital functions in multiple cancers. However, lncRNA functions in melanoma tumorigenesis and metastasis remain unclear. In this study, we investigated lncRNA and messenger RNA (mRNA) expression profiles in primary melanomas, metastatic melanomas and normal skin samples from the Gene Expression Omnibus database. We used GSE15605 as the training set (n = 74) and GSE7553 as the validation set (n = 58). In three comparisons (primary melanoma versus normal skin, metastatic melanoma versus normal skin, and metastatic melanoma versus primary melanoma), 178, 295 and 48 lncRNAs and 847, 1758, and 295 mRNAs were aberrantly expressed, respectively. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses to examine the differentially expressed mRNAs, and potential core lncRNAs were predicted by lncRNA-mRNA co-expression networks. Based on our results, 15 lncRNAs and 144 mRNAs were significantly associated with melanoma tumorigenesis and metastasis. A subsequent analysis suggested a critical role for a five-lncRNA signature during melanoma tumorigenesis and metastasis. Low expression of U47924.27 was significantly associated with decreased survival of patients with melanoma. To the best of our knowledge, this study is the first to explore the expression patterns of lncRNAs and mRNAs during melanoma tumorigenesis and metastasis by re-annotating microarray data from the Gene Expression Omnibus (GEO) microarray dataset. These findings reveal potential roles for lncRNAs during melanoma tumorigenesis and metastasis and provide a rich candidate reservoir for

  13. The path of messenger RNA through the ribosome.

    PubMed

    Yusupova, G Z; Yusupov, M M; Cate, J H; Noller, H F

    2001-07-27

    Using X-ray crystallography, we have directly observed the path of mRNA in the 70S ribosome in Fourier difference maps at 7 A resolution. About 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. The Shine-Dalgarno helix is bound in a large cleft between the head and the back of the platform. At the interface, only about eight nucleotides (-1 to +7), centered on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA. The mRNA enters the ribosome around position +13 to +15, the location of downstream pseudoknots that stimulate -1 translational frame shifting.

  14. Self-complementarity of messenger RNA's of periodic proteins

    NASA Technical Reports Server (NTRS)

    Ycas, M.

    1973-01-01

    It is shown that the mRNA's of three periodic proteins, collagen, keratin and freezing point depressing glycoproteins show a marked degree of self-complementarity. The possible origin of this self-complementarity is discussed.

  15. Self-complementarity of messenger RNA's of periodic proteins

    NASA Technical Reports Server (NTRS)

    Ycas, M.

    1973-01-01

    It is shown that the mRNA's of three periodic proteins, collagen, keratin and freezing point depressing glycoproteins show a marked degree of self-complementarity. The possible origin of this self-complementarity is discussed.

  16. Structural signatures of thermal adaptation of bacterial ribosomal RNA, transfer RNA, and messenger RNA.

    PubMed

    Jegousse, Clara; Yang, Yuedong; Zhan, Jian; Wang, Jihua; Zhou, Yaoqi

    2017-01-01

    Temperature adaptation of bacterial RNAs is a subject of both fundamental and practical interest because it will allow a better understanding of molecular mechanism of RNA folding with potential industrial application of functional thermophilic or psychrophilic RNAs. Here, we performed a comprehensive study of rRNA, tRNA, and mRNA of more than 200 bacterial species with optimal growth temperatures (OGT) ranging from 4°C to 95°C. We investigated temperature adaptation at primary, secondary and tertiary structure levels. We showed that unlike mRNA, tRNA and rRNA were optimized for their structures at compositional levels with significant tertiary structural features even for their corresponding randomly permutated sequences. tRNA and rRNA are more exposed to solvent but remain structured for hyperthermophiles with nearly OGT-independent fluctuation of solvent accessible surface area within a single RNA chain. mRNA in hyperthermophiles is essentially the same as random sequences without tertiary structures although many mRNA in mesophiles and psychrophiles have well-defined tertiary structures based on their low overall solvent exposure with clear separation of deeply buried from partly exposed bases as in tRNA and rRNA. These results provide new insight into temperature adaptation of different RNAs.

  17. Construction of a synthetic messenger RNA encoding a membrane protein

    PubMed Central

    1983-01-01

    We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV- infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes. PMID:6341380

  18. Defective control of pre-messenger RNA splicing in human disease.

    PubMed

    Chabot, Benoit; Shkreta, Lulzim

    2016-01-04

    Examples of associations between human disease and defects in pre-messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies.

  19. Defective control of pre–messenger RNA splicing in human disease

    PubMed Central

    Shkreta, Lulzim

    2016-01-01

    Examples of associations between human disease and defects in pre–messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies. PMID:26728853

  20. Regulation of chemoresistance via alternative messenger RNA splicing.

    PubMed

    Eblen, Scott T

    2012-04-15

    The acquisition of resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell's response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype.

  1. Messenger RNA exchange between scions and rootstocks in grafted grapevines

    USDA-ARS?s Scientific Manuscript database

    We demonstrated the existence of genome-scale mRNA exchange in grafted grapevines, a woody fruit species with significant economic importance. By using diagnostic SNPs derived from high throughput genome sequencing, we identified more than three thousand genes transporting mRNAs across graft junctio...

  2. Regulation of Chemoresistance Via Alternative Messenger RNA Splicing

    PubMed Central

    Eblen, Scott T.

    2012-01-01

    The acquisition of drug resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that have were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell’s response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype. PMID:22248731

  3. Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle.

    PubMed

    Sztuba-Solińska, Joanna; Stollar, Victor; Bujarski, Jozef J

    2011-04-10

    Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (-)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (-)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (-)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. How do messenger RNA splicing alterations drive myelodysplasia?

    PubMed Central

    2017-01-01

    Mutations in RNA splicing factors are the single most common class of genetic alterations in myelodysplastic syndrome (MDS) patients. Although much has been learned about how these mutations affect splicing at a global- and transcript-specific level, critical questions about the role of these mutations in MDS development and maintenance remain. Here we present the questions to be addressed in order to understand the unique enrichment of these mutations in MDS. PMID:28348147

  5. Labelling and imaging of single endogenous messenger RNA particles in vivo.

    PubMed

    Spille, Jan-Hendrik; Kubitscheck, Ulrich

    2015-10-15

    RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA life cycle from transcription, through the processing of nascent RNA, to the regulatory function of non-coding RNA and cytoplasmic translation of messenger RNA has been studied extensively using biochemical and molecular biology techniques. In this Commentary, we highlight how single molecule imaging and particle tracking can yield further insight into the dynamics of RNA particles in living cells. In the past few years, a variety of bright and photo-stable labelling techniques have been developed to generate sufficient contrast for imaging of single endogenous RNAs in vivo. New imaging modalities allow determination of not only lateral but also axial positions with high precision within the cellular context, and across a wide range of specimen from yeast and bacteria to cultured cells, and even multicellular organisms or live animals. A whole range of methods to locate and track single particles, and to analyze trajectory data are available to yield detailed information about the kinetics of all parts of the RNA life cycle. Although the concepts presented are applicable to all types of RNA, we showcase here the wealth of information gained from in vivo imaging of single particles by discussing studies investigating dynamics of intranuclear trafficking, nuclear pore transport and cytoplasmic transport of endogenous messenger RNA.

  6. Deregulated messenger RNA expression during T cell apoptosis.

    PubMed Central

    Kerkhoff, E; Ziff, E B

    1995-01-01

    The IL-2 dependent murine cytotoxic T cell line CTLL-2 undergoes programmed cell death when deprived of its specific cytokine. We analyzed the expression of cell cycle related genes after IL-2 deprivation. Here we show that a generalized decrease and re elevation of the levels of mRNA takes place as part of the apoptotic program. The levels of several mRNAs encoding cell cycle functions, including cyclin D2, cyclin D3, cyclin B1, c-myc and max all declined at 1.5-3 h following IL-2 deprivation. Notably, the maxmRNA, which was shown to be expressed in proliferating, growth arrested and differentiated cells, is down regulated with the same kinetics as the other mRNAs. Surprisingly, the mRNAs whose levels declined at 1.5-3 h rose again at 10-14 h, a time which closely followed the time of the first detection of apoptotic DNA degradation, at 8 h, but which precedes actual loss of viability, at 14 h, as measured by trypan blue exclusion. Of all analyzed genes only the expression of the S-phase specific histone H4 gene resists the initial decrease and declines gradually over the course of cell death. Measurement of c-Myc protein synthesis at a late stage of the apoptotic program revealed that the accumulated reinduced mRNA is not translated into protein. Because transcriptional regulation has been shown to be dependent on the chromatin structure, the reinduction may be triggered by relaxation of the chromatin caused by alterations in the chromatin structure of apoptotic cells. Images PMID:8532529

  7. Messenger RNA processing is altered in autosomal dominant leukodystrophy†

    PubMed Central

    Bartoletti-Stella, Anna; Gasparini, Laura; Giacomini, Caterina; Corrado, Patrizia; Terlizzi, Rossana; Giorgio, Elisa; Magini, Pamela; Seri, Marco; Baruzzi, Agostino; Parchi, Piero; Brusco, Alfredo; Cortelli, Pietro; Capellari, Sabina

    2015-01-01

    Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD. PMID:25637521

  8. Cortical rotation and messenger RNA localization in Xenopus axis formation.

    PubMed

    Houston, Douglas W

    2012-01-01

    In Xenopus eggs, fertilization initiates a rotational movement of the cortex relative to the cytoplasm, resulting in the transport of critical determinants to the future dorsal side of the embryo. Cortical rotation is mediated by microtubules, resulting in activation of the Wnt/β-catenin signaling pathway and expression of organizer genes on the dorsal side of the blastula. Similar cytoplasmic localizations resulting in β-catenin activation occur in many chordate embryos, suggesting a deeply conserved mechanism for patterning early embryos. This review summarizes the experimental evidence for the molecular basis of this model, focusing on recent maternal loss-of-function studies that shed light on two main unanswered questions: (1) what regulates microtubule assembly during cortical rotation and (2) how is Wnt/β-catenin signaling activated dorsally? In addition, as these processes depend on vegetally localized molecules in the oocyte, the mechanisms of RNA localization and novel roles for localized RNAs in axis formation are discussed. The work reviewed here provides a beginning framework for understanding the coupling of asymmetry in oogenesis with the establishment of asymmetry in the embryo. Copyright © 2012 Wiley Periodicals, Inc.

  9. Role of Auf1 in elimination of oxidatively damaged messenger RNA in human cells.

    PubMed

    Ishii, Takashi; Hayakawa, Hiroshi; Sekiguchi, Takeshi; Adachi, Noritaka; Sekiguchi, Mutsuo

    2015-02-01

    In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures. Both types of Auf1-deficient cells are viable, but exhibit growth retardation. The stability of messenger RNA for four different housekeeping genes was determined in Auf1-deficient and -proficient cells, treated with or without hydrogen peroxide. The level of oxidized messenger RNA was considerably higher in Auf1-deficient cells than in Auf1-proficient cells. Auf1 may play a role in the elimination of oxidized RNA, which is required for the maintenance of proper gene expression under conditions of oxidative stress.

  10. Use of Eukaryotic Native Small Ribosomal Subunits for the Translation of Globin Messenger RNA

    PubMed Central

    Freienstein, Christoph; Blobel, Günter

    1974-01-01

    A highly active in vitro system for the translation of globin mRNA, resulting in more than 10 rounds of translation, is described. The reconstituted system consists of native small ribosomal subunits of rabbit reticulocytes (as a source of initiation factors as well as small ribosomal subunits), large subunits derived from rat liver polysomes by the puromycin-KCl procedure, and a pH 5 fraction obtained from a Krebs ascites cell high speed supernatant. In this system no differences were found between globin messenger ribonucleoprotein and globin mRNA. Images PMID:4530315

  11. Messenger RNA fluctuations and regulatory RNAs shape the dynamics of a negative feedback loop

    NASA Astrophysics Data System (ADS)

    Rodríguez Martínez, María; Soriano, Jordi; Tlusty, Tsvi; Pilpel, Yitzhak; Furman, Itay

    2010-03-01

    Single-cell experiments of simple regulatory networks can markedly differ from cell population experiments. Such differences arise from stochastic events in individual cells that are averaged out in cell populations. For instance, while individual cells may show sustained oscillations in the concentrations of some proteins, such oscillations may appear damped in the population average. In this paper we investigate the role of RNA stochastic fluctuations as a leading force to produce a sustained excitatory behavior at the single-cell level. As opposed to some previous models, we build a fully stochastic model of a negative feedback loop that explicitly takes into account the RNA stochastic dynamics. We find that messenger RNA random fluctuations can be amplified during translation and produce sustained pulses of protein expression. Motivated by the recent appreciation of the importance of noncoding regulatory RNAs in post-transcription regulation, we also consider the possibility that a regulatory RNA transcript could bind to the messenger RNA and repress translation. Our findings show that the regulatory transcript helps reducing gene expression variability both at the single-cell level and at the cell population level.

  12. Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis.

    PubMed

    Hezaveh, Kebria; Kloetgen, Andreas; Bernhart, Stephan H; Mahapatra, Kunal Das; Lenze, Dido; Richter, Julia; Haake, Andrea; Bergmann, Anke K; Brors, Benedikt; Burkhardt, Birgit; Claviez, Alexander; Drexler, Hans G; Eils, Roland; Haas, Siegfried; Hoffmann, Steve; Karsch, Dennis; Klapper, Wolfram; Kleinheinz, Kortine; Korbel, Jan; Kretzmer, Helene; Kreuz, Markus; Küppers, Ralf; Lawerenz, Chris; Leich, Ellen; Loeffler, Markus; Mantovani-Loeffler, Luisa; López, Cristina; McHardy, Alice C; Möller, Peter; Rohde, Marius; Rosenstiel, Philip; Rosenwald, Andreas; Schilhabel, Markus; Schlesner, Matthias; Scholz, Ingrid; Stadler, Peter F; Stilgenbauer, Stephan; Sungalee, Stéphanie; Szczepanowski, Monika; Trümper, Lorenz; Weniger, Marc A; Siebert, Reiner; Borkhardt, Arndt; Hummel, Michael; Hoell, Jessica I

    2016-11-01

    MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project "Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing", we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.

  13. Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

    PubMed Central

    Hezaveh, Kebria; Kloetgen, Andreas; Bernhart, Stephan H; Mahapatra, Kunal Das; Lenze, Dido; Richter, Julia; Haake, Andrea; Bergmann, Anke K; Brors, Benedikt; Burkhardt, Birgit; Claviez, Alexander; Drexler, Hans G; Eils, Roland; Haas, Siegfried; Hoffmann, Steve; Karsch, Dennis; Klapper, Wolfram; Kleinheinz, Kortine; Korbel, Jan; Kretzmer, Helene; Kreuz, Markus; Küppers, Ralf; Lawerenz, Chris; Leich, Ellen; Loeffler, Markus; Mantovani-Loeffler, Luisa; López, Cristina; McHardy, Alice C; Möller, Peter; Rohde, Marius; Rosenstiel, Philip; Rosenwald, Andreas; Schilhabel, Markus; Schlesner, Matthias; Scholz, Ingrid; Stadler, Peter F; Stilgenbauer, Stephan; Sungalee, Stéphanie; Szczepanowski, Monika; Trümper, Lorenz; Weniger, Marc A; Siebert, Reiner; Borkhardt, Arndt; Hummel, Michael; Hoell, Jessica I.

    2016-01-01

    MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis. PMID:27390358

  14. Effects of local structural transformation of lipid-like compounds on delivery of messenger RNA.

    PubMed

    Li, Bin; Luo, Xiao; Deng, Binbin; Giancola, JoLynn B; McComb, David W; Schmittgen, Thomas D; Dong, Yizhou

    2016-02-26

    Lipid-like nanoparticles (LLNs) have shown great potential for RNA delivery. Lipid-like compounds are key components in LLNs. In this study, we investigated the effects of local structural transformation of lipid-like compounds on delivery of messenger RNA. Our results showed that position change of functional groups on lipid-like compounds can dramatically improve delivery efficiency. We then optimized formulation ratios of TNT-b10 LLNs, a lead material, increasing delivery efficiency over 2-fold. More importantly, pegylated TNT-b10 LLNs is stable for over four weeks and is over 10-fold more efficient than that of its counterpart TNT-a10 LLNs. Additionally, the optimal formulation O-TNT-b10 LLNs is capable of delivering mRNA encoding luciferase in vivo. These results provide useful insights into the design of next generation LLNs for mRNA delivery.

  15. Postage for the messenger: Designating routes for Nuclear mRNA Export

    PubMed Central

    Natalizio, Barbara J.; Wente, Susan R.

    2013-01-01

    Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

  16. Exploring the recovery and detection of messenger RNA and DNA from enhanced fingermarks in blood.

    PubMed

    Fox, A; Gittos, M; Harbison, S A; Fleming, R; Wivell, R

    2014-05-01

    Often in the examination of bloodstained fingermarks discussion occurs around whether to prioritise the fingerprint evidence or focus on the biological evidence. Collecting a sample for genetic profiling may result in the loss of ridge detail that could have been used for fingerprint comparison. Fingermark enhancement and recovery methods along with sample collection methods could also compromise downstream genetic analysis. Previous forensic casework has highlighted circumstances where, after enhancement had been performed, it would have been extremely valuable to both identify the body fluid and generate a DNA profile from the same sample. We enhanced depletion series of fingermarks made in blood, using single treatments consisting of aqueous amido black, methanol-based amido black, acid yellow and leucocrystal violet, and exposure to long wave UV light. We then extracted the DNA and RNA for profiling, to assess the recovery and detection of genetic material from the enhanced fingermarks. We have shown that genetic profiling of bloodstained fingermarks can be successful after chemical enhancement; however it may still be necessary to prioritise evidence types in certain circumstances. From our results it appears that even with visible bloodstained fingermarks, leucocrystal violet can reduce the effectiveness of subsequent messenger RNA profiling. Aqueous amido black and acid yellow also have adverse effects on messenger RNA profiling of depleted fingermarks with low levels of cellular material. These results help with forensic decision-making by expanding knowledge of the extent of the detrimental effects of blood-enhancement reagents on both DNA profiling and body fluid identification using messenger RNA profiling. Copyright © 2014 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

  17. Messenger RNA-based therapeutics for the treatment of apoptosis-associated diseases.

    PubMed

    Matsui, Akitsugu; Uchida, Satoshi; Ishii, Takehiko; Itaka, Keiji; Kataoka, Kazunori

    2015-10-28

    Gene therapy is a promising approach for treating diseases that are closely associated with excessive apoptosis, because the gene can effectively and sustainably introduce anti-apoptotic factors into cells. However, DNA delivery poses the risk of random genomic integration, leading to overexpression of the delivered gene and cancer development. Messenger RNA (mRNA) can evade integration events in target cells. We examined the use of mRNA-based therapeutics for introducing anti-apoptotic factors by using a mouse model of fulminant hepatitis. For introducing mRNA into the liver, a synthesised polymer-based carrier of polyplex nanomicelles was used for hydrodynamic intravenous injection. Using GFP as a reporter, we demonstrate that mRNA delivery induced efficient protein expression in almost 100% of liver cells, while plasmid DNA (pDNA) delivery provided a smaller percentage of GFP-positive cells. Analyses using Cy5-labelled mRNA and pDNA revealed that efficient expression by mRNA was attributed to a simple intracellular mechanism, without the need for nuclear entry. Consistent with this observation, Bcl-2 mRNA was more effective on reducing apoptosis in the liver of mice with fulminant hepatitis than Bcl-2 pDNA. Therefore, mRNA-based therapeutics combined with an effective delivery system such as polyplex nanomicelles is a promising treatment for intractable diseases associated with excessive apoptosis.

  18. SmpB contributes to reading frame selection in the translation of transfer-messenger RNA.

    PubMed

    Watts, Talina; Cazier, DeAnna; Healey, David; Buskirk, Allen

    2009-08-14

    Transfer-messenger RNA (tmRNA) acts first as a tRNA and then as an mRNA template to rescue stalled ribosomes in eubacteria. Together with its protein partner, SmpB (small protein B), tmRNA enters stalled ribosomes and transfers an Ala residue to the growing polypeptide chain. A remarkable step then occurs: the ribosome leaves the stalled mRNA and resumes translation using tmRNA as a template, adding a short peptide tag that destines the aborted protein for destruction. Exactly how the ribosome switches templates, resuming translation on tmRNA in the proper reading frame, remains unknown. Within the tmRNA sequence itself, five nucleotides (U85AGUC) immediately upstream of the first codon appear to direct frame selection. In particular, mutation of the conserved A86 results in severe loss of function both in vitro and in vivo. The A86C mutation causes translation to resume exclusively in the +1 frame. Several candidate binding partners for this upstream sequence have been identified in vitro. Using a genetic selection for tmRNA activity in Escherichia coli, we identified mutations in the SmpB protein that restore the function of A86C tmRNA in vivo. The SmpB mutants increase tagging in the normal reading frame and reduce tagging in the +1 frame. These results demonstrate that SmpB is functionally linked with the sequence upstream of the tmRNA template; both contribute to reading frame selection on tmRNA.

  19. Protein Kinase Target Discovery From Genome-Wide Messenger RNA Expression Profiling

    PubMed Central

    Ma’ayan, Avi; He, John C.

    2010-01-01

    Genome-wide messenger RNA profiling provides a snapshot of the global state of the cell under different experimental conditions such as diseased versus normal cellular states. However, because measurements are in the form of quantitative changes in messenger RNA levels, such experimental data does not provide direct understanding of the regulatory molecular mechanisms responsible for the observed changes. Identifying potential cell signaling regulatory mechanisms responsible for changes in gene expression under different experimental conditions or in different tissues has been the focus of many computational systems biology studies. Most popular approaches include promoter analysis, gene ontology, or pathway enrichment analysis, as well as reverse engineering of networks from messenger RNA expression data. Here we present a rational approach for identifying and ranking protein kinases that are likely responsible for observed changes in gene expression. By combining promoter analysis; data from various chromatin immunoprecipitation studies such as chromatin immunoprecipitation sequencing, chromatin immunoprecipitation coupled with paired-end ditag, and chromatin immunoprecipitation-on-chip; protein-protein interactions; and kinase-protein phosphorylation reactions collected from the literature, we can identify and rank candidate protein kinases for knock-down, or other types of functional validations, based on genome-wide changes in gene expression. We describe how protein kinase candidate identification and ranking can be made robust by cross-validation with phosphoproteomics data as well as through a literature-based text-mining approach. In conclusion, data integration can produce robust candidate rankings for understanding cell regulation through identification of protein kinases responsible for gene expression changes, and thus rapidly advancing drug target discovery and unraveling drug mechanisms of action. PMID:20687179

  20. Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

    PubMed Central

    Muller, Mandy

    2017-01-01

    During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonu- clease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs, including the transcript encoding interleukin-6 (IL-6), escape SOX-induced cleavage. IL-6 escape is mediated through a 3’ UTR RNA regulatory element that overrides the SOX targeting mechanism. Here, we reveal that this protective RNA element functions to broadly restrict cleavage by a range of homologous and non-homologous viral endonucleases. However, it does not impede cleavage by cellular endonucleases. The IL-6 protective sequence may be representative of a larger class of nuclease escape elements, as we identified a similar protective element in the GADD45B mRNA. The IL-6 and GADD45B-derived elements display similarities in their sequence, putative structure, and several associated RNA binding proteins. However, the overall composition of their ribonucleoprotein complexes appears distinct, leading to differences in the breadth of nucleases restricted. These findings highlight how RNA elements can selectively control transcript abundance in the background of widespread virus-induced mRNA degradation. PMID:28841715

  1. Developmental regulation of a proinsulin messenger RNA generated by intron retention

    PubMed Central

    Mansilla, Alicia; López-Sánchez, Carmen; de la Rosa, Enrique J; García-Martínez, Virginio; Martínez-Salas, Encarna; de Pablo, Flora; Hernández-Sánchez, Catalina

    2005-01-01

    Proinsulin gene expression regulation and function during early embryonic development differ remarkably from those found in postnatal organisms. The embryonic proinsulin protein content decreased from gastrulation to neurulation in contrast with the overall proinsulin messenger RNA increase. This is due to increasing levels of a proinsulin mRNA variant generated by intron 1 retention in the 5′ untranslated region. Inclusion of intron 1 inhibited proinsulin translation almost completely without affecting nuclear export or cytoplasmic decay. The novel proinsulin mRNA isoform expression was developmentally regulated and tissue specific. The proportion of intron retention increased from gastrulation to organogenesis, was highest in the heart tube and presomitic region, and could not be detected in the pancreas. Notably, proinsulin addition induced cardiac marker gene expression in the early embryonic stages when the translationally active transcript was expressed. We propose that regulated unproductive splicing and translation is a mechanism that regulates proinsulin expression in accordance with specific requirements in developing vertebrates. PMID:16179943

  2. Circulating thyroid stimulating hormone receptor messenger RNA and differentiated thyroid cancer: A diagnostic meta-analysis

    PubMed Central

    Kong, Chao-Yue; Li, Zhan-Ming; Wang, Li-Shun

    2017-01-01

    Thyroid stimulating hormone receptor messenger RNA (TSHR-mRNA) is over-expressed in thyroid cancer patients, which indicates that TSHR-mRNA is a potential biomarker of thyroid cancer. However, system evaluation for TSHR-mRNA as a diagnostic biomarker of thyroid cancer is deficient. The performance of TSHR-mRNA for thyroid cancer diagnosis was evaluated in this study. Three common international databases as well as a Chinese database were applied for literature researching. Quality assessment of the included literatures was conducted by the QUADAS-2 tool. Totally, 1027 patients from nine studies eligible for the meta-analysis were included in this study. Global sensitivity and specificity for the positivity of TSHR-mRNA in the thyroid cancer diagnosis is 72% and 82%. The value of AUC for this test performance was 0.84. Our meta-analysis suggests that TSHR-mRNA might be a potential biomarker to complete present diagnostic methods for early and precision diagnosis of thyroid cancer. Notably, this findings need validation thorough large-scale clinical studies. PMID:28036261

  3. In vivo messenger RNA introduction into the central nervous system using polyplex nanomicelle.

    PubMed

    Uchida, Satoshi; Itaka, Keiji; Uchida, Hirokuni; Hayakawa, Kentaro; Ogata, Toru; Ishii, Takehiko; Fukushima, Shigeto; Osada, Kensuke; Kataoka, Kazunori

    2013-01-01

    Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.

  4. SmpB contributes to reading frame selection in the translation of transfer-messenger RNA (tmRNA)

    PubMed Central

    Watts, Talina; Cazier, DeAnna; Healey, David; Buskirk, Allen

    2009-01-01

    Transfer-messenger RNA (tmRNA) acts first as a tRNA and then as an mRNA template to rescue stalled ribosomes in eubacteria. Together with its protein partner, SmpB, tmRNA enters stalled ribosomes and transfers an Ala residue to the growing polypeptide chain. A remarkable step then occurs: the ribosome leaves the stalled mRNA and resumes translation using tmRNA as a template, adding a short peptide tag that destines the aborted protein for destruction. Exactly how the ribosome switches templates, resuming translation on tmRNA in the proper reading frame, remains unknown. Within the tmRNA sequence itself, five nucleotides (U85AGUC) immediately upstream of the first codon appear to direct frame selection. In particular, mutation of the conserved A86 results in severe loss of function both in vitro and in vivo. The A86C mutation causes translation to resume exclusively in the +1 frame. Several candidate binding partners for this upstream sequence have been identified in vitro. Using a genetic selection for tmRNA activity in E. coli, we identified mutations in the SmpB protein that restore the function of A86C tmRNA in vivo. The SmpB mutants increase tagging in the normal reading frame and reduce tagging in the +1 frame. These results demonstrate that SmpB is functionally linked with the sequence upstream of the tmRNA template; both contribute to reading frame selection on tmRNA. PMID:19540849

  5. FATHEAD MINNOW VITELLOGENIN: COMPLEMENTARY DNA SEQUENCE AND MESSENGER RNA AND PROTEIN EXPRESSION AFTER 17B-ESTRADIOL TREATMENT

    EPA Science Inventory

    Induction of vitellogenin (VTG) in oviparous animals has been proposed as a sensitive indicator of invironmental contaminants that activate the estrogen receptor. In the present study, a sensitive ribonuclease protection assay (RPA) for VTG messenger RNA (mRNA) was developed for ...

  6. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    ERIC Educational Resources Information Center

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  7. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    ERIC Educational Resources Information Center

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  8. Expression and localization of lactotransferrin messenger RNA in the cortex of Alzheimer's disease.

    PubMed

    An, Li; Sato, Haruhisha; Konishi, Yoshihiro; Walker, Douglas G; Beach, Thomas G; Rogers, Joseph; Tooyama, Ikuo

    2009-03-20

    We and others have previously reported that lactotransferrin (LF), acting both as an iron-binding protein and inflammatory modulator, is greatly up-regulated in the brain of patients with Alzheimer's disease (AD). However, it remains unknown which type of cells express LF in the brain of AD. In this study, therefore, we investigated the expression and localization of LF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) and in situ hybridization histochemistry. Real-time PCR demonstrated that LF mRNA expression in the cortex of AD cases was significantly greater than that in control cases. LF mRNA-positive granules were observed in the cortex by in situ hybridization histochemistry, and the number of positive granules was increased in AD cases compared to controls. The double staining technique of LF mRNA in situ hybridisation and D-related human leukocyte antigen (HLA-DR) immunohistochemistry revealed that positive granules were localized in a subpopulation of HLA-DR-positive reactive microglia. In addition, LF mRNA-positive granules were observed in some cells that were negative for HLA-DR. These cells were also negative for CD4 and CD8 but positive for leukocyte common antigen (CD45RB), suggesting they were monocytes/macrophages. These results indicate that reactive microglia in the cerebral cortex and monocytes/macrophages infiltrating from the circulation might be responsible for synthesizing LF in AD brain.

  9. Comment on ``Length-dependent translation of messenger RNA by ribosomes''

    NASA Astrophysics Data System (ADS)

    Zhang, Yunxin

    2012-02-01

    In a recent paper by Valleriani [Phys. Rev. EPLEEE81539-375510.1103/PhysRevE.83.042903 83, 042903 (2011)], a simple model for the translation of messenger RNA (mRNA) is presented. Using this model, the protein translational ratio r, defined as the ratio of protein translation rate ωtl from mRNA to protein degradation rate ωp, is obtained. The key point in obtaining the translational ratio r is to get the protein translation rate ωtl. In Valleriani 's paper, ωtl is obtained as the mean value of the measured translation rate, which is the ratio of the synthesized protein number to the mRNA lifetime. However, in experiments, different methods might be used to obtain the value of ωtl. Therefore, to apply Valleriani 's model to more general experiments, in this Comment three methods to obtain the translation rate ωtl, and consequently the translational ratio r, are presented. Based on one of the methods which might be employed in most of the experiments, we find that the translational ratio r decays exponentially with mRNA length in prokaryotic cells, and decays reciprocally with mRNA length in eukaryotic cells. This result is slight different from that which was obtained in Valleriani 's paper.

  10. Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

    PubMed Central

    Shurtz, R; Dolev, S; Aboud, M; Salzberg, S

    1979-01-01

    When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts. PMID:117118

  11. Antidepressants upregulate messenger RNA levels of the neuroprotective enzyme superoxide dismutase (SOD1).

    PubMed Central

    Li, X M; Chlan-Fourney, J; Juorio, A V; Bennett, V L; Shrikhande, S; Bowen, R C

    2000-01-01

    OBJECTIVE: To investigate the effect of amitriptyline, bupropion, doxepin or venlafaxine on the gene expression of the neuroprotective enzyme superoxide dismutase (SOD1) in a catecholamine cell in vitro model. DESIGN: Molecular study of a cultured cell line. INTERVENTIONS: Rat pheochromocytoma (PC12) cells were incubated in 1 and 10 mumol/L of various antidepressant medications for 24 or 48 hours. OUTCOME MEASURES: Northern blot analysis. RESULTS: Amitriptyline up-regulated SOD1 messenger RNA in a time- and dose-dependent manner. The greatest up-regulation was following incubation with 10 mumol/L amitriptyline for 48 hours. The addition of bupropion, doxepin or venlafaxine to PC12 cell cultures also up-regulated SOD1 mRNA. CONCLUSIONS: These findings suggest that some antidepressants have the ability to positively regulate neuroprotective genes. Images Fig. 2 PMID:10721683

  12. Don't kill the messenger: Long-distance trafficking of mRNA molecules.

    PubMed

    Spiegelman, Ziv; Golan, Guy; Wolf, Shmuel

    2013-12-01

    The phloem sap contains numerous macromolecules such as proteins and RNAs, in addition to photoassimilates, amino acids and other small molecules. The transcription profile of messenger RNA (mRNA) molecules in the sieve tubes is unique and does not reflect the transcript profile in the neighboring companion cells. This discovery suggests tight regulation on cell-to-cell movement of mRNA molecules from the companion cells into the sieve tube. Heterografting experiments and RNA-detection methods have provided unequivocal evidence for the trafficking of several specific mRNA molecules between distant organs. Detection of various plant transcripts in their respective plant parasites further confirms this long-distance movement. The finding that several of these trafficked transcripts are involved in the control of developmental processes as well as responses to growth substances or environmental cues has led to a new paradigm that mRNA molecules act as non-cell-autonomous signaling agents operating in the vascular system. Trafficking of these molecules creates a communication network between distant organs that is required for coordinated development of the whole plant under adverse conditions. The generality of this concept, however, is still under debate, because the raison d'être for long-distance movement of mRNA is not clear. In this review we discuss the identity and potential function of phloem-sap mRNA molecules, the factors facilitating RNA transport, and the rationale for their action as long-distance signaling agents in the control of developmental processes.

  13. A quantitative method to identify microRNAs targeting a messenger RNA using a 3'UTR RNA affinity technique.

    PubMed

    Shi, Miao; Han, Weiguo; Spivack, Simon D

    2013-12-01

    The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3'UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitative polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA.

  14. Selective Pyramidal Cell Reduction of GABAA Receptor α1 Subunit Messenger RNA Expression in Schizophrenia

    PubMed Central

    Glausier, Jill R; Lewis, David A

    2011-01-01

    Levels of messenger RNA (mRNA) for the α1 subunit of the GABAA receptor, which is present in 60% of cortical GABAA receptors, have been reported to be lower in layer 3 of the prefrontal cortex (PFC) in subjects with schizophrenia. This subunit is expressed in both pyramidal cells and interneurons, and thus lower α1 subunit levels in each cell population would have opposite effects on net cortical excitation. We used dual-label in situ hybridization to quantify GABAA α1 subunit mRNA expression in calcium/calmodulin-dependent kinase II α (CaMKIIα)-containing pyramidal cells and glutamic acid decarboxylase 65 kDa (GAD65)-containing interneurons in layer 3 of the PFC from matched schizophrenia and healthy comparison subjects. In subjects with schizophrenia, mean GABAA α1 subunit mRNA expression was significantly 40% lower in pyramidal cells, but was not altered in interneurons. Lower α1 subunit mRNA expression in pyramidal cells was not attributable to potential confounding factors, and thus appeared to reflect the disease process of schizophrenia. These results suggest that pyramidal cell inhibition is reduced in schizophrenia, whereas inhibition of GABA neurons is maintained. The cell type specificity of these findings may reflect a compensatory response to enhance layer 3 pyramidal cell activity in the face of the diminished excitatory drive associated with the lower dendritic spine density on these neurons. PMID:21677653

  15. Messenger RNA profiling for forensic body fluid identification: research and applications.

    PubMed

    Wang, Zheng; Zhang, Su-hua; Di, Zhou; Zhao, Shu-min; Li, Cheng-tao

    2013-10-01

    Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts. However, the conventional body fluid identification methods are prone to various limitations, such as time consumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Recently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profiling, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibility with current DNA extraction and analysis procedure. In the current review, we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework.

  16. Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA

    PubMed Central

    Warren, Luigi; Ni, Yuhui; Wang, Jiwu; Guo, Xirong

    2012-01-01

    The therapeutic promise of induced pluripotent stem cells (iPSCs) has spurred efforts to circumvent genome alteration when reprogramming somatic cells to pluripotency. Approaches based on episomal DNA, Sendai virus, and messenger RNA (mRNA) can generate “footprint-free” iPSCs with efficiencies equaling or surpassing those attained with integrating viral vectors. The mRNA method uniquely affords unprecedented control over reprogramming factor (RF) expression while obviating a cleanup phase to purge residual traces of vector. Currently, mRNA-based reprogramming is relatively laborious due to the need to transfect daily for ~2 weeks to induce pluripotency, and requires the use of feeder cells that add complexity and variability to the procedure while introducing a route for contamination with non-human-derived biological material. We accelerated the mRNA reprogramming process through stepwise optimization of the RF cocktail and leveraged these kinetic gains to establish a feeder-free, xeno-free protocol which slashes the time, cost and effort involved in iPSC derivation. PMID:22984641

  17. Exercise does not influence myostatin and follistatin messenger RNA expression in young women.

    PubMed

    Jensky, Nicole E; Sims, Jennifer K; Dieli-Conwright, Christina M; Sattler, Fred R; Rice, Judd C; Schroeder, E Todd

    2010-02-01

    We evaluated changes in myostatin, follistatin, and MyoD messenger RNA (mRNA) gene expression using eccentric exercise (EE) and concentric exercise (CE) as probes to better understand the mechanisms of muscle hypertrophy in young women. Twelve women performed single-leg maximal eccentric (n = 6, 25 +/- 1 years, 59 +/- 7 kg) or concentric (n = 6, 24 +/- 1 years, 65 +/- 7 kg) isokinetic knee extension exercise for 7 sessions. Muscle biopsies were taken from the vastus lateralis at baseline, 8 hours after the first exercise session, and 8 hours after the seventh exercise session. In the EE group, there were no changes in myostatin and follistatin (p > or = 0.17); however, MyoD expression increased after 1 exercise bout (p = 0.02). In the CE group, there were no changes in myostatin, follistatin, or MyoD mRNA gene expression (p > or = 0.07). Differences between the EE and CE groups were not significant (p > or = 0.05). These data suggest that a single bout or multiple bouts of maximal EE or CE may not significantly alter myostatin or follistatin mRNA gene expression in young women. However, MyoD mRNA expression seems to increase only after EE.

  18. Messenger RNA sequence and the translation process --a particle transport perspective

    NASA Astrophysics Data System (ADS)

    Dong, Jiajia; Schmittmann, Beate; Zia, Royce K. P.

    2008-03-01

    The translation process in bacteria has been under intensive study. A key question concerns the quantitative effect of different elongation rates, associated with different codons, on the overall translation efficiency. Starting with a simple particle transport model, the totally asymmetric simple exclusion process (TASEP), we incorporate the essential components of the translation process: Ribosomes, cognate tRNA concentrations, and messenger RNA (mRNA) templates correspond to particles, hopping rates, and the underlying lattice, respectively. Using simulations and mean-field approximations to obtain the stationary currents (the protein production rates) associated with different mRNA sequences, we are especially interested in the effect of slow codons, i.e., codons which are associated with rare tRNAs and are therefore translated very slowly. As the first step, we look at a ``designed sequence'' with one and two slow codons and quantify the marked impact of their spatial distribution to the currents. Extending the results to several mRNA sequences taken from real genes, we argue that an effective translation rate including the information from the vicinity of each codon needs to be taken into consideration when seeking an efficient strategy to optimize the protein production.

  19. Serotonin transporter messenger RNA expression in neural crest-derived structures and sensory pathways of the developing rat embryo.

    PubMed

    Hansson, S R; Mezey, E; Hoffman, B J

    1999-03-01

    A growing body of evidence suggests that serotonin plays an important role in the early development of both neural and non-neural tissues from vertebrate and invertebrate species. Serotonin is removed from the extracellular space by the cocaine- and antidepressant-sensitive serotonin transporter, thereby limiting its action on receptors. In situ hybridization histochemistry was used to delineate serotonin transporter messenger RNA expression during rat embryonic development. Serotonin transporter messenger RNA was widely expressed beginning prior to organogenesis and throughout the second half of gestation. Strikingly, serotonin transporter messenger RNA was detected in neural crest cells, some of which respond to serotonin in vitro, and neural crest-derived tissues, such as autonomic ganglia, tooth primordia, adrenal medulla, chondrocytes and neuroepithelial cells, in the skin, heart, intestine and lung. Within the peripheral sensory pathways, two major cells types were serotonin transporter messenger RNA-positive: (i) sensory ganglionic neurons and (ii) neuroepithelial cells which serve as targets for the outgrowing sensory neurons. Several sensory organs (cochlear and retinal ganglionic cells, taste buds, whisker and hair follicles) contained serotonin transporter messenger RNA by late gestation. The expression of serotonin transporter messenger RNA throughout the sensory pathways from central nervous system relay stations [Hansson S. R. et al. (1997) Neuroscience 83, 1185-1201; Lebrand C. et al. (1996) Neuron 17, 823-835] to sensory nerves and target organs as shown in this study suggests that serotonin may regulate peripheral synaptogenesis, and thereby influence later processing of sensory stimuli. If the early detection of serotonin transporter messenger RNA in skin and gastrointestinal and airway epithelia correlates with protein activity, it may permit establishment of a serotonin concentration gradient across epithelia, either from serotonin in the

  20. Integrating microRNA and messenger RNA expression profiles in a rat model of deep vein thrombosis.

    PubMed

    Jin, Qian-Qian; Sun, Jun-Hong; Du, Qiu-Xiang; Lu, Xiao-Jun; Zhu, Xi-Yan; Fan, Hao-Liang; Hölscher, Christian; Wang, Ying-Yuan

    2017-10-01

    Deep vein thrombosis (DVT) is a disease involving multiple genes and systems. MicroRNAs (miRNAs) represent a class of non-coding small RNAs that post-transcriptionally suppress their target genes. The expression patterns of miRNA and messenger RNA (mRNA) in DVT remain poorly characterized. The aim of the present study was to evaluate miRNA and mRNA expression profiles in a stasis-induced DVT rat model. Male SD rats were randomly divided into three groups as follows: DVT, sham and control. The inferior vena cava (IVC) of rats was ligated to construct stasis-induced DVT models. Rats were sacrificed three days after ligation, and morphological changes in the vein tissues were observed by hematoxylin and eosin and Masson staining. The miRNA and mRNA expression profiles were evaluated by microarrays, followed by bioinformatics analysis. The microarray analysis identified 22 miRNAs and 487 mRNAs that were significantly differentially expressed between the experimental and control groups, and between the experimental and sham groups, but not between the control and sham groups (P≤0.05; ≥2.0‑fold change). By subsequent bioinformatics analysis, a 19 miRNA-98 mRNAs network was constructed in the stasis-induced DVT rat model. Notably, the majority of these miRNAs and mRNAs are reported to be expressed by endothelial cells (ECs) and are associated with the function of ECs. The results provide evidence indicating that the regulatory association of miRNA and mRNA points to key roles played by ECs in thrombosis. These findings advance our understanding of the molecular regulatory mechanisms underlying the pathophysiology of DVT.

  1. Role of a neuronal small non-messenger RNA: behavioural alterations in BC1 RNA-deleted mice.

    PubMed

    Lewejohann, L; Skryabin, B V; Sachser, N; Prehn, C; Heiduschka, P; Thanos, S; Jordan, U; Dell'Omo, G; Vyssotski, A L; Pleskacheva, M G; Lipp, H-P; Tiedge, H; Brosius, J; Prior, H

    2004-09-23

    BC1 RNA is a small non-messenger RNA common in dendritic microdomains of neurons in rodents. In order to investigate its possible role in learning and behaviour, we compared controls and knockout mice from three independent founder lines established from separate embryonic stem cells. Mutant mice were healthy with normal brain morphology and appeared to have no neurological deficits. A series of tests for exploration and spatial memory was carried out in three different laboratories. The tests were chosen as to ensure that different aspects of spatial memory and exploration could be separated and that possible effects of confounding variables could be minimised. Exploration was studied in a barrier test, in an open-field test, and in an elevated plus-maze test. Spatial memory was investigated in a Barnes maze and in a Morris water maze (memory for a single location), in a multiple T-maze and in a complex alley maze (route learning), and in a radial maze (working memory). In addition to these laboratory tasks, exploratory behaviour and spatial memory were assessed under semi-naturalistic conditions in a large outdoor pen. The combined results indicate that BC1 RNA-deficient animals show behavioural changes best interpreted in terms of reduced exploration and increased anxiety. In contrast, spatial memory was not affected. In the outdoor pen, the survival rates of BC1-depleted mice were lower than in controls. Thus, we conclude that the neuron-specific non-messenger BC1 RNA contributes to the aptive modulation of behaviour.

  2. Role of messenger RNA-ribosome complex in complementary DNA display.

    PubMed

    Naimuddin, Mohammed; Ohtsuka, Isao; Kitamura, Koichiro; Kudou, Motonori; Kimura, Shinnosuke

    2013-07-15

    In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. The Arabidopsis MOS4-associated Complex Promotes MicroRNA Biogenesis and Precursor Messenger RNA Splicing.

    PubMed

    Jia, Tianran; Zhang, Bailong; You, Chenjiang; Zhang, Yong; Zeng, Liping; Li, Shengjun; Johnson, Kaeli C M; Yu, Bin; Li, Xin; Chen, Xuemei

    2017-09-25

    In Arabidopsis thaliana, the MOS4-ASSOCIATED COMPLEX (MAC) is required for defense and development. The evolutionarily conserved, putative RNA helicase MAC7 is a component of the Arabidopsis MAC and the human MAC7 homolog, Aquarius, is implicated in pre-mRNA splicing. Here, we show that mac7-1, a partial loss-of-function mutant in MAC7, and two other MAC subunit mutants, mac3a mac3b and prl1 prl2 (pleiotropic regulatory locus), exhibit reduced microRNA (miRNA) levels, indicating that MAC promotes miRNA biogenesis. The mac7-1 mutant shows reduced primary miRNA (pri-miRNA) levels without affecting miRNA gene (MIR) promoter activity or the half-life of pri-miRNA transcripts. As a nuclear protein, MAC7 is not concentrated in dicing bodies, but it affects the localization of HYPONASTIC LEAVES1 (HYL1), a key protein in pri-miRNA processing, to dicing bodies. Immunoprecipitation of HYL1 retrieved eleven known MAC subunits, including MAC7, indicating association between HYL1 and MAC. We propose that MAC7 links MIR transcription to pri-miRNA processing. RNA-seq analysis showed that down-regulated genes in MAC subunit mutants are mostly involved in plant defense and stimulus responses, confirming a role of MAC in biotic and abiotic stress responses. We also discovered global intron retention defects in mutants in three subunits of MAC, thus linking MAC function to splicing in Arabidopsis. © 2017 American Society of Plant Biologists. All rights reserved.

  4. Reduced Cortical Cannabinoid 1 Receptor Messenger RNA and Protein Expression in Schizophrenia

    PubMed Central

    Eggan, Stephen M.; Hashimoto, Takanori; Lewis, David A.

    2010-01-01

    Context Cannabis use is associated with both impaired cognitive functions, including working memory, and an increased risk of schizophrenia. Schizophrenia is characterized by impairments in working memory that are associated with reduced γ-aminobutyric acid (GABA) neurotransmission in the dorsolateral prefrontal cortex. The cannabinoid 1 receptor (CB1R) is highly expressed in the dorsolateral prefrontal cortex, is contained in the axon terminals of a subpopulation of perisomatic-targeting GABA neurons, and, when activated, suppresses the release of GABA. Objective To determine the potential relationship between CB1R signaling and altered GABA neurotransmission in schizophrenia by evaluating CB1R messenger RNA (mRNA) and protein expression in the dorsolateral pre-frontal cortex. Design In situ hybridization and immunocytochemistry techniques were used to examine the cortical levels of CB1R mRNA and protein, respectively. Setting Brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner’s Office, Pittsburgh, Pennsylvania. Participants Postmortem brain specimens from 23 pairs of subjects with schizophrenia and age-, sex-, and postmortem interval–matched comparison subjects, as well as brain specimens from 18 macaque monkeys with long-term exposure to haloperidol, olanzapine, or placebo. Main Outcome Measures Optical density measures of CB1R mRNA expression and protein levels and correlations with previously reported glutamic acid decarboxylase 67 and cholecystokinin mRNA measures. Results Levels of CB1R mRNA were significantly lower by 14.8% in the subjects with schizophrenia. Similarly, CB1R protein levels, assessed by radioimmunocytochemistry and standard immunocytochemistry, were significantly decreased by 11.6% and 13.9%, respectively. Group differences in CB1R mRNA levels were significantly correlated with those in glutamic acid decarboxylase 67 and cholecystokinin mRNA levels. Expression of CB1R mRNA was not changed in

  5. Colored petri net modeling of small interfering RNA-mediated messenger RNA degradation

    PubMed Central

    Nickaeen, Niloofar; Moein, Shiva; Heidary, Zarifeh; Ghaisari, Jafar

    2016-01-01

    Background: Mathematical modeling of biological systems is an attractive way for studying complex biological systems and their behaviors. Petri Nets, due to their ability to model systems with various levels of qualitative information, have been wildly used in modeling biological systems in which enough qualitative data may not be at disposal. These nets have been used to answer questions regarding the dynamics of different cell behaviors including the translation process. In one stage of the translation process, the RNA sequence may be degraded. In the process of degradation of RNA sequence, small-noncoding RNA molecules known as small interfering RNA (siRNA) match the target RNA sequence. As a result of this matching, the target RNA sequence is destroyed. Materials and Methods: In this context, the process of matching and destruction is modeled using Colored Petri Nets (CPNs). The model is constructed using CPNs which allow tokens to have a value or type on them. Thus, CPN is a suitable tool to model string structures in which each element of the string has a different type. Using CPNs, long RNA, and siRNA strings are modeled with a finite set of colors. The model is simulated via CPN Tools. Results: A CPN model of the matching between RNA and siRNA strings is constructed in CPN Tools environment. Conclusion: In previous studies, a network of stoichiometric equations was modeled. However, in this particular study, we modeled the mechanism behind the silencing process. Modeling this kind of mechanisms provides us with a tool to examine the effects of different factors such as mutation or drugs on the process. PMID:27376039

  6. Unusual stability of messenger RNA in snake venom reveals gene expression dynamics of venom replenishment.

    PubMed

    Currier, Rachel B; Calvete, Juan J; Sanz, Libia; Harrison, Robert A; Rowley, Paul D; Wagstaff, Simon C

    2012-01-01

    Venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that venomous snakes possess a highly efficient venom production and delivery system to maintain their predatory arsenal. Here, we exploit the unusual stability of messenger RNA in venom to conduct, for the first time, quantitative PCR to characterise the dynamics of gene expression of newly synthesised venom proteins following venom depletion. Quantitative PCR directly from venom enables real-time dynamic studies of gene expression in the same animals because it circumvents the conventional requirement to sacrifice snakes to extract mRNA from dissected venom glands. Using qPCR and proteomic analysis, we show that gene expression and protein re-synthesis triggered by venom expulsion peaks between days 3-7 of the cycle of venom replenishment, with different protein families expressed in parallel. We demonstrate that venom re-synthesis occurs very rapidly following depletion of venom stores, presumably to ensure venomous snakes retain their ability to efficiently predate and remain defended from predators. The stability of mRNA in venom is biologically fascinating, and could significantly empower venom research by expanding opportunities to produce transcriptomes from historical venom stocks and rare or endangered venomous species, for new therapeutic, diagnostic and evolutionary studies.

  7. Messenger RNA- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity.

    PubMed

    Steichen, Clara; Luce, Eléanor; Maluenda, Jérôme; Tosca, Lucie; Moreno-Gimeno, Inmaculada; Desterke, Christophe; Dianat, Noushin; Goulinet-Mainot, Sylvie; Awan-Toor, Sarah; Burks, Deborah; Marie, Joëlle; Weber, Anne; Tachdjian, Gérard; Melki, Judith; Dubart-Kupperschmitt, Anne

    2014-06-01

    The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications. ©AlphaMed Press.

  8. Systemic delivery of factor IX messenger RNA for protein replacement therapy.

    PubMed

    Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B; Karmali, Priya P; Chivukula, Pad; Verma, Inder M

    2017-03-07

    Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.

  9. Messenger RNA- Versus Retrovirus-Based Induced Pluripotent Stem Cell Reprogramming Strategies: Analysis of Genomic Integrity

    PubMed Central

    Steichen, Clara; Luce, Eléanor; Maluenda, Jérôme; Tosca, Lucie; Moreno-Gimeno, Inmaculada; Desterke, Christophe; Dianat, Noushin; Goulinet-Mainot, Sylvie; Awan-Toor, Sarah; Burks, Deborah; Marie, Joëlle; Weber, Anne; Tachdjian, Gérard; Melki, Judith

    2014-01-01

    The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients’ iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications. PMID:24736403

  10. MicroRNA Seed Region Length Impact on Target Messenger RNA Expression and Survival in Colorectal Cancer.

    PubMed

    Mullany, Lila E; Herrick, Jennifer S; Wolff, Roger K; Slattery, Martha L

    2016-01-01

    microRNAs (miRNA) repress messenger RNAs post-transcriptionally through binding to the 3' UTR of the mRNA with the miRNA seed region. It has been purported that longer seed regions have a greater efficacy on mRNA repression. We tested this hypothesis by evaluating differential expression of miRNAs involved in regulating the immune response, an important mechanism in colorectal cancer (CRC), by seed length category. We subsequently evaluated differential expression of these miRNAs' targets in colonic tissue and the impact of these miRNAs on CRC survival. We determined sequence complementarity between each miRNA seed region and the 3' UTR of each experimentally verified mRNA target gene. We classified miRNAs into groups based on seed regions matching perfectly to a mRNA UTR with six bases beginning at position two, seven bases beginning at position one, seven bases beginning at position two, or eight bases beginning at position one. We analyzed these groups in terms of miRNA differential expression between carcinoma and normal colorectal mucosa, differential colonic target mRNA expression, and risk of dying from CRC. After correction for multiple comparisons, the proportion of the miRNAs that were associated with differential mRNA expression was 0% for the 6-mer, 13.64% for the 7α-mer group, 12.82% for the 7β-mer group, and 8.70% for the 8-mer group. The proportion of miRNAs associated with survival was 20% for the 6-mer group, 27.27% for the 7α-mer group, 10.23% for the 7β-mer group, and 21.74% for the 8-mer group. We did not see a linear relationship between seed length and miRNA expression dysregulation, mRNA expression, or survival. Our findings do not support the hypothesis the seed region length alone influences mRNA repression.

  11. The bacterial second messenger c-di-GMP: probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs.

    PubMed

    Shanahan, Carly A; Strobel, Scott A

    2012-12-14

    The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs.

  12. The bacterial second messenger c-di-GMP: Probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs

    PubMed Central

    Shanahan, Carly A.; Strobel, Scott A.

    2013-01-01

    The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs. PMID:23108253

  13. RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells.

    PubMed

    Uchida, Naoya; Washington, Kareem N; Mozer, Brian; Platner, Charlotte; Ballantine, Josiah; Skala, Luke P; Raines, Lydia; Shvygin, Anna; Hsieh, Matthew M; Mitchell, Lloyd G; Tisdale, John F

    2017-02-14

    Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34(+) cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

  14. The use of insulin like-growth factor II messenger RNA binding protein-3 in diagnostic pathology.

    PubMed

    Findeis-Hosey, Jennifer J; Xu, Haodong

    2011-03-01

    The histologic distinction between reactive processes and malignant neoplasms and between low-grade and high-grade tumors is not always straightforward and is sometimes extremely challenging. This is especially the case when the diagnostic material is a small biopsy specimen or a cytology specimen with scant cellularity. In addition, suboptimal processing and crush artifact may limit accurate diagnosis. A reliable diagnostic biomarker that preferentially highlights malignant processes and high-grade tumors would be very valuable in segregating these entities from reactive processes and low-grade lesions. Recent extensive studies have shown that an oncoprotein, insulin like-growth factor II messenger RNA binding protein-3, is not only a prognostic biomarker but also a diagnostic molecule. This review focuses on discussing the value of insulin like-growth factor II messenger RNA binding protein-3 in diagnostic pathology, with a focus on utilization of insulin like-growth factor II messenger RNA binding protein-3 in the discrimination of benign effusions from malignant effusions, malignant mesothelioma from mesothelial hyperplasia, carcinoids from high-grade neuroendocrine carcinomas, low-grade dysplasia from high-grade dysplasia, hepatocellular carcinoma from hepatic adenoma, cholangiocarcinoma and metastatic pancreatic ductal carcinoma from benign bile duct lesions, melanoma from nevi, and follicular thyroid carcinoma from follicular adenoma of the thyroid, as well as examining insulin like-growth factor II messenger RNA binding protein-3 expression in lymphomas of germinal center origin.

  15. Seasonal effect on sperm messenger RNA profile of domestic swine (Sus Scrofa).

    PubMed

    Yang, C C; Lin, Y S; Hsu, C C; Tsai, M H; Wu, S C; Cheng, W T K

    2010-05-01

    Seasonal infertility is a well-known problem in the modern swine (Sus scrofa) industry. The molecular mechanisms responsible for thermal effects on spermatogenesis are, however, just beginning to be elucidated. The existence of specific messenger RNA (mRNA) remnants contained within freshly ejaculated sperm has been identified in several species. Investigators have obtained differential RNA profiles of infertile men compared with fertile individuals; however, there are limited to the probes, which are mostly derived from nucleic acids of testicular tissues of either human or mice. The objective of this study was to investigate mRNA remnants from ejaculated sperm of the domestic swine and uncover important clues regarding the molecular regulation of spermatogenesis under environmental thermo-impacts. We utilized the remnant mRNA collected from swine ejaculated sperm as the target source to detect the global gene expression in summer and in winter by swine sperm-specific oligonucleotide microarray. Sixty-seven transcripts were differentially expressed with statistical differences between seasons of sperm samples collected, including forty-nine in winter (49/67) and eighteen in summer (18/67). There were only 33 of these transcripts that could be annotated to gene ontology hierarchy with the database of Homo sapiens and their functions mostly were involved in variety of metabolic processes. Moreover, these studies also confirmed that significant differences of gene expression profiles were found in swine sperm when comparisons were made between ejaculates collected during the winter and the summer season under the subtropical area such as Taiwan. Even though most of the genes found in our experiments are still poorly understood in terms of their true functions in spermatogenesis, bioinformatics analysis suggested that they are involved in a broad spectrum of biochemical processes including gamete generation. These concordant profiles should permit the development of a

  16. The neurofibromatosis type I messenger RNA undergoes base-modification RNA editing.

    PubMed Central

    Skuse, G R; Cappione, A J; Sowden, M; Metheny, L J; Smith, H C

    1996-01-01

    A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes. PMID:8602361

  17. Starvation and hypothyroidism exert an overlapping influence on rat hepatic messenger RNA activity profiles.

    PubMed Central

    Carr, F E; Seelig, S; Mariash, C N; Schwartz, H L; Oppenheimer, J H

    1983-01-01

    To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together

  18. Integrated microRNA and messenger RNA analysis in aortic stenosis

    PubMed Central

    Coffey, Sean; Williams, Michael J. A.; Phillips, L. Vicky; Galvin, Ivor F.; Bunton, Richard W.; Jones, Gregory T.

    2016-01-01

    Aortic valve stenosis (AS) is a major cause of morbidity and mortality, with no effective medical therapies. Investigation into the underlying biology of AS in humans is limited by difficulties in obtaining healthy valvular tissue for use as a control group. However, micro-ribonucleic acids (miRNAs) are stable in post-mortem tissue. We compared valve specimens from patients undergoing aortic valve replacement for AS to non-diseased cadaveric valves. We found 106 differentially expressed miRNAs (p < 0.05, adjusted for multiple comparisons) on microarray analysis, with highly correlated expression among up- and down-regulated miRNAs. Integrated miRNA/gene expression analysis validated the microarray results as a whole, while quantitative polymerase chain reaction confirmed downregulation of miR-122-5p, miR-625-5p, miR-30e-5p and upregulation of miR-21-5p and miR-221-3p. Pathway analysis of the integrated miRNA/mRNA network identified pathways predominantly involved in extracellular matrix function. A number of currently available therapies target products of upregulated genes in the integrated miRNA/mRNA network, with these genes being predominantly more peripheral members of the network. The identification of a group of tissue miRNA associated with AS may contribute to the development of new therapeutic approaches to AS. This study highlights the importance of systems biology-based approaches to complex diseases. PMID:27876829

  19. Integrated microRNA and messenger RNA analysis in aortic stenosis.

    PubMed

    Coffey, Sean; Williams, Michael J A; Phillips, L Vicky; Galvin, Ivor F; Bunton, Richard W; Jones, Gregory T

    2016-11-23

    Aortic valve stenosis (AS) is a major cause of morbidity and mortality, with no effective medical therapies. Investigation into the underlying biology of AS in humans is limited by difficulties in obtaining healthy valvular tissue for use as a control group. However, micro-ribonucleic acids (miRNAs) are stable in post-mortem tissue. We compared valve specimens from patients undergoing aortic valve replacement for AS to non-diseased cadaveric valves. We found 106 differentially expressed miRNAs (p < 0.05, adjusted for multiple comparisons) on microarray analysis, with highly correlated expression among up- and down-regulated miRNAs. Integrated miRNA/gene expression analysis validated the microarray results as a whole, while quantitative polymerase chain reaction confirmed downregulation of miR-122-5p, miR-625-5p, miR-30e-5p and upregulation of miR-21-5p and miR-221-3p. Pathway analysis of the integrated miRNA/mRNA network identified pathways predominantly involved in extracellular matrix function. A number of currently available therapies target products of upregulated genes in the integrated miRNA/mRNA network, with these genes being predominantly more peripheral members of the network. The identification of a group of tissue miRNA associated with AS may contribute to the development of new therapeutic approaches to AS. This study highlights the importance of systems biology-based approaches to complex diseases.

  20. Systemic delivery of factor IX messenger RNA for protein replacement therapy

    PubMed Central

    Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B.; Karmali, Priya P.; Chivukula, Pad; Verma, Inder M.

    2017-01-01

    Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable. PMID:28202722

  1. Comparison of circulating, hepatocyte specific messenger RNA and microRNA as biomarkers for chronic hepatitis B and C.

    PubMed

    Zhang, Xiaonan; Zhang, Zhanqing; Dai, Fahui; Shi, Bisheng; Chen, Liang; Zhang, Xinxin; Zang, Guoqing; Zhang, Jiming; Chen, Xiaorong; Qian, Fangxing; Hu, Yunwen; Yuan, Zhenghong

    2014-01-01

    Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patients from healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients' plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for

  2. DNA-water interactions distinguish messenger RNA genes from transfer RNA genes.

    PubMed

    Khandelwal, Garima; Jayaram, B

    2012-05-30

    Physicochemical properties of DNA sequences as a guide to developing insights into genome organization has received little attention. Here, we utilize the energetics of DNA to further advance the knowledge on its language at a molecular level. Specifically, we ask the question whether physicochemical properties of different functional units on genomes differ. We extract intramolecular and solvation energies of different DNA base pair steps from a comprehensive set of molecular dynamics simulations. We then investigate the solvation behavior of DNA sequences coding for mRNAs and tRNAs. Distinguishing mRNA genes from tRNA genes is a tricky problem in genome annotation without assumptions on length of DNA and secondary structure of the product of transcription. We find that solvation energetics of DNA behaves as an extremely efficient property in discriminating 2,063,537 genes coding for mRNAs from 56,251 genes coding for tRNAs in all (~1500) completely sequenced prokaryotic genomes.

  3. Gingival Toll-like receptor and cytokine messenger RNA levels in equine periodontitis and oral health.

    PubMed

    Kennedy, R; Lappin, D F; Dixon, P M; Bennett, D; Riggio, M P

    2017-05-01

    Equine periodontitis is a common and painful condition. However, the disease often goes unnoticed by owners and is thus a major welfare concern. The aetiopathogenesis of the condition remains poorly understood and has been investigated in few studies. The innate immune system is known to play an important role in human periodontitis, but its role in equine periodontitis has not been examined. To quantify the messenger (m)RNA levels of Toll-like receptors (TLRs) and cytokines in gingival tissue from orally healthy horses and those affected by periodontitis. Observational study. Gingival tissue samples were taken post-mortem from 13 horses with no clinical signs of oral disease and 20 horses with periodontitis. mRNA levels of TLR2, TLR4 and TLR9 and cytokines interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-4, IL-6, IL-10, IL-12, IL-17 and interferon-γ (IFN-γ) were determined using quantitative real-time PCR. The statistical significance of results was assessed using appropriate t tests. mRNA levels of all TLRs and cytokines were upregulated in equine periodontitis. Significant increases in mRNA levels of TLR2, TLR9, IL-4, IL-10, IL-12 (P≤0.05) and IFN-γ (P≤0.01) were observed for both unweighted and age-weighted analyses of diseased gingival tissue samples compared with healthy gingival samples. In comparisons of samples of periodontitis lesions with healthy gingival control samples from the same horse, significant increases in mRNA levels of TLR4, TLR9, IL-10, IFN-γ (P≤0.05), TLR2, IL-1β and IL-12p35 (P≤0.01) were observed. This study has provided an initial insight into the involvement of the immune system in equine periodontitis. Increased mRNA levels of TLR2, TLR4 and TLR9 indicate substantial microbial challenge in diseased gingival tissue. A mixed Th1/Th2/Th17 cytokine response is produced in equine periodontitis. Further studies are required to more fully characterise the role of the innate immune system in this disease. © 2016

  4. Genome-Wide Scleral Micro- and Messenger-RNA Regulation During Myopia Development in the Mouse.

    PubMed

    Metlapally, Ravikanth; Park, Han Na; Chakraborty, Ranjay; Wang, Kevin K; Tan, Christopher C; Light, Jacob G; Pardue, Machelle T; Wildsoet, Christine F

    2016-11-01

    MicroRNA (miRNAs) have been previously implicated in scleral remodeling in normal eye growth. They have the potential to be therapeutic targets for prevention/retardation of exaggerated eye growth in myopia by modulating scleral matrix remodeling. To explore this potential, genome-wide miRNA and messenger RNA (mRNA) scleral profiles in myopic and control eyes from mice were studied. C57BL/6J mice (n = 7; P28) reared under a 12L:12D cycle were form-deprived (FD) unilaterally for 2 weeks. Refractive error and axial length changes were measured using photorefraction and 1310-nm spectral-domain optical coherence tomography, respectively. Scleral RNA samples from FD and fellow control eyes were processed for microarray assay. Statistical analyses were performed using National Institute of Aging array analysis tool; group comparisons were made using ANOVA, and gene ontologies were identified using software available on the Web. Findings were confirmed using quantitative PCR in a separate group of mice (n = 7). Form-deprived eyes showed myopic shifts in refractive error (-2.02 ± 0.47 D; P < 0.01). Comparison of the scleral RNA profiles of test eyes with those of control eyes revealed 54 differentially expressed miRNAs and 261 mRNAs fold-change >1.25 (maximum fold change = 1.63 and 2.7 for miRNAs and mRNAs, respectively) (P < 0.05; minimum, P = 0.0001). Significant ontologies showing gene over-representation (P < 0.05) included intermediate filament organization, scaffold protein binding, detection of stimuli, calcium ion, G protein, and phototransduction. Significant differential expression of Let-7a and miR-16-2, and Smok4a, Prph2, and Gnat1 were confirmed. Scleral mi- and mRNAs showed differential expression linked to myopia, supporting the involvement of miRNAs in eye growth regulation. The observed general trend of relatively small fold-changes suggests a tightly controlled, regulatory mechanism for scleral gene expression.

  5. Genome-Wide Scleral Micro- and Messenger-RNA Regulation During Myopia Development in the Mouse

    PubMed Central

    Metlapally, Ravikanth; Park, Han Na; Chakraborty, Ranjay; Wang, Kevin K.; Tan, Christopher C.; Light, Jacob G.; Pardue, Machelle T.; Wildsoet, Christine F.

    2016-01-01

    Purpose MicroRNA (miRNAs) have been previously implicated in scleral remodeling in normal eye growth. They have the potential to be therapeutic targets for prevention/retardation of exaggerated eye growth in myopia by modulating scleral matrix remodeling. To explore this potential, genome-wide miRNA and messenger RNA (mRNA) scleral profiles in myopic and control eyes from mice were studied. Methods C57BL/6J mice (n = 7; P28) reared under a 12L:12D cycle were form-deprived (FD) unilaterally for 2 weeks. Refractive error and axial length changes were measured using photorefraction and 1310-nm spectral-domain optical coherence tomography, respectively. Scleral RNA samples from FD and fellow control eyes were processed for microarray assay. Statistical analyses were performed using National Institute of Aging array analysis tool; group comparisons were made using ANOVA, and gene ontologies were identified using software available on the Web. Findings were confirmed using quantitative PCR in a separate group of mice (n = 7). Results Form-deprived eyes showed myopic shifts in refractive error (−2.02 ± 0.47 D; P < 0.01). Comparison of the scleral RNA profiles of test eyes with those of control eyes revealed 54 differentially expressed miRNAs and 261 mRNAs fold-change >1.25 (maximum fold change = 1.63 and 2.7 for miRNAs and mRNAs, respectively) (P < 0.05; minimum, P = 0.0001). Significant ontologies showing gene over-representation (P < 0.05) included intermediate filament organization, scaffold protein binding, detection of stimuli, calcium ion, G protein, and phototransduction. Significant differential expression of Let-7a and miR-16-2, and Smok4a, Prph2, and Gnat1 were confirmed. Conclusions Scleral mi- and mRNAs showed differential expression linked to myopia, supporting the involvement of miRNAs in eye growth regulation. The observed general trend of relatively small fold-changes suggests a tightly controlled, regulatory mechanism for scleral gene expression. PMID

  6. Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

  7. Profiling of differential expression of messenger RNA in normal, benign, and metastatic prostate cell lines.

    PubMed

    Chakrabarti, Ratna; Robles, Liza D; Gibson, Jane; Muroski, Megan

    2002-12-01

    To understand the phenotypic changes associated with prostate cancer development and metastasis, we investigated differential gene expression in primary and established prostate cell lines used as models. We have used a differential display of messenger RNA (DDRT-PCR) technique using 168 primer combinations and total RNA from BPH-1, LNCaP, and PC3 cells to identify filter-based cDNA microarrays containing 18,376 nonredundant clones of genes and expressed sequence tags (EST) using mRNA from PrEC and MDAPCa2a cells to identify genes that are differentially expressed in normal, benign, and cancerous prostate cell lines. Twenty-five cDNA with a significant difference in expression of 76 candidate cDNA, as identified by DDRT-PCR and confirmed by slot-blot analysis, were selected for sequence analysis. Of these, 14 cDNA were further confirmed by Northern blot analysis. Analysis of the cDNA microarray data showed that a variety of genes/EST were up- or down-regulated in the metastatic prostate tumor cells and a majority of these genes encode cytoskeletal proteins and proteins with regulatory function. Expression profile of two EST was confirmed by reverse transcription polymerase chain reaction. We also have identified a number of genes exhibiting differential expression in prostate cancer cells, which were not known earlier to be involved in prostate cancer. This report provides a comparative analysis of differential gene expression between normal prostatic epithelial cells and prostate cancer cells, and a foundation to facilitate in-depth studies on the mechanism of prostate cancer development and metastasis.

  8. Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

  9. RNA toxins: mediators of stress adaptation and pathogen defense.

    PubMed

    Zhabokritsky, Alice; Kutky, Meherzad; Burns, Lydia A; Karran, Rajita A; Hudak, Katalin A

    2011-01-01

    RNA toxins are a group of enzymes primarily synthesized by bacteria, fungi, and plants that either cleave or depurinate RNA molecules. These proteins may be divided according to their RNA substrates: ribotoxins are nucleases that cleave ribosomal RNA (rRNA), ribosome inactivating proteins are glycosidases that remove a base from rRNA, messenger RNA (mRNA) interferases are nucleases that cleave mRNAs, and anticodon nucleases cleave transfer RNAs (tRNAs). These modifications to the RNAs may substantially alter gene expression and translation rates. Given that some of these enzymes cause cell death, it has been suggested that they function mainly in defense, either to kill competing cells or to elicit suicide and thereby limit pathogen spread from infected cells. Although good correlations have been drawn between their enzymatic functions and toxicity, recent work has shown that some RNA toxins cause apoptosis in the absence of damage to RNA and that defense against pathogens can be achieved without host cell death. Moreover, a decrease in cellular translation rate, insufficient to cause cell death, allows some organisms to adapt to stress and environmental change. Although ascribing effects observed in vitro to the roles of these toxins in nature has been challenging, recent results have expanded our understanding of their modes of action, and emphasized the importance of these toxins in development, adaptation to stress and defense against pathogens.

  10. The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive

    PubMed Central

    Calapez, Alexandre; Pereira, Henrique M.; Calado, Angelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C.; Carmo-Fonseca, Maria

    2002-01-01

    fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes. PMID:12473688

  11. The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

    PubMed Central

    Moser, Joanna J.; Fritzler, Marvin J.

    2010-01-01

    Background GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. Methodology/Principal Findings RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. Conclusions/Significance The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in

  12. Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

    PubMed

    Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively

  13. PMP22 messenger RNA levels in skin biopsies: testing the effectiveness of a Charcot-Marie-Tooth 1A biomarker.

    PubMed

    Nobbio, Lucilla; Visigalli, Davide; Radice, Davide; Fiorina, Elisabetta; Solari, Alessandra; Lauria, Giuseppe; Reilly, Mary M; Santoro, Lucio; Schenone, Angelo; Pareyson, Davide

    2014-06-01

    Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with increased gene dosage for PMP22. Therapeutic approaches are currently aiming at correcting PMP22 over-expression. It is unknown whether PMP22 can be used as a biological marker of disease progression and therapy efficacy. We performed quantitative real-time polymerase chain reaction on skin biopsies of 45 patients with CMT1A, obtained at study entry and after 24-months of treatment either with ascorbic acid or placebo. Data of a subgroup of patients were also compared with matched healthy subjects. Finally, we analysed PMP22 messenger RNA levels in sural nerve biopsies. We did not find significant differences in the levels of any known PMP22 transcripts in treated or untreated patients with CMT1A, thus confirming that ascorbic acid does not impact on the molecular features of CMT1A. Most importantly, we did not observe any correlation between PMP22 messenger RNA levels and the different clinical and electrophysiological outcome measures, underscoring the weakness of PMP22 to mirror the phenotypic variability of patients with CMT1A. We did not find increased PMP22 messenger RNA levels in skin and sural nerve biopsies of patients with CMT1A compared with relative controls. In conclusion, this study shows that ascorbic acid does not impact on PMP22 transcriptional regulation and PMP22 is not a suitable biomarker for CMT1A.

  14. Gap junctional communication between vascular cells. Induction of connexin43 messenger RNA in macrophage foam cells of atherosclerotic lesions.

    PubMed Central

    Polacek, D.; Lal, R.; Volin, M. V.; Davies, P. F.

    1993-01-01

    The structure and function of blood vessels depend on the ability of vascular cells to receive and transduce signals and to communicate with each other. One means by which vascular cells have been shown to communicate is via gap junctions, specifically connexin43. In atherosclerosis, the normal physical patterns of communication are disrupted by the subendothelial infiltration and accumulation of blood monocytes, which in turn can differentiate into resident foam cells. In this paper we report that neither freshly isolated human peripheral blood monocytes nor differentiated monocytes/macrophages exhibit functional gap junctional dye transfer in homo-cellular culture or in co-culture with endothelial cells or smooth muscle cells. By Northern analysis, neither freshly isolated blood monocytes nor pure cultures of differentiated monocyte/macrophages expressed gap junction messenger RNA. However, immunohistochemical staining followed by in situ hybridization on sections of human atherosclerotic carotid arteries revealed strong expression of gap junction connexin43 messenger RNA by macrophage foam cells. These results suggest that tissue-specific conditions present in atherosclerotic arteries induce expression of connexin43 messenger RNA in monocyte/macrophages. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8382009

  15. Human bitter perception correlates with bitter receptor messenger RNA expression in taste cells123

    PubMed Central

    Lipchock, Sarah V; Mennella, Julie A; Spielman, Andrew I; Reed, Danielle R

    2013-01-01

    Background: Alleles of the receptor gene TAS2R38 are responsible in part for the variation in bitter taste perception of 6-n-propylthiouracil (PROP) and structurally similar compounds (eg, glucosinolates in cruciferous vegetables). At low concentrations, people with the PAV (“taster” amino acid sequence) form of TAS2R38 perceive these bitter compounds, whereas most with the AVI (“nontaster” amino acid sequence) form do not; heterozygotes (PAV/AVI) show the widest range of bitter perception. Objectives: The objectives were to examine individual differences in expression of PAV-TAS2R38 messenger RNA (mRNA) among heterozygotes, to test the hypotheses that the abundance of allele-specific gene expression accounts for the variation in human bitter taste perception, and to relate to dietary intake of bitter-tasting beverages and foods. Design: Heterozygous individuals (n = 22) provided psychophysical evaluation of the bitterness of PROP, glucosinolate-containing broccoli juice, non–glucosinolate-containing carrot juice, and several bitter non-TAS2R38 ligands as well as dietary recalls. Fungiform taste papillae were examined for allele-specific TAS2R38 expression by using quantitative polymerase chain reaction. Results: PAV-TAS2R38 mRNA expression was measured in 18 of 22 heterozygous subjects. Relative expression varied widely and positively correlated with ratings of bitterness intensity of PROP (P = 0.007) and broccoli juice (P = 0.004) but not of the control solutions carrot juice (P = 0.26), NaCl (P = 0.68), caffeine (P = 0.24), or urea (P = 0.47). Expression amounts were related to self-reported recent and habitual caffeine intake (P = 0.060, P = 0.005); vegetable intake was too low to analyze. Conclusions: We provide evidence that PAV-TAS2R38 expression amount correlates with individual differences in bitter sensory perception and diet. The nature of this correlation calls for additional research on the molecular mechanisms associated with some individual

  16. A Novel Route Controlling Begomovirus Resistance by the Messenger RNA Surveillance Factor Pelota

    PubMed Central

    Lapidot, Moshe; Karniel, Uri; Gelbart, Dana; Fogel, Doron; Evenor, Dalia; Kutsher, Yaarit; Makhbash, Zion; Nahon, Sahadia; Shlomo, Haviva; Chen, Lea; Reuveni, Moshe; Levin, Ilan

    2015-01-01

    Tomato yellow leaf curl virus (TYLCV) is a devastating disease of tomato (Solanum lycopersicum) that can be effectively controlled by the deployment of resistant cultivars. The TYLCV-resistant line TY172 carries a major recessive locus for TYLCV resistance, designated ty-5, on chromosome 4. In this study, the association between 27 polymorphic DNA markers, spanning the ty-5 locus, and the resistance characteristics of individual plants inoculated with TYLCV in 51 segregating recombinant populations were analyzed. These analyses localized ty-5 into a 425 bp region containing two transversions: one in the first exon of a gene encoding the tomato homolog of the messenger RNA surveillance factor Pelota (Pelo), and a second in its proximal promoter. Analyses of susceptible and resistant lines revealed that the relative transcript level of the gene remained unchanged, regardless of whether the plants were infected with TYLCV or not. This suggests that the polymorphism discovered in the coding region of the gene controls the resistance. Silencing of Pelo in a susceptible line rendered the transgenic plants highly resistant, while in the resistant line TY172 had no effect on symptom development. In addition, over-expression of the susceptible allele of the gene in the resistant TY172 line rendered it susceptible, while over-expression of the resistant allele in susceptible plants had no effect. These results confirm that Pelo is the gene controlling resistance at the ty-5 locus. Pelo, implicated in the ribosome recycling-phase of protein synthesis, offers an alternative route to promote resistance to TYLCV and other viruses. PMID:26448569

  17. Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels.

    PubMed

    Hall, R E; Lee, C S; Alexander, I E; Shine, J; Clarke, C L; Sutherland, R L

    1990-12-15

    The relative expression in human breast cancer cells of messenger ribonucleic acids (mRNA) encoding different steroid hormone receptors is unknown. Accordingly, mRNA levels in total RNA extracted from 13 human breast cancer cell lines were measured by Northern analysis employing complementary DNA probes for the human oestrogen (ER), progesterone (PR), androgen (AR), vitamin D3 (VDR) and glucocorticoid receptors (GR). The 7 ER+ lines expressed a single 6.4 kilobases (kb) ER mRNA. Interestingly, low concentrations of ER mRNA were detected in the ER- cell lines, MDA-MB-330 and BT 20. PR mRNA, predominantly a 13.5 kb species, was expressed in the 6 lines known to be ER+, PR+ by radioligand binding; however, one ER+ cell line, MDA-MB-134, failed to express PR mRNA. A 10.5 kb AR mRNA was expressed at significantly higher levels in ER+ than ER- cell lines. All cell lines expressed a single 4.6 kb mRNA for VDR and a single 7.4 kb mRNA for GR. ER and PR mRNA levels were positively correlated (p = 0.011) and each was positively correlated with androgen receptor (AR) mRNA levels (p less than or equal to 0.009). ER, PR and AR mRNAs were negatively associated with GR levels (p less than or equal to 0.012), while ER and AR mRNA levels were negatively correlated with mRNA for the epidermal growth factor receptor. In contrast, levels of VDR mRNA were unrelated to the concentration of any other steroid receptor mRNA. Our data demonstrate the coordinate expression of ER, PR and AR genes, and an inverse relationship between sex steroid hormone receptor and GR gene expression in human breast cancer cell lines.

  18. Regulation of antifreeze protein messenger RNA and a female-specific messenger RNA in winter flounder: Evidence for temperature and photoperiod effects

    SciTech Connect

    Price, J.L.

    1988-01-01

    Northern blot hybridizations showed that mRNA{sub f} is female-specific, and that both mRNAs are liver-specific and expressed at high levels only during the fall and winter in the wild. When warm-acclimated flounder (18C) were acclimated over 2 days to low temperature (4 C) and short photoperiod in July and October, high levels of AF mRNA accumulated within 4-10 days. Low levels were found in fish at high temperature at any time, on long photoperiod after acclimation to low temperature in July, or continuously at 4 C from March until September. A temperature increase in January resulted in a dramatic decrease in the levels of AF mRNA within 5 days. Beta actin mRNA, mRNA{sub f} and most mRNAs translated in a reticulocyte lysate did not change in abundance after temperature increases or decreases. Synthesis of AF mRNA in liver slices was investigated by Northern blot hybridization and hybridization of {sup 32}P-labeled RNA to AF cDNA. Liver slices from cold-acclimated January fish expressed high levels of AF mRNA initially at 4 C and lower levels at 18 C. AF mRNA expression was turned off in tissue from warm-acclimated fish but was progressively induced after long 4 C incubations in vitro in livers from fall fish but not from spring fish. Temperature and photoperiod act either as zeitgebers, which set the phase of an endogenous rhythm, or as more direct signals that stimulate a transient accumulation of AF mRNA.

  19. Single-cell messenger RNA sequencing reveals rare intestinal cell types.

    PubMed

    Grün, Dominic; Lyubimova, Anna; Kester, Lennart; Wiebrands, Kay; Basak, Onur; Sasaki, Nobuo; Clevers, Hans; van Oudenaarden, Alexander

    2015-09-10

    Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5

  20. Content of N-6 methyl adenylic acid in heterogeneous nuclear and messenger RNA of HeLa cells.

    PubMed Central

    Lavi, U; Fernandez-Muñoz, R; Darnell, J E

    1977-01-01

    With the aid of a suitable thin layer chromatographic procedure, the N-6 methyl adenylic acid (m6A), content of a variety of 32P labeled RNA species from HeLa cells has been measured. Poly(A)-containing (poly(A)+) cytoplasmic RNA has on the average one m6Ap per 800 to 900 nucleotides. This value is independent of the length of the molecules. The proportion of m6Ap in poly(A)+ cytoplasmic RNA does not change between 4 and 18 hours of labeling with 32P, suggesting that the majority of the messenger RNA molecules may have a similar level of internal methylation regardless of their half-life. The non-polyadenylated, non-ribosomal cytoplasmic RNA fraction sedimenting from 10S TO 28S is less methylated with approximately one m6A per 2,700 nucleotides. Heterogeneous nuclear RNA molecules (DMSO treated) which sediment from 28S to 45S have approximately one m6Ap per 3,000 nucleotides. The hnRNA molecules sedimenting from 10S to 28S have one m6Ap per 1,800 nucleotides. Poly(A)+ nuclear RNA is enriched in m6A, containing 1 residue of m6A per 700 to 800 nucleotides, a value close to that obtained for the polyadenylated cytoplasmic RNA. Images PMID:866178

  1. Functional and structural characterization of the mammalian TREX-2 complex that links transcription with nuclear messenger RNA export

    PubMed Central

    Jani, Divyang; Lutz, Sheila; Hurt, Ed; Laskey, Ronald A.; Stewart, Murray; Wickramasinghe, Vihandha O.

    2012-01-01

    Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs. PMID:22307388

  2. Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond.

    PubMed

    Vallazza, Britta; Petri, Sebastian; Poleganov, Marco A; Eberle, Florian; Kuhn, Andreas N; Sahin, Ugur

    2015-01-01

    In recent years, the interest in using messenger RNA (mRNA) as a therapeutic means to tackle different diseases has enormously increased. This holds true not only for numerous preclinical studies, but mRNA has also entered the clinic to fight cancer. The advantages of using mRNA compared to DNA were recognized very early on, e.g., the lack of risk for genomic integration, or the expression of the encoded protein in the cytoplasm without the need to cross the nuclear membrane. However, it was generally assumed that mRNA is just not stable enough to give rise to sufficient expression of the encoded protein. Yet, an initially small group of mRNA aficionados could demonstrate that the stability of mRNA and the efficiency, by which the encoded protein is translated, can be significantly increased by selecting the right set of cis-acting structural elements (including the 5'-cap, 5'- and 3'-untranslated regions, poly(A)-tail, and modified building blocks). In parallel, significant advances in RNA packaging and delivery have been made, extending the potential for this molecule. This paved the way for further work to prove mRNA as a promising therapeutic for multiple diseases. Here, we review the developments to optimize mRNA regarding stability, translational efficiency, and immune-modulating properties to enhance its functionality and efficacy as a therapeutic. Furthermore, we summarize the current status of preclinical and clinical studies that use mRNA for cancer immunotherapy, for the expression of functional proteins as so-called transcript (or protein) replacement therapy, as well as for induction of pluripotent stem cells.

  3. Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives.

    PubMed

    Bujarski, Jozef J

    2013-01-01

    RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA-RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

  4. Effects of messenger RNA structure and other translational control mechanisms on major histocompatibility complex-I mediated antigen presentation

    PubMed Central

    Murat, Pierre; Tellam, Judy

    2015-01-01

    Effective T-cell surveillance of antigen-presenting cells is dependent on the expression of an array of antigenic peptides bound to major histocompatibility complex (MHC) class I (MHC-I) or class II (MHC-II) molecules. Pathogens co-evolving with their hosts exploit crucial translational regulatory mechanisms in order to evade host immune recognition and thereby sustain their infection. Evasion strategies that downregulate viral protein synthesis and thereby restrict antigen presentation to cytotoxic T-cells through the endogenous MHC-I pathway have been implicated in the pathogenesis of viral-associated malignancies. An understanding of the mechanisms by which messenger RNA (mRNA) structure modulates both viral mRNA translation and the antigen processing machinery to escape immune surveillance, will stimulate the development of alternative therapeutic strategies focused on RNA-directed drugs designed to enhance immune responses against infected cells. In this review, we discuss regulatory aspects of the MHC-I pathway and summarize current knowledge of the role attributed by mRNA structure and other translational regulatory mechanisms in immune evasion. In particular we highlight the impact of recently identified G-quadruplex structures within virally encoded transcripts as unique regulatory signals for translational control and antigen presentation. WIREs RNA 2015, 6:157–171. doi: 10.1002/wrna.1262 PMID:25264139

  5. Proopiomelanocortin messenger RNA levels are increased in the anterior pituitary of the sheep fetus after adrenalectomy in late gestation.

    PubMed

    McMillen, I C; Antolovich, G C; Mercer, J E; Perry, R A; Silver, M

    1990-09-01

    We have investigated the effect of bilateral adrenalectomy at 116-119 days' gestation on the levels of the messenger (m) RNA for proopiomelanocortin (POMC) in the anterior pituitary of the fetal sheep and in the ovine placentome during late gestation (134-136 days' gestation). After fetal adrenalectomy there was a significant (p less than 0.001) and sustained increase in circulating ACTH concentrations in the adrenalectomised group (1,838 +/- 155 ng/l at 130-136 days) when compared with the intact control group (131 +/- 25 ng/l at 130-136 days). The mean levels of POMCmRNA relative to 18S RNA were also significantly higher (p less than 0.001) in the adrenalectomised fetal sheep pituitaries (2.8 +/- 0.12; n = 4) than in the intact/control fetal sheep pituitaries (1.31 +/- 0.13; n = 4). In contrast to the findings in the anterior pituitary, POMCmRNA was not detected in RNA extracted from the placentomes of either the adrenalectomised or intact fetal sheep. There was also a significant arteriovenous difference in ACTH concentrations in the umbilical circulation in both adrenalectomised and intact fetal sheep at 134-136 days' gestation. This study demonstrates therefore that the fetal adrenals act to suppress POMCmRNA levels in late gestation and also that the increase in circulating ACTH after adrenalectomy originates from the pituitary and not the placentome.

  6. Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

    PubMed Central

    Bujarski, Jozef J.

    2013-01-01

    RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA–RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA–RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented. PMID:23533000

  7. DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

    PubMed

    Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

    2016-01-01

    Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  8. Adipose tissue angiopoietin-like protein 4 messenger RNA changes with altered energy balance in lactating Holstein cows.

    PubMed

    Koltes, D A; Spurlock, D M

    2012-11-01

    Negative energy balance at the onset of lactation is unfavorably associated with fitness traits in high-producing dairy cows. Angiopoietin-like protein 4 (ANGPTL4) is an adipokine that has been associated with the regulation of lipid metabolism through the inhibition of lipoprotein lipase activity and regulation of lipolysis. Expression of ANGPTL4 messenger RNA (mRNA) increases during early lactation, but its regulation with changing energy status is currently unknown. Accordingly, the objective of this study was to determine whether ANGPTL4 mRNA abundance is responsive to declining energy balance induced by the transition from pregnancy to lactation, feed restriction, and GH administration in lactating dairy cows. The mRNA abundance of leptin, adiponectin, and adiponectin receptor 2 were also measured to compare adipokine mRNA profiles during changes in energy metabolism. Repeated adipose tissue biopsies were taken from different cows during transition from late pregnancy to lactation (n = 26), feed restriction (n = 19), and GH administration (n = 20). As expected, milk yield increased with the onset of lactation and GH administration (P < 0.01) but declined during feed restriction. Energy balance declined in each experiment, resulting in negative energy balance at the onset of lactation and after feed restriction. Abundance of ANGPTL4 mRNA expression increased 2- to 6-fold with declining energy balance in each experiment. Leptin mRNA declined with feed restriction, and adiponectin mRNA decreased with the onset of lactation. The consistency and magnitude of the increase in ANGPTL4 mRNA across multiple models of altered energy balance identifies it as an adipokine that is uniquely responsive to changes in energy balance in the lactating dairy cow. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges.

    PubMed

    Steinle, Heidrun; Behring, Andreas; Schlensak, Christian; Wendel, Hans Peter; Avci-Adali, Meltem

    2017-01-01

    Several diseases are caused by missing or defective synthesis of proteins due to genetic or acquired disorders. In recent years, in vitro transcribed (IVT) messenger RNA (mRNA)-based therapy for de novo protein expression in cells has increased in importance. Thereby, desired proteins can be produced in cells by exogenous delivery of IVT mRNA, which does not integrate into the host genome and results in transient production of target proteins. Due to the lack of genomic integration, the risk of mutation and tumor development is minimized. Different approaches using IVT mRNA have been applied to alter the expression profiles of cells by the production of proteins. IVT mRNAs encoding transcription factors have led to the highly efficient induction of pluripotency in somatic cells and generated induced pluripotent stem cells that are free of viral vector components. Furthermore, specific IVT mRNA cocktails containing more than one specific IVT mRNA can be used to directly induce the differentiation into a desired cell type. In theory, every desired mRNA can be produced in vitro and used to enable extrinsic biosynthesis of target proteins in each cell type. Cells can be engineered by IVT mRNA to express antigens on dendritic cells for vaccination and tumor treatment, surface receptors on stem cells for increased homing to distinct areas, and to produce industrial grade human growth factors. In this review, we focus on the progress and challenges in mRNA-based cell engineering approaches. Stem Cells 2017;35:68-79. © 2016 AlphaMed Press.

  10. Effects of messenger RNA structure and other translational control mechanisms on major histocompatibility complex-I mediated antigen presentation.

    PubMed

    Murat, Pierre; Tellam, Judy

    2015-01-01

    Effective T-cell surveillance of antigen-presenting cells is dependent on the expression of an array of antigenic peptides bound to major histocompatibility complex (MHC) class I (MHC-I) or class II (MHC-II) molecules. Pathogens co-evolving with their hosts exploit crucial translational regulatory mechanisms in order to evade host immune recognition and thereby sustain their infection. Evasion strategies that downregulate viral protein synthesis and thereby restrict antigen presentation to cytotoxic T-cells through the endogenous MHC-I pathway have been implicated in the pathogenesis of viral-associated malignancies. An understanding of the mechanisms by which messenger RNA (mRNA) structure modulates both viral mRNA translation and the antigen processing machinery to escape immune surveillance, will stimulate the development of alternative therapeutic strategies focused on RNA-directed drugs designed to enhance immune responses against infected cells. In this review, we discuss regulatory aspects of the MHC-I pathway and summarize current knowledge of the role attributed by mRNA structure and other translational regulatory mechanisms in immune evasion. In particular we highlight the impact of recently identified G-quadruplex structures within virally encoded transcripts as unique regulatory signals for translational control and antigen presentation.

  11. The cytoskeletal framework of sea urchin eggs and embryos: developmental changes in the association of messenger RNA.

    PubMed

    Moon, R T; Nicosia, R F; Olsen, C; Hille, M B; Jeffery, W R

    1983-02-01

    Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.

  12. Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish.

    PubMed

    Kobayashi, Y; Peterson, B C; Waldbieser, G C

    2015-04-01

    This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P < 0.01). Expression of UCP2b mRNA tended to be lower (P < 0.10) in fast growing fish in the middle of the study although levels were similar at the beginning and the end of the study. In the fed vs fasted study, expression of UCP2b mRNA in muscle was increased (P < 0.05) in fish assigned to 30 d of fasting. Our results suggest that, based on the nucleotide and amino acid sequence similarities and tissue mRNA distribution, catfish UCP2b may be the analog to UCP3. Moreover, our results suggest selection toward growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms.

  13. Whole blood cells loaded with messenger RNA as an anti-tumor vaccine.

    PubMed

    Phua, Kyle K L; Boczkowski, David; Dannull, Jens; Pruitt, Scott; Leong, Kam W; Nair, Smita K

    2014-06-01

    The use of a cell-based vaccine composed of autologous whole blood cells loaded with mRNA is described. Mice immunized with whole blood cells loaded with mRNA encoding antigen develop anti-tumor immunity comparable to DC-RNA immunization. This approach offers a simple and affordable alternative to RNA-based cellular therapy by circumventing complex, laborious and expensive ex vivo manipulations required for DC-based immunizations.

  14. Regulation of neurotrophin and trkA, trkB and trkC tyrosine kinase receptor messenger RNA expression in kindling.

    PubMed

    Bengzon, J; Kokaia, Z; Ernfors, P; Kokaia, M; Leanza, G; Nilsson, O G; Persson, H; Lindvall, O

    1993-03-01

    Levels of messenger RNA for nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and the tyrosine kinase receptors trkA, trkB and trkC have been studied using in situ hybridization in the rat brain 2 h and four weeks after kindling-induced seizures. Epileptiform activity evoked by hippocampal stimulation and exceeding 70 s lead to a concomitant and transient increase of brain- derived neurotrophic factor, nerve growth factor, trkB and trkC messenger RNA expression in dentate granule cells after both focal and generalized seizures. Brain-derived neurotrophic factor messenger RNA levels were also increased bilaterally in the CA1-CA3 regions, amygdala and the piriform, entorhinal, perirhinal, retrosplenial and temporal cortices after generalized seizures. The magnitude of the increases was similar throughout the development of kindling and in the fully kindled brain. No changes of trkA messenger RNA were observed. In amygdalar kindling, elevated brain-derived neurotrophic factor messenger RNA levels developed more rapidly in the amygdala-piriform cortex than after stimulation in the hippocampus but changes in the hippocampal formation were only seen in few animals. Intraventricular 6-hydroxydopamine or a bilateral fimbria-fornix lesion did not alter basal expression or seizure-evoked changes in messenger RNA levels for neurotrophins or trk receptors but increased the number of animals exhibiting elevated levels after the first stimulation, probably due to a prolongation of seizure activity. Both in sham-operated and fimbria-fornix-lesioned rats seizure activity caused a marked reduction of neurotrophin-3 messenger RNA levels in dentate granule cells. The results indicate that activation of the brain-derived neurotrophic factor gene, at least in dentate granule cells, is an "all-or-none" type of response and dependent on the duration but not the severity of seizures or the stage of kindling epileptogenesis. Changes in brain-derived neurotrophic

  15. Efficient ex vivo delivery of chemically modified messenger RNA using lipofection and magnetofection.

    PubMed

    Badieyan, Zohreh Sadat; Pasewald, Tamara; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2017-01-22

    Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used ∼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery.

  16. Messenger RNA delivery of a cartilage-anabolic transcription factor as a disease-modifying strategy for osteoarthritis treatment.

    PubMed

    Aini, Hailati; Itaka, Keiji; Fujisawa, Ayano; Uchida, Hirokuni; Uchida, Satoshi; Fukushima, Shigeto; Kataoka, Kazunori; Saito, Taku; Chung, Ung-il; Ohba, Shinsuke

    2016-01-05

    Osteoarthritis (OA) is a chronic degenerative joint disease and a major health problem in the elderly population. No disease-modifying osteoarthritis drug (DMOAD) has been made available for clinical use. Here we present a disease-modifying strategy for OA, focusing on messenger RNA (mRNA) delivery of a therapeutic transcription factor using polyethylene glycol (PEG)-polyamino acid block copolymer-based polyplex nanomicelles. When polyplex nanomicelles carrying the cartilage-anabolic, runt-related transcription factor (RUNX) 1 mRNA were injected into mouse OA knee joints, OA progression was significantly suppressed compared with the non-treatment control. Expressions of cartilage-anabolic markers and proliferation were augmented in articular chondrocytes of the RUNX1-injected knees. Thus, this study provides a proof of concept of the treatment of degenerative diseases such as OA by the in situ mRNA delivery of therapeutic transcription factors; the presented approach will directly connect basic findings on disease-protective or tissue-regenerating factors to disease treatment.

  17. Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

    SciTech Connect

    Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-01

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection of target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.

  18. Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes.

    PubMed

    Fenton, Owen S; Kauffman, Kevin J; Kaczmarek, James C; McClellan, Rebecca L; Jhunjhunwala, Siddharth; Tibbitt, Mark W; Zeng, Manhao D; Appel, Eric A; Dorkin, Joseph R; Mir, Faryal F; Yang, Jung H; Oberli, Matthias A; Heartlein, Michael W; DeRosa, Frank; Langer, Robert; Anderson, Daniel G

    2017-09-01

    B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Messenger RNA delivery of a cartilage-anabolic transcription factor as a disease-modifying strategy for osteoarthritis treatment

    PubMed Central

    Aini, Hailati; Itaka, Keiji; Fujisawa, Ayano; Uchida, Hirokuni; Uchida, Satoshi; Fukushima, Shigeto; Kataoka, Kazunori; Saito, Taku; Chung, Ung-il; Ohba, Shinsuke

    2016-01-01

    Osteoarthritis (OA) is a chronic degenerative joint disease and a major health problem in the elderly population. No disease-modifying osteoarthritis drug (DMOAD) has been made available for clinical use. Here we present a disease-modifying strategy for OA, focusing on messenger RNA (mRNA) delivery of a therapeutic transcription factor using polyethylene glycol (PEG)-polyamino acid block copolymer-based polyplex nanomicelles. When polyplex nanomicelles carrying the cartilage-anabolic, runt-related transcription factor (RUNX) 1 mRNA were injected into mouse OA knee joints, OA progression was significantly suppressed compared with the non-treatment control. Expressions of cartilage-anabolic markers and proliferation were augmented in articular chondrocytes of the RUNX1-injected knees. Thus, this study provides a proof of concept of the treatment of degenerative diseases such as OA by the in situ mRNA delivery of therapeutic transcription factors; the presented approach will directly connect basic findings on disease-protective or tissue-regenerating factors to disease treatment. PMID:26728350

  20. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

    SciTech Connect

    Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

    1984-03-01

    Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared.

  1. Dark induction of haem oxygenase messenger RNA by haematoporphyrin derivative and zinc phthalocyanine; agents for photodynamic therapy.

    PubMed

    Bressoud, D; Jomini, V; Tyrrell, R M

    1992-07-30

    Haematoporphyrin derivative is one of the main drugs currently used in clinical trials involving photodynamic therapy of cancer, and zinc phthalocyanine is being considered as one of several possible alternatives. We show that incubation of cultured human fibroblasts populations with either of the two drugs will lead to a sharp increase in the accumulation of the messenger RNA corresponding to haem oxygenase. Only cells incubated with haematoporphyrin derivative show additional enhancement of expression of this specific gene on exposure to red light. Since haem oxygenase induction appears to be a specific stress response that may be involved in cellular defence, such observations should be confirmed under conditions which would allow the clinical implications to be fully evaluated.

  2. A quantitative method to identify microRNAs targeting a messenger RNA using a 3′UTR RNA affinity technique

    PubMed Central

    Shi, Miao; Han, Weiguo; Spivack, Simon D.

    2014-01-01

    The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3′UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitatvie polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA. PMID:23938772

  3. Erythromycin-induced stabilization of ermA messenger RNA in Staphylococcus aureus and Bacillus subtilis.

    PubMed

    Sandler, P; Weisblum, B

    1988-10-20

    Erythromycin-induced stabilization of ermA mRNA was studied in Staphylococcus aureus, its original host background, and in Bacillus subtilis, subcloned on plasmid vectors. By RNA blot analysis it was shown that 40 nM-erythromycin specifically increased the chemical half-life of ermA mRNA from 2.5 to 17.5 minutes whereas the half-life of cat-86 mRNA was not increased by erythromycin. While expression of ermA has been shown to be induced by erythromycin at the level of translation, our studies with three ermA constitutive mutants demonstrated that mRNA stabilization in growing cells occurred independently of induced gene expression, suggesting that the stabilized mRNA was not functional for protein synthesis. Studies of ermA/lacZ fusions demonstrated that the 5' end of the mRNA was sufficient to confer stabilization. Translation of specific amino acid codons in a leader peptide located at the extreme 5' end of the mRNA was required for the erythromycin-induced stabilization as a frameshift mutation introduced into the leader peptide determinant abolished stabilization. By S1 mapping, no differences were detected in the length of the 5' or 3' end of ermA mRNA with the addition of erythromycin, indicating that the stabilized transcript was not processed at its ends.

  4. Affinity Purification of Binding miRNAs for Messenger RNA Fused with a Common Tag

    PubMed Central

    Wei, Ke; Yan, Feng; Xiao, Hui; Yang, Xiaoxu; Xie, Guie; Xiao, Ye; Wang, Tingting; Xun, Yu; Huang, Zhaoqin; Han, Mei; Zhang, Jian; Xiang, Shuanglin

    2014-01-01

    Prediction of microRNA–mRNA interaction typically relies on bioinformatic methods, but these methods only suggest the possibility of microRNA binding and may miss important interactions as well as falsely predict others. A major obstacle to the miRNA research has been the lack of experimental procedures for the identification of miRNA–mRNA interactions. Recently, a few studies have attempted to explore experimental methods to isolate and identify miRNA targets or miRNAs targeting a single gene. Here, we developed an more convenient experimental approach for the isolation and identification of miRNAs targeting a single gene by applying short biotinylated DNA anti-sense oligonucleotides mix to enhanced green fluorescent protein (EGFP) mRNA which was fused to target gene mRNA. This method does not require a design of different anti-sense oligonucleotides to any mRNA. This is a simple and an efficient method to potentially identify miRNAs targeting specific gene mRNA combined with chip screen. PMID:25153630

  5. Genetic analysis of the structure and function of transfer messenger RNA pseudoknot 1.

    PubMed

    Tanner, Douglas R; Dewey, Jonathan D; Miller, Mickey R; Buskirk, Allen R

    2006-04-14

    tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

  6. Activation of ribosomal and messenger RNA synthesis in excised Jerusalem artichoke tuber slices.

    PubMed

    Byrne, H; Setterfield, G

    1977-01-01

    RNA synthesis was studied in Jerusalem artichoke (Helianthus tuberosus L.) tuber slices immediately following excision and during the early period of aging in water. Incorporation of [(3)H]adenosine into RNA was detected as early as 20 min after excision. Measurement of the specific activities of RNA (cpm/μg) and of ATP showed that RNA synthesis proceeded at a constant rate for the first several hours of aging and then increased moderately. [(3)H]adenosine was incorporated into polysomes throughout the aging period examined. Sucrose gradient fractionation of EDTA-dissociated polysomes showed that during the first 2 h of aging most of this incorporation was not into ribosome subunits but into presumed mRNA. Autoradiographic analysis of [(3)H]adenosine labelled nuclei showed that this was caused, at least in part, by a delay in the onset of rRNA synthesis synthesized during this time chromatographed as poly(A)-RNA on oligo(dT)-cellulose, indicating that a large part of the mRNA was not polyadenylated.

  7. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.

    PubMed

    Listerman, Imke; Sapra, Aparna K; Neugebauer, Karla M

    2006-09-01

    Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

  8. Photoperiodic regulation of hypothalamic neuropeptide messenger RNA expression: effect of pinealectomy and neuroanatomical location.

    PubMed

    Duncan, M J

    1998-06-01

    Seasonal changes in daylength (photoperiod) affect many aspects of mammalian physiology and behavior, including reproduction, metabolism, thermoregulation, and sleep. The circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) regulates these photoperiodic changes. Our studies of the Siberian hamster SCN have shown that two types of neuropeptide-containing neurons, vasopressin (AVP) and vasoactive intestinal peptide (VIP) neurons, respond to short photoperiod by decreasing mRNA expression. The present studies investigated whether photoperiodic inhibition of mRNA expression also occurs in somatostatin-synthesizing neurons in the SCN, depends upon the pineal gland, and occurs in neurons in other hypothalamic nuclei. Juvenile Siberian hamsters exposed to either long photoperiod (16 h light/day) or short photoperiod (10 h light/day) for 2 weeks after weaning, were used for these studies. Coronal sections throughout the SCN were prepared and processed for in situ hybridization. The results showed that photoperiod decreased the expression of AVP mRNA and VIP mRNA in the SCN, as seen previously, but not somatostatin mRNA. Furthermore, pinealectomy did not attenuate the short photoperiod inhibition of AVP mRNA and VIP mRNA expression in the SCN. Also, short photoperiod inhibition of AVP mRNA expression was found in the paraventricular and supraoptic nuclei, as well as in the SCN. These results show that short photoperiod inhibition of mRNA expression is partially selective among the neuropeptides, but is not restricted to the SCN. Furthermore, these findings suggest that photoperiodic regulation of neuropeptide mRNA expression is independent of pineal melatonin secretion and gonadal steroid secretion. Copyright 1998 Elsevier Science B.V. All rights reserved.

  9. Progestin regulation of estrogen receptor messenger RNA in human breast cancer cells.

    PubMed

    Alexander, I E; Shine, J; Sutherland, R L

    1990-06-01

    Progestin antagonism of estrogen action is thought to be due, at least in part, to progestin down-regulation of the estrogen receptor (ER). The molecular mechanisms subserving this effect, and the functional consequences in terms of target cell sensitivity to estrogens, are poorly understood. The present study was undertaken to address these issues with particular emphasis on progestin regulation of ER gene expression at the mRNA level. The T-47D human breast cancer cell line was treated with the synthetic progestin, ORG 2058, and the resultant changes in ER mRNA and ER levels determined by Northern analysis and radioligand binding, respectively. Treatment of T-47D cells with ORG 2058 resulted in rapid down-regulation of ER mRNA levels to a nadir of 35-40% of control by 6 h. This fall in ER mRNA levels was accompanied by a slower but more sustained fall in ER binding to a nadir of 20% of control at 24 h. Between 12 and 24 h ER mRNA levels recovered partially while ER ligand binding continued to fall. At 48 h both ER mRNA and ER concentrations remained depressed, although the latter to a greater extent. ER mRNA half-life was determined by [3H]uridine incorporation to be approximately 60 min and was unaffected by progestin treatment during the early rapid phase of ER mRNA down-regulation. These data demonstrate that progestins cause rapid down-regulation of the ER mRNA and suggest that during the early rapid phase of this effect, reduced transcription of the ER gene rather than altered ER mRNA half-life mediate this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Identification of genes expressed in response to phytoplasma infection in leaves of Prunus armeniaca by messenger RNA differential display.

    PubMed

    Carginale, Vincenzo; Maria, Giovanna; Capasso, Clemente; Ionata, Elena; La Cara, Francesco; Pastore, Maria; Bertaccini, Assunta; Capasso, Antonio

    2004-05-12

    The messenger RNA (mRNA) differential display technique was applied to the identification and isolation of genes whose transcription was altered in leaves of Prunus armeniaca infected by European stone fruit yellows (ESFY) phytoplasma belonging to ribosomal subgroup 16SrX-B. Four genes whose steady-state levels of expression significantly changed in response to phytoplasma infection were isolated and identified. The results obtained show that two group of genes are affected by phytoplasma infection in apricot leaves. The first group comprises genes that are up-regulated by phytoplasma presence: in particular, a gene encoding the heat-shock protein HSP-70, a gene encoding a metallothionein (MT) and another homologous to the EST 673 cDNA clone of P. armeniaca, whose function was unknown. The other gene identified in our analysis is down-regulated by phytoplasma presence. It encodes a protein having homology to an amino acid transporter of Arabidopsis thaliana. Our findings demonstrate the usefulness of mRNA differential display approach for the detection of plant metabolic pathways affected by phytoplasma infection.

  11. Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.

    PubMed

    Quinet, E M; Agellon, L B; Kroon, P A; Marcel, Y L; Lee, Y C; Whitlock, M E; Tall, A R

    1990-02-01

    Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.

  12. Changes in hepatic messenger RNA for phosphoenolpyruvate carboxykinase (GTP) during development

    PubMed Central

    Ruiz, Josefa P. Garcia; Ingram, Robert; Hanson, Richard W.

    1978-01-01

    Phosphoenolpyruvate carboxykinase (GTP) [GTP;oxaloacetate carboxy-lyase(transphosphorylating); EC 4.1.1.32] is absent in rat liver cytosol during fetal life and is synthesized initially at birth. De novo synthesis of the enzyme can be induced prematurely by injection of dibutyryl cyclic AMP or glucagon into fetal animals in utero. In this study a wheat germ translation assay was used to quantitate the level of total functional mRNA for phosphoenolpyruvate carboxykinase in the liver of fetal rats at 21 days of pregnancy under different induction situations. The translatable mRNA for the enzyme was marginally detectable in fetal rat liver. Administration of either glucagon or dibutyryl cyclic AMP to fetal rats in utero caused a marked induction of functional mRNA for this enzyme. Three hours after administration of dibutyryl cyclic AMP, the level of translatable mRNA increased almost 23-fold, but by 6 hr the level dropped approximately 60%. Administration of actinomycin D prior to dibutyryl cyclic AMP in 21-day fetal rats prevented the appearance of newly synthesized poly(A)-containing RNA in the cytoplasm as well as the induction of translatable mRNA for phosphoenolpyruvate carboxykinase. In animals delivered prematurely and maintained for varying periods, the translatable mRNA for the enzyme accumulated in the liver at a rate comparable to that observed for enzyme synthesis. PMID:212740

  13. Expression of cytokine messenger RNA after heart transplantation: relationship with rejection and serum cytokines.

    PubMed

    Grant, S C; Guy, S P; Lamb, W R; Brooks, N H; Brenchley, P E; Hutchinson, I V

    1996-10-15

    Different groups of cytokines may initiate or inhibit the rejection process. We used the polymerase chain reaction to study the expression of cytokine mRNA for interleukin (IL)-2, -4, -6 and -10, tumor necrosis factor-alpha, and interferon-gamma in 187 biopsy specimens from 24 human cardiac transplant recipients 5-555 days after transplantation. Cytokine levels in the serum were also measured. Cytokine mRNA was detected in 38.5% of biopsy specimens. IL-10 mRNA was detected more frequently with mild or absent rejection (11.6% in grades 0 and 1 - vs. 1.4% in grades 2 and 3, P=0.01). Up to 90 days after transplantation, IL-2 mRNA was detected more frequently with moderate rejection (13% in grades 2 and 3 vs. 0% in grades 0 and 1, P=0.076), and IL-4 mRNA was detected more frequently with mild or absent rejection (16% in grades 0 and 1 - vs. 0% in grades 2 and 3, P=0.061). More than 90 days after transplantation, IL-2 mRNA was detected more frequently with mild or absent rejection (10% in grades 0 and 1 vs. 0% in grades 2 and 3, P=0.078). Serum IL-4 levels corresponding to biopsy specimens positive for IL-4 mRNA were higher than those corresponding to specimens negative for IL-4 mRNA (59 pg/ml vs. 32 pg/ml medians, P=0.028). Our results suggest that IL-10 and possibly IL-4 (T helper 2 cytokines) may suppress graft rejection, whereas IL-2 (T helper 1 cytokine) may promote cellular rejection. In addition, cytokine profiles may change with length of time after transplantation. The association of elevated serum levels of IL-4 with increased expression of intragraft IL-4 mRNA may suggest release of this cytokine from the graft into the circulation.

  14. Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

    PubMed Central

    Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

    2010-01-01

    The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression. PMID:20699122

  15. In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups

    SciTech Connect

    Burbach, J.P.; Voorhuis, T.A.; van Tol, H.H.; Ivell, R.

    1987-05-29

    Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded /sup 35/S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at these various sites.

  16. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

    PubMed Central

    Boni, I V; Isaeva, D M; Musychenko, M L; Tzareva, N V

    1991-01-01

    Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome. Images PMID:2011495

  17. Improvements in immunoprecipitation of specific messenger RNA. Isolation of highly purified conalbumin mRNA in high yield.

    PubMed

    Payvar, F; Schimke, R T

    1979-11-01

    We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non-specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (E) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation. We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.

  18. Translation of thyroglobulin 33S messenger RNA as a means of determining thyroglobulin quaternary structure.

    PubMed

    Vassart, G; Refetoff, S; Brocas, H; Dinsart, C; Dumont, J E

    1975-10-01

    Thyroglobulin is a 19S protein of approximately 660,000 daltons and unknown quaternary structure. We have previously shown that a 33S mRNA purified from mammalian thyroids promoted synthesis in the Xenopus oocyte of a peptide immunologically related to thyroglobulin. Chemical identity to the native protein is now presented by means of a tryptic peptide analysis. Moreover, the 33S mRNA is shown to contain all the information required for the synthesis of a complete 19S thyroglobulin molecule. Gel filtration in Sepharose under denaturing conditions indicates that the reduced polypeptide encoded by the 33S mRNA is larger than 210,000 daltons. A model of a dimeric thyroglobulin with about 300,000 dalton subunits is presented.

  19. All histological types of primary human rhabdomyosarcoma express alpha-cardiac and not alpha-skeletal actin messenger RNA.

    PubMed Central

    Schürch, W.; Bochaton-Piallat, M. L.; Geinoz, A.; d'Amore, E.; Laurini, R. N.; Cintorino, M.; Bégin, L. R.; Boivin, Y.; Gabbiani, G.

    1994-01-01

    Eleven human primary rhabdomyosarcomas (RMSs), including all histological variants, were analyzed morphologically, immunohistochemically for intermediate filament proteins and actin isoforms, and by means of Northern blots with probes specific for total actin, alpha-skeletal (SK), alpha-cardiac (CARD), and alpha-smooth muscle actin messenger (m)RNAs. All tumors disclosed ultrastructural evidence of skeletal muscle features with terminal differentiation in three cases. The RMSs contained immunohistochemically the intermediate filament proteins vimentin and desmin and reacted positively with the alpha-sarcomeric actin antibody, which recognizes alpha-SK and alpha-CARD actin isoforms. All RMSs reacted with the total actin probe, recognizing at 2.1 kb cytoplasmic actin mRNAs and at 1.7 kb alpha-actin mRNAs. With the specific probes, all RMSs expressed alpha-CARD actin mRNA, four neoplasms expressed also alpha-smooth muscle actin mRNA, whereas the probe for alpha-SK actin mRNA never produced a signal except in one case, in which the tumor masses were intermingled with non-neoplastic preexistent striated muscle fibers. Because alpha-CARD and alpha-smooth muscle actins are transiently expressed during normal skeletal muscle development, RMSs seem to follow normal skeletal myogenesis without completing the final step, consisting of alpha-SK actin mRNA expression. The use of Northern blots for alpha-CARD actin as an adjunct to conventional techniques may be helpful for the precise identification of primary RMSs compared to other soft tissue neoplasms. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8160781

  20. Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos.

    PubMed

    Biase, Fernando Henrique; Martelli, Lúcia; Puga, Renato; Giuliatti, Silvana; Santos-Biase, Weruska Karyna Freitas; Fonseca Merighe, Giovana Krempel; Meirelles, Flávio Vieira

    2010-05-15

    To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Comparison between oocyte groups and between early embryo stages. Laboratories of embryo manipulation and molecular biology from Departamento de Genética (FMRP) and Departamento de Ciências Básicas (FZEA)--University of São Paulo. Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good- and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  1. Frequency distribution of pre-messenger RNA sequences in polyadenylated and non-polyadenylated nuclear RNA from Friend cells.

    PubMed Central

    Balmain, A; Minty, A J; Birnie, G D

    1980-01-01

    Hybridisation of cDNA probes for abundant and rare polysomal polyadenylated RNAs with polyadenylated and non-polyadenylated nuclear RNA from Friend cells indicated that the abundant polysomal polyadenylated RNA sequences were present at a higher concentration in the nucleus than rare polysomal sequences, but at a reduced range of concentrations. The ratio of the concentrations of abundant and rare sequences was about 3 in non-polyadenylated nuclear RNA, 9 in polyadenylated nuclear RNA and 13 in polysomal polyadenylated RNA. This suggests that polyadenylation may play a role in the quantitative selection of sequences for transport to the cytoplasm. Polyadenylation cannot be the only signal for transport, since a highly complex population of nucleus-confined polyadenylated molecules exists, each of which is present on average at less than one copy per cell. PMID:7433127

  2. Polysomes, messenger RNA, and growth in soybean stems during development and water deficit. [Glycine max (L. )

    SciTech Connect

    Mason, H.S.; Mullet, J.E.; Boyer, J.S. )

    1988-01-01

    The polysome status and populations of polysomal mRNA were examined in different regions of dark-grown soybean (Glycine max (L.) Merr.) stems that contained either dividing, elongating, or mature (non-growing) cells. There was a developmental gradient of polysome content in which the dividing tissue had the highest levels and the mature tissue the lowest. A few hours after transplanting the seedlings to vermiculite having low water content (water potential {psi}{sub w} = {minus}0.29 megapascals), stem growth rate decreased to 30% of well-watered controls and the polysome content decreased most in the dividing and elongating tissues. After 24 to 36 hours, stem growth and polysome content recovered gradually. In vitro translation products of polysomal mRNA from dividing, elongating or mature tissue were examined on two-dimensional gels. In well-watered controls, each of the stem regions was enriched in a small subset of the polysomal mRNA population, probably because of developmentally regulated gene expression. Exposing plants to low {psi}{sub w} for 24 hours induced a change in the relative abundance of a small number of polysomal mRNAs in the elongating and mature tissues, but not in the dividing tissue. After 24 to 72 hours at low {psi}{sub w}, the changes in polysomal mRNA population were reversed in the elongating tissue.

  3. Rational design of avian metapneumovirus live attenuated vaccines by inhibiting viral messenger RNA cap methyltransferase

    USDA-ARS?s Scientific Manuscript database

    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis, is a non-segmented negative-sense RNA virus belonging to the family of Paramyxoviridae, the subfamily Pneumovirinae, and the genus Metapneumovirus. aMPV is the causative agent of respiratory tract infection and ...

  4. Nucleotide sequence from the coding region of rabbit β-globin messenger RNA

    PubMed Central

    Proudfoot, N.J.

    1976-01-01

    A sequence of 89 nucleotides from rabbit β-globin mRNA has been determined and is shown to code for residues 107 to 137 of the β-globin protein. In addition, a sequence heterogeneity has been identified within this 89 nucleotide long sequence which corresponds to a known polymorphic variant of rabbit β-globin. Images PMID:61580

  5. Thyroglobulin messenger RNA: translation of a 33-S mRNA into a peptide immunologically related to thyroglobulin.

    PubMed

    Vassart, G; Brocas, H; Lecocq, R; Dumont, J E

    1975-06-16

    Poly(UC)--Sepharose chromatography of the RNA extracted from a thyroid fraction sedimenting between 800 X g and 27000 X g allows the purification of two RNA fractions amounting each to 1% of the applied material. The first one is loosely bound to the column from which it is eluted at 25 degrees C. It is mainly composed of 16-S and 12-S RNA comprising no poly(A) sequences. This could correspond to mitochondrial rRNA. The second one, which is eluted at 50 degrees C, is poly(A)-rich and represents 33-S and 17--18-S RNA species. The 33-S RNA resists heating at 80 degrees C, suggesting that it is composed of one polynucleotide chain. When injected into Xenopus oocytes, the 33-S RNA specifically promotes the synthesis of a peptide with an apparent molecular weight of 185000 and an apparent sedimentation coefficient of 10-S. This peptide is immunologically related to thyroglobulin and could represent its main precursor. Under the conditions tested it does not polymerize spontaneously into 19-S thyroglobulin, suggesting that assembly of the molecule could require specific, post-translational alterations of the precursor and/or the presence of additional lighter subunits.

  6. Polysomes, Messenger RNA, and Growth in Soybean Stems during Development and Water Deficit 1

    PubMed Central

    Mason, Hugh S.; Mullet, John E.; Boyer, John S.

    1988-01-01

    The polysome status and populations of polysomal mRNA were examined in different regions of dark-grown soybean (Glycine max [L.] Merr.) stems that contained either dividing, elongating, or mature (nongrowing) cells. There was a developmental gradient of polysome content in which the dividing tissue had the highest levels and the mature tissue the lowest. A few hours after transplanting the seedlings to vermiculite having low water content (water potential Ψw = −0.29 megapascals), stem growth rate decreased to 30% of well-watered controls and the polysome content decreased most in the dividing and elongating tissues. After 24 to 36 hours, stem growth and polysome content recovered gradually. In vitro translation products of polysomal mRNA from dividing, elongating or mature tissue were examined on two-dimensional gels. In well-watered controls, each of the stem regions was enriched in a small subset of the polysomal mRNA population, probably because of developmentally regulated gene expression. Exposing plants to low Ψw for 24 hours induced a change in the relative abundance of a small number of polysomal mRNAs in the elongating and mature tissues, but not in the dividing tissue. After 24 to 72 hours at low Ψw, the changes in polysomal mRNA population were reversed in the elongating tissue. The data indicate that changes in stem growth at low water potential are associated with changes in polysome status and polysomal mRNA in the elongating tissue. Images Fig. 3 Fig. 4 Fig. 5 PMID:16665977

  7. Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum.

    PubMed Central

    Ganguly, T C; Liu, Y; Hyde, J F; Hagenbuch, B; Meier, P J; Vore, M

    1994-01-01

    We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity. Images Figure 2 PMID:7945260

  8. A nerve growth factor-regulated messenger RNA encodes a new intermediate filament protein

    PubMed Central

    1988-01-01

    Differential screening of a cDNA library from the PC12 rat pheochromocytoma cell line previously revealed a clone, clone 73, whose corresponding mRNA is induced by nerve growth factor (NGF). Induction parallels NGF-stimulated PC12 differentiation from a chromaffinlike phenotype to a sympathetic neuronlike phenotype. We report that DNA sequence analysis reveals that clone 73 mRNA encodes an intermediate filament (IF) protein whose predicted amino acid sequence is distinct from the known sequences of other members of the IF protein family. The sequence has highest homology with desmin and vimentin and includes the highly conserved central alpha-helical rod domain with the characteristic heptad repeat of hydrophobic residues, but has lower homology in the amino-terminal head and carboxyl-terminal tail domains. The head domain contains a large number of serine residues which are potential phosphorylation sites. The expression of clone 73 in vivo in the nervous system of the adult rat was investigated by in situ hybridization of clone 73 probes to tissue sections. The mRNA is expressed at high levels in ganglia of the peripheral nervous system, including the superior cervical ganglion (sympathetic), ciliary ganglion (parasympathetic), and dorsal root ganglion (sensory). In the central nervous system, motor nuclei of cranial nerves III, IV, V, VI, VII, X, and XII as well as ventral horn motor neurons and a restricted set of other central nervous system nuclei express the clone 73 mRNA. Tissues apart from those of the nervous system did not in general express the mRNA, with only very low levels detected in adrenal gland. We discuss the implications of these results for the mechanism of NGF- induced PC12 cell differentiation, the pathways of neuronal development in vivo, and the possible function of the clone 73 IF protein and its relationship to other IF proteins. PMID:3339087

  9. ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors

    SciTech Connect

    Ray, B.K.; Lawson, T.G.; Kramer, J.C.; Cladaras, M.H.; Grifo, J.A.; Abramson, R.D.; Merrick, W.C.; Thach, R.E.

    1985-06-25

    Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with /sup 32/P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or ''unwind'' mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. These results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.

  10. Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

    USGS Publications Warehouse

    Harms, Craig A.; Ottinger, Christopher A.; Kennedy-Stoskopf, S.

    2000-01-01

    Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis× male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

  11. Basic fibroblast growth factor messenger RNA is expressed strongly at the acute stage of cerebral contusion.

    PubMed

    Iwamoto, Y; Yamaki, T; Murakami, N; Sugawa, N; Yoshino, E; Ueda, S; Nosaka, K; Nishino, H; Iwashima, A

    1994-01-01

    Basic fibroblast growth factor (bFGF) has a neurotrophic effect both in vitro and in vivo, and is considered to play an important role in the maintenance of neuronal functions in the normal brain. Neural damage in brain contusion progresses after the primary injury of trauma because of cerebral hemodynamic and metabolic impairment including intracranial hemorrhage and/or brain swelling. Northern blot analysis of bFGF mRNA was performed in rats after cerebral contusion produced by our modified fluid percussion device. Expression of bFGF mRNA increased significantly on the second day after trauma. A possible role of bFGF is functioning to protect the critical neurons from secondary neural damage in cerebral contusion.

  12. Messenger RNA is a functional component of a chromatin insulator complex.

    PubMed

    Matzat, Leah H; Dale, Ryan K; Lei, Elissa P

    2013-10-01

    Chromatin insulators are DNA protein complexes situated throughout the genome capable of demarcating independent transcriptional domains. Previous studies point to an important role for RNA in gypsy chromatin insulator function in Drosophila; however, the identity of these putative insulator-associated RNAs is not currently known. Here we utilize RNA-immunoprecipitation and high throughput sequencing (RIP-seq) to isolate RNAs stably associated with gypsy insulator complexes. Strikingly, these RNAs correspond to specific sense-strand, spliced and polyadenylated mRNAs, including two insulator protein transcripts. In order to assess the functional significance of these associated mRNAs independent of their coding function, we expressed untranslatable versions of these transcripts in developing flies and observed both alteration of insulator complex nuclear localization as well as improvement of enhancer-blocking activity. Together, these data suggest a novel, noncoding mechanism by which certain mRNAs contribute to chromatin insulator function.

  13. Messenger RNA is a functional component of a chromatin insulator complex

    PubMed Central

    Matzat, Leah H; Dale, Ryan K; Lei, Elissa P

    2013-01-01

    Chromatin insulators are DNA protein complexes situated throughout the genome capable of demarcating independent transcriptional domains. Previous studies point to an important role for RNA in gypsy chromatin insulator function in Drosophila; however, the identity of these putative insulator-associated RNAs is not currently known. Here we utilize RNA-immunoprecipitation and high throughput sequencing (RIP-seq) to isolate RNAs stably associated with gypsy insulator complexes. Strikingly, these RNAs correspond to specific sense-strand, spliced and polyadenylated mRNAs, including two insulator protein transcripts. In order to assess the functional significance of these associated mRNAs independent of their coding function, we expressed untranslatable versions of these transcripts in developing flies and observed both alteration of insulator complex nuclear localization as well as improvement of enhancer-blocking activity. Together, these data suggest a novel, noncoding mechanism by which certain mRNAs contribute to chromatin insulator function. PMID:23917615

  14. Detection and Quantification of N (6)-Methyladenosine in Messenger RNA by TLC.

    PubMed

    Bodi, Zsuzsanna; Fray, Rupert G

    2017-01-01

    The base-modified nucleotide, N (6)-methyladenosine, is a relatively abundant modification found in the mRNA of most higher eukaryotes. Methylation levels can change dependent upon environmental conditions, cell differentiation state, or following knockdown of members of the methylase complex, and it is often useful to directly measure and compare N (6)-methyladenosine levels between samples. Two dimensional chromatography of radiolabeled nucleotides, following specific nuclease treatments, provides a robust, sensitive, and reproducible assay for this modification.

  15. The selective elimination of messenger RNA underlies the mitosis-meiosis switch in fission yeast.

    PubMed

    Yamamoto, Masayuki

    2010-01-01

    The cellular programs for meiosis and mitosis must be strictly distinguished but the mechanisms controlling the entry to meiosis remain largely elusive in higher organisms. In contrast, recent analyses in yeast have shed new light on the mechanisms underlying the mitosis-meiosis switch. In this review, the current understanding of these mechanisms in the fission yeast Schizosaccharomyces pombe is discussed. Meiosis-inducing signals in this microbe emanating from environmental conditions including the nutrient status converge on the activity of an RRM-type RNA-binding protein, Mei2. This protein plays pivotal roles in both the induction and progression of meiosis and has now been found to govern the meiotic program in a quite unexpected manner. Fission yeast contains an RNA degradation system that selectively eliminates meiosis-specific mRNAs during the mitotic cell cycle. Mmi1, a novel RNA-binding protein of the YTH-family, is essential for this process. Mei2 tethers Mmi1 and thereby stabilizes the transcripts necessary for the progression of meiosis.

  16. Effect of ribonucleic acid (RNA) isolation methods on putative reference genes messenger RNA abundance in human spermatozoa.

    PubMed

    Barragán, M; Martínez, A; Llonch, S; Pujol, A; Vernaeve, V; Vassena, R

    2015-07-01

    Although the male gamete participates in a significant proportion of infertility cases, there are currently no proven molecular markers of sperm quality. The search for significant gene expression markers is partially hindered by the lack of a recognized set of reference genes (RGs) to normalize reverse transcription quantitative PCR (RT-qPCR) data across studies. The aim of this study is to define a set of RGs in assisted reproduction patients undergoing different sample collection and RNA isolation methods. Twenty-two normozoospermic men were included in the study. From each man, semen was either cryopreserved by slow freezing or analyzed fresh, and, for each, RNA was extracted with either phenol-free or phenol-based methods. In two cases, both methods were used to isolate RNA. Twenty putative RGs were analyzed and their mRNA abundance across samples was estimated by RT-qPCR. To determine the genes whose steady-state mRNA abundance remains unchanged, three different algorithms (geNorm, BestKeeper and NormFinder) were applied to the qPCR data. We found that RGs such as GAPDH or ACTB, useful in other biological contexts, cannot be used as reference for human spermatozoa. It is possible to compare gene expression from fresh and cryopreserved sperm samples using the same isolation method, while the mRNA abundance of expressed genes becomes different depending on the RNA isolation technique employed. In our conditions, the most appropriate RGs for RT-qPCR analysis were RPLP1, RPL13A, and RPLP2. Published discrepancies in gene expression studies in human spermatozoa may be due in part to inappropriate RGs selection, suggesting a possible different interpretation of PCR data in several reports, which were normalized using unstable RGs.

  17. Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*

    PubMed Central

    Holberger, Laura E.; Hayes, Christopher S.

    2009-01-01

    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL+ cells. Additionally, tmRNA·SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA·SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA·SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA·SmpB activity. We propose that tmRNA·SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants. PMID:19776006

  18. Expression profile, clinical significance, and biological function of insulin-like growth factor 2 messenger RNA-binding proteins in non-small cell lung cancer.

    PubMed

    Shi, Run; Yu, Xinnian; Wang, Yajing; Sun, Jing; Sun, Qi; Xia, Wenjie; Dong, Gaochao; Wang, Anpeng; Gao, Zhaojia; Jiang, Feng; Xu, Lin

    2017-04-01

    Insulin-like growth factor 2 messenger RNA-binding proteins have been described to associate with malignant process in many cancers. However, the role of insulin-like growth factor 2 messenger RNA-binding protein family has not been thoroughly elucidated in non-small cell lung cancer. This study was to investigate the expression profile, clinical significance, and biological function of insulin-like growth factor 2 messenger RNA-binding proteins family in non-small cell lung cancer. The expression levels of IGF2BP1-IGF2BP3 in tumor and adjacent normal tissues were determined, and association with clinicopathological features and overall survival was investigated by analyzing The Cancer Genome Atlas lung cancer database. Proliferation, migration, invasion assays, and flow-cytometry analysis were used to investigate the biological function in vitro. Insulin-like growth factor 2 messenger RNA-binding protein expression levels were significantly increased in non-small cell lung cancer compared to adjacent normal lung tissues. Chi-square test indicated that IGF2BP1 and IGF2BP3 expressions correlated with some meaningful clinical characteristics in non-small cell lung cancer. Kaplan-Meier analysis showed that high-level expression of IGF2BP1 or IGF2BP3 predicted poor overall survival in lung adenocarcinoma patients. Multivariate regression analysis showed that high level of IGF2BP3 was an independent risk factor for poor prognosis in lung adenocarcinoma patients (hazard ratio = 1.616, p = 0.017). In vitro, knockdown of IGF2BP3 inhibited lung adenocarcinoma cell proliferation by inducing cell cycle arrest and apoptosis, and undermined abilities of migration and invasion, and overexpression of IGF2BP3 could promote malignant phenotypes in lung adenocarcinoma cells. Our study revealed that expression of insulin-like growth factor 2 messenger RNA-binding proteins was widely upregulated and correlated with some certain clinicopathological features in non-small cell

  19. A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA

    PubMed Central

    Esposito, Maria Valeria; Nunziato, Marcella; Starnone, Flavio; Telese, Antonella; Calabrese, Alessandra; D’Aiuto, Giuseppe; Pucci, Pietro; D’Aiuto, Massimiliano; Baralle, Francisco; D’Argenio, Valeria; Salvatore, Francesco

    2016-01-01

    About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient’s RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient’s transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset. PMID:28009814

  20. Messenger RNA expression of chicken CLOCK gene in the response to Campylobacter jejuni inoculation.

    PubMed

    Liu, Xiaoyi; Liu, Liying; Zhang, Maozhi; Yang, Ning; Qi, Yukai; Sun, Yu; Li, Xianyao

    2015-09-01

    Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial gastroenteritis worldwide. Previous research has shown that circadian rhythm plays a critical role in host response to C. jejuni colonization. The CLOCK gene is one of the core genes regulating circadian rhythms and shows significant expression on 7 d post-C. jejuni inoculation. The objective of this study was to investigate temporal and spatial expression of chicken CLOCK gene post-C. jejuni inoculation. Cecal and splenic RNA were isolated from 2 distinct chicken breeds and used to compare the mRNA expression of CLOCK gene between inoculated and noninoculated chickens within each breed and between breeds within each of inoculated and noninoculated groups. Our results showed that the CLOCK gene was significantly down-regulated at 20 h postinoculation (hpi) in cecum and spleen in Jiningbairi chicken. CLOCK gene was significantly down-regulated at 4 and 16 hpi and up-regulated at 8 hpi in cecum and spleen in specific pathogen free white leghorn noninoculated chicken. The findings suggested that expression of CLOCK gene was significantly changed post C. jejuin inoculation. This change was affected by genetic background, tissue, and time points postinoculation. © 2015 Poultry Science Association Inc.

  1. Structural analysis of the messenger RNA cap-binding protein: presence of phosphate, sulfhydryl, and disulfide groups

    SciTech Connect

    Not Available

    1986-01-05

    The messenger-RNA cap-binding protein (CBP) was isolated from human erythrocyte, rabbit erythrocyte, and rabbit reticulocyte lysate by affinity chromatography on 7-methylguanosine 5'-triphosphate-Sepharose. The specific activity of binding to capped oligonucleotides was similar for the human erythrocyte and rabbit reticulocyte CBPs. Isoelectric focusing of human and rabbit preparations revealed that each was composed of up to five species. The pI values of human and rabbit CBPs ranged from 5.7 to 6.5. The predominant form in erythrocytes had a pI of 6.3 while in reticulocytes, two major species, having pI values of 5.9 and 6.3, were present. Labeling of rabbit reticulocytes with (/sup 32/P)orthophosphate revealed that the pI 5.9 but not the pI 6.3 form contained phosphate. All of the phosphate was found in phosphoserine residues. The amino acid compositions of human erythrocyte and rabbit reticulocyte CBPs were quite similar. Both proteins had 7 tryptophanyl and 6 cysteinyl residues. Labeling with (1-/sup 14/C)iodoacetic acid under native and denaturing conditions provided evidence that 2 of the cysteinyl residues are present in the reduced form and 4 in disulfide bridges.

  2. Aging and sequential resistance exercise bout effects on housekeeping gene messenger RNA expression in human skeletal muscle.

    PubMed

    Sunderland, Kyle L; Roberts, Michael D; Dalbo, Vincent J; Kerksick, Chad M

    2013-01-01

    The purpose of this study was to investigate how age and 1 week of conventional resistance exercise affects commonly used housekeeping gene (HKG) messenger RNAs (mRNAs) in skeletal muscle. Ten college-aged (18-25 years) and 10 older (60-76 years) men completed 3 lower-body resistance exercise bouts on Monday, Wednesday, and Friday, and muscle samples were obtained before bout 1 (T1), 48 hours after the first (T2) and second bouts (T3), and 24 hours after the third bout (T4). Raw Ct values indicated that β-actin and cyclophilin were more highly expressed in older vs. younger males (p < 0.01) at T1. When normalizing each HKG mRNA to the other 4 HKG mRNAs, CYC increased at T3 and glyceraldehyde-3-phosphate dehydrogenase decreased at T2 (p < 0.05) in younger men. This is one of the few studies to suggest that explicit HKG mRNAs should be used depending upon age group and resistance exercise intervention.

  3. Leishmania pifanoi: kinetics of messenger RNA expression during amastigote to promastigote transformation in vitro.

    PubMed

    Campbell, S M; Rainey, P M

    1993-08-01

    Conditions have been developed which induce axenically grown Leishmania pifanoi amastigotes to transform into the promastigote stage in a highly reproducible fashion. Transformation was induced by a temperature shift from 31 to 22 degrees C, was inhibited by high cell concentration (> or = 40 x 10(6) cells/ml), and was unaffected by pH from 5.5-7.2. Morphologic transformation was first evident at 8 hr after induction, had occurred in > 50% of cells by 24 hr, and was > 90% complete by 48 hr. This system enabled study of the kinetics of mRNA expression during the transformation of Leishmania. The differentially expressed mRNAs for ATPase 1a and 1b, alpha- and beta-tubulin, P100/11E, Pro-1, and pLm 2, 7, 14, and 16 exhibited complex patterns of temporal expression, suggesting a highly regulated process. Differentiation on the biochemical level was evident within an hour and continued throughout the course of morphologic transformation. In addition, transformed L. pifanoi promastigotes in the plateau growth phase expressed genes characteristic of metacyclic promastigotes. Axenically cultured L. pifanoi should provide an excellent model for the study of differentiation in Leishmania.

  4. Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

    PubMed

    Ito, Akira; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Aoyama, Tomoki; Kuroki, Hiroshi

    2015-01-01

    Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis.

  5. Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats.

    PubMed Central

    Lehman-McKeeman, L D; Andrews, G K; Klaassen, C D

    1988-01-01

    Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone. Images Fig. 4. PMID:3342021

  6. Emergence of the β-CASP ribonucleases: highly conserved and ubiquitous metallo-enzymes involved in messenger RNA maturation and degradation.

    PubMed

    Dominski, Zbigniew; Carpousis, Agamemnon J; Clouet-d'Orval, Béatrice

    2013-01-01

    The β-CASP ribonucleases, which are found in the three domains of life, have in common a core of 460 residues containing seven conserved sequence motifs involved in the tight binding of two catalytic zinc ions. A hallmark of these enzymes is their ability to catalyze both endo- and exo-ribonucleolytic degradation. Exo-ribonucleolytic degradation proceeds in the 5' to 3' direction and is sensitive to the phosphorylation state of the 5' end of a transcript. Recent phylogenomic analyses have shown that the β-CASP ribonucleases can be partitioned into two major subdivisions that correspond to orthologs of eukaryal CPSF73 and bacterial RNase J. We discuss the known functions of the CPSF73 and RNase J orthologs, their association into complexes, and their structure as it relates to mechanism of action. Eukaryal CPSF73 is part of a large multiprotein complex that is involved in the maturation of the 3' end of RNA Polymerase II transcripts and the polyadenylation of messenger RNA. RNase J1 and J2 are paralogs in Bacillus subtilis that are involved in the degradation of messenger RNA and the maturation of non-coding RNA. RNase J1 and J2 co-purify as a heteromeric complex and there is recent evidence that they interact with other enzymes to form a bacterial RNA degradosome. Finally, we speculate on the evolutionary origin of β-CASP ribonucleases and on their functions in Archaea. Orthologs of CPSF73 with endo- and exo-ribonuclease activity are strictly conserved throughout the archaea suggesting a role for these enzymes in the maturation and/or degradation of messenger RNA. This article is part of a Special Issue entitled: RNA Decay mechanisms. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Bisphenol S alters embryonic viability, development, gallbladder size, and messenger RNA expression in chicken embryos exposed via egg injection.

    PubMed

    Crump, Doug; Chiu, Suzanne; Williams, Kim L

    2016-06-01

    Amid concerns about the toxicological effects and environmental prevalence of bisphenol A (BPA), efforts to find suitable, safer replacement alternatives are essential. Bisphenol S (BPS) is a potential chemical substitute for BPA; however, few studies are available confirming that it has a more desirable ecotoxicological profile. In the present study, BPS was injected into the air cell of unincubated, fertilized chicken embryos at 6 concentrations ranging from 0 μg/g to 207 μg/g egg to determine effects on pipping success, development, hepatic messenger ribonucleic acid (mRNA) expression, thyroid hormone levels, and circulating bile acid concentrations. Concentrations of BPS increased in a dose-dependent manner in whole-embryo homogenates, and exposure to the highest dose, 207 μg/g, resulted in decreased pipping success (estimated median lethal dose  = 279 μg/g; 95% confidence interval = 161-486 μg/g). Exposure to BPS also reduced growth metrics including embryo mass and tarsus length, whereas the most pronounced phenotypic effect was the concentration-dependent, significant increase in gallbladder size at concentrations ≥52.8 μg/g. These adverse phenotypic outcomes were associated with the modulation of gene targets from a chicken ToxChip polymerase chain reaction array, which are involved with xenobiotic metabolism, lipid homeostasis, bile acid synthesis, and the thyroid hormone pathway. Expression levels of 2 estrogen-responsive genes, apolipoprotein II and vitellogenin, were too low at the sampling time point assessed (i.e., pipping embryos) to quantify changes, and no effects were observed on circulating free thyroxine or bile acid concentrations. The present study provides novel, whole-animal toxicological data for a BPA replacement alternative that is not well characterized. Environ Toxicol Chem 2016;35:1541-1549. © 2015 SETAC. © 2015 SETAC.

  8. Plant serine/arginine-rich proteins: roles in precursor messenger RNA splicing, plant development, and stress responses.

    PubMed

    Reddy, Anireddy S N; Shad Ali, Gul

    2011-01-01

    Global analyses of splicing of precursor messenger RNAs (pre-mRNAs) have revealed that alternative splicing (AS) is highly pervasive in plants. Despite the widespread occurrence of AS in plants, the mechanisms that control splicing and the roles of splice variants generated from a gene are poorly understood. Studies on plant serine/arginine-rich (SR) proteins, a family of highly conserved proteins, suggest their role in both constitutive splicing and AS of pre-mRNAs. SR proteins have a characteristic domain structure consisting of one or two RNA recognition motifs at the N-terminus and a C-terminal RS domain rich in arginine/serine dipeptides. Plants have many more SR proteins compared to animals including several plant-specific subfamilies. Pre-mRNAs of plant SR proteins are extensively alternatively spliced to increase the transcript complexity by about six-fold. Some of this AS is controlled in a tissue- and development-specific manner. Furthermore, AS of SR pre-mRNAs is altered by various stresses, raising the possibility of rapid reprogramming of the whole transcriptome by external signals through regulation of the splicing of these master regulators of splicing. Most SR splice variants contain a premature termination codon and are degraded by up-frameshift 3 (UPF3)-mediated nonsense-mediated decay (NMD), suggesting a link between NMD and regulation of expression of the functional transcripts of SR proteins. Limited functional studies with plant SRs suggest key roles in growth and development and plant responses to the environment. Here, we discuss the current status of research on plant SRs and some promising approaches to address many unanswered questions about plant SRs.

  9. Primary structure of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

    SciTech Connect

    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

    1986-10-01

    The messenger RNA coding the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase in an enriched fraction of poly(A/sup +/)-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA.

  10. Analysis of aberrant pre-messenger RNA splicing resulting from mutations in ATP8B1 and efficient in vitro rescue by adapted U1 small nuclear RNA.

    PubMed

    van der Woerd, Wendy L; Mulder, Johanna; Pagani, Franco; Beuers, Ulrich; Houwen, Roderick H J; van de Graaf, Stan F J

    2015-04-01

    ATP8B1 deficiency is a severe autosomal recessive liver disease resulting from mutations in the ATP8B1 gene characterized by a continuous phenotypical spectrum from intermittent (benign recurrent intrahepatic cholestasis; BRIC) to progressive familial intrahepatic cholestasis (PFIC). Current therapeutic options are insufficient, and elucidating the molecular consequences of mutations could lead to personalized mutation-specific therapies. We investigated the effect on pre-messenger RNA splicing of 14 ATP8B1 mutations at exon-intron boundaries using an in vitro minigene system. Eleven mutations, mostly associated with a PFIC phenotype, resulted in aberrant splicing and a complete absence of correctly spliced product. In contrast, three mutations led to partially correct splicing and were associated with a BRIC phenotype. These findings indicate an inverse correlation between the level of correctly spliced product and disease severity. Expression of modified U1 small nuclear RNAs (snRNA) complementary to the splice donor sites strongly improved or completely rescued splicing for several ATP8B1 mutations located at donor, as well as acceptor, splice sites. In one case, we also evaluated exon-specific U1 snRNAs that, by targeting nonconserved intronic sequences, might reduce possible off-target events. Although very effective in correcting exon skipping, they also induced retention of the short downstream intron. We systematically characterized the molecular consequences of 14 ATP8B1 mutations at exon-intron boundaries associated with ATP8B1 deficiency and found that the majority resulted in total exon skipping. The amount of correctly spliced product inversely correlated with disease severity. Compensatory modified U1 snRNAs, complementary to mutated donor splice sites, were able to improve exon definition very efficiently and could be a novel therapeutic strategy in ATP8B1 deficiency as well as other genetic diseases. © 2014 by the American Association for the Study

  11. Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique

    PubMed Central

    Hassan, Tidi; Smith, Stephen G. J.; Gaughan, Kevin; Oglesby, Irene K.; O’Neill, Shane; McElvaney, Noel G.; Greene, Catherine M.

    2013-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. Although numerous bioinformatic prediction models exist to identify miRNA–mRNA interactions, experimental validation of bona fide interactions can be difficult and laborious. Few methods can comprehensively identify miRNAs that target a single mRNA. We have developed an experimental approach to search for miRNAs targeting any mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide. This method identifies miRNAs targeting the full length of the mRNA. The method was tested using three separate mRNA targets: alpha-1 antitrypsin (AAT) mRNA, interleukin-8 mRNA and secretory leucoprotease inhibitor mRNA. AAT mRNA-specific and total miRNAs from three different cell lines (monocytic THP-1, bronchial epithelial 16HBE14o− and liver HepG2 cells) were profiled, and validation studies revealed that AAT mRNA-specific miRNAs functionally target the AAT mRNA in a cell-specific manner, providing the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression. Interleukin-8 and secretory leucoprotease inhibitor mRNAs and their cognate miRNAs were also successfully captured using this approach. This is a simple and an efficient method to potentially identify miRNAs targeting sequences within the full length of a given mRNA transcript. PMID:23325846

  12. Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes.

    PubMed

    Cavalla, Franco; Biguetti, Claudia; Jain, Sameer; Johnson, Cleverick; Letra, Ariadne; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

    2017-09-01

    Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets. Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant. Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG. Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory

  13. In vitro Study of a Novel Stent Coating Using Modified CD39 Messenger RNA to Potentially Reduce Stent Angioplasty-Associated Complications

    PubMed Central

    Abraham, Meike-Kristin; Nolte, Andrea; Reus, Rebekka; Behring, Andreas; Zengerle, Diane; Avci-Adali, Meltem; Hohmann, Jan David; Peter, Karlheinz; Schlensak, Christian; Wendel, Hans Peter; Krajewski, Stefanie

    2015-01-01

    Background Stent angioplasty provides a minimally invasive treatment for atherosclerotic vessels. However, no treatment option for atherosclerosis-associated endothelial dysfunction, which is accompanied by a loss of CD39, is available, and hence, adverse effects like thromboembolism and restenosis may occur. Messenger RNA (mRNA)-based therapy represents a novel strategy, whereby de novo synthesis of a desired protein is achieved after delivery of a modified mRNA to the target cells. Methods and Findings Our study aimed to develop an innovative bioactive stent coating that induces overexpression of CD39 in the atherosclerotic vessel. Therefore, a modified CD39-encoding mRNA was produced by in vitro transcription. Different endothelial cells (ECs) were transfected with the mRNA, and CD39 expression and functionality were analyzed using various assays. Furthermore, CD39 mRNA was immobilized using poly(lactic-co-glycolic-acid) (PLGA), and the transfection efficiency in ECs was analyzed. Our data show that ECs successfully translate in vitro-generated CD39 mRNA after transfection. The overexpressed CD39 protein is highly functional in hydrolyzing ADP and in preventing platelet activation. Furthermore, PLGA-immobilized CD39 mRNA can be delivered to ECs without losing its functionality. Summary In summary, we present a novel and promising concept for a stent coating for the treatment of atherosclerotic blood vessels, whereby patients could be protected against angioplasty-associated complications. PMID:26381750

  14. Enzymatic amplification of translation inhibition of rabbit beta-globin mRNA mediated by anti-messenger oligodeoxynucleotides covalently linked to intercalating agents.

    PubMed Central

    Cazenave, C; Loreau, N; Thuong, N T; Toulmé, J J; Hélène, C

    1987-01-01

    The effects of anti-messenger oligodeoxynucleotides, covalently linked to an intercalating agent, on translation of rabbit beta-globin mRNA, were investigated both in wheat germ extract and in microinjected Xenopus oocytes. A specific inhibition of beta-globin synthesis was observed in both expression systems with a modified 11-mer covalently linked to an acridine derivative. In injected oocytes a more efficient block was observed with this modified oligonucleotide than with its unsubstituted homolog. This was ascribed to stacking interactions of the intercalating agent with base pairs which provide an additional stabilization of the [mRNA/DNA] hybrid. We demonstrated that in wheat germ extract, the modified and unmodified oligonucleotides behaved similarly due to the presence of a high RNaseH activity. RNaseH was also present, although to a lesser extent, in the oocyte cytoplasm. This anti-messenger DNA-induced degradation of target mRNA resulted in amplified efficiency of hybrid-arrested translation. This additional mechanism might provide anti-sense DNAs with an advantage over anti-sense RNAs. Images PMID:3037483

  15. Mercury's Messenger

    ERIC Educational Resources Information Center

    Chapman, Clark R.

    2004-01-01

    Forty years after Mariner 2, planetary exploration has still only just begun, and many more missions are on drawing boards, nearing the launch pad, or even en route across interplanetary space to their targets. One of the most challenging missions that will be conducted this decade is sending the MESSENGER spacecraft to orbit the planet Mercury.…

  16. Mercury's Messenger

    ERIC Educational Resources Information Center

    Chapman, Clark R.

    2004-01-01

    Forty years after Mariner 2, planetary exploration has still only just begun, and many more missions are on drawing boards, nearing the launch pad, or even en route across interplanetary space to their targets. One of the most challenging missions that will be conducted this decade is sending the MESSENGER spacecraft to orbit the planet Mercury.…

  17. Active and Accurate trans-Translation Requires Distinct Determinants in the C-terminal Tail of SmpB Protein and the mRNA-like Domain of Transfer Messenger RNA (tmRNA)*♦

    PubMed Central

    Camenares, Devin; Dulebohn, Daniel P.; Svetlanov, Anton; Karzai, A. Wali

    2013-01-01

    Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants. PMID:23986442

  18. Active and accurate trans-translation requires distinct determinants in the C-terminal tail of SmpB protein and the mRNA-like domain of transfer messenger RNA (tmRNA).

    PubMed

    Camenares, Devin; Dulebohn, Daniel P; Svetlanov, Anton; Karzai, A Wali

    2013-10-18

    Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants.

  19. Involvement of second messengers in the signaling pathway of vitellogenesis-inhibiting hormone and their effects on vitellogenin mRNA expression in the whiteleg shrimp, Litopenaeus vannamei.

    PubMed

    Bae, Sun-Hye; Okutsu, Tomoyuki; Tsutsui, Naoaki; Kang, Bong Jung; Chen, Hsiang-Yin; Wilder, Marcy N

    2017-01-03

    We incubated fragments of Litopenaeus vannamei ovary to investigate second messengers involved in the regulation of vitellogenin (vg) mRNA levels. The use of 100nM recombinant vitellogenesis-inhibiting hormone (VIH) (corresponding to recombinant L. vannamei sinus gland peptide-G: rLiv-SGP-G) significantly reduced vg mRNA expression in sub-adults after 8h incubation to less than 20% of the control. The concentration of intracellular cyclic guanosine monophosphate (cGMP) increased 3.2-fold relative to the control after 2h incubation with rLiv-SGP-G. However, it reached levels 18-fold relative to the control after 0.5h incubation with rLiv-SGP-G where 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) was also added. Moreover, vg mRNA expression was significantly reduced to less than 50% of the control after 24h incubation with 1μM A23187 (a calcium ionophore). Thus, rLiv-SGP-G and calcium ionophore reduced vg mRNA expression in in vitro-cultured ovary, and cGMP may be involved in the signaling pathway of VIH. Overall, the above results suggest that vg mRNA expression might be inhibited in vitro by increasing intracellular cGMP and Ca(2+) in L. vannamei ovary.

  20. Detection of carcinoembryonic antigen messenger RNA-expressing cells in peripheral blood 7 days after curative surgery is a novel prognostic factor in colorectal cancer.

    PubMed

    Sadahiro, Sotaro; Suzuki, Toshiyuki; Maeda, Yuji; Yurimoto, Satoshi; Yasuda, Seiei; Makuuchi, Hiroyasu; Kamijo, Akemi; Murayama, Chieko

    2007-03-01

    The significance of detection of circulating cancer cells in blood during surgery in patients with colorectal cancer (CRC) remains controversial. Experimental study revealed that the cancer cells injected from the vein disappeared completely until 7 days. The aim of this study was to clarify that the detection of circulating cancer cells in blood taken later than 7 days after curative surgery may be a prognostic factor. Two hundred consecutive patients with CRC who underwent potentially curative surgery were the subjects. Peripheral blood was collected between 7 and 10 days after resection. Cancer cells were detected using reverse transcriptase-polymerase chain reaction targeting carcinoembryonic antigen (CEA) messenger RNA (mRNA). The median follow-up period was 52 months (range: 34-69 months). The overall positive incidence of CEA mRNA was 22%. Detection of CEA mRNA was not significantly related to conventional clinicopathological findings. Recurrence has been confirmed in 55 patients (28%). The recurrence rate was significantly higher in patients with rectal cancer, deep penetration, lymph node metastasis, preoperative chemoradiotherapy and positive CEA mRNA. The CEA mRNA positive patients showed significantly poorer disease free survival (DFS) and overall survival (OS) than the negative patients (DFS, P = 0.007; OS, P = 0.04). Multivariate analysis revealed that the positive expression of CEA mRNA (P < 0.01) as well as the tumor location and TNM stage classification was identified as the significant risk factors for recurrence. Detection of CEA mRNA expressing cells in peripheral blood 7 days after curative surgery is a novel independent factor predicting recurrence in patients with CRC.

  1. Comparative Diagnosis of Human Bocavirus 1 Respiratory Infection With Messenger RNA Reverse-Transcription Polymerase Chain Reaction (PCR), DNA Quantitative PCR, and Serology.

    PubMed

    Xu, Man; Arku, Benedict; Jartti, Tuomas; Koskinen, Janne; Peltola, Ville; Hedman, Klaus; Söderlund-Venermo, Maria

    2017-05-15

    Human bocavirus (HBoV) 1 can cause life-threatening respiratory tract infection in children. Diagnosing acute HBoV1 infection is challenging owing to long-term airway persistence. We assessed whether messenger RNA (mRNA) detection would correlate better than DNA detection with acute HBoV1 infection. Paired serum samples from 121 children with acute wheezing were analyzed by means of serology. Quantitative polymerase chain reaction (PCR) and reverse-transcription (RT) PCR were applied to nasopharyngeal swab (NPS) samples from all acutely HBoV1-infected children and from controls with nonacute infection. By serology, 16 of 121 children (13.2%) had acute HBoV1 infection, all of whom had HBoV1 DNA in NPS samples, and 12 of 16 (75%) had HBoV1 mRNA. Among 25 children with nondiagnostic results, 6 had HBoV1 DNA in NPS samples, and 1 had mRNA. All 13 mRNA-positive samples exhibited high DNA loads (≥106 copies/mL). No mRNA persisted for 2 weeks, whereas HBoV1 DNA persisted for 2 months in 4 children; 1 year later all 15 samples were DNA negative. Compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). In contrast, mRNA RT-PCR had low clinical sensitivity (75%) but high specificity (96%). A combination of HBoV1 serology and nasopharyngeal DNA quantitative PCR and mRNA RT-PCR should be used for accurate diagnosis of HBoV1 infection.

  2. Labeling of eukaryotic messenger RNA 5' terminus with phosphorus -32: use of tobacco acid pyrophosphatase for removal of cap structures

    SciTech Connect

    Lockard, R.E.; Rieser, L.; Vournakis, J.N.

    1981-01-01

    In recent years, there has been a growing appreciation of the potential applications of 5'-/sup 32/P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity (gamma-/sup 32/P)ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure.

  3. Effect of cyclic strain on tensile properties of a naturally derived, decellularized tendon scaffold seeded with allogeneic tenocytes and associated messenger RNA expression.

    PubMed

    Whitlock, Patrick W; Seyler, Thorsten M; Northam, Casey N; Smith, Thomas L; Poehling, Gary G; Koman, L Andrew; Van Dyke, Mark E

    2013-01-01

    Naturally derived tendon scaffolds have the potential to improve the treatment of flexor tendon injuries. Seeded and unseeded tendon scaffolds were maintained in the presence or absence of physiologic strain for 7 days. After 7 days, the tensile properties and associated messenger RNA expression were compared. Seeded scaffolds maintained in the absence of strain had significantly lower tensile properties than unseeded tendons and fresh-frozen tendons. The loss of tensile properties was associated with elevated matrix metalloproteinase-2 and collagen III expression. Tensile properties of seeded scaffolds maintained in the presence of strain for 7 days after seeding did not differ from those of fresh-frozen tendons. This study demonstrates that the tensile properties of seeded, naturally derived tendon scaffolds will degrade rapidly in the absence of cyclic strain. Seeded scaffolds used for tendon reconstruction should be maintained under cyclic strain to maintain essential tensile properties.

  4. Erythropoietin messenger RNA levels in developing mice and transfer of /sup 125/I-erythropoietin by the placenta

    SciTech Connect

    Koury, M.J.; Bondurant, M.C.; Graber, S.E.; Sawyer, S.T.

    1988-07-01

    Erythropoietin (EP) mRNA was measured in normal and anemic mice during fetal and postnatal development. Normal fetal livers at 14 d of gestation contained a low level of EP mRNA. By day 19 of gestation, no EP mRNA was detected in normal or anemic fetal livers or normal fetal kidneys, but anemic fetal kidneys had low levels of EP mRNA. Newborn through adult stage mice responded to anemia by accumulating renal and hepatic EP mRNA. However, total liver EP mRNA was considerably less than that of the kidneys. Juvenile animals, 1-4 wk old, were hyperresponsive to anemia in that they produced more EP mRNA than adults. Moreover, nonanemic juveniles had readily measured renal EP mRNA, whereas the adult level was at the lower limit of detection. Because of the very low level of fetal EP mRNA, placental transfer of EP was evaluated. When administered to the pregnant mouse, /sup 125/I-EP was transferred in significant amounts to the fetuses. These results indicate that in mice the kidney is the main organ of EP production at all stages of postnatal development and that adult kidney may also play some role in providing EP for fetal erythropoiesis via placental transfer of maternal hormone.

  5. [Frequency of the occurrence of spliced variants of the messenger RNA DR3/LARD in herpesviral infection].

    PubMed

    Utkin, O V; Starikova, V D; Novikov, V V

    2014-01-01

    Analysis of frequency of the occurrence of membrane and soluble forms of the mRNA DR3/LARD in blood in herpesviral infection of various etiology was studied. Four forms of the mRNA DR3/LARD were detected with various frequencies in blood cells of healthy volunteers. Patients with herpesviral infection of various etiology were studied using RT-PCR. Two forms encoded membrane molecules (mRNA LARD 1a, mRNA DR3beta) and two other forms accorded soluble forms of receptor (mRNA LARD 3, mRNA soluble DR3beta). It was revealed that the frequency of the occurrence of mRNA soluble DR3beta form decreased in patients with the varicella zoster virus (VZV) infection in comparison with healthy volunteers. However, the patients with the Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection did not display significant change in occurrence of mRNA soluble DR3beta form. As a whole, changes in frequency of occurrence of spliced variants of mRNA DR3/LARD are directed toward modulation of apoptosis and restraint antiviral immune response.

  6. Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1β on primordial follicle activation and survival in vitro.

    PubMed

    Passos, J R S; Costa, J J N; da Cunha, E V; Silva, A W B; Ribeiro, R P; de Souza, G B; Barroso, P A A; Dau, A M P; Saraiva, M V A; Gonçalves, P B D; van den Hurk, R; Silva, J R V

    2016-01-01

    This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Tyrosinase messenger RNA in peripheral blood is related to poor survival in patients with metastatic melanoma following interleukin-2-based immunotherapy.

    PubMed

    Schmidt, Henrik; Sorensen, Boe S; Fode, Kirsten; Nexo, Ebba; von der Maase, Hans

    2005-10-01

    This study was conducted to examine the prognostic impact of four biomarkers [tyrosinase and MART-1 messenger RNA (mRNA), S100beta protein and lactate dehydrogenase (LDH)] in patients with metastatic melanoma, together with established clinical factors. Tyrosinase and MART-1 mRNA were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). S100beta was measured using a commercially available immunoassay, and LDH was analysed conventionally. All markers were measured in blood samples before interleukin-2-based immunotherapy in 85 patients with metastatic melanoma. LDH, S100beta, tyrosinase, number of metastatic sites, location of metastatic sites and performance status were all significant factors for survival in univariate analyses. In multivariate analysis, tyrosinase [hazard ratio (HR)=1.6; 95% confidence interval (CI), 1.1-2.6; P=0.04] and LDH (HR=2.0; 95% CI, 1.1-3.5; P=0.02) were both independent prognostic factors for survival. A combination variable of tyrosinase and LDH remained independently associated with survival (P=0.04) after adjusting for the American Joint Committee on Cancer (AJCC) stage IV classification in a multivariate analysis involving both models. It can be concluded that tyrosinase mRNA and elevated LDH are independent prognostic factors for poor survival in this group of 85 patients. Additional studies are needed before the prognostic value of tyrosinase mRNA in metastatic melanoma can be firmly established. Further evaluation of the combined measurement of tyrosinase mRNA and LDH is warranted.

  8. Comparative study of candidate housekeeping genes for quantification of target gene messenger RNA expression by real-time PCR in patients with inflammatory bowel disease.

    PubMed

    Bamias, Giorgos; Goukos, Dimitris; Laoudi, Eyfrosyni; Balla, Iliana G; Siakavellas, Spyros I; Daikos, George L; Ladas, Spiros D

    2013-12-01

    Mucosal expression of immunological mediators is modified in inflammatory bowel disease (IBD). Quantification of target gene messenger RNA (mRNA) transcripts depends on the normalization to a housekeeping or reference gene. Stability of housekeeping gene expression is critical for the accurate measurement of transcripts of the target gene. No studies have addressed the optimization of reference gene performance for mRNA studies in healthy intestinal mucosa and during mucosal inflammation. RNA was extracted from endoscopically obtained intestinal biopsies from healthy control subjects and patients with active IBD or non-IBD inflammatory diseases. Comparative analysis of 10 candidate housekeeping genes for quantitative real-time PCR was carried out according to predefined criteria, including use of the Web-based RefFinder platform. We demonstrate that intestinal inflammation may significantly affect the stability of mucosal expression of housekeeping genes. Commonly used controls, such as glyceraldehyde-3-phosphate dehydrogenase, β-actin, or β2-microglobulin displayed high variability within the control group and/or between the healthy and inflamed mucosae. In contrast, we have identified novel genes with optimal stability, which may be used as appropriate housekeeping controls. The ribosomal proteins encoding genes (RPLPO and RPS9) were the most stable because their expression was not affected by interindividual differences, the presence of inflammation, or intestinal location. Normalization ofthe mRNA expression of mucosal tumor necrosis factor-α was highly dependent on the specific reference gene and varied significantly when normalized to genes with high or low stability. Validation for optimal performance of the housekeeping gene is required for target mRNA quantification in healthy intestine and IBD-associated lesions. Suboptimal reference gene expression may explain conflicting results from published studies on IBD gene expression.

  9. Tumor necrosis factor alpha-308 and -238 polymorphisms in rheumatoid arthritis. Association with messenger RNA expression and sTNF-alpha.

    PubMed

    Oregón-Romero, Edith; Vázquez-Del Mercado, Mónica; Ruiz-Quezada, Sandra Luz; Navarro-Hernández, Rosa Elena; Rangel-Villalobos, Héctor; Martínez-Bonilla, Gloria; Bernard-Medina, Ana Guilaisne; Armendáriz-Borunda, Juan; García-Bañuelos, Jesús; Muñoz-Valle, José Francisco

    2008-10-01

    Rheumatoid arthritis (RA) is characterized by a progressive joint damage mediated mainly by tumor necrosis factor alpha (TNFalpha). We investigated the relationship of TNFalpha-308 and -238 polymorphisms with messenger RNA (mRNA) expression and soluble TNFalpha (sTNFalpha) in 50 RA and 100 healthy subjects (HS). Clinical and laboratory assessments were performed. Spanish Health Assessment Questionnaire Disability Index, Spanish version of Arthritis Impact Measurement Scales and Disease Activity Score using 28 joint count indices were applied to RA patients. The TNFalpha-308 and -238 polymorphisms were performed by polymerase chain reaction and restriction fragment length polymorphism techniques. The mRNA expression of TNFalpha was quantified by real-time polymerase chain reaction. The sTNFalpha levels were measured by enzyme-linked immunosorbent assay. The TNFalpha-308 polymorphism showed an increased frequency of guanine (G)/adenine (A) genotype in RA versus HS (P = 0.03; 95% confidence interval, 1.05-8.08; odds ratio, 2.9) and also the A allele was more frequent in RA patients versus HS (P = 0.04; 95% confidence interval, 1.01-7.29; odds ratio, 2.7). The G/G genotype and also the G allele were more frequent in HS. No significant difference was observed in TNFalpha-238 polymorphism. Rheumatoid arthritis patients showed high TNFalpha mRNA expression (1.33-fold). The G/G genotype was associated with high mRNA and sTNFalpha levels in both TNFalpha polymorphisms. The correlation of sTNFalpha levels with C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, Spanish Health Assessment Questionnaire Disability Index, and Spanish version of Arthritis Impact Measurement Scales, was observed. The TNFalpha-308 polymorphism is a susceptibility marker to RA. The G/G genotype is associated with a high mRNA and soluble TNFalpha expression.

  10. Purification of 14S Messenger RNA of Immunoglobulin Light Chain That Codes for a Possible Light-Chain Precursor

    PubMed Central

    Mach, B.; Faust, C.; Vassalli, P.

    1973-01-01

    Polysomes released from microsomes of MOPC 41 mouse myeloma were used to prepare a poly(A)-containing fraction of RNA by chromatography on poly-(dT)-cellulose. From that fraction, a 14S RNA species was purified to a single peak by successive sucrose gradient centrifugations, followed by acrylamide gel electrophoresis. The RNA has an apparent molecular weight of 380,000 (1100 nucleotides), as estimated from the electrophoretic analyses. In a reticulocyte lysate this RNA directs the synthesis of a protein that migrates more slowly in sodium dodecylsulfate-acrylamide gels than does the light chain secreted by the same tumor. This difference in migration corresponds to a size difference appropriate for polypeptide chain about 20 amino acids longer than the light chain. The tryptic peptides of this protein correspond to those of the secreted light chain, except for the presence of two additional peptides from the product synthesized in vitro and for the absence of one light-chain peptide. The purified RNA is, therefore, the mRNA of the light chain, and it seems to code for a precursor protein slightly larger than the light chain. From the estimated size of the 14S mRNA, it appears that only 65% of the RNA is translated. Images PMID:4510289

  11. Complex Degradation Processes Lead to Non-Exponential Decay Patterns and Age-Dependent Decay Rates of Messenger RNA

    PubMed Central

    Deneke, Carlus; Lipowsky, Reinhard; Valleriani, Angelo

    2013-01-01

    Experimental studies on mRNA stability have established several, qualitatively distinct decay patterns for the amount of mRNA within the living cell. Furthermore, a variety of different and complex biochemical pathways for mRNA degradation have been identified. The central aim of this paper is to bring together both the experimental evidence about the decay patterns and the biochemical knowledge about the multi-step nature of mRNA degradation in a coherent mathematical theory. We first introduce a mathematical relationship between the mRNA decay pattern and the lifetime distribution of individual mRNA molecules. This relationship reveals that the mRNA decay patterns at steady state expression level must obey a general convexity condition, which applies to any degradation mechanism. Next, we develop a theory, formulated as a Markov chain model, that recapitulates some aspects of the multi-step nature of mRNA degradation. We apply our theory to experimental data for yeast and explicitly derive the lifetime distribution of the corresponding mRNAs. Thereby, we show how to extract single-molecule properties of an mRNA, such as the age-dependent decay rate and the residual lifetime. Finally, we analyze the decay patterns of the whole translatome of yeast cells and show that yeast mRNAs can be grouped into three broad classes that exhibit three distinct decay patterns. This paper provides both a method to accurately analyze non-exponential mRNA decay patterns and a tool to validate different models of degradation using decay data. PMID:23408982

  12. Gap junctional connexin messenger RNA expression in the ovine uterus and placenta: effects of estradiol-17β-treatment, early pregnancy stages, and embryo origin.

    PubMed

    Johnson, M L; Redmer, D A; Reynolds, L P; Grazul-Bilska, A T

    2017-01-01

    Gap junctions play a major role in direct, contact-dependent cell-cell communication, and they have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues. Gap junctional channels, composed of connexin (Cx) proteins, have been detected and shown to be influenced by hormones (eg, estrogen and progesterone) in uterine and placental tissues in several species. We hypothesized that (1) the messenger RNA (mRNA) for Cx26, Cx32, Cx37, and Cx43 is expressed in the uterus of ovariectomized sheep treated with estradiol-17β (E2) and in ovine placenta during early pregnancy, (2) E2-treatment of ovariectomized ewes would cause time-specific changes in Cx26, Cx32, Cx37, and Cx43 mRNA expression (experiment 1), and (3) expression of these 4 Cx would vary across the days of early pregnancy (experiment 2) and will be affected by embryo origin (ie, after application of assisted reproductive technologies [ARTs]; experiment 3). Thus, we collected uterine tissues at 0 to 24 h after E2 treatments (experiment 1), and placental tissues during days 14 to 30 of early pregnancy after natural (NAT) breeding (experiment 2) and on day 22 of early pregnancy established after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA, parthenotes; experiment 3). In experiment 1, the expression of Cx26, Cx37, and Cx43 mRNA increased (P < 0.05) and Cx32 mRNA decreased (P < 0.06) in both caruncular and intercaruncular tissues after E2 treatment. In experiment 2, during early pregnancy, there were significant changes (P < 0.01) across days in the expression of Cx26, Cx37, and Cx43 mRNA in the maternal placenta, accompanied by changes (P < 0.001) in Cx37 and Cx43 mRNA in the fetal placenta. In experiment 3, in maternal placenta, Cx32 mRNA expression was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared to the NAT group; but

  13. Messenger RNA stability of parathyroid hormone-related protein regulated by transforming growth factor-beta1.

    PubMed

    Sellers, R S; Capen, C C; Rosol, Thomas J

    2002-02-25

    Humoral hypercalcemia of malignancy (HHM), a paraneoplastic syndrome associated with epithelial cancers, including squamous cell carcinoma (SCC), is due to expression and secretion of parathyroid hormone-related protein (PTHrP). Transforming growth factor-beta1 (TGFbeta1), expressed by many tumors, has been demonstrated in vitro to increase the half-life of PTHrP mRNA. In this study, oral squamous carcinoma cells (SCC2/88) had a two-fold increase in PTHrP mRNA stability (from 45 to 90 min) in response to treatment with TGFbeta1. In order to examine the mechanism of TGFbeta1-mediated PTHrP mRNA stability, a cell-free assay of mRNA degradation was utilized in which the degradation of in vitro-transcribed mRNA incubated with cytoplasmic protein extracts from SCC2/88 treated with vehicle or TGFbeta1 was measured. In this assay, full-length PTHrP mRNA was not significantly stabilized in TGFbeta1-treated samples when compared to vehicle treated samples. However, there was a striking (>5-fold) increase in PTHrP mRNA half-life in TGFbeta1-treated samples when PTHrP mRNA lacked the 3'-untranslated region (3'-UTR). In contrast, the degradation of 3'-UTR-truncated PTHrP mRNA using the cell-free assay was not altered in vehicle-treated samples. UV cross-linking of PTHrP mRNA and cytoplasmic proteins from cells treated with either vehicle or TGFbeta1 revealed numerous mRNA-binding proteins. TGFbeta1 treatment resulting in decreased binding of 33, 31, 27, 20 and 18 kDa binding proteins to the terminal coding region. These studies revealed that TGFbeta1-induced PTHrP mRNA stability might be, in part, the result of cis-acting sequences within the coding region of the PTHrP mRNA.

  14. Anabolic-androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice.

    PubMed

    Ambar, G; Chiavegatto, S

    2009-03-01

    Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly,the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion,high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

  15. IRES-mediated translation of cellular messenger RNA operates in eIF2α- independent manner during stress

    PubMed Central

    Thakor, Nehal; Holcik, Martin

    2012-01-01

    Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2α. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2α phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia, serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However, the molecular mechanism that allows IRES-mediated translation to continue in an eIF2α-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis, XIAP, IRES to address this question. Using toeprinting assay, western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2α is phosphorylated both in vitro and in vivo. During normal growth condition eIF2α-dependent translation on the IRES is preferred. However, IRES-mediated translation switches to eIF5B-dependent mode when eIF2α is phosphorylated as a consequence of cellular stress. PMID:21917851

  16. Characterizing the Effects of Inorganic Acid and Alkaline Shock on the Staphylococcus aureus Transcriptome and Messenger RNA Turnover

    PubMed Central

    Anderson, Kelsi L.; Roux, Christelle M.; Olson, Matthew W.; Luong, Thanh T.; Lee, Chia Y.; Olson, Robert; Dunman, Paul M.

    2010-01-01

    Staphylococcus aureus pathogenesis can be partially attributed to its ability to adapt to otherwise deleterious host-associated stresses. Here, Affymetrix GeneChips® were used to examine the S. aureus responses to inorganic acid and alkaline shock and to assess whether stress dependent changes in mRNA turnover are likely to facilitate the organism’s ability to tolerate pH challenge. Results indicate that S. aureus adapts to pH shock by eliciting responses expected of cells coping with pH alteration, including neutralizing cellular pH, DNA repair, amino acid biosynthesis and virulence factor expression. Further, the S. aureus response to alkaline conditions is strikingly similar to that of stringent response induced cells. Indeed, we show that alkaline shock stimulates accumulation of the stringent response activator (p)ppGpp. Results also revealed that pH shock significantly alters the mRNA properties of the cell. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. A set of small stable RNA molecules were induced in response to acid or alkaline shock conditions and may mediate adaptation to pH stress. PMID:21039920

  17. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat.

    PubMed Central

    Yamamoto, M; Igarashi, T; Muramatsu, M; Fukagawa, M; Motokura, T; Ogata, E

    1989-01-01

    To examine the effects of serum calcium concentrations on PTH biosynthesis, rats were made hyper- (serum total calcium, approximately 3.5 mM) or hypocalcemic (approximately 1.25 mM) and steady-state levels of PTH mRNA in parathyroid cells were measured by the primer extension method using a 32P-labeled synthetic oligomer. PTH mRNA levels increased about twofold in the rats made slightly hypocalcemic by infusion of calcium-free solution and decreased slightly in those made hypercalcemic by CaCl2 infusion (120-150 mumol/h) compared with the levels present in nonfasting control rats. Infusion of calcitonin (0.5 U/h) or EGTA (90 mumol/h) with calcium-free solution increased PTH mRNA levels further (two- to sevenfold) above the levels present in animals infused with calcium-free solution alone. These changes in PTH mRNA levels were observed after 48- but not 24-h infusion, and there was an inverse correlation between PTH mRNA levels and serum calcium concentrations. The results suggest that changes in serum calcium concentrations in the near physiological range regulate the biosynthesis of PTH by affecting steady-state levels of PTH mRNA when hypercalcemia or hypocalcemia continues for a relatively long period. Images PMID:2493484

  18. Destabilization of ERBB2 transcripts by targeting 3' untranslated region messenger RNA associated HuR and histone deacetylase-6.

    PubMed

    Scott, Gary K; Marx, Corina; Berger, Crystal E; Saunders, Laura R; Verdin, Eric; Schäfer, Stefan; Jung, Manfred; Benz, Christopher C

    2008-07-01

    In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies.

  19. Differential display and suppressive subtractive hybridization used to identify granulosa cell messenger rna associated with bovine oocyte developmental competence.

    PubMed

    Robert, C; Gagné, D; Bousquet, D; Barnes, F L; Sirard, M A

    2001-06-01

    The main objective of this study was to identify mRNA expressed in the granulosa cells characterizing differentiated follicles bearing developmentally competent bovine oocytes. Analytical comparisons were made on mRNA pools of granulosa cells using differential display reverse transcription polymerase chain reaction (DDRT) analysis and suppressive subtractive hybridization (SSH). With DDRT, mRNA patterns of granulosa cells from small (< 4 mm) and large (> 8 mm) follicles cultured in the presence or absence of LH were compared to identify mRNA associated with follicular size or with the LH response. Nine clones were sequenced, and two were identified. One of the clones, DRAK 1, was associated with the presence of LH in the medium. Other comparisons directed toward the identification of mRNA associated with the presence of a competent oocyte were done on granulosa cells collected in vivo from superstimulated heifers. With the DDRT analysis, four clones associated with the oocyte developmental competence status were identified. With the SSH analysis, four clones specific to the presence of an incompetent oocyte were sequenced and none were identified, whereas 49 clones specific to the presence of a competent oocyte were sequenced and 18 were identified. Among these clones, early growth response 1, sprouty 2, cytochrome C oxidase, matrix metalloproteinase inducer, matrix metalloproteinase, epiregulin, prostaglandin receptor, and progesterone receptor were the most relevant to the ovarian physiology being examined.

  20. Follicular progesterone concentrations and messenger RNA expression of MATER and OCT-4 in immature bovine oocytes as predictors of developmental competence.

    PubMed

    Urrego, R; Herrera-Puerta, E; Chavarria, N A; Camargo, O; Wrenzycki, C; Rodriguez-Osorio, N

    2015-04-15

    The ability of bovine embryos to develop to the blastocyst stage and to implant and generate healthy offspring depends greatly on the competence of the oocyte. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesize and store substantial amounts of messenger RNA. Higher developmental competence of bovine oocytes has been associated with both the expression of a cohort of developmental genes and the concentration of sex steroids in the follicular fluid. The aim of this study was to identify differences in the expression of FST in cumulus cells and OCT-4 and MATER in oocytes and the influence of the follicular progesterone and follicular estrogen concentration on the competence of bovine oocytes retrieved 30 minutes or 4 hours after slaughter. Cumulus-oocyte complexes (COCs) were left in postmortem ovaries for 30 minutes (group I) or 4 hours (group II) at 30 °C. Aspirated oocytes were then subjected to IVM, IVF, and IVC or were evaluated for MATER and OCT-4 messenger RNA abundance by quantitative real-time polymerase chain reaction. Total RNA was isolated from pools of 100 oocytes for each experimental replicate. Progesterone and estradiol concentration in follicular fluid was evaluated by immunoassay using an IMMULITE 2000 analyzer. Three repeats of in vitro embryo production were performed with a total of 455 (group I) and 470 (group II) COCs. There were no significant differences between the cleavage rates (72 hours postinsemination [hpi]) between both groups (63.5% vs. 69.1%). However, blastocyst (168 hpi) and hatching (216 hpi) rates were higher (P < 0.05) in group II compared with those of group I (21.3% vs. 30.7% and 27.6% vs. 51.5%, respectively). Group II oocytes exhibited the highest MATER and OCT-4 abundance (P < 0.05). Follicular estradiol concentration was not different between both the groups, whereas the progesterone concentration was lower (P ≤ 0.05) in group II follicles. These

  1. Development and regional expression of beta nerve growth factor messenger RNA and protein in the rat central nervous system.

    PubMed Central

    Whittemore, S R; Ebendal, T; Lärkfors, L; Olson, L; Seiger, A; Strömberg, I; Persson, H

    1986-01-01

    The presence of nerve growth factor (NGF) mRNA and protein in the rat central nervous system is documented. Blot-hybridization analysis showed an abundance of NGF mRNA in the hippocampus, cerebral cortex, and olfactory bulb. Enzyme immunoassay confirmed significant levels of a NGF-like protein in the hippocampus and cerebral cortex. Bioassay of a NGF-like immunoaffinity-purified protein from these regions was physiologically indistinguishable from NGF. Immunohistochemistry revealed a widespread distribution of NGF-like reactivity in the adult brain, preferentially in fiber tracts. NGF mRNA accumulation began at birth, with adult levels reached 3 weeks postnatally. Enzyme immunoassay detected the presence of a NGF-like protein in the embryonic rat brain. Postnatally, the level of NGF-like protein reached a maximum at 3 weeks. Additionally, a distinct fetal form of NGF may exist. Images PMID:3456170

  2. Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.

    PubMed

    Schubert, Mario; Lapouge, Karine; Duss, Olivier; Oberstrass, Florian C; Jelesarov, Ilian; Haas, Dieter; Allain, Frédéric H-T

    2007-09-01

    Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.

  3. Evidence for the role of double-helical structures in the maturation of simian virus-40 messenger RNA.

    PubMed

    Chiu, N H; Bruszewski, W B; Salzman, N P

    1980-01-11

    Simian Virus-40 infected BSC-1 cells were pretreated with glucosamine and briefly pulsed with [3H]-uridine. The labeling can be halted instantaneously by the addition of cold uridine and glucosamine. Under these pulse-chase conditions, the inhibitory effects of the intercalating agent proflavine on the processing of prelabeled nuclear RNA precursors were examined in vivo. Proflavine inhibits the cleavage of viral nuclear RNA precursors. However, turnover of the mature viral mRNAs in the cytoplasm is not inhibited. The effect of proflavine on processing is not a secondary consequence of its inhibition of protein synthesis. The data suggest that base-paired secondary structures in the primary transcripts are important processing signals in the generation of viral mRNA molecules.

  4. Inhibition of transcription and translation of globin messenger RNA in dimethyl sulfoxide-stimulated Friend erythroleukemic cells treated with interferon.

    PubMed Central

    Rossi, G B; Dolei, A; Cioé, L; Benedetto, A; Matarese, G P; Belardelli, F

    1977-01-01

    The addition of appropriate doses of interferon (IF) to cultures of Friend erythroleukemic cells inhibits dimethyl sulfoxide (Me2SO)-stimulated erythroid differentiation. In this study, the synthesis of heme, hemoglobin, and globin mRNA in Me2SO-stimulated cultures, with or without IF added, was compared. Although the hemoglobin content in Me2SO+IF-treated cultures was reduced 6- to 9-fold compared to that of cultures treated with Me2SO alone, there was less than a 2-fold decrease in the amount of heme accumulated. Globin mRNA, although unchanged in size or base sequence, was reduced in content in the Me2SO+IF cultures. The level of reduction of globin mRNA was insufficient to account for the lack of globin synthesis. Thus, it appears that IF may operate on two levels--one involving the transcription of globin mRNA and the other involving its translation. PMID:266723

  5. Association of Cocaine- and Amphetamine-Regulated Transcript (CART) Messenger RNA Level, Food Intake, and Growth in Channel Catfish

    USDA-ARS?s Scientific Manuscript database

    Cocaine-and Amphetamine-Regulated Transcript (CART) is a potent hypothalamic anorectic peptide in mammals and fish. We hypothesized that increased food intake is associated with changes in expression of CART mRNA within the brain of channel catfish. Objectives were to clone the CART gene, examine ...

  6. Spatial distribution of CYP2B1/2 messenger RNA within the rat liver acinus following exposure to the inducers phenobarbital and dieldrin.

    PubMed

    Dail, Mary B; Burgess, Shane C; Meek, Edward C; Wagner, Jennifer; Baravik, Jeffrey; Chambers, Janice E

    2007-09-01

    Traditionally, the liver has been considered a homogeneous organ, but literature suggests that the cytochromes P450 are differentially distributed among the hepatocytes and that the pattern of this distribution is altered by various xenobiotics. In this study, the CYP2B1/2 messenger RNA (mRNA) in the hepatocytes was compared following treatment of rats with either of two inducers, phenobarbital (PB), or dieldrin. Adult male Sprague-Dawley-derived rats were treated with either ip PB in saline at 80 mg/kg/day for 5 days or dieldrin in corn oil by oral gavage at 2.5 mg/kg/day for 13 days. Control rats received ip saline or po corn oil for the same time. Laser capture microdissection (LCM) followed by duplex quantitative real-time reverse transcriptase PCR was used to measure the CYP2B1/2 mRNA produced in bands of hepatocytes isolated from three locations along the sinusoidal path. The amounts of mRNA present in whole liver subsamples were also analyzed. CYP2B1/2 enzyme activity was determined by assaying 16beta-hydroxytestosterone formation. Whole liver mRNA samples exhibited significant induction in CYP2B1/2 transcript levels: sixfold for PB and 2200-fold for dieldrin. All the LCM band samples also showed significant fold induction in CYP2B1/2 mRNA compared to controls. Dieldrin caused marked increases in CYP2B1/2 mRNA levels in the direction of blood flow through the acinus: periportal, 300-fold; midzonal, 600-fold; and centrilobular, 1700-fold. A different pattern of induction was observed in the PB-treated rats: periportal, 1800-fold; midzonal, 8800-fold; and centrilobular, 1600-fold. The present study indicates the differences in spatial responses that can be exhibited within the liver following exposure to various xenobiotics. It also indicates the importance of examining xenobiotic metabolism in the liver in light of its nonhomogeneous, zoned microenvironment.

  7. Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries

    PubMed Central

    Hershko-Shalev, Tal; Odenheimer-Bergman, Ahuva; Elgrably-Weiss, Maya; Ben-Zvi, Tamar; Govindarajan, Sutharsan; Seri, Hemda; Papenfort, Kai; Vogel, Jörg; Altuvia, Shoshy

    2016-01-01

    While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein. PMID:27057757

  8. Distribution of C-type natriuretic peptide and its messenger RNA in rat central nervous system and peripheral tissue.

    PubMed

    Minamino, N; Aburaya, M; Kojima, M; Miyamoto, K; Kangawa, K; Matsuo, H

    1993-11-30

    In rat, the highest concentration of immunoreactive (ir-) C-type natriuretic peptide (CNP) was found in the central nervous system, as is the case in pig and human. Although its concentration in peripheral tissue was much lower than that in brain, CNP was present mainly as CNP-53 in ileum-jejunum, colon-cecum, stomach, kidney, lung, testis and submaxillary gland, but not in heart. By Northern blot analysis, CNP mRNA was detected in ileum-jejunum, testis, thymus, adrenal gland and submaxillary gland as well as in brain and spinal cord. CNP mRNA was further verified by reverse transcription-polymerase chain reaction to be present in most peripheral tissue, including aorta and bone marrow. These results indicate that CNP is synthesized in peripheral tissue and possibly functions as a local regulator in addition to acting as a neuropeptide in the central nervous system.

  9. Intercellular adhesion molecule-1 expression in experimental alcoholic liver disease: relationship to endotoxemia and TNF alpha messenger RNA.

    PubMed

    Nanji, A A; Griniuviene, B; Yacoub, L K; Fogt, F; Tahan, S R

    1995-02-01

    We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship among intercellular adhesion molecule-1 (ICAM-1) expression, tumor necrosis factor-alpha (TNF-alpha), plasma endotoxin, and inflammatory changes in the liver. Rats were fed different dietary fats (saturated fat, corn oil, and fish oil) with ethanol; control rats were fed isocaloric amounts of dextrose instead of ethanol. At sacrifice the following were evaluated: liver pathologic changes, TNF-alpha mRNA by reverse transcription-PCR, plasma endotoxin, and ICAM-1 by immunohistochemistry and immunoblot analysis. Upregulation of ICAM-1 in endothelial lining cells in central and portal veins was observed in rats showing evidence of pathologic changes. Rats fed fish oil and ethanol, which exhibited the most severe inflammation, also showed hepatocyte ICAM-1 staining. The presence of ICAM-1 staining, in general, correlated with the level of TNF-alpha mRNA expression and plasma endotoxin levels. Upregulation of ICAM-1 in rats fed ethanol may contribute to the inflammatory changes seen in this model. The association between ICAM-1 upregulation and endotoxin and TNF-alpha mRNA suggests a role for these mediators in the inflammatory process in alcoholic liver injury.

  10. Activity, synthesis, storage, and messenger RNA of cyclooxygenase in intrauterine tissues of guinea pigs near term and during labor.

    PubMed

    Schellenberg, J-C; Shelling, A N; Van Ee, C C

    2003-04-01

    Whether the reported gestation-dependent increase in cyclooxygenase activity in gestational tissues is due to an accumulation of cyclooxygenase in vivo or an increasing capacity to synthesize cyclooxygenase in vitro is unknown. In this study in guinea pigs, COX activity was estimated from the net production rates of prostaglandins E(2) and F(2alpha) in the presence of optimal substrate concentrations. Cyclooxygenase activity in amnion increased between 45 days of gestation and labor in microsomes (150-fold in relation to PGF(2alpha) production and 116-fold in relation to PGE(2) production) and in tissue explants (42-fold in relation to PGF(2alpha) production). The capacity for de novo synthesis of cyclooxygenase after aspirin treatment increased nine-fold between 45 days of gestation and labor in amnion explants. Comparison of COX activity in amnion explants with or without prior aspirin treatment showed that COX activity is at least three-fold higher in controls than would be expected if the activity was due to de novo synthesis alone. Cyclooxygenase-2 mRNA predominated in amnion but neither cyclooxygenase-2 nor cyclooxygenase-1 mRNA levels (semi-quantitative RT-PCR) changed significantly. This suggests that the gestation-dependent increase in cyclooxygenase activity in guinea pig amnion is due in part to accumulation of cyclooxygenase in vivo, that COX-2 predominates, and that COX activity is not correlated with levels of COX mRNA.

  11. Distribution of precursor amyloid-. beta. -protein messenger RNA in human cerebral cortex: relationship to neurofibrillary tangles and neuritic plaques

    SciTech Connect

    Lewis, D.A.; Higgins, G.A.; Young, W.G.; Goldgaber, D.; Gajdusek, D.C.; Wilson, M.C.; Morrison, J.H.

    1988-03-01

    Neurofibrillary tangles (NFT) and neuritic plaques (NP), two neuropathological markers of Alzheimer disease, may both contain peptide fragments derived from the human amyloid ..beta.. protein. However, the nature of the relationship between NFT and NP and the source of the amyloid ..beta.. proteins found in each have remained unclear. The authors used in situ hybridization techniques to map the anatomical distribution of precursor amyloid-..beta..-protein mRNA in the neocortex of brains from three subjects with no known neurologic disease and from five patients with Alzheimer disease. In brains from control subjects, positively hybridizing neurons were present in cortical regions and layers that contain a high density of neuropathological markers in Alzheimer disease, as well as in those loci that contain NP but few NFT. Quantitative analyses of in situ hybridization patterns within layers III and V of the superior frontal cortex revealed that the presence of high numbers of NFT in Alzheimer-diseased brains was associated with a decrease in the number of positively hybridizing neurons compared to controls and Alzheimer-diseased brains with few NFT. These findings suggest that the expression of precursor amyloid-..beta..-protein mRNA may be a necessary but is clearly not a sufficient prerequisite for NFT formation. In addition, these results may indicate that the amyloid ..beta.. protein, present in NP in a given region or layer of cortex, is not derived from the resident neuronal cell bodies that express the mRNA for the precursor protein.

  12. 5'-heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events.

    PubMed

    McCormick, J A; Lyons, V; Jacobson, M D; Noble, J; Diorio, J; Nyirenda, M; Weaver, S; Ester, W; Yau, J L; Meaney, M J; Seckl, J R; Chapman, K E

    2000-04-01

    Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5'-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 1(6) and 1(10)) were expressed in all tissues examined, together present in 77-87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 1(7). Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predominant exon 1(10), suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.

  13. Translational recognition of the 5'-terminal 7-methylguanosine of globin messenger RNA as a function of ionic strength.

    PubMed

    Chu, L Y; Rhoads, R E

    1978-06-13

    The translation of rabbit globin mRNA in cell-free systems derived from either wheat germ or rabbit reticulocyte was studied in the presence of various analogues of the methylated 5' terminus (cap) as a function of ionic strength. Inhibition by these analogues was strongly enhanced by increasing concentrations of KCl, K(OAc), Na(OAc), or NH4(OAc). At appropriate concentrations of K(OAc), both cell-free systems were equally sensitive to inhibition by m7GTP. At 50 mM K(OAc), the reticulocyte system was not sensitive to m7GMP or m7GTP, but at higher concentrations up to 200 mM K(OAc), both nucleotides caused strong inhibition. The compound in m7G5'ppp5'Am was inhibitory at all concentrations of K(OAc) ranging from 50 to 200 mM, although more strongly so at the higher concentrations. Over the same range of nucleotide concentrations, the compounds GMP, GTP, and G5'ppp5'Am were not inhibitors. The mobility on sodium dodecyl sulfate-polyacrylamide electrophoresis of the translation product was that of globin at all K(OAc) concentrations in the presence of m7GTP. Globin mRNA from which the terminal m7GTP group had been removed by chemical treatment (periodate-cyclohexylamine-alkaline phosphatase) or enzymatic treatment (tobacco acid pyrophosphatase-alkaline phosphatase) was translated less efficiently than untreated globin mRNA at higher K(OAc) concentrations, but retained appreciable activity at low K(OAc) concentrations.

  14. Immunohistochemical identification of messenger RNA-related proteins in basophilic inclusions of adult-onset atypical motor neuron disease.

    PubMed

    Fujita, Kengo; Ito, Hidefumi; Nakano, Satoshi; Kinoshita, Yoshimi; Wate, Reika; Kusaka, Hirofumi

    2008-10-01

    This report concerns an immunohistochemical investigation on RNA-related proteins in the basophilic inclusions (BIs) from patients with adult-onset atypical motor neuron disease. Formalin-fixed, paraffin-embedded sections of the motor cortex and the lumbar spinal cord were examined. The BIs appeared blue in color with H&E and Nissl stain, and pink with methylgreen-pyronin stain. Ribonuclease pretreatment abolished the methylgreen-pyronin staining, suggesting that the BIs contained RNA. Immunohistochemically, the BIs were distinctly labeled with the antibodies against poly(A)-binding protein 1, T cell intracellular antigen 1, and ribosomal protein S6. These proteins are essential constituents of stress granules. In contrast, the BIs were not immunoreactive for ribosomal protein L28 and decapping enzyme 1, which are core components of transport ribonucleoprotein particles and processing bodies, respectively. Moreover, the BIs were not immunopositive for TDP-43. Our results imply that translation attenuation could be involved in the processes of BI formation in this disorder.

  15. Modification of alternative messenger RNA splicing of fibroblast growth factor receptors in human cardiac allografts during rejection.

    PubMed Central

    Zhao, X M; Frist, W H; Yeoh, T K; Miller, G G

    1994-01-01

    Accelerated coronary atherosclerosis in cardiac transplants (cardiac allograft vasculopathy, CAV) is characterized by coronary intimal hyperplasia. Acidic fibroblast growth factor (aFGF) is a potent mitogen for vascular smooth muscle cells and endothelial cells, and its expression is increased in cardiac allografts, suggesting it may play a role in the pathogenesis of CAV. The activity of aFGF is dependent on binding to transmembrane receptors. To investigate whether receptors for aFGF are also induced after transplantation, polymerase chain reaction, in situ hybridization, and immunohistochemistry were used to analyze expression of four receptors for aFGF (FGFR1-FGFR4). Expression of mRNA encoding extracellular immunoglobulin-like domains of FGFR1 was increased 35-fold in cardiac allografts compared with normal hearts and was predominantly present in cardiac myocytes and vascular structures. Alternatively spliced mRNA that encodes transmembrane forms of FGFR1, which contain the signal-transducing tyrosine kinase domains, was induced in allografts during rejection, in infiltrating cells, vascular structures, and myocytes. In vitro experiments showed that differential expression of FGF receptor isoforms was induced by aFGF, and also by IL-6 and TGF-beta, which are expressed in cardiac allografts during rejection. The results show that expression of both aFGF and its receptors is altered in cardiac allografts and suggest that these events are important in the pathogenesis of CAV. Images PMID:7521891

  16. Experimental murine myopia induces collagen type Iα1 (COL1A1) DNA methylation and altered COL1A1 messenger RNA expression in sclera

    PubMed Central

    Zhou, Xiangtian; Ji, Fengtao; An, Jianhong; Zhao, Fuxin; Shi, Fanjun; Huang, Furong; Li, Yuan; Jiao, Shiming; Yan, Dongsheng; Chen, Xiaoyan; Chen, JiangFan

    2012-01-01

    Purpose To investigate whether myopia development is associated with changes of scleral DNA methylation in cytosine-phosphate-guanine (CpG) sites in the collagen 1A1 (COL1A1) promoter and messenger RNA (mRNA) levels following murine form deprivation myopia. Methods Fifty-seven C57BL/6 mice (postnatal day 23) were randomly assigned to four groups: (1) monocular form deprivation (MD) in which a diffuser lens was placed over one eye for 28 days; (2) normal controls without MD; (3) MD recovery in which the diffuser lens was removed for seven days; and (4) MD recovery normal controls. The DNA methylation pattern in COL1A1 promoter and exon 1 was determined by bisulfite DNA sequencing, and the COL1A1 mRNA level in sclera was determined by quantitative PCR. Results MD was found to induce myopia in the treated eyes. Six CpG sites in the promoter and exon 1 region of COL1A1 were methylated with significantly higher frequency in the treated eyes than normal control eyes (p<0.05), with CpG island methylation in MD-contralateral eyes being intermediate. Consistent with the CpG methylation, scleral COL1A1 mRNA was reduced by 57% in the MD-treated eyes compared to normal controls (p<0.05). After seven days of MD recovery, CpG methylation was significantly reduced (p=0.01). The methylation patterns returned to near normal level in five CpG sites, but the sixth was hypomethylated compared to normal controls. Conclusions In parallel with the development of myopia and the reduced COL1A1 mRNA, the frequency of methylation in CpG sites of the COL1A1 promoter/exon 1 increased during MD and returned to near normal during recovery. Thus, hypermethylation of CpG sites in the promoter/exon 1 of COL1A1 may underlie reduced collagen synthesis at the transcriptional level in myopic scleras. PMID:22690110

  17. Dietary iron supplements and Moringa oleifera leaves influence the liver hepcidin messenger RNA expression and biochemical indices of iron status in rats.

    PubMed

    Saini, R K; Manoj, P; Shetty, N P; Srinivasan, K; Giridhar, P

    2014-07-01

    In this study, the effects of iron depletion and repletion on biochemical and molecular indices of iron status were investigated in growing male Wistar rats. We hypothesized that iron from Moringa leaves could overcome the effects of iron deficiency and modulate the expression of iron-responsive genes better than conventional iron supplements. Iron deficiency was induced by feeding rats an iron-deficient diet for 10 weeks, whereas control rats were maintained on an iron-sufficient diet (35.0-mg Fe/kg diet). After the depletion period, animals were repleted with different source of iron, in combination with ascorbic acid. Iron deficiency caused a significant (P < .05) decrease in serum iron and ferritin levels by 57% and 40%, respectively, as compared with nondepleted control animals. Significant changes in the expression (0.5- to100-fold) of liver hepcidin (HAMP), transferrin, transferrin receptor-2, hemochromatosis type 2, ferroportin 1, ceruloplasmin, and ferritin-H were recorded in iron-depleted and iron-repleted rats, as compared with nondepleted rats (P < .05). Dietary iron from Moringa leaf was found to be superior compared with ferric citrate in overcoming the effects of iron deficiency in rats. These results suggest that changes in the relative expression of liver hepcidin messenger RNA can be used as a sensitive molecular marker for iron deficiency.

  18. Common acute lymphoblastic leukemia antigen: partial characterization by in vivo labeling and isolation of its messenger RNA

    SciTech Connect

    Heinsohn, S.; Kabisch, H.

    1987-01-01

    Common acute lymphoblastic leukemia (ALL) antigen (CALLA)-like proteins were detected by in vivo labeling experiments carried out with human lymphoblastoid cell line KM3 and also in cell-free translation, directed by CALLA-specific mRNA prepared from immunoadsorbed KM3 polysomes. The CALLA-like structure found in both systems shows an Mr of 95kDa. Additional CALLA-like proteins could be identified in the in vivo experiments with calculated Mrs of 40kDa in the cells and 85 and 38kDa in the culture medium. In the cell-free translation system, an additional product of Mr 80kDa could be detected.

  19. Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm

    SciTech Connect

    Wang, S.Z.

    1985-01-01

    Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A)/sup +/ mRNA were found to direct into vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A)/sup +/ mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with /sup 32/P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not. Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons.

  20. Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells

    PubMed Central

    Pato, A; Eisenberg, G; Machlenkin, A; Margalit, A; Cafri, G; Frankenburg, S; Merims, S; Peretz, T; Lotem, M; Gross, G

    2015-01-01

    Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte–macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy. PMID:26212048

  1. Messenger RNA expression and immunolocalization of psoriasin in the goat mammary gland and its milk concentration after an intramammary infusion of lipopolysaccharide.

    PubMed

    Zhang, G W; Lai, S J; Yoshimura, Y; Isobe, N

    2014-10-01

    Psoriasin (S100A7) is a member of the S100 protein family of calcium-binding proteins and plays a crucial role in local host defenses. The present study aimed to identify the expression of S100A7 in the goat mammary gland and in milk. The goat S100A7 coding DNA sequence was identified using direct sequencing. An S100A7 antibody was raised in rabbits by immunization with a synthetic S100A7 peptide consisting of 13 amino acids. Messenger RNA expression and protein localization in different regions of a healthy mammary gland were detected by reverse transcription-polymerase chain reaction and immunohistochemistry. Changes in the concentration of S100A7 in the milk after an intramammary infusion of Escherichia coli lipopolysaccharide (LPS) were examined by an enzyme immunoassay. The goat S100A7 peptide had 98% and 86% sequence similarity to that of sheep and bovines, respectively. The S100A7 mRNA expression was higher in the teat and udder skin than in the cistern and parenchyma of the mammary gland. Immunoreactive S100A7 was localized in the epithelial cells of the alveolus and gland cistern, and stratified squamous epithelium of the teat. Psoriasin as a secreted protein was detectable in healthy milk, and an intramammary LPS infusion increased the concentration of S100A7 in the milk. The results suggest that S100A7 is produced in the epithelial cells of the mammary gland and is secreted into the milk.

  2. In situ changes in the relative abundance of human epidermal cytokine messenger RNA levels following exposure to the poison ivy/oak contact allergen urushiol.

    PubMed

    Boehm, K D; Yun, J K; Strohl, K P; Trefzer, U; Häffner, A; Elmets, C A

    1996-06-01

    Abstract: Epidermal keratinocytes in culture have been shown to produce many cytokines, and their proteins have been identified in skin tissue samples. It has therefore been assumed that these cytokines are transcribed in vivo by the epidermis in response to contact allergens. In this report, in situ hybridization was used to detect the messenger RNAs for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) in samples of human skin prior to and at various times after application of urushiol, the immunogenic component of poison ivy/oak. In sensitive subjects, IL-1 alpha and TNF-alpha mRNAs showed a progressive increase in transcript levels that paralleled the clinical and histological features of the inflammatory process. The time-course of the IL-1 beta response differed from that of IL-1 alpha and TNF-alpha, in that there was an early (by 6 h after urushiol administration) elevation in IL-1 beta mRNA that occurred before there was evidence of inflammation and had returned to background levels by 72 h when the reaction had reached its peak. In contrast to urushiol-sensitive subjects, urushiol-anergic individuals did not exhibit an increase in IL-1 alpha, IL-1 beta or TNF-alpha mRNA levels. The data provide evidence for an in vivo role for epidermal IL-1 alpha, IL-1 beta and TNF-alpha transcription in the regulation of IL-1 beta and TNF-alpha polypeptide levels in the epidermis in response to this common contact allergen.

  3. Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.

    PubMed

    Pato, A; Eisenberg, G; Machlenkin, A; Margalit, A; Cafri, G; Frankenburg, S; Merims, S; Peretz, T; Lotem, M; Gross, G

    2015-11-01

    Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy. © 2015 British Society for Immunology.

  4. An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

    PubMed Central

    Chamorro, M; Parkin, N; Varmus, H E

    1992-01-01

    Synthesis of the pol gene products of most retroviruses requires ribosomes to shift frame once or twice in the -1 direction while translating gag-pol mRNA. The viral signals for frameshifting include a heptanucleotide sequence on which the shift occurs and higher-order RNA structure just downstream of the shift site. We have made site-directed mutations in two stems (S1 and S2) of a putative RNA pseudoknot that begins 7 nucleotides 3' of the previously identified shift site (A AAA AAC) in the gag-pro region of mouse mammary tumor virus (MMTV) RNA. The mutants confirm the predicted structure, show that loss of either S1 or S2 impairs frameshifting, and exclude alternative RNA structures as significant for frameshifting. The importance of the MMTV pseudoknot has been further demonstrated by showing that shift sites from two other retroviruses function more efficiently in the position of the MMTV site than in their native contexts. However, the MMTV pseudoknot cannot promote detectable frameshifting in the absence of a recognizable upstream shift site. In addition, the species of tRNA that reads the second codon in the shift site appears to be a critical determinant, since changing the 7th nucleotide in the MMTV gag-pro shift site from C to A, U, or G severely impairs frameshifting. Images PMID:1309954

  5. Alternative messenger RNA splicing of autophagic gene Beclin 1 in human B-cell acute lymphoblastic leukemia cells.

    PubMed

    Niu, Yu-Na; Liu, Qing-Qing; Zhang, Su-Ping; Yuan, Na; Cao, Yan; Cai, Jin-Yang; Lin, Wei-Wei; Xu, Fei; Wang, Zhi-Jian; Chen, Bo; Wang, Jian-Rong

    2014-01-01

    Beclin 1 is a key factor for initiation and regulation of autophagy, which is a cellular catabolic process involved in tumorigenesis. To investigate the role of alternative splicing of Beclin1 in the regulation of autophagy in leukemia cells, Beclin1 mRNA from 6 different types of cell lines and peripheral blood mononuclear cells from 2 healthy volunteers was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants were analyzed by DNA sequencing. A transcript variant of Beclin 1 gene carrying a deletion of exon 11, which encoded a C-terminal truncation of Beclin 1 isoform, was found. The alternative isoform was assessed by bioinformatics, immunoblotting and subcellular localization. The results showed that this variable transcript is generated by alternative 3' splicing, and its translational product displayed a reduced activity in induction of autophagy by starvation, indicating that the spliced isoform might function as a dominant negative modulator of autophagy. Our findings suggest that the alternative splicing of Beclin 1 might play important roles in leukemogenesis regulated by autophagy.

  6. Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein.

    PubMed

    Ferranti, P; Addeo, F; Malorni, A; Chianese, L; Leroux, C; Martin, P

    1997-10-01

    The identity of multiple forms of caprine alpha(s1)-casein in variants A, B, and C has been determined by structural characterisation using mass spectrometry, automated Edman degradation and peptide mapping. Mature goat alpha(s1)-casein exists as a mixture of at least four molecular species which differ in peptide chain length. The main component corresponds to the 199-residues form already described. The other three, in lesser amounts, were shorter forms of alpha(s1)-casein and differed for the deleted peptides 141-148, as shown previously for ovine alpha(s1)-casein, peptide 110-117, or Gln78. Analysis of alpha(s1)-casein mRNA from milk somatic cells demonstrated that these forms originated from skipping events at the level of exon 13 (codifying for peptide 110-117) and 16 (codifying for peptide 141-148) and from the presence of a cryptic splice site within exon 11 (whose first CAG triplet encodes Gln78) during primary transcript processing. The finding of these splicing abnormalities in the three common variants A, B, and C suggests that this is a general feature of alpha(s1)-casein in goat. A further source of heterogeneity of caprine alpha(s1)-casein was identified in the discrete phosphorylation of seryl residues. Eight serine residues (at positions 44, 46, 64 to 68 and 75) are fully phosphorylated (except in variant A because of the replacement Glu77-->Gln which prevents phosphorylation of Ser75). Conversely, Ser115 and Ser41 are phosphorylated only to about 50% and 20%, respectively. Ser12, although located in a consensus triplet, is never phosphorylated, similarly to the ovine alpha(s1)-casein variants. These results confirm that there are stabilised mechanisms of simultaneous synthesis of alpha(s1)-casein at different length and of post-translational modification in both caprine and ovine species.

  7. Localization of brain 5α-reductase messenger RNA in mice selectively bred for high chronic alcohol withdrawal severity.

    PubMed

    Roselli, Charles E; Finn, Timothy J; Ronnekleiv-Kelly, Sean M; Tanchuck, Michelle A; Kaufman, Katherine R; Finn, Deborah A

    2011-12-01

    Several lines of evidence suggest that fluctuations in endogenous levels of the γ-aminobutyric acid (GABA)ergic neurosteroid allopregnanolone (ALLO) represent one mechanism for regulation of GABAergic inhibitory tone in the brain, with an ultimate impact on behavior. Consistent with this idea, there was an inverse relationship between ALLO levels and symptoms of anxiety and depression in humans and convulsive activity in rodents during alcohol withdrawal. Our recent studies examined the activity and expression of 5α-reductase (Srd5a1), the rate-limiting enzyme in the biosynthesis of ALLO, during alcohol withdrawal in mice selectively bred for high chronic alcohol withdrawal (Withdrawal Seizure-Prone [WSP]) and found that Srd5a1 was downregulated in the cortex and hippocampus over the time course of dependence and withdrawal. The purpose of the present studies was to extend these findings and more discretely map the regions of Srd5a1 expression in mouse brain using radioactive in situ hybridization in WSP mice that were ethanol naïve, following exposure to 72h ethanol vapor (dependent) or during peak withdrawal. In naïve animals, expression of Srd5a1 was widely distributed throughout the mouse brain, with highest expression in specific regions of the cerebral cortex, hippocampus, thalamus, hypothalamus, and amygdala. In dependent animals and during withdrawal, there was no change in Srd5a1 expression in cortex or hippocampus, which differed from our recent findings in dissected tissues. These results suggest that local Srd5a1 mRNA expression in WSP brain may not change in parallel with local ALLO content or withdrawal severity. Published by Elsevier Inc.

  8. Onapristone (ZK299) and mifepristone (RU486) regulate the messenger RNA and protein expression levels of the progesterone receptor isoforms A and B in the bovine endometrium.

    PubMed

    Rekawiecki, Robert; Kowalik, Magdalena K; Kotwica, Jan

    2015-08-01

    The aim of this study was to examine whether progesterone (P(4)) and its antagonists, onapristone (ZK299) and mifepristone (RU486), affect the levels of PGRA and PGRB messenger RNA (mRNA) and protein in the cow uterus which may be important in understanding whether the final physiological effect evoked by an antagonist depends on PGR isoform bound to the antagonist. Endometrial slices on Days 6 to 10 and 17 to 20 of the estrous cycle were treated for 6 or 24 hours for mRNA and protein expression analysis, respectively, with P4, ZK299, or RU486 at a dose of 10(-4), 10(-5), or 10(-6) M. In the samples on Days 6 to 10 of the estrous cycle, PGRAB mRNA was stimulated by P(4) (10(-4) M; P < 0.01) and RU486 (10(-6); P < 0.001) and was decreased by ZK299 (10(-5); P < 0.05). In contrast, PGRB mRNA was decreased by the all P(4) (P < 0.01) and ZK299 (P < 0.001) doses and by two of the RU486 doses (10(-4) M; P < 0.01 and 10(-5) M; P < 0.01). In samples on Days 17 to 20 of the estrous cycle, PGRAB mRNA was stimulated by RU486 (10(-5) M; P < 0.001). PGRB mRNA was decreased by P(4) (10(-4) and 10(-5) M; P < 0.001), ZK299 (10(-4) and 10(-5) M; P < 0.001), and RU486 (10(-4) M; P < 0.01 and 10(-6) M; P < 0.001) and was increased by ZK299 (10(-6) M; P < 0.001) and RU486 (10(-5) M; P < 0.001). In samples on Days 6 to 10 of the estrous cycle, PGRB protein levels were decreased (P < 0.05) by all three ZK299 doses and by two of the RU486 doses (10(-4) M; P < 0.05 and 10(-5) M; P < 0.01). In contrast, in samples on Days 17 to 20, both PGRA and PGRB protein levels were decreased by ZK299 stimulation (10(-5) M; P < 0.05 and 10(-5) M; P < 0.01, respectively), whereas only PGRA protein levels were increased by RU486 (10(-5) M; P < 0.01). Both ZK299 and RU486 may exhibit both agonist and antagonist properties depending on which receptor isoform they affect. As a result, an increase or decrease in the expression of a particular PGR isoform will be observed.

  9. Differential effects of common variants in SCN2A on general cognitive ability, brain physiology, and messenger RNA expression in schizophrenia cases and control individuals.

    PubMed

    Dickinson, Dwight; Straub, Richard E; Trampush, Joey W; Gao, Yuan; Feng, Ningping; Xie, Bin; Shin, Joo Heon; Lim, Hun Ki; Ursini, Gianluca; Bigos, Kristin L; Kolachana, Bhaskar; Hashimoto, Ryota; Takeda, Masatoshi; Baum, Graham L; Rujescu, Dan; Callicott, Joseph H; Hyde, Thomas M; Berman, Karen F; Kleinman, Joel E; Weinberger, Daniel R

    2014-06-01

    One approach to understanding the genetic complexity of schizophrenia is to study associated behavioral and biological phenotypes that may be more directly linked to genetic variation. To identify single-nucleotide polymorphisms associated with general cognitive ability (g) in people with schizophrenia and control individuals. Genomewide association study, followed by analyses in unaffected siblings and independent schizophrenia samples, functional magnetic resonance imaging studies of brain physiology in vivo, and RNA sequencing in postmortem brain samples. The discovery cohort and unaffected siblings were participants in the National Institute of Mental Health Clinical Brain Disorders Branch schizophrenia genetics studies. Additional schizophrenia cohorts were from psychiatric treatment settings in the United States, Japan, and Germany. The discovery cohort comprised 339 with schizophrenia and 363 community control participants. Follow-up analyses studied 147 unaffected siblings of the schizophrenia cases and independent schizophrenia samples including a total of an additional 668 participants. Imaging analyses included 87 schizophrenia cases and 397 control individuals. Brain tissue samples were available for 64 cases and 61 control individuals. We studied genomewide association with g, by group, in the discovery cohort. We used selected genotypes to test specific associations in unaffected siblings and independent schizophrenia samples. Imaging analyses focused on activation in the prefrontal cortex during working memory. Brain tissue studies yielded messenger RNA expression levels for RefSeq transcripts. The schizophrenia discovery cohort showed genomewide-significant association of g with polymorphisms in sodium channel gene SCN2A, accounting for 10.4% of g variance (rs10174400, P = 9.27 × 10(-10)). Control individuals showed a trend for g/genotype association with reversed allelic directionality. The genotype-by-group interaction was also genomewide

  10. Gauge Messenger Models

    SciTech Connect

    Kim, Hyung Do

    2006-11-28

    We consider gauge messenger models in which X and Y gauge bosons and gauginos are messengers of supersymmetry breaking. In simple gauge messenger models, all the soft parameters except {mu} and B{mu} are calculated in terms of a single scale parameter MSUSY which is proportional to F / MGUT. Unique prediction on dark matter in gauge messenger models is discussed. (Based on hep-ph/0601036 and hep-ph/0607169)

  11. Glutathione Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Transcription via WRKY33 and 1-Aminocyclopropane-1-Carboxylate Oxidase by Modulating Messenger RNA Stability to Induce Ethylene Synthesis during Stress.

    PubMed

    Datta, Riddhi; Kumar, Deepak; Sultana, Asma; Hazra, Saptarshi; Bhattacharyya, Dipto; Chattopadhyay, Sharmila

    2015-12-01

    Glutathione (GSH) plays a fundamental role in plant defense-signaling network. Recently, we have established the involvement of GSH with ethylene (ET) to combat environmental stress. However, the mechanism of GSH-ET interplay still remains unexplored. Here, we demonstrate that GSH induces ET biosynthesis by modulating the transcriptional and posttranscriptional regulations of its key enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Transgenic Arabidopsis (Arabidopsis thaliana) plants with enhanced GSH content (AtECS) exhibited remarkable up-regulation of ACS2, ACS6, and ACO1 at transcript as well as protein levels, while they were down-regulated in the GSH-depleted phytoalexin deficient2-1 (pad2-1) mutant. We further observed that GSH induced ACS2 and ACS6 transcription in a WRKY33-dependent manner, while ACO1 transcription remained unaffected. On the other hand, the messenger RNA stability for ACO1 was found to be increased by GSH, which explains our above observations. In addition, we also identified the ACO1 protein to be a subject for S-glutathionylation, which is consistent with our in silico data. However, S-glutathionylation of ACS2 and ACS6 proteins was not detected. Further, the AtECS plants exhibited resistance to necrotrophic infection and salt stress, while the pad2-1 mutant was sensitive. Exogenously applied GSH could improve stress tolerance in wild-type plants but not in the ET-signaling mutant ethylene insensitive2-1, indicating that GSH-mediated resistance to these stresses occurs via an ET-mediated pathway. Together, our investigation reveals a dual-level regulation of ET biosynthesis by GSH during stress.

  12. A high throughput messenger RNA differential display screen identifies discrete domains of gene expression and novel patterning processes along the developing neural tube

    PubMed Central

    Chambers, David; Mason, Ivor

    2006-01-01

    Background During early development the vertebrate neural tube is broadly organized into the forebrain, midbrain, hindbrain and spinal cord regions. Each of these embryonic zones is patterned by a combination of genetic pathways and the influences of local signaling centres. However, it is clear that much remains to be learned about the complete set of molecular cues that are employed to establish the identity and intrinsic neuronal diversity of these territories. In order to address this, we performed a high-resolution messenger RNA differential display screen to identify molecules whose expression is regionally restricted along the anteroposterior (AP) neuraxis during early chick development, with particular focus on the midbrain and hindbrain vesicles. Results This approach identified 44 different genes, with both known and unknown functions, whose transcription is differentially regulated along the AP axis. The identity and ontological classification of these genes is presented. The wide variety of functional classes of transcripts isolated in this screen reflects the diverse spectrum of known influences operating across these embryonic regions. Of these 44 genes, several have been selected for detailed in situ hybridization analysis to validate the screen and accurately define the expression domains. Many of the identified cDNAs showed no identity to the current databases of known or predicted genes or ESTs. Others represent genes whose embryonic expression has not been previously reported. Expression studies confirmed the predictions of the primary differential display data. Moreover, the nature of identified genes, not previously associated with regionalisation of the brain, identifies novel potential mechanisms in that process. Conclusion This study provides an insight into some of the varied and novel molecular networks that operate during the regionalization of embryonic neural tissue and expands our knowledge of molecular repertoire used during

  13. Regional age-related changes in neuronal nitric oxide synthase (nNOS), messenger RNA levels and activity in SAMP8 brain

    PubMed Central

    Colas, Damien; Gharib, Abdallah; Bezin, Laurent; Morales, Anne; Guidon, Gérard; Cespuglio, Raymond; Sarda, Nicole

    2006-01-01

    Background Nitric oxide (NO) is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs) acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8) mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS) is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS). To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control) animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx-) levels, in three brain areas (n = 7 animals in each group). Calibrated reverse transcriptase (RT) and real-time polymerase chain reaction (PCR) and biochemical procedures were used. Results We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1. Conclusion Concomitant variations occurring in NO levels derived from n

  14. Regional age-related changes in neuronal nitric oxide synthase (nNOS), messenger RNA levels and activity in SAMP8 brain.

    PubMed

    Colas, Damien; Gharib, Abdallah; Bezin, Laurent; Morales, Anne; Guidon, Gérard; Cespuglio, Raymond; Sarda, Nicole

    2006-12-21

    Nitric oxide (NO) is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs) acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8) mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS) is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS). To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control) animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx-) levels, in three brain areas (n = 7 animals in each group). Calibrated reverse transcriptase (RT) and real-time polymerase chain reaction (PCR) and biochemical procedures were used. We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1. Concomitant variations occurring in NO levels derived from nNOS and iNOS at an early age

  15. Heterogeneous cationic liposomes modified with 3β-{N-[(N',N'-dimethylamino)ethyl]carbamoyl}cholesterol can induce partial conformational changes in messenger RNA and regulate translation in an Escherichia coli cell-free translation system.

    PubMed

    Suga, Keishi; Tanabe, Tomoyuki; Umakoshi, Hiroshi

    2013-02-12

    The effect of cationic liposomes (CLs) on messenger RNA(mRNA) conformation and translation was studied, focusing on membrane heterogeneity. CLs, composed of 1,2-dioleoyl-sn-glycerol-3-phosphocholine/1,2-dioleoyl-3-timethylammonium propane (DOPC/DOTAP) and DOPC/3β-{N-[(N',N'-dimethylamino)ethyl]carbamoyl}cholesterol (DOPC/DC-Ch), inhibited mRNA translation in an Escherichia coli cell-free translation system. Analysis of the membrane fluidity and polarity indicated a heterogeneous DOPC/DC-Ch (70/30) membrane, while other CLs exhibited homogeneous disordered membranes. mRNA adsorbed onto DOPC/DC-Ch liposomes showed translational activity, while DOPC/DOTAP liposomes inhibited mRNA translation in proportion to its adsorption onto membranes. Dehydration of DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) was observed in the presence of mRNA but not in the case of zwitterionic DOPC liposomes, indicating that mRNA binds in regions between the phosphate [-PO(2)(-)-] and carbonyl [-C=O-] moieties of lipids. UV resonance Raman spectroscopy suggests that adenine, cytosine, and guanine interact with DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) but not with DOPC. Circular dichroism indicates that DOPC/DOTAP (70/30) extensively denatured the mRNA. In contrast, heterogeneous DOPC/DC-Ch (70/30) induced partial conformational changes but maintained the translational activity of mRNA.

  16. Lack of pharmacokinetic interaction of mipomersen sodium (ISIS 301012), a 2'-O-methoxyethyl modified antisense oligonucleotide targeting apolipoprotein B-100 messenger RNA, with simvastatin and ezetimibe.

    PubMed

    Yu, Rosie Z; Geary, Richard S; Flaim, Joann D; Riley, Gina C; Tribble, Diane L; vanVliet, André A; Wedel, Mark K

    2009-01-01

    Mipomersen sodium (ISIS 301012) is a 20-mer phosphorothioate antisense oligonucleotide that is complementary to human apolipoprotein B-100 (apoB-100) messenger RNA and subsequently reduces translation of ApoB-100 protein, the major apolipoprotein of very low-density lipoprotein, intermediate-density lipoprotein and low-density lipoprotein (LDL). Mipomersen sodium is currently being studied in phase II/III clinical studies to determine its clinical utility as add-on therapy to HMG-CoA reductase inhibitors or other lipid-lowering agents in subjects with hypercholesterolaemia. The aim of this study was to characterize the pharmacokinetic interactions of mipomersen sodium with simvastatin and ezetimibe. Another aim was to evaluate the ability of mipomersen sodium to inhibit major cytochrome P450 (CYP) isoenzymes in vitro. In a phase I clinical study, ten healthy subjects per cohort received a single oral dose of simvastatin 40 mg or ezetimibe 10 mg followed by four 2-hour intravenous doses of mipomersen sodium 200 mg over an 8-day period, with simvastatin 40 mg or ezetimibe 10 mg being administered again with the last dose of mipomersen sodium. Mipomersen sodium pharmacokinetic profiles were assessed following the first dose (mipomersen sodium alone) and the last dose (mipomersen sodium in combination with simvastatin or ezetimibe). Plasma samples for measurement of simvastatin, simvastatin acid, and free and total ezetimibe concentrations were collected at various timepoints following their first and last oral dosing. A comparative pharmacokinetic analysis was performed to determine if there were any effects resulting from coadministration of mipomersen sodium with these lipid-lowering drugs. In addition to the clinical pharmacokinetic analysis, the ability of mipomersen sodium to inhibit the major CYP isoform enzymes (namely CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was evaluated in cryo-preserved human hepatocytes in vitro. The area under the plasma concentration

  17. Induction of differentiation of the human myeloid cell line, ML3, by tumour necrosis factor and interferon-gamma is accompanied by enhanced expression of the CD4 protein and messenger RNA.

    PubMed

    Cassatella, M A; Trinchieri, G; Hassan, N F; Hartman, L; Sorio, C; Berton, G

    1992-05-01

    Tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma) induce differentiation of human myeloid cell lines along the monocytic lineage. In this study we investigated the effects of TNF and IFN-gamma on the expression of the CD4 protein and messenger RNA (mRNA) in the two myeloid cell lines, ML3 and HL-60. We observed that CD4 antigen expression on ML3 cells is almost undetectable and that TNF and IFN-gamma induced CD4 antigen expression on these cells. HL-60 cells express surface CD4 antigen at high density and treatment with TNF and IFN-gamma caused a decrease of CD4 expression. We also investigated the expression of CD4 mRNA in ML3 and HL-60 cells. ML3 constitutively express, albeit at low levels, CD4 mRNA. TNF induced CD4 mRNA in ML3 cells and IFN-gamma synergistically potentiated the effect of TNF, thus indicating that the enhanced expression of the CD4 protein on ML3 cells is due, at least in part, to an enhanced accumulation of the CD4 mRNA. CD4 mRNA is constitutively expressed in HL-60 cells at high levels. TNF and IFN-gamma, alone or in combination, did not cause any significant change of CD4 mRNA expression in HL-60 cells, thus indicating that decrease of surface CD4, which accompanies differentiation with these cytokines, is likely due to alterations of the CD4 protein synthesis and/or transport to the plasma membrane. These results provide evidence that myeloid cell lines are heterogeneous in expression of CD4, and that in ML3 cells, which constitutively express low levels of CD4 mRNA and undetectable amounts of surface CD4, the predominant effect of the two cytokines is to induce both CD4 mRNA and protein.

  18. Transmembrane Signalling: Membrane messengers

    NASA Astrophysics Data System (ADS)

    Cockroft, Scott L.

    2017-05-01

    Life has evolved elaborate means of communicating essential chemical information across cell membranes. Inspired by biology, two new artificial mechanisms have now been developed that use synthetic messenger molecules to relay chemical signals into or across lipid membranes.

  19. About The ESO Messenger

    NASA Astrophysics Data System (ADS)

    Kjär, K.

    2000-06-01

    The present Messenger is the hundredth issue to be published. This may be a good moment to look back to the beginning and to the development of this publication. The idea of an internal ESO newsletter was born in the early seventies. Using the words of Professor Blaauw, Director General of ESO at that time and the person who launched The Messenger in its orbit, the purpose of The ESO Messenger should be “first of all, to promote the participation of ESO staff in what goes on in the Organization, especially at places of duty other than our own. Moreover, The Messenger may serve to give the world outside some impression of what happens inside ESO. The need for more internal communication is felt by many of the staff. The dispersion of our resources over several countries in widely separated continents demands a special effort to keep us aware of what is going on at the other establishments...”

  20. MESSENGER Laser Altimeter

    NASA Image and Video Library

    MESSENGER's Mercury Laser Altimeter sends out laser pulses that hit the ground and return to the instrument. The amount of light that returns for each pulse gives the reflectance at that point on t...

  1. Expression of messenger RNA for transforming growth factor-beta1 and for transforming growth factor-beta receptors in peripheral blood of systemic lupus erythematosus patients treated with low doses of quinagolide.

    PubMed

    Hrycek, Antoni; Kusmierz, Dariusz; Dybała, Tomasz; Swiatkowska, Longina

    2007-02-01

    The objective of this study was to determine the expression of transforming growth factor-beta1 messenger RNA (TGF-beta1 mRNA) and the expression of mRNA for TGF-beta receptors (TGF-beta Rs mRNA) in whole peripheral blood of consecutive (treated from several months to several years) systemic lupus erythematosus (SLE) patients (21 women). A further aim of this study was to evaluate the association between expression of the above mentioned parameters in relation to the form of applied therapy (9 patients treated with quinagolide and 12 with quinagolide plus prednisone, azathioprine or cyclosporine A). The control group consisted of 15 healthy women. Most of the patients had mild SLE with SLE disease activity index (SLEDAI) score < 10 at time when blood samples were collected. Laboratory measurements included real-time polymerase chain reaction (RT-QPCR). The expression levels of TGF-beta1 mRNA and mRNA for TGF-beta RII and RIII were significantly lower in patients whereas the expression level of TGF-beta RI was statistically significantly higher in SLE patients than in the controls. A very high positive correlation between TGF-beta1 mRNA expression and expression levels of TGF-beta Rs mRNA was found. In compared subgroups selected according to the form of the applied therapy no statistically significant differences were observed. We conclude that the TGF-beta signaling pathway can be altered in circulating leukocytes derived from treated patients with SLE and that the assumed forms of the applied therapy in the group of patients under consideration are accompanied by similarity in the expression level of transcripts for TGF-beta1 and TGF-beta Rs determined in whole blood. In our investigations, we cannot exclude the influence of the disease itself on the obtained results.

  2. Olive leaf extract suppresses messenger RNA expression of proinflammatory cytokines and enhances insulin receptor substrate 1 expression in the rats with streptozotocin and high-fat diet-induced diabetes.

    PubMed

    Liu, Ya-Nan; Jung, Ji-Hye; Park, Hyunjin; Kim, HyunSook

    2014-05-01

    Type 2 diabetes, characterized by hyperglycemia and hyperlipidemia, is a metabolic disease resulting from defects in both insulin secretion and insulin resistance. Recently, olive leaf has been reported as an anti-inflammatory, antioxidant, and antidiabetic agent. This study sought to investigate whether olive leaf extract can improve the insulin resistance and inflammation response in rats with type 2 diabetes induced by high-fat diet and streptozotocin. After administering olive leaf extract for 8 weeks (200 and 400 mg/kg body weight), rats given the higher dose showed significantly lower blood glucose, serum total cholesterol, and triglyceride levels compared with those of diabetic control rats (P < .05). Results of oral glucose tolerance tests, homeostasis model assessment of insulin resistance, and messenger RNA (mRNA) expression of tumor necrosis factor α and interleukin (IL) 6 in the liver show significantly decreased glucose level in rats given either dose of olive leaf extract (P < .05). Both olive leaf extract-treated groups showed significantly increased insulin receptor substrate 1 expression (P < .05). Tumor necrosis factor α, IL-6 and IL-1β mRNA expressions in epididymis adipose tissue were significantly lower in rats that received higher dose of olive leaf extract (P < .05). Lymphocyte infiltration was not observed in these rats. The results suggest that olive leaf extract may attenuate insulin resistance by suppressing mRNA expression of proinflammatory cytokines and elevating of insulin receptor substrate 1 expression.

  3. High-fat diet-induced reduction of peroxisome proliferator-activated receptor-γ coactivator-1α messenger RNA levels and oxidative capacity in the soleus muscle of rats with metabolic syndrome.

    PubMed

    Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Takeda, Isao; Tsuda, Kinsuke; Ishihara, Akihiko

    2012-02-01

    Animal models of type 2 diabetes exhibit reduced peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) messenger RNA (mRNA) levels, which are associated with decreased oxidative capacity, in skeletal muscles. In contrast, animal models with metabolic syndrome show normal PGC-1α mRNA levels. We hypothesized that a high-fat diet decreases PGC-1α mRNA levels in skeletal muscles of rats with metabolic syndrome, reducing muscle oxidative capacity and accelerating metabolic syndrome or inducing type 2 diabetes. We examined mRNA levels and fiber profiles in the soleus muscles of rats with metabolic syndrome (SHR/NDmcr-cp [cp/cp]; CP) fed a high-fat diet. Five-week-old CP rats were assigned to a sedentary group (CP-N) that was fed a standard diet (15.1 kJ/g, 23.6% protein, 5.3% fat, and 54.4% carbohydrates) or a sedentary group (CP-H) that was fed a high-fat diet (21.6 kJ/g, 23.6% protein, 34.9% fat, and 25.9% carbohydrates) and were housed for 10 weeks. Body weight, energy intake, and systolic blood pressure were higher in the CP-H group than in the CP-N group. Nonfasting glucose, triglyceride, total cholesterol, and leptin levels were higher in the CP-H group than in the CP-N group. There was no difference in insulin levels between the CP-N and CP-H groups. Muscle PGC-1α mRNA levels and succinate dehydrogenase activity were lower in the CP-H group than in the CP-N group. We concluded that a high-fat diet reduces PGC-1α mRNA levels and oxidative capacity in skeletal muscles and accelerates metabolic syndrome.

  4. Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma

    PubMed Central

    2010-01-01

    Background The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients. PMID:21040522

  5. Mercury MESSENGER Stamp Unveiling

    NASA Image and Video Library

    2011-05-03

    Daughters of NASA astronaut Alan Shepard, Laura Shepard Churchley, left, Alice Wackermann and Julie Jenkins, right, speak during an unveiling ceremony of two USPS stamps that commemorate and celebrate 50 years of US Spaceflight and the MESSENGER program during an event, Wednesday, May 4, 2011 at the NASA Kennedy Space Center in Cape Canaveral, Fla. One stamp commemorates NASA’s Project Mercury, America’s first manned spaceflight program, and NASA astronaut Alan Shepard’s historic flight on May 5, 1961, aboard spacecraft Freedom 7. The other stamp draws attention to NASA’s unmanned MESSENGER mission, a scientific investigation of the planet Mercury. On March 17, 2011, MESSENGER became the first spacecraft to enter into orbit around Mercury. Photo Credit: (NASA/Bill Ingalls)

  6. Mercury MESSENGER Stamp Unveiling

    NASA Image and Video Library

    2011-05-03

    Daughters of NASA astronaut Alan Shepard, Laura Shepard Churchley, standing left, Alice Wackermann and Julie Jenkins, standing right, speak during an unveiling ceremony of two USPS stamps that commemorate and celebrate 50 years of US Spaceflight and the MESSENGER program during an event, Wednesday, May 4, 2011 at the NASA Kennedy Space Center in Cape Canaveral, Fla. One stamp commemorates NASA’s Project Mercury, America’s first manned spaceflight program, and NASA astronaut Alan Shepard’s historic flight on May 5, 1961, aboard spacecraft Freedom 7. The other stamp draws attention to NASA’s unmanned MESSENGER mission, a scientific investigation of the planet Mercury. On March 17, 2011, MESSENGER became the first spacecraft to enter into orbit around Mercury. Photo Credit: (NASA/Bill Ingalls)

  7. Mercury MESSENGER Stamp Unveiling

    NASA Image and Video Library

    2011-05-03

    United States Postal Service Vice President of Finance Steve Masse, left, and NASA Mercury Astronaut Scott Carpenter, unveil two USPS stamps to commemorate and celebrate 50 years of US Spaceflight and the MESSENGER program during an event, Wednesday, May 4, 2011 at the NASA Kennedy Space Center in Cape Canaveral, Fla. One stamp commemorates NASA’s Project Mercury, America’s first manned spaceflight program, and NASA astronaut Alan Shepard’s historic flight on May 5, 1961, aboard spacecraft Freedom 7. The other stamp draws attention to NASA’s unmanned MESSENGER mission, a scientific investigation of the planet Mercury. On March 17, 2011, MESSENGER became the first spacecraft to enter into orbit around Mercury. Photo Credit: (NASA/Bill Ingalls)

  8. Mercury MESSENGER Stamp Unveiling

    NASA Image and Video Library

    2011-05-03

    NASA Administrator Charles Boldin speaks during an unveiling ceremony of two USPS stamps that commemorate and celebrate 50 years of US Spaceflight and the MESSENGER program during an event, Wednesday, May 4, 2011 at the NASA Kennedy Space Center in Cape Canaveral, Fla. One stamp commemorates NASA’s Project Mercury, America’s first manned spaceflight program, and NASA astronaut Alan Shepard’s historic flight on May 5, 1961, aboard spacecraft Freedom 7. The other stamp draws attention to NASA’s unmanned MESSENGER mission, a scientific investigation of the planet Mercury. On March 17, 2011, MESSENGER became the first spacecraft to enter into orbit around Mercury. Photo Credit: (NASA/Bill Ingalls)

  9. Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

    PubMed

    Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle

    2016-07-15

    In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  10. Translation of globin messenger RNA modified by benzo(a)pyrene 7,8-dihydrodiol 9,10-oxide in a wheat germ cell-free system

    SciTech Connect

    Grunberger, D.; Pergolizzi, R.G.; Jones, R.E.

    1980-01-25

    Poly(U/sub 3/G) and rabbit globin mRNA were modified with the active carcinogenic metabolite of benzo(a)pyrene, (+-)-7..beta..,8..cap alpha..-dihydroxy-9..cap alpha..,10..cap alpha..-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and the effects of modification on translation in a cell-free protein synthesizing system were studied. High performance liquid chromatography of modified nucleosides from enzymatically hydrolyzed globin mRNA reveals that the active carcinogen formed two adducts with guanosine, four adducts with adenosine, and one adduct probably with cytidine residues. When globin mRNA with 0.4 carcinogen residues/molecule is used as a template, incorporation of amino acids into proteins is inhibited by 50%, and mRNA with 2.4 residues has only 10% of the template activity compared to unmodified molecules. On the other hand, modification of poly(U/sub 3/G) has no effect on its template activity. Since no significant formation of smaller peptides in the protein synthesizing system programmed with modified mRNA is detected, it is suggested that the carcinogen does not block the elongation step in mRNA translation. However, glycerol gradient centrifugation of initiation complexes reveals that modified globin mRNA does not form initiation complexes with ribosomes as effectively as does the unmodified globin mRNA. These results suggest that modification significantly reduces the ability of mRNA to be translated by affecting the initiation step in protein synthesis.

  11. Effect of repeated alcohol exposure during the third trimester-equivalent on messenger RNA levels for interleukin-1β, chemokine (C-C motif) ligand 2, and interleukin 10 in the developing rat brain after injection of lipopolysaccharide.

    PubMed

    Topper, Lauren A; Valenzuela, C Fernando

    2014-12-01

    Microglia undergo maturation during the third trimester of human development (equivalent to the first 1-2 weeks of postnatal life in rodents), during which these cells may be particularly sensitive to insult. Alcohol exposure during this period can activate the neuroimmune system, an effect that may contribute to the pathophysiology of fetal alcohol spectrum disorders. Here, we investigated whether repeated alcohol exposure during the third trimester-equivalent in rats has a priming effect on the neuroimmune response to injection of bacterial lipopolysaccharide (LPS). Pups were exposed to alcohol in vapor chambers for 4 h daily from postnatal day (PD)2 to PD16 (peak blood alcohol concentrations ∼150 mg/dL). On PD17, rats were injected with either saline or LPS (50 μg/kg) and the frontal cortex, cerebellar vermis, and dentate gyrus were collected 2 h later. Messenger RNA (mRNA) levels for the pro-inflammatory agents interleukin 1β (IL-1β) and chemokine (C-C) motif ligand 2 (CCL2), as well as levels of the anti-inflammatory cytokine interleukin 10 (IL-10), were measured using reverse transcriptase polymerase chain reaction. LPS consistently increased IL-1β and CCL2 mRNA levels in the dentate gyrus, frontal cortex, and cerebellum of both male and female rats. Furthermore, the LPS-induced increase of IL-1β mRNA levels was significantly blunted in the frontal cortex of alcohol-exposed female rats. Conversely, LPS only minimally affected IL-10 mRNA expression and there were no significant differences between air- and alcohol-exposed rats. Taken together with the literature regarding the effect of third-trimester alcohol exposure on the neuroimmune system, our findings suggest that chronic exposure to lower levels is less disruptive to the neuroimmune system than binge-like exposure to high doses of alcohol.

  12. Placental development during early pregnancy in sheep: estrogen and progesterone receptor messenger RNA expression in pregnancies derived from in vivo-produced and in vitro-produced embryos.

    PubMed

    Reynolds, L P; Haring, J S; Johnson, M L; Ashley, R L; Redmer, D A; Borowicz, P P; Grazul-Bilska, A T

    2015-10-01

    Sex steroids are important regulators of angiogenesis and growth in reproductive tissues, including the placenta. In experiment (exp.) 1, to examine the expression of a suite of sex steroid receptors throughout early pregnancy, maternal (caruncular [CAR]) and fetal (fetal membranes [FM]) placental tissues were collected on days 14 to 30 after mating and on day 10 after estrus (nonpregnant controls). In exp. 2, to examine the hypothesis that assisted reproductive technology would affect the expression of the same suite of sex steroid receptors, pregnancies were achieved through natural mating (NAT) or transfer of embryos from natural mating (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA), and CAR and FM were collected on day 22. In exp. 1, for CAR messenger RNA (mRNA) expression of estrogen receptors (ESR) 1 and 2, nuclear (n) progesterone receptors (PGR) and membrane (m) PGRα, β, and γ were affected (P < 0.02) by pregnancy stage, as were ESR1, nPGR, and mPGRα, β, and γ for FM (P < 0.03). In exp. 2, for CAR, mRNA expression of ESR1 and nPGR was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared with NAT. For FM, mRNA expression of ESR1 tended to be greater (P = 0.10) in the IVA group compared with NAT and NAT-ET, and GPER1 was greater (P < 0.05) in NAT-ET and IVF compared with NAT. These data establish the normal pattern of sex steroid receptor mRNA expression in maternal and fetal placenta during early pregnancy in sheep, and in addition, suggest that altered expression of placental sex steroid receptors may be an early event leading to poor placental vascularization and growth after assisted reproductive technology.

  13. Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry.

    PubMed Central

    Guillier, Maude; Allemand, Frédéric; Raibaud, Sophie; Dardel, Frédéric; Springer, Mathias; Chiaruttini, Claude

    2002-01-01

    In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling. All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene. L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene. This interaction results in the formation of a pseudoknot. Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro. The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene. By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions. Therefore, these two regions define two L20-binding sites in the operator. Using mutations and deletions of rpml'-'lacZ fusions, we show that both sites are essential for repression in vivo. However L20 can bind to each site independently in vitro. One site is similar to the L20-binding site on 23S rRNA. Here we propose that L20 recognizes its mRNA and its rRNA in similar way. PMID:12166643

  14. Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus.

    PubMed

    Sukjumlong, S; Persson, E; Dalin, A-M; Janson, V; Sahlin, L

    2009-06-01

    The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.

  15. Changes in brain ribonuclease (BRB) messenger RNA in granulosa cells (GCs) of dominant vs subordinate ovarian follicles of cattle and the regulation of BRB gene expression in bovine GCs.

    PubMed

    Dentis, J L; Schreiber, N B; Gilliam, J N; Schutz, L F; Spicer, L J

    2016-04-01

    Brain ribonuclease (BRB) is a member of the ribonuclease A superfamily that is constitutively expressed in a range of tissues and is the functional homolog of human ribonuclease 1. This study was designed to characterize BRB gene expression in granulosa cells (GCs) during development of bovine dominant ovarian follicles and to determine the hormonal regulation of BRB in GCs. Estrous cycles of Holstein cows (n = 18) were synchronized, and cows were ovariectomized on either day 3 to 4 or day 5 to 6 after ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GCs were collected for RNA isolation and quantitative polymerase chain reaction. Follicles were categorized as small (1-5 mm; pooled per ovary), medium (5-8 mm; individually collected), or large (8.1-17 mm; individually collected) based on surface diameter. Estradiol (E2) and progesterone (P4) levels were measured by radioimmunoassay (RIA) in FFL. Abundance of BRB messenger RNA (mRNA) in GCs was 8.6- to 11.8-fold greater (P < 0.05) in small (n = 31), medium (n = 66), and large (n = 33) subordinate E2-inactive (FFL E2 < P4) follicles than in large (n = 16) dominant E2-active (FFL E2 > P4) follicles. In the largest 4 follicles, GCs BRB mRNA abundance was negatively correlated (P < 0.01) with FFL E2 (r = -0.65) and E2:P4 ratio (r = -0.46). In experiment 2, GCs from large (8-22 mm diameter) and small (1-5 mm diameter) follicles were treated with insulin-like growth factor 1 (IGF1; 0 or 30 ng/mL) and/or tumor necrosis factor alpha (0 or 30 ng/mL); IGF1 increased (P < 0.05) BRB mRNA abundance, and tumor necrosis factor alpha decreased (P < 0.001) the IGF1-induced BRB mRNA abundance in large-follicle GCs. In experiment 3 to 6, E2, follicle-stimulating hormone, fibroblast growth factor 9, cortisol, wingless 3A, or sonic hedgehog did not affect (P > 0.10) abundance of BRB mRNA in GCs; thyroxine and luteinizing hormone increased (P < 0.05), whereas

  16. Messenger RNA encoding a glutathione-S-transferase responsible for herbicide tolerance in maize is induced in response to safener treatment.

    PubMed

    Wiegand, R C; Shah, D M; Mozer, T J; Harding, E I; Diaz-Collier, J; Saunders, C; Jaworski, E G; Tiemeier, D C

    1986-07-01

    Glutathione-S-transferases (GST's) in maize represent a family of enzymes which conjugate glutathione to several major classes of pre-emergent, selective herbicides. Chemicals termed safeners have been demonstrated to increase the tolerance of maize toward such herbicides when the maize seed has been previously treated with safeners. It has subsequently been shown that corresponding increases in glutathione-S-transferase species occur. To determine whether these compounds act at a transcriptional level we have used synthetic oligonucleotide probes to isolate cDNA clones encoding the major GST polypeptide subunit, designated GST A. The identity of the clones has been confirmed by hybrid-selected mRNA translation and immunoprecipitation using antibodies made against this GST species as well as by production of active GST in yeast cells transformed with an expression vector containing the cloned DNA. GST A has been found to be encoded in a mRNA of 1.1 kb. Sequencing of cDNA products obtained by primer extension of maize mRNA using our oligonucleotide probes is consistent with this mRNA corresponding to the isolated cDNA clone. Using the clone as a probe for Northern analysis we have found a three to four-fold increase in the steady state level of this mRNA in maize tissue grown from safener-treated seeds. The level of safener which gives this induction is comparable to that required to obtain herbicide tolerance in the field.

  17. Analysis of GNAS1 and overlapping transcripts identifies the parental origin of mutations in patients with sporadic Albright hereditary osteodystrophy and reveals a model system in which to observe the effects of splicing mutations on translated and untranslated messenger RNA.

    PubMed

    Rickard, Sarah J; Wilson, Louise C

    2003-04-01

    Albright hereditary osteodystrophy (AHO) is caused by heterozygous deactivating GNAS1 mutations. There is a parent-of-origin effect. Maternally derived mutations are usually associated with resistance to parathyroid hormone termed "pseudohypoparathyroidism type Ia." Paternally derived mutations are associated with AHO but usually normal hormone responsiveness, known as "pseudo-pseudohypoparathyroidism." These observations can be explained by tissue-specific GNAS1 imprinting. Regulation of the genomic region that encompasses GNAS1 is complex. At least three upstream exons that splice to exon 2 of GNAS1 and that are imprinted have been reported. NESP55 is exclusively maternally expressed, whereas exon 1A and XL alphas are exclusively paternally expressed. We set out to identify the parental origin of GNAS1 mutations in patients with AHO by searching for their mutation in the overlapping transcripts. This information would be of value in patients with sporadic disease, for predicting their endocrine phenotype and planning follow-up. In doing so, we identified mutations that resulted in nonsense-mediated decay of the mutant Gs alpha transcript but that were detectable in NESP55 messenger RNA (mRNA), probably because they lie within its 3' untranslated region. Analysis of the NESP55 transcripts revealed the creation of a novel splice site in one patient and an unusual intronic mutation that caused retention of the intron in a further patient, neither of which could be detected by analysis of the Gs alpha complementary DNA. This cluster of overlapping transcripts represents a useful model system in which to analyze the effects that mutant sequence has on mRNA-in particular, splicing-and the mechanisms of nonsense-mediated mRNA decay.

  18. Increased messenger RNA levels of the mesenchymal cadherin-11 in the peripheral blood of systemic sclerosis patients correlate with diffuse skin involvement.

    PubMed

    Christopoulos, Panagiotis F; Bournia, Vasiliki-Kalliopi; Panopoulos, Stylianos; Vaiopoulos, Aristeides; Koutsilieris, Michael; Sfikakis, Petros P

    2015-01-01

    Cadherin-11 is a cell-cell adhesion molecule also involved in cellular migration and invasion. Experimental studies implicated this molecule in inflammatory arthritis and fibrosing conditions. Moreover, cadherin-11 protein is hyper-expressed on fibroblasts and macrophages in the skin of systemic sclerosis (SSc) patients, whereas the respective mRNA levels correlate with skin thickness. Herein, we searched for possible cadherin-11 expression also in cells that circulate in SSc peripheral blood. Cadherin-11 mRNA was quantified by real-time reverse transcription-polymerase chain reaction in 3 ml blood samples obtained from 71 SSc patients (aged 53±2 years, 65 women) and 35 control non-SSc patients with Raynaud's phenomenon. Cadherin-11 mRNA transcripts were detected in blood samples from 39% of patients with diffuse SSc, versus 16% of those with limited SSc, versus 6% and 16% of patients with idiopathic or associated with other connective tissue diseases Raynaud's phenomenon, respectively (p=0.049). Cadherin-11 mRNA levels in SSc patients were increased by 3.74-fold comparing to controls (p=0.036). By multivariate logistic regression analysis we found that diffuse skin involvement correlated, independently of age, gender, disease duration, lung involvement, digital ulcers, inflammatory indices or anti-Scl-70 autoantibody presence, with cadherin-11 mRNA positivity (p=0.028), but also with increased cadherin-11 mRNA levels (≥3-fold of non-SSc levels, p=0.011). Cadherin-11 may be hyper-expressed in the peripheral blood of diffuse SSc patients. Studies on the origin and possible pathogenic function of these circulating cells may shed light into the complex disease pathogenesis and further support the notion that cadherin-11 is a potential therapeutic target in SSc.

  19. Proteasomal Activity Is Required to Initiate and to Sustain Translational Activation of Messenger RNA Encoding the Stem-Loop-Binding Protein During Meiotic Maturation in Mice1

    PubMed Central

    Yang, Qin; Allard, Patrick; Huang, Michael; Zhang, Wenling; Clarke, Hugh J.

    2009-01-01

    Developmentally regulated translation plays a key role in controlling gene expression during oogenesis. In particular, numerous mRNA species are translationally repressed in growing oocytes and become translationally activated during meiotic maturation. While many studies have focused on a U-rich sequence, termed the cytoplasmic polyadenylation element (CPE), located in the 3′-untranslated region (UTR) and the CPE-binding protein (CPEB) 1, multiple mechanisms likely contribute to translational control in oocytes. The stem-loop-binding protein (SLBP) is expressed in growing oocytes, where it is required for the accumulation of nonpolyadenylated histone mRNAs, and then accumulates substantially during meiotic maturation. We report that, in immature oocytes, Slbp mRNA carries a short poly(A) tail, and is weakly translated, and that a CPE-like sequence in the 3′-UTR is required to maintain this low activity. During maturation, Slbp mRNA becomes polyadenylated and translationally activated. Unexpectedly, proteasomal activity is required both to initiate and to sustain translational activation. This proteasomal activity is not required for the polyadenylation of Slbp mRNA during early maturation; however, it is required for a subsequent deadenylation of the mRNA that occurs during late maturation. Moreover, although CPEB1 is degraded during maturation, inhibiting its degradation by blocking mitogen-activated protein kinase 1/3 activity does not prevent the accumulation of SLBP, indicating that CPEB1 is not the protein whose degradation is required for translational activation of Slbp mRNA. These results identify a new role for proteasomal activity in initiating and sustaining translational activation during meiotic maturation. PMID:19759367

  20. Expression of messenger RNA coding for 5-HT receptor, alpha and beta adrenoreceptor (subtypes) during oestrus and dioestrus in the bovine uterus.

    PubMed

    Ontsouka, E C; Reist, M; Graber, H; Blum, J W; Steiner, A; Hirsbrunner, G

    2004-12-01

    Serotoninergic and adrenergic receptors (5-HTR and AR) are involved in the regulation of uterine contractility. The objective of this study was to compare mRNA levels of 5-HTR(1A), 5-HTR(1B), 5-HTR(1D), 5-HTR(1F), 5-HTR(2A), 5-HTR(2B), 5-HTR(2C), 5-HTR(4) and alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), and beta(1), beta(2), beta(3)-AR in oestrus and dioestrus, and at three uterine locations (tip, middle and base) using quantitative real-time polymerase chain reaction. Uterine specimens consisting of endometrium and myometrium including vessels and serosa were collected from cows in oestrus (n = 10) and dioestrus (n = 15) respectively. Levels of 5-HTR and AR mRNA were expressed relative to the geometric mean of ribosomal RNA (18S), ubiquitin and glyceraldehyde phosphate dehydrogenase by the mean values of geNorm algorithm. 5-HTR(1A), 5-HTR(2C) and beta(3)-AR mRNA could not be detected in uterine tissues. The mRNA levels of 5-HTR(1F) and 5-HTR(2B) were lower (P < 0.05), but of 5-HTR(4) were higher (P < 0.05) in oestrus than in dioestrus. The mRNA levels of alpha(1A)-AR, alpha(2AD)-AR, alpha(2B)-AR were lower (P < 0.05), but of alpha(2C)-AR and beta(2)-AR were higher (P < 0.05) in oestrus than dioestrus. The mRNA levels of 5-HTR(1B) and 5-HTR(1D) (oestrus) and of alpha(2AD)-AR (dioestrus) differed among uterine locations (base > middle > tip; P < 0.05). The mRNA expression of 5-HTR and AR (subtypes) in bovine uterus was associated with cycle activity and varied according to uterine location. Additional studies on protein level will be carried out in order to elucidate the role of these receptor families on uterine contractility, which may then help to clarify clinical relevance.

  1. In situ nucleic acid hybridization of pyruvate dehydrogenase complex-E2 in primary biliary cirrhosis: pyruvate dehydrogenase complex-E2 messenger RNA is expressed in hepatocytes but not in biliary epithelium.

    PubMed

    Harada, K; Van de Water, J; Leung, P S; Coppel, R L; Nakanuma, Y; Gershwin, M E

    1997-01-01

    Pyruvate dehydrogenase-E2, or a cross-reactive molecule, has been shown by a variety of immunohistochemical methods to be present in increased amounts in biliary epithelial cells (BEC) in primary biliary cirrhosis (PBC). In this study, to further understand the nature of the immunoreactive molecule in BEC, we examined the expression of pyruvate dehydrogenase complex-E2 (PDC-E2) messenger RNA (mRNA) and PDC-E2 protein in sections of livers from patients and controls to help identify the molecule found in BEC. We performed in situ hybridization using an antisense probe against the major epitope of PDC-E2. The data were very striking and suggested that there was no increased production of PDC-E2 in BEC. For example, in livers from patients with PBC, PDC-E2 mRNA was found in periportal hepatocytes in 16 of 17 cases (94%). In contrast, interlobular bile ducts and septal bile ducts had detectable levels of PDC-E2 mRNA in only 1 of 17 (6%) and 3 of 8 (38%) cases, respectively. Interestingly, proliferating bile ductules contained detectable levels of mRNA in 12 of 15 cases (80%). In control liver, periportal hepatocytes were positive in 15 of 17 cases (88%). Interlobular bile ducts, septal bile ducts, and proliferating bile ductules expressed mRNA signals in 4 of 17 (24%), 2 of 10 (20%), and 14 of 16 (88%), respectively. When formalin-fixed, paraffin-embedded sections were examined by immunohistochemical staining with anti-PDC-E2 monoclonal antibody (mAb) C355.1, the interlobular bile ducts showed typical aberrant apical staining in all 10 PBC cases, but 0 of 9 liver controls. Periportal hepatocytes, proliferating bile ductules and infiltrating mononuclear cells stained with C355.1 but in a characteristic mitochondrial staining pattern. The presence of a PDC-E2-like molecule recognized by C355.1 is not reflected by the expression levels of PDC-E2 mRNA in the BEC of patients with PBC.

  2. Down-regulation of P2U-purinergic nucleotide receptor messenger RNA expression during in vitro differentiation of human myeloid leukocytes by phorbol esters or inflammatory activators.

    PubMed

    Martin, K A; Kertesy, S B; Dubyak, G R

    1997-01-01

    HL-60 human promyelocytic leukocytes express G protein-coupled P2U-purinergic nucleotide receptors (P2UR or P2Y2R) that activate inositol phospholipid hydrolysis and Ca24 mobilization in response to ATP or UTP. We examined the expression of functional P2UR and P2UR mRNA levels during in vitro differentiation of HL-60 cells by dibutyryl-cAMP (Bt2cAMP), which induces a granulocyte/neutrophil phenotype, or by phorbol-12-myristate-13-acetate (PMA), which induces a monocyte/macrophage phenotype. Both P2UR function and P2UR mRNA levels were only modestly attenuated during granulocytic differentiation by Bt2cAMP. In contrast, P2UR function, as assayed by either Ca2+ mobilization or inositol trisphosphate generation, was greatly reduced in PMA-differentiated cells. This inhibition of P2UR function was strongly correlated with PMA-induced decreases in P2UR mRNA levels, as assayed by Northern blot analysis or reverse transcription-polymerase chain reaction-based quantification. Although PMA induced an early, transient up-regulation of P2UR mRNA, this was rapidly followed by a sustained decrease in P2UR mRNA to a level 5-10-fold lower than that in undifferentiated HL-60 cells. The half-life of the P2UR transcript in HL-60 cells was approximately 60 min, and this was not affected by acute exposure (< or = 4 hr) to Bt2cAMP or PMA. PMA down-regulated P2UR mRNA in THP-1 monocytes and HL-60 granulocytes but not in A431 human epithelial cells or human keratinocytes. P2UR mRNA was also down-regulated in THP-1 monocytes differentiated into inflammatory macrophages by gamma-interferon and endotoxin. These data indicate that myeloid leukocytes possess tissue-specific mechanisms for the rapid modulation of P2UR expression and function during differentiation and inflammatory activation.

  3. Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen, EBNA1

    PubMed Central

    Tellam, Judy T.; Lekieffre, Lea; Zhong, Jie; Lynn, David J.; Khanna, Rajiv

    2012-01-01

    Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to

  4. Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1.

    PubMed

    Tellam, Judy T; Lekieffre, Lea; Zhong, Jie; Lynn, David J; Khanna, Rajiv

    2012-12-01

    Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to

  5. Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

    PubMed Central

    Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

    1994-01-01

    Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

  6. Diphosphoinositol Polyphosphates: Metabolic Messengers?

    PubMed Central

    Shears, Stephen B.

    2009-01-01

    The diphosphoinositol polyphosphates (“inositol pyrophosphates”) are a specialized subgroup of the inositol phosphate signaling family. This review proposes that many of the current data concerning the metabolic turnover and biological effects of the diphosphoinositol polyphosphates are linked by a common theme: these polyphosphates act as metabolic messengers. This review will also discuss the latest proposals concerning possible molecular mechanisms of action of this intriguing class of molecules. PMID:19439500

  7. Mercury MESSENGER Stamp Unveiling

    NASA Image and Video Library

    2011-05-03

    From left, NASA Deputy Director, Planetary Science Division, Science Mission Directorate, Jim Adams, NASA Kennedy Space Center Director of Education and External Relations Cheryl Hurst, United States Postal Service Vice President of Finance Steve Masse, NASA Mercury Astronaut Scott Carpenter, NASA Administrator Charles Boldin, Daughters of NASA astronaut Alan Shepard, Alice Wackermann, Laura Shepard Churchley, and Julie Jenkins, and NASA Kennedy Space Center Director Robert Cabana pose for a photograph during an unveiling ceremony of two USPS stamps that commemorate and celebrate 50 years of US Spaceflight and the MESSENGER program during an event, Wednesday, May 4, 2011 at the NASA Kennedy Space Center in Cape Canaveral, Fla. One stamp commemorates NASA’s Project Mercury, America’s first manned spaceflight program, and NASA astronaut Alan Shepard’s historic flight on May 5, 1961, aboard spacecraft Freedom 7. The other stamp draws attention to NASA’s unmanned MESSENGER mission, a scientific investigation of the planet Mercury. On March 17, 2011, MESSENGER became the first spacecraft to enter into orbit around Mercury. Photo Credit: (NASA/Bill Ingalls)

  8. Altered microtubule-associated tau messenger RNA isoform expression in livers of griseofulvin- and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-treated mice.

    PubMed

    Kenner, L; Zatloukal, K; Stumptner, C; Eferl, R; Denk, H

    1999-03-01

    Tau proteins belong to the family of microtubule-associated proteins (MAPs), which so far have been mostly detected in neuronal cells. Different domains on the protein serve different functions. By alternative splicing, several mRNAs and tau isoforms are created from one gene, which contain these functionally important domains to various degrees, and thus differ in their microtubule-related properties. In the present article, several novel observations are reported. Tau mRNA and proteins have been identified and further characterized in mouse liver. It is shown on the basis of mRNA determinations that at least three tau isoforms differing particularly with respect to their amino-terminal domains are present in mouse liver. The major and predominant isoform (isoform 1) lacks portions encoded by exons 2 and 3, which are responsible for cross-talk of microtubules with their environment ("projection domain"). Moreover, mRNA encoding tau protein with four repeats of the microtubule binding domain predominate in embryonal as well as adult mouse liver in contrast to brain, in which a shift from the predominant three-repeat isoform to the four-repeat isoform characterizes the transition from the embryonic to the adult stage. Intoxication with griseofulvin (GF) or 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) significantly affects in a reversible manner the levels of tau mRNA as well as isoform ratios in mouse liver, but not in mouse brain. Tau mRNAs are significantly increased in intoxicated mouse livers. Moreover, a shift to isoform 1 lacking exons 2 and 3 occurs. However, the increase in liver tau protein was less than expected from increased mRNA levels, which could be the result of translational or posttranslational regulation. The consequences on microtubular function are as yet unclear, but impairment can be expected because the overexpressed tau mRNA isoform lacks the domain that mediates interaction of microtubules with their environment. On the other hand, the

  9. Messenger RNA Detection in Leukemia Cell lines by Novel Metal-Tagged in situ Hybridization using Inductively Coupled Plasma Mass Spectrometry

    PubMed Central

    Ornatsky, Olga I.; Baranov, Vladimir I.; Bandura, Dmitry R.; Tanner, Scott D.; Dick, John

    2006-01-01

    Conventional gene expression profiling relies on using fluorescent detection of hybridized probes. Physical characteristics of fluorophores impose limitations on achieving a highly multiplex gene analysis of single cells. Our work demonstrates the feasibility of using metal-tagged in situ hybridization for mRNA detection by inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS as an analytical detector has a number of unique and relevant properties: 1) metals and their stable isotopes generate non-overlapping distinct signals that can be detected simultaneously; 2) these signals can be measured over a wide dynamic range; 3) ICP-MS is quantitative and very sensitive. We used commercial antibodies conjugated to europium (Eu) and gold together with biotinylated oligonucleotide probes reacted with terbium-labeled streptavidin to demonstrate simultaneous mRNA and protein detection by ICP-MS in leukemia cells. PMID:23662035

  10. Messenger RNA Detection in Leukemia Cell lines by Novel Metal-Tagged in situ Hybridization using Inductively Coupled Plasma Mass Spectrometry.

    PubMed

    Ornatsky, Olga I; Baranov, Vladimir I; Bandura, Dmitry R; Tanner, Scott D; Dick, John

    2006-01-01

    Conventional gene expression profiling relies on using fluorescent detection of hybridized probes. Physical characteristics of fluorophores impose limitations on achieving a highly multiplex gene analysis of single cells. Our work demonstrates the feasibility of using metal-tagged in situ hybridization for mRNA detection by inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS as an analytical detector has a number of unique and relevant properties: 1) metals and their stable isotopes generate non-overlapping distinct signals that can be detected simultaneously; 2) these signals can be measured over a wide dynamic range; 3) ICP-MS is quantitative and very sensitive. We used commercial antibodies conjugated to europium (Eu) and gold together with biotinylated oligonucleotide probes reacted with terbium-labeled streptavidin to demonstrate simultaneous mRNA and protein detection by ICP-MS in leukemia cells.

  11. Influences of sex, incubation temperature, and environmental quality on gonadal estrogen and androgen receptor messenger RNA expression in juvenile American alligators (Alligator mississippiensis).

    PubMed

    Moore, Brandon C; Milnes, Matthew R; Kohno, Satomi; Katsu, Yoshinao; Iguchi, Taisen; Guillette, Louis J

    2010-01-01

    Gonadal steroid hormone receptors play a vital role in transforming ligand signals into gene expression. We have shown previously that gonads from wild-caught juvenile alligators express greater levels of estrogen receptor 1 (ESR1) than estrogen receptor 2 (ESR2). Furthermore, sexually dimorphic ESR2 mRNA expression (female > male) observed in animals from the reference site (Lake Woodruff, FL, USA) was lost in alligators from the contaminated Lake Apopka (FL, USA). We postulated that environmental contaminant exposure could influence gonadal steroid hormone receptor expression. Here, we address questions regarding gonadal estrogen and androgen receptor (AR) mRNA expression in 1-yr-old, laboratory-raised alligators. What are relative expression levels within gonads? Do these levels vary between sexes or incubation temperatures? Can contaminant exposure change these levels? We observed a similar pattern of expression (ESR1 > AR > ESR2) in ovary and testis. However, both incubation temperature and environment modulated expression. Males incubated at 33.5 degrees C expressed greater AR levels than females incubated at 30 degrees C; dimorphic expression was not observed in animals incubated at 32 degrees C. Compared to Lake Woodruff alligators, Lake Apopka animals of both sexes showed lesser ESR2 mRNA expression levels. Employing cluster analyses, we integrated these receptor expression patterns with those of steroidogenic factors. Elevated ESR2 and CYP19A1 expressions were diagnostic of alligator ovary, whereas elevated HSD3B1, CYP11A1, and CYP17A1 expressions were indicative of testis. In contrast, AR, ESR1, and NR5A1 showed variable expressions that were not entirely associated with sex. These findings demonstrate that the mRNA expression of receptors required for steroid hormone signaling are modified by exposure to environmental factors, including temperature and contaminants.

  12. Influences of Sex, Incubation Temperature, and Environmental Quality on Gonadal Estrogen and Androgen Receptor Messenger RNA Expression in Juvenile American Alligators (Alligator mississippiensis)1

    PubMed Central

    Moore, Brandon C.; Milnes, Matthew R.; Kohno, Satomi; Katsu, Yoshinao; Iguchi, Taisen; Guillette, Louis J.

    2009-01-01

    Gonadal steroid hormone receptors play a vital role in transforming ligand signals into gene expression. We have shown previously that gonads from wild-caught juvenile alligators express greater levels of estrogen receptor 1 (ESR1) than estrogen receptor 2 (ESR2). Furthermore, sexually dimorphic ESR2 mRNA expression (female > male) observed in animals from the reference site (Lake Woodruff, FL, USA) was lost in alligators from the contaminated Lake Apopka (FL, USA). We postulated that environmental contaminant exposure could influence gonadal steroid hormone receptor expression. Here, we address questions regarding gonadal estrogen and androgen receptor (AR) mRNA expression in 1-yr-old, laboratory-raised alligators. What are relative expression levels within gonads? Do these levels vary between sexes or incubation temperatures? Can contaminant exposure change these levels? We observed a similar pattern of expression (ESR1 > AR > ESR2) in ovary and testis. However, both incubation temperature and environment modulated expression. Males incubated at 33.5°C expressed greater AR levels than females incubated at 30°C; dimorphic expression was not observed in animals incubated at 32°C. Compared to Lake Woodruff alligators, Lake Apopka animals of both sexes showed lesser ESR2 mRNA expression levels. Employing cluster analyses, we integrated these receptor expression patterns with those of steroidogenic factors. Elevated ESR2 and CYP19A1 expressions were diagnostic of alligator ovary, whereas elevated HSD3B1, CYP11A1, and CYP17A1 expressions were indicative of testis. In contrast, AR, ESR1, and NR5A1 showed variable expressions that were not entirely associated with sex. These findings demonstrate that the mRNA expression of receptors required for steroid hormone signaling are modified by exposure to environmental factors, including temperature and contaminants. PMID:19759368

  13. Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

    PubMed Central

    Eissa, Nour; Kermarrec, Laëtitia; Hussein, Hayam; Bernstein, Charles N.; Ghia, Jean-Eric

    2017-01-01

    2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis is an experimental model that mimics Crohn’s disease. Appropriateness of reference genes is crucial for RT-qPCR. This is the first study to determine the stability of reference gene expression (RGE) in mice treated with DNBS. DNBS experimental Colitis was induced in male C57BL/6 mice. RNA was extracted from colon tissue and comprehensive analysis of 13 RGE was performed according to predefined criteria. Relative colonic TNF-α and IL-1β mRNA levels were calculated. Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest fluctuation within the inflamed and control groups. Conversely, ribosomal protein large P0 (Rplp0), non-POU domain containing (Nono), TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. TNF-α and IL-1β mRNA levels was dependent on the reference gene used and varied from significant when the most stable genes were used to non-significant when the least stable genes were used. The appropriate choice of RGE is critical to guarantee satisfactory normalization of RT-qPCR data when using DNBS-Model. We recommend using Rplp0, Nono, Tbp, Hprt and Eef2 instead of common reference genes. PMID:28186172

  14. Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

    PubMed Central

    Ylä-Herttuala, S; Rosenfeld, M E; Parthasarathy, S; Sigal, E; Särkioja, T; Witztum, J L; Steinberg, D

    1991-01-01

    Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor. Images PMID:2010531

  15. Messenger RNA profiling of rabbit quadriceps femoris after repeat injections of botulinum toxin: Evidence for a dynamic pattern without further structural alterations.

    PubMed

    Hart, David A; Fortuna, Rafael; Herzog, Walter

    2017-08-18

    Onabotulinum toxin type A (BoNT-A) is widely used clinically, but it may cause adverse effects. Earlier studies showed repeat BoNT-A injections did not cause progressive atrophy or function loss. The purpose of this study was to assess the influence of repeat BoNT-A injections into rabbit muscle on subsequent molecular alterations. Twenty-two rabbits received 0, 1, 2, or 3 BoNT-A injections in the quadriceps femoris muscle at 3-month intervals and were euthanized 6 months after the last injection. Aliquots of both injected and contralateral muscle were frozen, and the total RNA quantified. Total RNA per illigram wet weight tissue was significantly elevated compared with control after 1, 2, or 3 BoNT-A injections. Analysis of mRNA levels for inflammatory molecules, proteinases, adipokines, and mesenchymal stem cells were elevated with increasing BoNT-A injections in injected leg and contralateral leg. Future studies should focus on the safety and possible complications of repeat BoNT-A treatments. Muscle Nerve, 2017. © 2017 The Authors Muscle & Nerve Published by Wiley Periodicals, Inc.

  16. Taurine and beta-alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA.

    PubMed

    Horikoshi, T; Asanuma, A; Yanagisawa, K; Anzai, K; Goto, S

    1988-09-01

    The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.

  17. Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis.

    PubMed Central

    Hamosh, A; Trapnell, B C; Zeitlin, P L; Montrose-Rafizadeh, C; Rosenstein, B J; Crystal, R G; Cutting, G R

    1991-01-01

    Cystic fibrosis (CF) is the most common, lethal inherited disorder in the Caucasian population. We have recently reported two African-American patients with nonsense mutations in each CF gene and severe pancreatic disease, but mild pulmonary disease. In order to examine the effect of these nonsense mutations on CF gene expression, bronchial and nasal epithelial cells were obtained from one of these patients (no. 246), a compound heterozygote for nonsense mutations R553X and W1316X; a healthy normal individual; a patient (no. 528) homozygous for the common CF mutation (delta F508); and a CF patient (no. 272) who carries the R553X mutation and a missense mutation, S549N. When mRNA from bronchial cells of the normal individual, the delta F508 homozygote, and the S549N/R553X compound heterozygote was reverse transcribed and amplified by polymerase chain reaction using primers derived from the CF gene, DNA fragments of the predicted size were observed. However, patient no. 246 with nonsense mutations in each CF gene has no detectable cystic fibrosis transmembrane conductance regulator (CFTR) messenger RNA, and therefore should have severely diminished, and possibly absent, CFTR protein. Furthermore, less than 2% of the CFTR transcripts in nasal epithelial cells from patient no. 272 (S549N/R553X) were derived from the gene with the nonsense mutation. We conclude that severe reduction in CFTR mRNA causes CF, but can have different consequences in the lung and pancreas. Images PMID:1721624

  18. Analysis of GNAS1 and Overlapping Transcripts Identifies the Parental Origin of Mutations in Patients with Sporadic Albright Hereditary Osteodystrophy and Reveals a Model System in Which to Observe the Effects of Splicing Mutations on Translated and Untranslated Messenger RNA

    PubMed Central

    Rickard, Sarah J.; Wilson, Louise C.

    2003-01-01

    Albright hereditary osteodystrophy (AHO) is caused by heterozygous deactivating GNAS1 mutations. There is a parent-of-origin effect. Maternally derived mutations are usually associated with resistance to parathyroid hormone termed “pseudohypoparathyroidism type Ia.” Paternally derived mutations are associated with AHO but usually normal hormone responsiveness, known as “pseudo-pseudohypoparathyroidism.” These observations can be explained by tissue-specific GNAS1 imprinting. Regulation of the genomic region that encompasses GNAS1 is complex. At least three upstream exons that splice to exon 2 of GNAS1 and that are imprinted have been reported. NESP55 is exclusively maternally expressed, whereas exon 1A and XLαs are exclusively paternally expressed. We set out to identify the parental origin of GNAS1 mutations in patients with AHO by searching for their mutation in the overlapping transcripts. This information would be of value in patients with sporadic disease, for predicting their endocrine phenotype and planning follow-up. In doing so, we identified mutations that resulted in nonsense-mediated decay of the mutant Gsα transcript but that were detectable in NESP55 messenger RNA (mRNA), probably because they lie within its 3′ untranslated region. Analysis of the NESP55 transcripts revealed the creation of a novel splice site in one patient and an unusual intronic mutation that caused retention of the intron in a further patient, neither of which could be detected by analysis of the Gsα complementary DNA. This cluster of overlapping transcripts represents a useful model system in which to analyze the effects that mutant sequence has on mRNA—in particular, splicing—and the mechanisms of nonsense-mediated mRNA decay. PMID:12624854

  19. Atrazine inhibits pulsatile gonadotropin-releasing hormone (GnRH) release without altering GnRH messenger RNA or protein levels in the female rat.

    PubMed

    Foradori, Chad D; Zimmerman, Arthur D; Hinds, Laura R; Zuloaga, Kristen L; Breckenridge, Charles B; Handa, Robert J

    2013-01-01

    Atrazine (ATR) is a commonly used pre-emergence/early postemergence herbicide. Previous work has shown that exposure to high doses of ATR in rats results in blunting of the hormone-induced luteinizing hormone (LH) surge and inhibition of pulsatile LH release without significantly reducing pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) agonist. Accompanying the reduction in the LH surge was an attenuation of GnRH neuronal activation. These findings suggest that ATR exposure may be acting to inhibit GnRH release. In this study, we examined GnRH directly to determine the effect of high doses of ATR on GnRH pulsatile release, gene expression, and peptide levels in the female rat. Ovariectomized adult female Wistar rats were treated with ATR (200 mg/kg) or vehicle for 4 days via gavage. Following the final treatment, GnRH release was measured from ex vivo hypothalamic explants for 3 h. In another experiment, animals were administered either vehicle or ATR (50, 100, or 200 mg/kg) daily for 4 days. Following treatment, in situ hybridization was performed to examine total GnRH mRNA and the primary GnRH heterogeneous nuclear RNA transcript. Finally, GnRH immunoreactivity and total peptide levels were measured in hypothalamic tissue of treated animals. ATR treatment resulted in no changes to GnRH gene expression, peptide levels, or immunoreactivity but a reduction in GnRH pulse frequency and an increased pulse amplitude. These findings suggest that ATR acts to inhibit the secretory dynamics of GnRH pulses without interfering with GnRH mRNA and protein synthesis.

  20. RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP.

    PubMed

    Kellenberger, Colleen A; Wilson, Stephen C; Sales-Lee, Jade; Hammond, Ming C

    2013-04-03

    Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.

  1. Temporal Variation for the Expression of Catalase in DROSOPHILA MELANOGASTER: Correlations between Rates of Enzyme Synthesis and Levels of Translatable Catalase-Messenger RNA

    PubMed Central

    Bewley, Glenn C.; Mackay, William J.; Cook, Julia L.

    1986-01-01

    Two variants that alter the temporal expression of catalase have been isolated from a set of third chromosome substitution lines. Each variant has been mapped to a cytogenetic interval flanked by the visible markers st (3-44.0) and cu (3-50.0) at a map position of 47.0, which is within or near the interval 75D-76A previously identified as containing the catalase structural gene on the bases of dosage responses to segmental aneuploidy. Each variant operates by modulating the rate of enzyme synthesis and the level of translatable catalase-mRNA. PMID:3091448

  2. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene.

    PubMed

    Rouvier, E; Luciani, M F; Mattéi, M G; Denizot, F; Golstein, P

    1993-06-15

    To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.

  3. A carvacrol-thymol blend decreased intestinal oxidative stress and influenced selected microbes without changing the messenger RNA levels of tight junction proteins in jejunal mucosa of weaning piglets.

    PubMed

    Wei, H-K; Xue, H-X; Zhou, Z X; Peng, J

    2017-02-01

    Recent studies indicate that intestinal oxidative stress and microbiota imbalance is involved in weaning-induced intestinal dysfunction in piglets. We have investigated the effect of feeding a carvacrol-thymol blend supplemented diet on intestinal redox status, selected microbial populations and the intestinal barrier in weaning piglets. The piglets (weaned at 21 days of age) were randomly allocated to two groups with six pens per treatment and 10 piglets per pen. At weaning day (21 days of age), six piglets were sacrificed before weaning to serve as the preweaning group. The weaned group was fed with a basal diet, while the weaned-CB group was fed with the basal diet supplemented with 100 mg/kg carvacrol-thymol (1 : 1) blend for 14 days. On day 7 post-weaning, six piglets from each group were sacrificed to determine intestinal redox status, selected microbial populations, messenger RNA (mRNA) transcript levels of proinflammatory cytokines and biomarkers of intestinal barrier function. Weaning resulted in intestinal oxidative stress, indicated by the increased concentration of reactive oxygen species and thiobarbituric acid-reactive substances present in the intestine. Weaning also reduced the population of Lactobacillus genus and increased the populations of Enterococcus genus and Escherichia coli in the jejunum, and increased mRNA levels of tumor necrosis factor α (TNF-α), interleukin 1β and interleukin 6 (IL-6). In addition, decreased mRNA levels of zonula occludens and occludin in the jejunal mucosa and increased plasma diamine oxidase concentrations indicated that weaning induced dysfunction of the intestinal barrier. On day 7 post-weaning, supplementation with the carvacrol-thymol blend restored weaning-induced intestinal oxidative stress. Compared with the weaned group, the weaned-CB group had an increased population of Lactobacillus genus but reduced populations of Enterococcus genus and E. coli in the jejunum and decreased mRNA levels of TNF-α. The

  4. MESSENGER: Science payload status

    NASA Astrophysics Data System (ADS)

    McNutt, R.; Solomon, S.; Gold, R.

    2003-04-01

    MESSENGER is a NASA Discovery mission to reach Mercury and orbit that planet for an Earth year, gathering data with a miniaturized scientific payload. The MESSENGER project is now entering the integration and test phase as the spacecraft is assembled and the instruments are calibrated and delivered to the spacecraft. The Gamma-Ray and Neutron spectrometer (GRNS) and X-Ray Spectrometer (XRS) experienced detector changes in order to increase the signal-to-noise ratio (based upon more experience with similar instrumentation on the Near Earth Asteroid Rendezvous, NEAR-Shoemaker, mission and on Mars Odyssey). The gamma-ray portion of GRNS uses a high-purity germanium crystal cooled to ˜90K and surrounded by an active shield to detect characteristic gamma-rays from the planet. The neutron spectrometer uses Li-glass and plastic scintillators to detect and separate thermal, epithermal, and fast neutrons. The XRS spectrometer uses three gas-filled proportional counters looking at the planet and a solar monitor to measure X-ray fluorescence lines from the planet's surface. These instruments thus provide information on elemental abundances. The optical remote-sensing instruments map the planet in several spectral bands (Mercury Dual Imaging System -- MDIS), measure surface spectral reflectance in the visible and infra-red and exospheric emission lines in the ultraviolet and visible (Mercury Atmospheric and Surface Composition Spectrometer -- MASCS), and measure surface topography (Mercury Laser Altimeter -- MLA). The combination of altimetry with MLA and radio-science (RS) measurements will allow maps of the gravitational field of the planet and inference of the planet's obliquity and physical amplitude. The combination of boom-mounted magnetometer (MAG) and combined Energetic Particle and Plasma Spectrometer (EPPS) allows internal and external sources of magnetic field to be separated, providing knowledge of both Mercury's internal structure and its magnetosphere and

  5. Mouse testis brain RNA-binding protein/translin selectively binds to the messenger RNA of the fibrous sheath protein glyceraldehyde 3-phosphate dehydrogenase-S and suppresses its translation in vitro.

    PubMed

    Yang, Juxiang; Chennathukuzhi, Vargheese; Miki, Kiyoshi; O'Brien, Deborah A; Hecht, Norman B

    2003-03-01

    The testis brain RNA-binding protein (TB-RBP/translin) is a DNA- and RNA-binding protein with multiple functions. As an RNA-binding protein, TB-RBP binds to conserved sequence elements often present in the 3' untranslated regions (UTRs) of specific mRNAs modulating their translation and transport. To identify additional mRNA targets of TB-RBP, immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) assays were carried out using an affinity-purified antibody to TB-RBP with testicular extracts. Gapds mRNA was found to be selectively precipitated in a TB-RBP-mRNA complex. Consistent with the delayed translation of GAPDS and the subcellular ribonucleoprotein location of TB-RBP, polysomal gradient analysis showed that most of the Gapds mRNA in adult testis extracts was present in the nonpolysomal fractions. In vitro translation assays revealed that Gapds mRNA translation was inhibited by recombinant TB-RBP or by a TB-RBP mutant protein, Nb, capable of binding RNA. No inhibition was seen with mutant forms of TB-RBP lacking domains required for RNA binding, including the TB-RBP Cb mutant and the C-terminal-truncated form of TB-RBP that disrupts the leucine zipper. As an additional indicator of the specificity of TB-RBP inhibition of Gapds mRNA translation, a putative TB-RBP binding H-element was deleted from the 5' UTR of the Gapds mRNA. No translational inhibition by recombinant TB-RBP was seen with Gapds mRNA lacking the H element. These data suggest that TB-RBP is involved in the posttranscriptional regulation of Gapds gene expression during spermiogenesis. Moreover, the Gapds mRNA is the first mRNA shown to have a functional TB-RBP binding site in its 5' UTR.

  6. Somatostatin inhibits stem cell factor messenger RNA expression by Sertoli cells and stem cell factor-induced DNA synthesis in isolated seminiferous tubules.

    PubMed

    Goddard, I; Bauer, S; Gougeon, A; Lopez, F; Giannetti, N; Susini, C; Benahmed, M; Krantic, S

    2001-12-01

    Immature porcine Sertoli cells have been reported to be targets for the regulatory peptide somatostatin (SRIF), which inhibits the basal and FSH-induced proliferation of Sertoli cells through a decrease of cAMP production. In the present study, we show that SRIF inhibits both basal and FSH-stimulated expression of the stem cell factor (SCF), a Sertoli cell-specific gene. The SRIF-mediated inhibition of forskolin-triggered, but not of 8-bromoadenosine-cAMP-triggered, SCF mRNA expression demonstrates the involvement of adenylyl cyclase in underlying peptide actions. Moreover, these effects require functional coupling of specific plasma membrane receptors to adenylyl cyclase via inhibitory G proteins, because pertussis toxin prevents SRIF-mediated inhibition of SCF mRNA expression. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays suggest the involvement of sst2 receptors in SRIF actions on Sertoli cells. The biological relevance of these data is supported by an SRIF-mediated decrease in SCF-induced incorporation of [(3)H]thymidine in isolated seminiferous tubules. In situ hybridization and confocal microscopy show that, in seminiferous tubules only, spermatogonia display both c-kit and sst2 receptors. Taken together, these results suggest that SCF-stimulated DNA synthesis can be inhibited by SRIF in spermatogonia, but not in Sertoli and peritubular cells. Combined RT-PCR and immunohistochemical approaches point toward spermatogonia and Leydig cells as the source of testicular SRIF. These data argue in favor of paracrine/autocrine SRIF actions in testis.

  7. Effect of N-Feruloylserotonin and Methotrexate on Severity of Experimental Arthritis and on Messenger RNA Expression of Key Proinflammatory Markers in Liver

    PubMed Central

    Poništ, Silvester; Mihálová, Danica; Nosáľ, Radomír; Harmatha, Juraj; Hrádková, Iveta; Šišková, Katarína; Bezáková, Lýdia

    2016-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease, leading to progressive destruction of joints and extra-articular tissues, including organs such as liver and spleen. The purpose of this study was to compare the effects of a potential immunomodulator, natural polyphenol N-feruloylserotonin (N-f-5HT), with methotrexate (MTX), the standard in RA therapy, in the chronic phase of adjuvant-induced arthritis (AA) in male Lewis rats. The experiment included healthy controls (CO), arthritic animals (AA), AA given N-f-5HT (AA-N-f-5HT), and AA given MTX (AA-MTX). N-f-5HT did not affect the body weight change and clinical parameters until the 14th experimental day. Its positive effect was rising during the 28-day experiment, indicating a delayed onset of N-f-5HT action. Administration of either N-f-5HT or MTX caused reduction of inflammation measured as the level of CRP in plasma and the activity of LOX in the liver. mRNA transcription of TNF-α and iNOS in the liver was significantly attenuated in both MTX and N-f-5HT treated groups of arthritic rats. Interestingly, in contrast to MTX, N-f-5HT significantly lowered the level of IL-1β in plasma and IL-1β mRNA expression in the liver and spleen of arthritic rats. This speaks for future investigations of N-f-5HT as an agent in the treatment of RA in combination therapy with MTX. PMID:27556049

  8. Messenger RNA stockpile of cyclin B, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor receptor Ib, and p53 in the rainbow trout oocyte in relation with developmental competence.

    PubMed

    Aegerter, Sandrine; Jalabert, Bernard; Bobe, Julien

    2004-02-01

    In the present study, correlations between the oocyte messenger RNA (mRNA) stockpile of Cyclin B, insulin-like growth factor I (IGF-I), insulin-like growth factor (IGF-II), insulin-like growth factor receptor Ib (IGFR Ib), and p53 transcripts and the developmental competence of the oocyte were studied. For this purpose, post-ovulatory ageing was used as a tool to generate oocytes of varying developmental competence. Mature female rainbow trout were held at 12 degrees C and periodically checked for ovulation. Oocytes were collected from each female at ovulation and 5, 14, 21 days later. For each collected egg batch, the abundance of several mRNAs in the oocyte was analyzed by real-time PCR and embryo development was monitored after fertilization. Egg quality was estimated not only through embryonic survival but also by studying the occurrence of specific morphological abnormalities. The present study showed that oocyte post-ovulatory ageing was associated with variations of the relative abundance of several studied transcripts within the oocyte. In addition, the abundance of specific mRNAs could be correlated with either the embryonic survival or the occurrence of malformations. Thus, the abundance of IGFR Ib and Cyclin B transcripts in the oocyte was correlated with the occurrence of morphological abnormalities observed at yolk-sac resorption (negatively for IGFR Ib and positively for Cyclin B), while the maternal stockpile of IGF-I, IGF-II, and IGFR Ib mRNAs was positively correlated with embryonic survival. Copyright 2004 Wiley-Liss, Inc.

  9. Dynamical Messengers for Gauge Mediation

    SciTech Connect

    Hook, Anson; Torroba, Gonzalo; /SLAC /Stanford U., Phys. Dept.

    2011-08-17

    We construct models of indirect gauge mediation where the dynamics responsible for breaking supersymmetry simultaneously generates a weakly coupled subsector of messengers. This provides a microscopic realization of messenger gauge mediation where the messenger and hidden sector fields are unified into a single sector. The UV theory is SQCD with massless and massive quarks plus singlets, and at low energies it flows to a weakly coupled quiver gauge theory. One node provides the primary source of supersymmetry breaking, which is then transmitted to the node giving rise to the messenger fields. These models break R-symmetry spontaneously, produce realistic gaugino and sfermion masses, and give a heavy gravitino.

  10. MESSENGER: Exploring Mercury's Magnetosphere

    NASA Technical Reports Server (NTRS)

    Slavin, James A.

    2008-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. Mercury's magnetosphere is unique in many respects. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only - 1000 to 2000 km above the surface. For this reason there are no closed dri-fi paths for energetic particles and, hence, no radiation belts; the characteristic time scales for wave propagation and convective transport are short possibly coupling kinetic and fluid modes; magnetic reconnection at the dayside magnetopause may erode the subsolar magnetosphere allowing solar wind ions to directly impact the dayside regolith; inductive currents in Mercury's interior should act to modify the solar In addition, Mercury's magnetosphere is the only one with its defining magnetic flux tubes rooted in a planetary regolith as opposed to an atmosphere with a conductive ionosphere. This lack of an ionosphere is thought to be the underlying reason for the brevity of the very intense, but short lived, approx. 1-2 min, substorm-like energetic particle events observed by Mariner 10 in Mercury's magnetic tail. In this seminar, we review what we think we know about Mercury's magnetosphere and describe the MESSENGER science team's strategy for obtaining answers to the outstanding science questions surrounding the interaction of the solar wind with Mercury and its small, but dynamic magnetosphere.

  11. Relative messenger RNA abundance in bovine oocytes collected in vitro or in vivo before and 20 hr after the preovulatory luteinizing hormone surge.

    PubMed

    Lonergan, Patrick; Gutiérrez-Adán, Alfonso; Rizos, Dimitrios; Pintado, Belen; de la Fuente, Julio; Boland, Maurice P

    2003-11-01

    In the cyclic cow, final maturation of the ovulatory follicle is initiated by the preovulatory luteinizing hormone (LH) surge. During the subsequent 24 hr period, the oocyte nucleus undergoes meiotic progression to metaphase II and several changes in cytoplasmic organization take place. We have previously shown that oocytes recovered at the time of the LH peak and matured in vitro are less competent to reach the blastocyst stage than their counterparts recovered 20 hr later following in vivo maturation, despite both groups undergoing IVF and culture in parallel. The objective of this study was to compare, using real-time quantitative RT-PCR, the relative abundance of various developmentally important gene transcripts in these oocytes. The groups used were mature bovine oocytes originating from: (1) 2-6 mm follicles from slaughterhouse ovaries; (2) preovulatory follicles punctured by ovum pick-up just before the LH surge (i.e., immature) and matured in vitro; or (3) preovulatory follicles punctured 20 hr later, just prior to ovulation (i.e., in vivo matured). In addition, immature oocytes from 2-6 mm follicles were examined. We examined the relative mRNA expression of five enzymes involved in protection against free oxygen radicals (mitochondrial Mn-superoxide dismutase, MnSOD, cytosolic Cu/Zn superoxide dismutase, Cu/ZnSOD, gamma-glutamyl-cysteine transferase, GCS, glutathione peroxidase, GPX, sarcosine oxidase, SOX), a transcript involved in follicular development (growth differentiation factor-9, GDF-9), transcripts involved in glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH, glucose transporter type-1 and -8, Glut-1, Glut-8) and genes involved in cell cycle events, Cyclin A and B, and poly(A) polymerase (PAP). Transcripts for all genes were detected, irrespective of oocyte origin. While differences were not significant in all cases, variations in levels of transcript abundance between the groups were related to developmental competence. In

  12. Insulin-like growth factor II messenger RNA-binding protein-3 is an indicator of malignant phyllodes tumor of the breast.

    PubMed

    Takizawa, Katsumi; Yamamoto, Hidetaka; Taguchi, Kenichi; Ohno, Shinji; Tokunaga, Eriko; Yamashita, Nami; Kubo, Makoto; Nakamura, Masafumi; Oda, Yoshinao

    2016-09-01

    The aim of this study was to elucidate the clinicopathological and prognostic significance of the expressions of insulin-like growth factor II mRNA-binding protein-3 (IMP3) and epidermal growth factor receptor (EGFR) in phyllodes tumors (PTs). Immunohistochemical staining for IMP3 and EGFR was performed in 130 cases of primary PTs (83 benign, 28 borderline, 19 malignant), 34 recurrent/metastatic PTs, and 26 fibroadenomas (FAs). Among the primary tumors, a high expression of IMP3 was significantly more frequently present in malignant PTs (17/19, 89%) than in the FAs (0/26, 0%), benign PTs (0/83, 0%) and borderline PTs (3/28, 11%). The recurrent and metastatic lesions of malignant PTs also showed high IMP3 expression (3/5 [60%] and 6/6 [100%], respectively). Most malignant PTs showed strong IMP3 expression at the interductal area or more diffusely, whereas weak and focal (low) expression of IMP3 was limited to the periductal area in FAs and benign PTs. EGFR overexpression was significantly correlated with tumor grade and high IMP3 expression. Overexpressions of IMP3 and EGFR were significantly associated with shorter periods of metastasis-free and disease-free survival. The results suggest that high expressions of IMP3 and EGFR with a characteristic staining pattern may be helpful for both identifying malignant PT and predicting the prognosis of these tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse.

    PubMed

    Frohman, M A; Downs, T R; Chomczynski, P; Frohman, L A

    1989-10-01

    We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. 12 CFR 7.1012 - Messenger service.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 1 2012-01-01 2012-01-01 false Messenger service. 7.1012 Section 7.1012 Banks... Bank Powers § 7.1012 Messenger service. (a) Definition. For purposes of this section, a “messenger... establish and operate a messenger service, or use, with its customers, a third party messenger service....

  15. 12 CFR 7.1012 - Messenger service.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 1 2011-01-01 2011-01-01 false Messenger service. 7.1012 Section 7.1012 Banks... Bank Powers § 7.1012 Messenger service. (a) Definition. For purposes of this section, a “messenger... establish and operate a messenger service, or use, with its customers, a third party messenger service....

  16. 12 CFR 7.1012 - Messenger service.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Messenger service. 7.1012 Section 7.1012 Banks... Bank Powers § 7.1012 Messenger service. (a) Definition. For purposes of this section, a “messenger... establish and operate a messenger service, or use, with its customers, a third party messenger service....

  17. 12 CFR 7.1012 - Messenger service.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 1 2013-01-01 2013-01-01 false Messenger service. 7.1012 Section 7.1012 Banks... Bank Powers § 7.1012 Messenger service. (a) Definition. For purposes of this section, a “messenger... establish and operate a messenger service, or use, with its customers, a third party messenger service....

  18. 12 CFR 7.1012 - Messenger service.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Messenger service. 7.1012 Section 7.1012 Banks... Bank Powers § 7.1012 Messenger service. (a) Definition. For purposes of this section, a “messenger... establish and operate a messenger service, or use, with its customers, a third party messenger service....

  19. The MESSENGER science payload

    NASA Astrophysics Data System (ADS)

    Gold, Robert E.; McNutt, Ralph L., Jr.; Solomon, Sean C.; MESSENGER Team

    2003-11-01

    The MESSENGER spacecraft will orbit Mercury and gather data for one Earth year with a miniaturized scientific payload. The MESSENGER project is in the integration and test phase in mid 2003. Seven assembled and calibrated instruments are mounted on the spacecraft. The Gamma-Ray and Neutron Spectrometer has a Gamma-Ray Spectrometer to measure atomic composition with a high-purity germanium detector and a Neutron Spectrometer that uses lithium-glass and boron-loaded plastic scintillators for sensing thermal, epithermal, and fast neutrons. The X-Ray Spectrometer measures Mercury surface elemental abundances by examining solar-flare-induced X-ray fluorescence lines. Three gas-filled proportional counters detect the X-ray fluorescence lines from the planet's surface, and a solid-state solar monitor measures the X-ray input to the planet. The Mercury Dual Imaging System (MDIS) has both wide-field and narrow-field cameras to map the surface of the planet. MDIS is also multi-spectral, with a 12-position filter wheel for the wide-field camera. The Mercury Atmospheric and Surface Composition Spectrometer measures both surface spectral reflectance in the visible and near infrared and exospheric emission lines in the ultraviolet and visible. The Mercury Laser Altimeter (MLA) determines the range to the planet with a resolution of 0.3 m. MLA will be combined with the radio-science investigation to map the gravitational field of the planet and determine the obliquity and physical libration amplitude. A magnetometer, mounted on a 3.6-m boom, will map the internal and external magnetic field. The Energetic Particle and Plasma Spectrometer will measure particles accelerated in the magnetosphere and the interactions of the magnetosphere with the solar wind. MDIS has its own pivot platform. All of the other instruments are fixed to the spacecraft. Pointing is accomplished by steering the entire spacecraft. All of the instruments are designed to deal with the extreme thermal

  20. A Protein of Molecular Weight 78,000 Bound to the Polyadenylate Region of Eukaryotic Messenger RNAs

    PubMed Central

    Blobel, Günter

    1973-01-01

    Two distinct proteins were found to be tightly bound to the heterogeneous messenger RNAs associated with polysomes in mouse L cells and rat hepatocytes. The molecular weight (78,000) of the larger of these two proteins is identical to that of the protein previously found associated with rabbit globin messenger RNA. This protein is shown to be bound to the adenylate-rich region of messenger RNA. The molecular weight of the smaller protein associated with messenger RNAs from hepatic and L-cell polysomes is similar to that found in globin messenger ribonucleoprotein. Images PMID:4515002

  1. Messenger RNA-based therapeutics for brain diseases: An animal study for augmenting clearance of beta-amyloid by intracerebral administration of neprilysin mRNA loaded in polyplex nanomicelles.

    PubMed

    Lin, Chin-Yu; Perche, Federico; Ikegami, Masaru; Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2016-08-10

    Alzheimer's disease (AD) pathogenesis is considered to be the metabolic imbalance between anabolism and clearance of amyloid-beta (Aβ), and the strategy of breaking the equilibrium between soluble and insoluble forms of Aβ is likely to help prevent the progression of AD. Neprilysin (NEP) plays a major role in the clearance of Aβ in the brain, and its supplementation using viral vectors has shown to decrease Aβ deposition and prevent pathogenic changes in the brain. In this study, we developed a new therapeutic strategy by mRNA-based gene introduction. mRNA has the advantages of negligible risk of random integration into genome and not needing to be transcribed precludes the need for nuclear entry. This allows efficient protein expression in slowly-dividing or non-dividing cells, such as neural cells. We constructed mRNA encoding the mouse NEP protein and evaluated its ability degrade Aβ. In vitro transfection of NEP mRNA to primary neurons exhibited Amyloid Precursor Protein (APP) degradation activity superior to that of NEP encoding plasmid DNA. We then evaluated the in vivo activity of NEP mRNA by intracerebroventricular (i.c.v.) infusion using a cationic polymer-based PEGylated nanocarrier to form polyplex nanomicelles, which have been shown to have a high potential to deliver mRNA to various target tissues and organs. Nanomicelles carrying a GFP-NEP fusion mRNA produced efficient protein expression in a diffuse manner surrounding the ventricular space. An ELISA evaluation revealed that the mRNA infusion significantly augmented NEP level and effectively reduced the concentration of Aβ that had been supplemented in the mouse brain. To the best of our knowledge, this is the first study to demonstrate the therapeutic potential of introducing exogenous mRNA for the treatment of brain diseases, opening the new era of mRNA-based therapeutics.

  2. Insulin-like growth factor I messenger RNA and protein are expressed in the human lymph node and distinctly confined to subtypes of macrophages, antigen-presenting cells, lymphocytes and endothelial cells.

    PubMed

    Oberlin, Dominique; Fellbaum, Christian; Eppler, Elisabeth

    2009-11-01

    Insulin-like growth factor I (IGF-I) is a potent hormone that stimulates growth and differentiation and inhibits apoptosis in numerous tissues. Preliminary evidence suggests that IGF-I exerts differentiating, mitogenic and restoring activities in the immune system but the sites of synthesis of local IGF-I are unknown. Identification of these sites would allow the functional role of local IGF-I to be clarified. The presence of IGF-I in non-immune cells suggests that it acts as a trophic factor, while its occurrence in subtypes of lymphocytes or antigen-presenting cells indicates paracrine/autocrine direct regulatory involvement of IGF-I in the human immune response. The present study investigated the location of IGF-I messenger RNA and protein on archival human lymph node samples by in situ hybridization, immunohistochemistry and double immunofluorescence staining using an IGF-I probe and antisera specific for human IGF-I and CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (macrophages), CD21 (follicular dendritic cells), S100 (interdigitating dendritic cells) and podoplanin (fibroblastic reticular cells). Numerous cells within the B- and T-cell compartments expressed the IGF-I gene, and the majority of these cells were identified as macrophages. Solitary follicular dendritic cells exhibited IGF-I. A few T lymphocytes, and no B lymphocytes, contained IGF-I immunoreactive material. Furthermore, IGF-I immunoreactive cells outside the follicles that did not react with CD3, CD20, S100 or podoplanin markers were identified as high-endothelial venule (HEV) cells. From this we conclude that the main task of IGF-I in human non-tumoral lymph node may be autocrine and paracrine regulation of the differentiation, stimulation and survival of lymphocytes, antigen-presenting cells and macrophages and the differentiation and maintenance of HEV cells.

  3. Nested reverse transcriptase-polymerase chain reactions targeting the messenger RNA of icl2, hspx, and rRNAP1 genes to detect viable Mycobacterium tuberculosis directly from clinical specimens.

    PubMed

    Lakshmipathy, Dhanurekha; Kulandai, Lily Therese; Ramasubban, Gayathri; Hajib Narahari Rao, Madhavan; Rathinam, Sridhar; Narasimhan, Meenakshi

    2015-12-01

    There is an urgent need for a rapid and reliable test to detect actively multiplying Mycobacterium tuberculosis directly from clinical specimens for an early initiation of the appropriate antituberculous treatment. This study was aimed at the optimization and application of nested reverse transcriptase-PCR (nRT-PCR) targeting the messenger RNA of the icl2, hspx, and rRNAP1 genes directly from sputum specimens, and their evaluation against the culture by the BACTEC MicroMGIT mycobacterial culture system. 203 Sputum samples from clinically suspected tuberculosis patients and 30 control specimens (clinically proven viral or bacterial infections other than tuberculosis) were included in this study. The mycobacterial culture was performed by the BACTEC MicroMGIT system following the manufacturer's instructions. The primers for nRT-PCRs targeting icl2, hspx, and rRNAP1 genes were indigenously designed using the Primer-BLAST software, and optimized for sensitivity and specificity. The icl2, hspx, and rRNAP1 genes were able to pick up 63.9%, 67.2%, and 58.75%, respectively, of culture-negative sputum specimens collected from clinically suspected tuberculosis patients. However, three (1.4%) were negative for nRT-PCR, but M. tuberculosis culture positive. All the 30 controls were negative for culture by the BACTEC MicroMGIT method and all three nRT-PCR. The novel nRT-PCRs targeting icl2, hspx, and rRNAP1 genes developed in this study are rapid and reliable diagnostic tools to detect viable M. tuberculosis directly from sputum specimens. However, further study by including a larger number of sputum specimens needs to be carried out to ascertain the diagnostic utility of the novel nRT-PCRs optimized in the study.

  4. Glutathione Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Transcription via WRKY33 and 1-Aminocyclopropane-1-Carboxylate Oxidase by Modulating Messenger RNA Stability to Induce Ethylene Synthesis during Stress1[OPEN

    PubMed Central

    Kumar, Deepak; Hazra, Saptarshi; Chattopadhyay, Sharmila

    2015-01-01

    Glutathione (GSH) plays a fundamental role in plant defense-signaling network. Recently, we have established the involvement of GSH with ethylene (ET) to combat environmental stress. However, the mechanism of GSH-ET interplay still remains unexplored. Here, we demonstrate that GSH induces ET biosynthesis by modulating the transcriptional and posttranscriptional regulations of its key enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Transgenic Arabidopsis (Arabidopsis thaliana) plants with enhanced GSH content (AtECS) exhibited remarkable up-regulation of ACS2, ACS6, and ACO1 at transcript as well as protein levels, while they were down-regulated in the GSH-depleted phytoalexin deficient2-1 (pad2-1) mutant. We further observed that GSH induced ACS2 and ACS6 transcription in a WRKY33-dependent manner, while ACO1 transcription remained unaffected. On the other hand, the messenger RNA stability for ACO1 was found to be increased by GSH, which explains our above observations. In addition, we also identified the ACO1 protein to be a subject for S-glutathionylation, which is consistent with our in silico data. However, S-glutathionylation of ACS2 and ACS6 proteins was not detected. Further, the AtECS plants exhibited resistance to necrotrophic infection and salt stress, while the pad2-1 mutant was sensitive. Exogenously applied GSH could improve stress tolerance in wild-type plants but not in the ET-signaling mutant ethylene insensitive2-1, indicating that GSH-mediated resistance to these stresses occurs via an ET-mediated pathway. Together, our investigation reveals a dual-level regulation of ET biosynthesis by GSH during stress. PMID:26463088

  5. MESSENGER: Exploring Mercury's Magnetosphere

    NASA Technical Reports Server (NTRS)

    Slavin, James A.; Krimigis, Stamatios M.; Acuna, Mario H.; Anderson, Brian J.; Baker, Daniel N.; Koehn, Patrick L.; Korth, Haje; Levi, Stefano; Mauk, Barry H.; Solomon, Sean C.; Zurbuchen, Thomas H.

    2005-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet s miniature magnetosphere since the brief flybys of Mariner 10. Mercury s magnetosphere is unique in many respects. The magnetosphere of Mercury is among the smallest in the solar system; its magnetic field typically stands off the solar wind only - 1000 to 2000 km above the surface. For this reason there are no closed drift paths for energetic particles and, hence, no radiation belts. The characteristic time scales for wave propagation and convective transport are short and kinetic and fluid modes may be coupled. Magnetic reconnection at the dayside magnetopause may erode the subsolar magnetosphere allowing solar wind ions to impact directly the regolith. Inductive currents in Mercury s interior may act to modify the solar wind interaction by resisting changes due to solar wind pressure variations. Indeed, observations of these induction effects may be an important source of information on the state of Mercury s interior. In addition, Mercury s magnetosphere is the only one with its defining magnetic flux tubes rooted in a planetary regolith as opposed to an atmosphere with a conductive ionospheric layer. This lack of an ionosphere is probably the underlying reason for the brevity of the very intense, but short-lived, - 1-2 min, substorm-like energetic particle events observed by Mariner 10 during its first traversal of Mercury s magnetic tail. Because of Mercury s proximity to the sun, 0.3 - 0.5 AU, this magnetosphere experiences the most extreme driving forces in the solar system. All of these factors are expected to produce complicated interactions involving the exchange and re-cycling of neutrals and ions between the solar wind, magnetosphere, and regolith. The electrodynamics of Mercury s magnetosphere are expected to be equally complex, with strong forcing by the solar wind, magnetic reconnection at the magnetopause and in the tail, and the pick-up of planetary ions all

  6. MESSENGER'S First Flyby of Mercury

    NASA Technical Reports Server (NTRS)

    Slavin, James A.

    2008-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only - 1000 to 2000 km above the surface. An overview of the MESSENGER mission and its January 14th close flyby of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER'S first flyby on January 14th, 2008 will be discussed with an emphasis on the magnetic field and charged particle measurements.

  7. The MESSENGER Spacecraft

    NASA Astrophysics Data System (ADS)

    Leary, James C.; Conde, Richard F.; Dakermanji, George; Engelbrecht, Carl S.; Ercol, Carl J.; Fielhauer, Karl B.; Grant, David G.; Hartka, Theodore J.; Hill, Tracy A.; Jaskulek, Stephen E.; Mirantes, Mary A.; Mosher, Larry E.; Paul, Michael V.; Persons, David F.; Rodberg, Elliot H.; Srinivasan, Dipak K.; Vaughan, Robin M.; Wiley, Samuel R.

    2007-08-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft was designed and constructed to withstand the harsh environments associated with achieving and operating in Mercury orbit. The system can be divided into eight subsystems: structures and mechanisms (e.g., the composite core structure, aluminum launch vehicle adapter, and deployables), propulsion (e.g., the state-of-the-art titanium fuel tanks, thruster modules, and associated plumbing), thermal (e.g., the ceramic-cloth sunshade, heaters, and radiators), power (e.g., solar arrays, battery, and controlling electronics), avionics (e.g., the processors, solid-state recorder, and data handling electronics), software (e.g., processor-supported code that performs commanding, data handling, and spacecraft control), guidance and control (e.g., attitude sensors including star cameras and Sun sensors integrated with controllers including reaction wheels), radio frequency telecommunications (e.g., the spacecraft antenna suites and supporting electronics), and payload (e.g., the science instruments and supporting processors). This system architecture went through an extensive (nearly four-year) development and testing effort that provided the team with confidence that all mission goals will be achieved.

  8. MESSENGER Final Image

    NASA Image and Video Library

    2015-04-30

    Today, the MESSENGER spacecraft sent its final image. Originally planned to orbit Mercury for one year, the mission exceeded all expectations, lasting for over four years and acquiring extensive datasets with its seven scientific instruments and radio science investigation. This afternoon, the spacecraft succumbed to the pull of solar gravity and impacted Mercury's surface. The image shown here is the last one acquired and transmitted back to Earth by the mission. The image is located within the floor of the 93-kilometer-diameter crater Jokai. The spacecraft struck the planet just north of Shakespeare basin. Date acquired: April 30, 2015 Image Mission Elapsed Time (MET): 72716050 Image ID: 8422953 Instrument: Narrow Angle Camera (NAC) of the Mercury Dual Imaging System (MDIS) Center Latitude: 72.0° Center Longitude: 223.8° E Resolution: 2.1 meters/pixel Scale: This image is about 1 kilometers (0.6 miles) across Incidence Angle: 57.9° Emission Angle: 56.5° Phase Angle: 40.7° http://photojournal.jpl.nasa.gov/catalog/PIA19448

  9. NASA Now: MESSENGER in Orbit

    NASA Image and Video Library

    Dr. Larry Evans, Senior Scientist for MESSENGER, discusses the difficulty of getting to Mercury, the challenges of visiting a planet so close to the sun and what we hope to discover when the spacec...

  10. MESSENGER Departing Earth Artist Concept

    NASA Image and Video Library

    2004-08-03

    Artist impression of NASA MErcury Surface, Space ENvironment, GEochemistry, and Ranging MESSENGER spacecraft as it leaves Earth, following its Aug. 3, 2004 launch from Cape Canaveral Air Force Station, Fla. aboard a Delta II rocket.

  11. MESSENGER: Exploring Mercury's Magnetosphere

    NASA Astrophysics Data System (ADS)

    Slavin, James A.; Krimigis, Stamatios M.; Acuña, Mario H.; Anderson, Brian J.; Baker, Daniel N.; Koehn, Patrick L.; Korth, Haje; Livi, Stefano; Mauk, Barry H.; Solomon, Sean C.; Zurbuchen, Thomas H.

    2007-08-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) mission to Mercury offers our first opportunity to explore this planet’s miniature magnetosphere since the brief flybys of Mariner 10. Mercury’s magnetosphere is unique in many respects. The magnetosphere of Mercury is among the smallest in the solar system; its magnetic field typically stands off the solar wind only ˜1000 to 2000 km above the surface. For this reason there are no closed drift paths for energetic particles and, hence, no radiation belts. Magnetic reconnection at the dayside magnetopause may erode the subsolar magnetosphere, allowing solar wind ions to impact directly the regolith. Inductive currents in Mercury’s interior may act to modify the solar wind interaction by resisting changes due to solar wind pressure variations. Indeed, observations of these induction effects may be an important source of information on the state of Mercury’s interior. In addition, Mercury’s magnetosphere is the only one with its defining magnetic flux tubes rooted beneath the solid surface as opposed to an atmosphere with a conductive ionospheric layer. This lack of an ionosphere is probably the underlying reason for the brevity of the very intense, but short-lived, ˜1-2 min, substorm-like energetic particle events observed by Mariner 10 during its first traversal of Mercury’s magnetic tail. Because of Mercury’s proximity to the sun, 0.3-0.5 AU, this magnetosphere experiences the most extreme driving forces in the solar system. All of these factors are expected to produce complicated interactions involving the exchange and recycling of neutrals and ions among the solar wind, magnetosphere, and regolith. The electrodynamics of Mercury’s magnetosphere are expected to be equally complex, with strong forcing by the solar wind, magnetic reconnection, and pick-up of planetary ions all playing roles in the generation of field-aligned electric currents. However, these field

  12. Lack of pharmacokinetic interaction for ISIS 113715, a 2'-0-methoxyethyl modified antisense oligonucleotide targeting protein tyrosine phosphatase 1B messenger RNA, with oral antidiabetic compounds metformin, glipizide or rosiglitazone.

    PubMed

    Geary, Richard S; Bradley, JoAnn D; Watanabe, Tanya; Kwon, Younggil; Wedel, Mark; van Lier, Jan J; VanVliet, André A

    2006-01-01

    ISIS 113715 is a 20-mer phosphorothioate antisense oligonucleotide (ASO) that is complementary to the protein tyrosine phosphatase 1B (PTP-1B) messenger RNA and subsequently reduces translation of the PTP-1B protein, a negative regulator of insulin receptor. ISIS 113715 is currently being studied in early phase II clinical studies to determine its ability to improve or restore insulin receptor sensitivity in patients with type 2 diabetes mellitus. Future work will investigate the combination of ISIS 113715 with antidiabetic compounds. In vitro ultrafiltration human plasma protein binding displacement studies and a phase I clinical study were used to characterise the potential for pharmacokinetic interaction of ISIS 113715 and three marketed oral antidiabetic agents. ISIS 113715 was co-incubated with glipizide and rosiglitazone in whole human plasma and tested for increased free drug concentrations. In a phase I clinical study, 23 healthy volunteers received a single oral dose of an antidiabetic compound (either metformin, glipizide or rosiglitazone) both alone and together with subcutaneous ISIS 113715 200 mg in a sequential crossover design. A comparative pharmacokinetic analysis was performed to determine if there were any effects that resulted from coadministration of ISIS 113715 with these antidiabetic compounds. In vitro human plasma protein binding displacement studies showed only minor effects on rosiglitazone and no effect on glipizide when co-incubated with ISIS 113715. The results of the phase I clinical study further indicate that there were no measurable changes in glipizide (5 mg), metformin (500 mg) or rosiglitazone (2 mg) exposure parameters, maximum plasma concentration and the area under the concentration-time curve, or pharmacokinetic parameter, elimination half-life when coadministered with ISIS 113715. Furthermore, there was no effect of ISIS 113715, administered in combination with metformin, on the urinary excretion of metformin. Conversely

  13. Isoleucine and Valine Metabolism in Escherichia coli K-12: Detection and Measurement of ilv-Specific Messenger Ribonucleic Acid

    PubMed Central

    Haar, R. A. Vonder; Umbarger, H. E.

    1974-01-01

    Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of messenger RNA transcribed from the ilv gene cluster of Escherichia coli K-12. Strains with derepressed levels of the isoleucine and valine biosynthetic enzymes owing to linked or unlinked genetic lesions were found to exhibit ilv messenger RNA levels from 1.5- to 4-fold higher than did their isogenic parents. When grown under conditions that specifically repressed the synthesis of isoleucine- and valine-forming enzymes, most strains exhibited drastically reduced ilv messenger RNA levels. Hybridization performed with the separated strands of ilv DNA showed that all the ilv genes are transcribed from the same strand, the “l strand” of λφ80CI857St68dilv DNA. Sucrose gradient analyses of RNA extracted from cells starved for isoleucine, valine, or leucine resulted in the detection of at least two distinct types of ilv messenger RNA. PMID:4616946

  14. The MESSENGER Spacecraft and Payload

    NASA Astrophysics Data System (ADS)

    Gold, R. E.; Solomon, S. C.; McNutt, R. L., Jr.; Santo, A. G.

    2002-01-01

    The MErcury, Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) mission will send the first spacecraft to orbit the planet Mercury. A Mercury orbiter mission is challenging from thermal and mass perspectives. MESSENGER overcomes these challenges while avoiding esoteric technologies by using an innovative approach with commonly available materials, minimal moving parts, and maximum heritage. The key concepts are a ceramic-cloth thermal shade, an integrated lightweight structure, a high performance propulsion system, and a solar array incorporating optical solar reflectors. A miniaturized set of seven instruments, along with the spacecraft telecommunications system, satisfy all scientific objectives of the mission. The payload includes a combined wide-angle and narrow-angle imaging system; amma-ray, neutron, and X-ray spectrometers for remote geochemical sensing; a vector magnetometer; a laser altimeter; a combined ultraviolet-visible and visible-infrared spectrometer to detect atmospheric species and map mineralogical absorption features; and an energetic particle and plasma spectrometer to characterize ionized species in the magnetosphere. MESSENGER construction is nearly complete and the integration and test phase is just beginning. Launch is March 2004.

  15. Geodesy at Mercury with MESSENGER

    NASA Technical Reports Server (NTRS)

    Smith, David E.; Zuber, Maria t.; Peale, Stanley J.; Phillips, Roger J.; Solomon, Sean C.

    2006-01-01

    In 2011 the MESSENGER (MErcury Surface, Space ENvironment, GEochemistry, and Ranging) spacecraft will enter Mercury orbit and begin the mapping phase of the mission. As part of its science objectives the MESSENGER mission will determine the shape and gravity field of Mercury. These observations will enable the topography and the crustal thickness to be derived for the planet and will determine the small libration of the planet about its axis, the latter critical to constraining the state of the core. These measurements require very precise positioning of the MESSENGER spacecraft in its eccentric orbit, which has a periapsis altitude as low as 200 km, an apoapsis altitude near 15,000 km, and a closest approach to the surface varying from latitude 60 to about 70 N. The X-band tracking of MESSENGER and the laser altimetry are the primary data that will be used to measure the planetary shape and gravity field. The laser altimeter, which has an expected range of 1000 to 1200 km, is expected to provide significant data only over the northern hemisphere because of MESSENGER's eccentric orbit. For the southern hemisphere, radio occultation measurements obtained as the spacecraft passes behind the planet as seen from Earth and images obtained with the imaging system will be used to provide the long-wavelength shape of the planet. Gravity, derived from the tracking data, will also have greater resolution in the northern hemisphere, but full global models for both topography and gravity will be obtained at low harmonic order and degree. The limiting factor for both gravity and topography is expected to be knowledge of the spacecraft location. Present estimations are that in a combined tracking, altimetry, and occultation solution the spacecraft position uncertainty is likely to be of order 10 m. This accuracy should be adequate for establishing an initial geodetic coordinate system for Mercury that will enable positioning of imaged features on the surface, determination of

  16. Melatonin: a universal time messenger.

    PubMed

    Erren, Thomas C; Reiter, Russel J

    2015-01-01

    Temporal organization plays a key role in humans, and presumably all species on Earth. A core building block of the chronobiological architecture is the master clock, located in the suprachi asmatic nuclei [SCN], which organizes "when" things happen in sub-cellular biochemistry, cells, organs and organisms, including humans. Conceptually, time messenging should follow a 5 step-cascade. While abundant evidence suggests how steps 1 through 4 work, step 5 of "how is central time information transmitted througout the body?" awaits elucidation. Step 1: Light provides information on environmental (external) time; Step 2: Ocular interfaces between light and biological (internal) time are intrinsically photosensitive retinal ganglion cells [ipRGS] and rods and cones; Step 3: Via the retinohypothalamic tract external time information reaches the light-dependent master clock in the brain, viz the SCN; Step 4: The SCN translate environmental time information into biological time and distribute this information to numerous brain structures via a melanopsin-based network. Step 5: Melatonin, we propose, transmits, or is a messenger of, internal time information to all parts of the body to allow temporal organization which is orchestrated by the SCN. Key reasons why we expect melatonin to have such role include: First, melatonin, as the chemical expression of darkness, is centrally involved in time- and timing-related processes such as encoding clock and calendar information in the brain; Second, melatonin travels throughout the body without limits and is thus a ubiquitous molecule. The chemial conservation of melatonin in all tested species could make this molecule a candidate for a universal time messenger, possibly constituting a legacy of an all-embracing evolutionary history.

  17. RNA epigenetics

    PubMed Central

    Liu, Nian; Pan, Tao

    2014-01-01

    Summary Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modification. PMID:24768686

  18. Alterations in polyribosome and messenger ribonucleic acid metabolism and messenger ribonucleoprotein utilization in osmotically stressed plant seedlings

    SciTech Connect

    Mason, H.S.

    1986-01-01

    Polyribosome aggregation state in growing tissues of barley and wheat leaf of stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf of stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of /sup 32/PO/sub 4//sup 3 -/ into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A)/sup +/RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A)/sup +/RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described.

  19. MESSENGER Observations of Mercury's Magnetosphere

    NASA Technical Reports Server (NTRS)

    Slavin, James A.

    2010-01-01

    During MESSENGER's second and third flybys of Mercury on October 6, 2008 and September 29, 2009, respectively, southward interplanetary magnetic field (IMF) produced intense reconnection signatures in the dayside and nightside magnetosphere and markedly different system-level responses. The IMF during the second flyby was continuously southward and the magnetosphere appeared very active, with large magnetic field components normal to the magnetopause and the generation of flux transfer events at the magnetopause and plasmoids in the tail current sheet every 30 to 90 s. However, the strength and direction of the tail magnetic field was stable. In contrast, the IMF during the third flyby varied from north to south on timescales of minutes. Although the MESSENGER measurements were limited during that encounter to the nightside magnetosphere, numerous examples of plasmoid release in the tail were detected, but they were not periodic. Instead, plasmoid release was highly correlated with four large enhancements of the tail magnetic field (i.e. by factors > 2) with durations of approx. 2 - 3 min. The increased flaring of the magnetic field during these intervals indicates that the enhancements were caused by loading of the tail with magnetic flux transferred from the dayside magnetosphere. New analyses of the second and third flyby observations of reconnection and its system-level effects provide a basis for comparison and contrast with what is known about the response of the Earth s magnetosphere to variable versus steady southward IMF.

  20. Details of MESSENGER Impact Location

    NASA Image and Video Library

    2015-04-29

    These graphics show the current best prediction of the location and time of NASA MESSENGER impact on Mercury surface. These current best estimates are: Date: 30 April 2015 Time: 3:26:02 pm EDT 19:26:02 UTC Latitude: 54.4° N Longitude: 210.1° E. Traveling at 3.91 kilometers per second (over 8,700 miles per hour), the MESSENGER spacecraft will collide with Mercury's surface, creating a crater estimated to be 16 meters (52 feet) in diameter. View this image to learn about the named features and geology of this region on Mercury. Instruments: Mercury Dual Imaging System (MDIS) and Mercury Laser Altimeter (MLA) Top Image Latitude Range: 49°-59° N Top Image Longitude Range: 204°-217° E Topography in Top Image: Exaggerated by a factor of 5.5. Colors in Top Image: Coded by topography. The tallest regions are colored red and are roughly 3 kilometers (1.9 miles) higher than low-lying areas such as the floors of impact craters, colored blue. Scale in Top Image: The large crater on the left side of the image is Janacek, with a diameter of 48 kilometers (30 miles) http://photojournal.jpl.nasa.gov/catalog/PIA19443

  1. Differential expression in glioblastoma multiforme and cerebral hemangioblastoma of cytoplasmic proteins that bind two different domains within the 3'-untranslated region of the human glucose transporter 1 (GLUT1) messenger RNA.

    PubMed Central

    Tsukamoto, H; Boado, R J; Pardridge, W M

    1996-01-01

    The glucose transporter 1 (GLUT1) protein is underexpressed in human glioblastoma multiforme and is overexpressed in human cerebral hemangioblastoma. To gain in-sight into possible posttranscriptional mechanisms regulating the expression of the GLUT1 protein in human brain tumors, cytosolic proteins were prepared from these two tumors and used in RNase T1 protection assays that employed [32P]human GLUT1 synthetic RNA prepared from transcription plasmids. Gel shift mobility assays and ultra-violet light cross-linking studies demonstrated the formation of specific RNA/protein complexes that migrated with a mol mass of 120, 44, and 41 kD. RNase T1 mapping and oligodeoxynucleotide competition studies showed that the 120 kD complex was comprised of an RNA fragment that localized to nucleotides 2186-2203 of the GLUT1 mRNA. The 44 kD complex contained an adenosine-uridine-rich RNA fragment that localized to nucleotides 1885-1906 of the human GLUT1 mRNA, and the formation of this complex was inhibited by synthetic RNA enriched in adenosine-uridine sequences. The 44 kD complex was selectively downregulated in hemangioblastoma as compared to glioblastoma multiforme. These studies demonstrate that human brain tumors have differential regulation of cytosolic proteins that specifically interact with two different domains in the 3'-untranslated region of the GLUT1 mRNA, which may serve to mediate the posttranscriptional regulation of GLUT1 gene expression in these tumors. PMID:8675694

  2. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  3. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  4. Cotranscriptional Recruitment of RNA Exosome Cofactors Rrp47p and Mpp6p and Two Distinct Trf-Air-Mtr4 Polyadenylation (TRAMP) Complexes Assists the Exonuclease Rrp6p in the Targeting and Degradation of an Aberrant Messenger Ribonucleoprotein Particle (mRNP) in Yeast*

    PubMed Central

    Stuparevic, Igor; Mosrin-Huaman, Christine; Hervouet-Coste, Nadège; Remenaric, Mateja; Rahmouni, A. Rachid

    2013-01-01

    The cotranscriptional mRNA processing and packaging reactions that lead to the formation of export-competent messenger ribonucleoprotein particles (mRNPs) are under the surveillance of quality control steps. Aberrant mRNPs resulting from faulty events are retained in the nucleus with ensuing elimination of their mRNA component. The molecular mechanisms by which the surveillance system recognizes defective mRNPs and stimulates their destruction by the RNA degradation machinery are still not completely elucidated. Using an experimental approach in which mRNP formation in yeast is disturbed by the action of the bacterial Rho helicase, we have shown previously that the targeting of Rho-induced aberrant mRNPs is mediated by Rrp6p, which is recruited cotranscriptionally in association with Nrd1p following Rho action. Here we investigated the specific involvement in this quality control process of different cofactors associated with the nuclear RNA degradation machinery. We show that, in addition to the main hydrolytic action of the exonuclease Rrp6p, the cofactors Rrp47p, Mpp6p as well as the Trf-Air-Mtr4 polyadenylation (TRAMP) components Trf4p, Trf5p, and Air2p contribute significantly by stimulating the degradation process upon their cotranscriptional recruitment. Trf4p and Trf5p are apparently recruited in two distinct TRAMP complexes that both contain Air2p as component. Surprisingly, Rrp47p appears to play an important role in mutual protein stabilization with Rrp6p, which highlights a close association between the two partners. Together, our results provide an integrated view of how different cofactors of the RNA degradation machinery cooperate to target and eliminate aberrant mRNPs. PMID:24047896

  5. Experiment K-7-22: Growth Hormone Regulation Synthesis and Secretion in Microgravity. Part 2; Hypothalamic Growth Hormone-Releasing Factor, Somatostatin Immunoreactivity, and Messenger RNA Levels in Microgravity

    NASA Technical Reports Server (NTRS)

    Sawchenko, P. E.; Arias, C.; Krasnov, I.; Grindeland, R. E.; Vale, W.

    1994-01-01

    Immunohistochemical analyses of hypothalamic hormones carried out on tissue from rats flown on an earlier flight (Cosmos 1887) suggested preferential effects on hypophysiotropic principles involved in the regulation of growth hormone secretion and synthesis. We found that staining in the median eminence for peptides that provide both stimulatory (growth hormone-releasing factor, or GRF) and inhibitory (somatostatin, SS) influences on growth hormone secretion were depressed in flight animals relative to synchronous controls, while staining for other neuroendocrine peptides, cortocotropin-releasing factor and arginine vasopressin, were similar in these two groups. While this suggests some selective impact of weightlessness on the two principal central nervous system regulators of growth hormone dynamics, the fact that both GRF- and SS-immunoreactivity (IR) appeared affected in the same direction is somewhat problematic, and makes tentative any intimation that effects on CNS control mechanisms may be etiologically significant contributors to the sequelae of reduced growth hormone secretion seen in prolonged space flight. To provide an additional, and more penetrating, analysis we attempted in hypothalamic material harvested from animals flown on Cosmos 2044 to complement immunohistochemical analyses of GRF and SS staining with quantitative, in situ assessments of messenger RNAs encoding the precursors for both these hormones.

  6. Experiment K-7-22: Growth Hormone Regulation Synthesis and Secretion in Microgravity. Part 2; Hypothalamic Growth Hormone-Releasing Factor, Somatostatin Immunoreactivity, and Messenger RNA Levels in Microgravity

    NASA Technical Reports Server (NTRS)

    Sawchenko, P. E.; Arias, C.; Krasnov, I.; Grindeland, R. E.; Vale, W.

    1994-01-01

    Immunohistochemical analyses of hypothalamic hormones carried out on tissue from rats flown on an earlier flight (Cosmos 1887) suggested preferential effects on hypophysiotropic principles involved in the regulation of growth hormone secretion and synthesis. We found that staining in the median eminence for peptides that provide both stimulatory (growth hormone-releasing factor, or GRF) and inhibitory (somatostatin, SS) influences on growth hormone secretion were depressed in flight animals relative to synchronous controls, while staining for other neuroendocrine peptides, cortocotropin-releasing factor and arginine vasopressin, were similar in these two groups. While this suggests some selective impact of weightlessness on the two principal central nervous system regulators of growth hormone dynamics, the fact that both GRF- and SS-immunoreactivity (IR) appeared affected in the same direction is somewhat problematic, and makes tentative any intimation that effects on CNS control mechanisms may be etiologically significant contributors to the sequelae of reduced growth hormone secretion seen in prolonged space flight. To provide an additional, and more penetrating, analysis we attempted in hypothalamic material harvested from animals flown on Cosmos 2044 to complement immunohistochemical analyses of GRF and SS staining with quantitative, in situ assessments of messenger RNAs encoding the precursors for both these hormones.

  7. Chicken granulosa cells show differential expression of epidermal growth factor (EGF) and luteinizing hormone (LH) receptor messenger RNA and differential responsiveness to EGF and LH dependent upon location of granulosa cells to the germinal disc.

    PubMed

    Yao, H H; Bahr, J M

    2001-06-01

    Granulosa cells in the chicken follicle exhibit different phenotypes according to their location relative to the germinal disc (GD). Granulosa cells proximal to the GD (referred to as proximal granulosa cells) are more proliferative, whereas granulosa cells distal to the GD (referred to as distal granulosa cells) are more differentiated. We have shown that epidermal growth factor (EGF) derived from the GD stimulated proliferation of granulosa cells proximal to the GD, whereas extraovarian LH promoted differentiation. We tested the hypothesis that phenotypic differences of granulosa cells are the result of differential responsiveness of granulosa cells to EGF and LH. We found that both granulosa and theca layers of chicken preovulatory follicles expressed mRNA for EGF receptor (EGFr) by polymerase chain reaction (PCR) analysis. However, only the granulosa layer showed differential expression of EGFr and LH receptor (LHr) mRNA. Competitive reverse transcription-PCR revealed that proximal granulosa cells expressed more EGFr mRNA but less LHr mRNA than distal granulosa cells. In addition, proximal granulosa cells proliferated more in response to EGF than their distal counterparts. We further demonstrated that EGF decreased LHr mRNA expression by granulosa cells in a dose-dependent manner, whereas EGF and LH had no effect on EGFr mRNA expression except at one dose of LH (15 ng/ml) that stimulated EGFr mRNA expression. Our findings suggest that EGF derived from the GD influences the phenotypes of granulosa cells. Granulosa cells proximal to the GD exhibit a proliferative phenotype possibly because they are exposed to and are more responsive to GD-derived EGF. Furthermore, GD-derived EGF decreases LHr mRNA expression by proximal granulosa cells and therefore results in less differentiated granulosa cell phenotype. In contrast, granulosa cells distal to the GD are not under the influence of EGF and exhibit a more differentiated phenotype.

  8. Neutralino Dark Matter in Gauge Messenger Models

    SciTech Connect

    Bae, Kyu Jung

    2008-11-23

    The lightest neutralino is one of the best candidate for dark matter. In gauge messenger models, It is generic that bino and wino masses are almostly degenerate. Because of this, neutralino annihilation becomes more efficient. Also, gauge messenger models have squeezed mass spectrum so that there are many resonance and co-annihilation regions, and can give correct amount of neutralino relic density.

  9. Astroparticles: Messengers from Outer Space

    NASA Astrophysics Data System (ADS)

    Desiati, Paolo

    2016-07-01

    Since Galileo pointed a spyglass toward the sky, 400 years ago, observations empowered by man-made instrumentation have provided us with an enormous leap in the knowledge of how the Universe functions. More and more powerful optical telescopes made it possible for us to reach the farthest corners of space. At the same time, the advances in microphysics and the discovery of the electromagnetic spectrum, made it possible to directly look at the Universe in a way that our eyes cannot see. The discoveries of the intimate structure of matter, of subatomic particles and of how they interact with each other, have led astronomers to use the smallest objects in Nature to observe the farthest reaches of the otherwise invisible Universe. Not unlike Galileo, today we observe Outer Space with visible light and beyond, across the entire electromagnetic spectrum, from long wavelength radio waves to short wavelength gamma rays. But also with instruments detecting cosmic rays (the atomic nuclei we know on Earth) neutrinos (neutral subatomic particles that interact very weakly with matter) and gravitational waves (perturbations of spacetime predicted by General Relativity). Each cosmic messenger provides us with a unique piece of information about their source and the history of their journey to us. Modern astrophysics has the challenging goal to collect as much information as possible from all those messengers, to reconstruct the story of the Universe and how it became what it is today. This journey started with the unsettling discovery that we are only one minuscule dot in the immensity of the Universe and yet we are able to observe objects that are far in space and time. This journey is yet to complete its course, and the more we advance our knowledge, the more we need to understand. This interdisciplinary talk provides an overview of this journey and the future perspectives.

  10. Two messenger RNA isoforms of the gonadotrophin-releasing hormone receptor, generated by alternative splicing and/or promoter usage, are differentially expressed in rainbow trout gonads during gametogenesis.

    PubMed

    Madigou, Thierry; Uzbekova, Svetlana; Lareyre, Jean-Jacques; Kah, Olivier

    2002-10-01

    The recent cloning of a gonadotrophin-releasing hormone receptor (GnRH-R) cDNA from rainbow trout showed that it contains several in-frame ATG codons, one of which, ATG2, corresponds to that found in other species. However, an upstream codon, ATG1, could give rise to a protein with a larger extracellular domain. Using S1 nuclease assay and a method combining primer extension and RACE-PCR, we characterized a second population of mRNA, termed mRNA-2, with a distinct 5'untranslated region and lacking ATG1. The genomic origin of the two mRNAs was determined by establishing the complete gene structure, which shows, for the first time in a vertebrate species that an alternative splicing and promoter usage generate two GnRH-R mRNA variants whose 5' extremities are encoded by two different exons. The analysis of the tissue distribution indicated that mRNA-2 presents a broader pattern of expression and is detected at higher levels than mRNA-1. Interestingly, it was found that those two mRNAs are differentially expressed in male and female gonads during gametogenesis. In particular, the variations of mRNA-1 levels parallel those of sGnRH expression during spermatogenesis, indicating that tissue-specific processing of the GnRH-R mRNA may underlie the effects of GnRH as a paracrine/autocrine regulator of gonadal functions. Copyright 2002 Wiley-Liss, Inc.

  11. Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide

    PubMed Central

    Inchaustegui Gil, Diana P.; Clayton, Christine

    2016-01-01

    The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA. PMID:26808308

  12. Molecular Diagnosis for Nodal Metastasis in Endoscopically Managed Cervical Cancer: The Accuracy of the APTIMA Test to Detect High-risk Human Papillomavirus Messenger RNA in Sentinel Lymph Nodes.

    PubMed

    Köhler, Christhardt; Le, Xin; Dogan, Nasuh Utku; Pfiffer, Tatiana; Schneider, Achim; Marnitz, Simone; Bertolini, Julia; Favero, Giovanni

    2016-01-01

    To evaluate the feasibility and accuracy of a commercially available test to detect E6/E7 mRNA of 14 subtypes of high-risk HPVs (APTIMA; Hologic, Bedford, MA) in the sentinel lymph nodes of CC patients laparoscopically operated. Prospective pilot study. The study was conducted in the Department of Advanced Operative and Oncologic Gynecology, Asklepios Hospital, Hamburg, Germany. 54 women with HPV-positive CC submitted to laparoscopic sentinel node biopsy alone or sentinel node biopsy followed by systematic pelvic and/or para-aortic endoscopic lymphadenectomy. All removed sentinel lymph nodes (SLNs) underwent sample collection by cytobrush for the APTIMA assay before frozen section. Results obtained with the HPV mRNA test were compared with the definitive histopathological analysis of the SLNs and additional lymph nodes removed. A total of 125 SLNs (119 pelvic and 6 paraaortic) were excised with a mean number of 2.3 SLNs per patient. Final histopathologic analysis confirmed nodal metastases in 10 SLNs from 10 different patients (18%). All the histologically confirmed metastatic lymph nodes were also HPV E6/E7 mRNA positive, resulting in a sensitivity of 100%. Four histologically free sentinel nodes were positive for HPV E6/E7 mRNA, resulting in a specificity of 96.4%. The HPV E6/E7 mRNA assay in the SLNs of patients with CC is feasible and highly accurate. The detection of HPV mRNA in 4 women with negative SLNs might denote a shift from microscopic identification of metastasis to the molecular level. The prognostic value of this findings awaits further verification. Copyright © 2016. Published by Elsevier Inc.

  13. MESSENGER: Exploring the Innermost Planet

    NASA Astrophysics Data System (ADS)

    Solomon, S. C.

    2011-12-01

    One of Earth's closest planetary neighbors, Mercury remained comparatively unexplored for the more than three decades that followed the three flybys of the innermost planet by the Mariner 10 spacecraft in 1974-75. Mariner 10 imaged 45% of Mercury's surface at about 1 km/pixel average resolution, confirmed Mercury's anomalously high bulk density and implied large fractional core size, discovered Mercury's internal magnetic field, documented that H and He are present in the planet's tenuous exosphere, and made the first exploration of Mercury's magnetosphere and solar wind environment. Ground-based astronomers later reported Na, K, and Ca in Mercury's exosphere; the presence of deposits in the floors of polar craters having radar characteristics best matched by water ice; and strong evidence from the planet's forced libration amplitude that Mercury has a fluid outer core. Spacecraft exploration of Mercury resumed with the selection for flight, under NASA's Discovery Program, of the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) mission. Launched in 2004, MESSENGER flew by the innermost planet three times in 2008-2009 en route to becoming the first spacecraft to orbit Mercury in March of this year. MESSENGER's first chemical remote sensing measurements of Mercury's surface indicate that the planet's bulk silicate fraction differs from those of the other inner planets, with a low-Fe surface composition intermediate between basalts and ultramafic rocks and best matched among terrestrial rocks by komatiites. Moreover, surface materials are richer in the volatile constituents S and K than predicted by most planetary formation models. Global image mosaics and targeted high-resolution images (to resolutions of 10 m/pixel) reveal that Mercury experienced globally extensive volcanism, including large expanses of plains emplaced as flood lavas and widespread examples of pyroclastic deposits likely emplaced during explosive eruptions of volatile

  14. The Mercury exosphere after MESSENGER

    NASA Astrophysics Data System (ADS)

    Killen, Rosemary; McClintock, William; Vervack, Ronald; Merkel, Aimee; Burger, Matthew; Cassidy, Timothy; Sarantos, Menelaos

    2016-07-01

    The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft observed sodium, calcium and magnesium emisison in Mercury's exosphere on a near-daily basis for >16 Mercury years. The MASCS observations showed that calcium in Mercury's exosphere is persistently concentrated in the dawn hemisphere and is of extreme temperature (>50,000 K). The column abundance varies seasonally, and is extremely repeatable each Mercury year. In addition, the calcium exhibits a persistent maximum not at perihelion but 20° after perihelion, an enhancement that was shown to be coincident with the probable intersection of Mercury's orbit with a dust stream originating at Comet Encke. Any mechanism producing the Mercurian Ca exosphere must explain the facts that the Ca is extremely hot, that it is seen almost exclusively on the dawnside of the planet, and that its content varies seasonally, not sporadically. Energization of the Ca atoms was suggested to originate through dissociation of Ca-bearing molecules ejected by meteoritic impacts. Magnesium was also observed on a daily basis throughout the MESSENGER orbital phase. Mg has its own spatial and temporal pattern, peaking at mid-morning instead of early morning like Ca, and exhibiting a warm thermal profile, about 5000 K, unlike the extreme temperature of Ca which is an order of magnitude hotter. Although Mercury's sodium exosphere has been observed from the ground for many decades, the MASCS observations showed that, like calcium, the sodium exosphere is dominated by seasonal variations, not sporadic variations. However a conundrum exists as to why ground-based observations show highly variable high-latitude variations that eluded the MASCS. The origin of a persistent south polar enhancement has not been explained. The more volatile element, Na, is again colder, about 1200 K, but not thermally accommodated to the surface temperature. A

  15. The Magnetometer Instrument on MESSENGER

    NASA Astrophysics Data System (ADS)

    Anderson, Brian J.; Acuña, Mario H.; Lohr, David A.; Scheifele, John; Raval, Asseem; Korth, Haje; Slavin, James A.

    2007-08-01

    The Magnetometer (MAG) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) mission is a low-noise, tri-axial, fluxgate instrument with its sensor mounted on a 3.6-m-long boom. The boom was deployed on March 8, 2005. The primary MAG science objectives are to determine the structure of Mercury’s intrinsic magnetic field and infer its origin. Mariner 10 observations indicate a planetary moment in the range 170 to 350 nT R {M/3} (where R M is Mercury’s mean radius). The uncertainties in the dipole moment are associated with the Mariner 10 trajectory and variability of the measured field. By orbiting Mercury, MESSENGER will significantly improve the determination of dipole and higher-order moments. The latter are essential to understanding the thermal history of the planet. MAG has a coarse range, ±51,300 nT full scale (1.6-nT resolution), for pre-flight testing, and a fine range, ±1,530 nT full scale (0.047-nT resolution), for Mercury operation. A magnetic cleanliness program was followed to minimize variable and static spacecraft-generated fields at the sensor. Observations during and after boom deployment indicate that the fixed residual field is less than a few nT at the location of the sensor, and initial observations indicate that the variable field is below 0.05 nT at least above about 3 Hz. Analog signals from the three axes are low-pass filtered (10-Hz cutoff) and sampled simultaneously by three 20-bit analog-to-digital converters every 50 ms. To accommodate variable telemetry rates, MAG provides 11 output rates from 0.01 s-1 to 20 s-1. Continuous measurement of fluctuations is provided with a digital 1-10 Hz bandpass filter. This fluctuation level is used to trigger high-time-resolution sampling in eight-minute segments to record events of interest when continuous high-rate sampling is not possible. The MAG instrument will provide accurate characterization of the intrinsic planetary field, magnetospheric structure, and

  16. MESSENGER'S First and Second Flybys of Mercury

    NASA Technical Reports Server (NTRS)

    Slavin, James A.

    2009-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only approximately 1000 km above the surface. An overview of the MESSENGER mission and its January 14th and October 6th, 2008 close flybys of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER will be discussed with an emphasis on the magnetic field and charged particle measurements.

  17. Effect of Thymine Starvation on Messenger Ribonucleic Acid Synthesis in Escherichia coli

    PubMed Central

    Luzzati, Denise

    1966-01-01

    Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435–1446. 1966.—During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation. PMID:5332402

  18. Molecular defect in siblings with prolidase deficiency and absence or presence of clinical symptoms. A 0.8-kb deletion with breakpoints at the short, direct repeat in the PEPD gene and synthesis of abnormal messenger RNA and inactive polypeptide.

    PubMed Central

    Tanoue, A; Endo, F; Akaboshi, I; Oono, T; Arata, J; Matsuda, I

    1991-01-01

    Prolidase deficiency is an autosomal recessive disorder with highly variable symptoms, including mental retardation, skin lesions, and abnormalities of collagenous tissues. In Japanese female siblings with polypeptide negative prolidase deficiency, and with different degrees of severity of skin lesions, we noted an abnormal mRNA with skipping of 192 bp sequence corresponding to exon 14 in lymphoblastoid cells taken from these patients. Transfection and expression analyses using the mutant prolidase cDNA revealed that a mutant protein translated from the abnormal mRNA had an Mr of 49,000 and was enzymatically inactive. A 774-bp deletion, including exon 14 was noted in the prolidase gene. The deletion had termini within short, direct repeats ranging in size of 7 bp (CCACCCT). The "slipped mispairing" mechanism may predominate in the generation of the deletion at this locus. This mutation caused a 192-bp in-frame deletion of prolidase mRNA and was inherited from the consanguineous parents. The same mutation caused a different degree of clinical phenotype of prolidase deficiency in this family, therefore factor(s) not related to the PEPD gene product also contribute to development of the clinical symptoms. Identification of mutations in the PEPD gene from subjects with prolidase deficiency provides further insight into the physiological role and structure-function relationship of this biologically important enzyme. Images PMID:2010534

  19. Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin–kieselguhr

    PubMed Central

    Johnson, Roger

    1970-01-01

    Chromatography on methylated albumin–kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The `chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source. PMID:4321853

  20. MESSENGER Team Presents Latest Science Results

    NASA Image and Video Library

    2009-04-30

    This mosaic was assembled using NAC images acquired as the MESSENGER spacecraft approached the planet during the mission second Mercury flyby The Rembrandt impact basin is seen at the center of the mosaic.

  1. An evaluation of the etiology of reduced CYP1A1 messenger RNA expression in the Atlantic tomcod from the Hudson River, New York, USA, using reverse transcriptase polymerase chain reaction analysis.

    PubMed

    Roy, N K; Courtenay, S; Yuan, Z; Ikonomou, M; Wirgin, I

    2001-05-01

    Adult Atlantic tomcod, Microgadus tomcod, from the Hudson River, New York State, USA, exhibit reduced inducibility of hepatic cytochrome P4501A1 (CYP1A1) mRNA compared with adult tomcod from the cleaner Miramichi River, New Brunswick, Canada, when treated with coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, little difference in CYP1A1 inducibility is observed between tomcod from these two rivers when treated with polycyclic aromatic hydrocarbons (PAHs). We sought to determine if impaired hepatic CYP1A1 inducibility in Hudson River tomcod results from a multigenerational, genetic adaptation or a single generational, physiological acclimation. Embryos and larvae from controlled experimental crosses of Hudson River and Miramichi River parents were exposed for 24 h to water-borne PCB congener 77 (10 ppm), benzo[a]pyrene (BaP; 10 ppm), or dimethysulfoxide, and CYP1A1 expression was assessed in individual larva using competitive reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The CYP1A1 mRNA was significantly induced in larvae from both populations by BaP (47- and 52-fold) and PCB 77 (9- and 22-fold), although levels of expression were higher in offspring of Miramichi matings. Most important, CYP1A1 mRNA was significantly induced by PCB 77 in larvae from Hudson River parents. Concentrations of dioxin, furan, and PCB congeners were measured in livers and eggs of female tomcod from these two locales to quantify the extent of maternal transfer of contaminants. For both rivers, wet-weight contaminant concentrations were significantly higher (4-7 times) in livers than in eggs of the same females, suggesting that a threshold level of contaminants may have to be reached before CYP1A1 transcription is impaired. We conclude that reduced inducibility of hepatic CYP1A1 mRNA in adult tomcod from the Hudson River is most consistent with single-generational acclimation.

  2. Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch

    SciTech Connect

    Kulshina, Nadia; Baird, Nathan J.; Ferré-D'Amaré, Adrian R.

    2009-12-03

    The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

  3. Cytochrome P450 side-chain cleavage (P450scc) in the hen ovary. I. Regulation of P450scc messenger RNA levels and steroidogenesis in theca cells of developing follicles.

    PubMed

    Kowalski, K I; Tilly, J L; Johnson, A L

    1991-12-01

    We have recently shown that granulosa cells from hen ovarian follicles, collected at a stage of development 2-3 wk prior to ovulation (e.g. 6-8 mm in diameter) are steroidogenically inactive. Therefore, the hypothesis tested in the present studies was that theca cells from follicles at this stage of development must contain sufficient levels of functional cytochrome P450 side-chain cleavage (P450scc) enzyme to produce the progestin precursor required for the synthesis of androgens and estrogens. Northern blot analysis of total theca RNA collected from 6-8-mm follicles indicated the presence of a single P450scc mRNA transcript of approximately 2 kb whose expression was increased following an 8-h preincubation with 200 ng/ml ovine LH (oLH) or 10 microM forskolin. Western blot analysis of crude mitochondrial protein revealed a band of immunoreactive P450scc protein of approximately 53 kDa that was determined to be capable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. In the second set of studies, conducted to examine the cellular regulation of steroidogenesis in isolated theca cells of 6-8-mm follicles, theca cells were found to produce measurable basal levels of cAMP, progesterone, androstenedione, and estradiol following a 3-h incubation of 5 x 10(5) cells. Furthermore, significant dose-dependent increases in steroidogenesis were observed in response to oLH (0.2-200 ng/ml), chicken FSH (cFSH; 20-200 ng/ml), cholera toxin (0.002-20 ng/ml), and 8-bromo-cAMP (0.1-3.33 mM). Phorbol 12-myristate 13-acetate (PMA; 10-167 nM) also stimulated dose-dependent increases in basal progesterone, androstenedione, and estradiol production. In addition, while PMA had no effect on oLH (200 ng/ml)-promoted cAMP accumulation, or on oLH (20 ng/ml)- or 8-bromo-cAMP (1 mM)-stimulated progesterone production, it attenuated oLH-induced and 8-bromo-cAMP-induced androstenedione and estradiol accumulation. We conclude that theca cells from 6-8-mm follicles possess mRNA

  4. Messenger Ribonucleic Acid Synthesis and Degradation in Escherichia coli During Inhibition of Translation

    PubMed Central

    Pato, Martin L.; Bennett, Peter M.; Von Meyenburg, Kaspar

    1973-01-01

    Various aspects of the coupling between the movement of ribosomes along messenger ribonucleic acids (mRNA) and the synthesis and degradation of mRNA have been investigated. Decreasing the rate of movement of ribosomes along an mRNA does not affect the rate of movement of some, and possibly most, of the RNA polymerases transcribing the gene coding for that mRNA. Inhibiting translation with antibiotics such as chloramphenicol, tetracycline, or fusidic acid protects extant mRNA from degradation, presumably by immobilizing ribosomes, whereas puromycin exposes mRNA to more rapid degradation than normal. The promoter distal (3′) portion of mRNA, synthesized after ribosomes have been immobilized by chloramphenicol on the promoter proximal (5′) portion of the mRNA, is subsequently degraded. PMID:4583248

  5. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    PubMed

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  6. Higgs mass from neutrino-messenger mixing

    NASA Astrophysics Data System (ADS)

    Byakti, Pritibhajan; Khosa, Charanjit K.; Mummidi, V. S.; Vempati, Sudhir K.

    2017-03-01

    The discovery of the Higgs particle at 125 GeV has put strong constraints on minimal messenger models of gauge mediation, pushing the stop masses into the multi-TeV regime. Extensions of these models with matter-messenger mixing terms have been proposed to generate a large trilinear parameter, A t , relaxing these constraints. The detailed survey of these models [1, 2] so far considered messenger mixings with only MSSM superfields. In the present work, we extend the survey to MSSM with inverse-seesaw mechanism. The neutrino-sneutrino corrections to the Higgs mass in the inverse seesaw model are not significant in the minimal gauge mediation model, unless one considers messenger-matter interaction terms. We classify all possible models with messenger-matter interactions and perform thorough numerical analysis to find out the promising models. We found that out of the 17 possible models 9 of them can lead to Higgs mass within the observed value without raising the sfermion masses significantly. The successful models have stop masses ˜1.5 TeV with small or negligible mixing and yet a light CP even Higgs at 125 GeV.

  7. The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

    PubMed

    Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik; Sowd, Gregory A; Qian, Juan; Hao, Caili; Dasgupta, Twishasri; Engelman, Alan N; Skowronski, Jacek

    2016-05-27

    HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

  8. Sweet spot supersymmetry and composite messengers

    NASA Astrophysics Data System (ADS)

    Ibe, Masahiro; Kitano, Ryuichiro

    2008-05-01

    Sweet spot supersymmetry is a phenomenological effective Lagrangian of weak scale supersymmetry with a certain set of natural assumptions. This framework is designed to avoid problems in low-energy phenomenology and cosmology of supersymmetric models. We discuss a class of dynamical models of supersymmetry breaking and its mediation, whose low-energy effective description falls into this framework. Hadron fields in the dynamical models play a role of the messengers of the supersymmetry breaking. As is always true in the models of the sweet spot supersymmetry, the messenger scale is predicted to be 105 GeV ≲Mmess ≲1010 GeV. Various values of the effective number of messenger fields Nmess are possible depending on the choice of the gauge group.

  9. Sweet Spot Supersymmetry and Composite Messengers

    SciTech Connect

    Ibe, Masahiro; Kitano, Ryuichiro

    2007-10-30

    Sweet spot supersymmetry is a phenomenologically and cosmologically perfect framework to realize a supersymmetric world at short distance. We discuss a class of dynamical models of supersymmetry breaking and its mediation whose low-energy effective description falls into this framework. Hadron fields in the dynamical models play a role of the messengers of the supersymmetry breaking. As is always true in the models of the sweet spot supersymmetry, the messenger scale is predicted to be 10{sup 5} GeV {approx}< M{sub mess} {approx}< 10{sup 10} GeV. Various values of the effective number of messenger fields N{sub mess} are possible depending on the choice of the gauge group.

  10. An allosteric self-splicing ribozyme triggered by a bacterial second messenger.

    PubMed

    Lee, Elaine R; Baker, Jenny L; Weinberg, Zasha; Sudarsan, Narasimhan; Breaker, Ronald R

    2010-08-13

    Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5' untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

  11. Intercultural Learning via Instant Messenger Interaction

    ERIC Educational Resources Information Center

    Jin, Li; Erben, Tony

    2007-01-01

    This paper reports on a qualitative study investigating the viability of instant messenger (IM) interaction to facilitate intercultural learning in a foreign language class. Eight students in a Chinese as a foreign language (CFL) class participated in the study. Each student was paired with a native speaker (NS) of Chinese, and each pair…

  12. MESSENGER Gets Closer to Mercury than Ever Before

    NASA Image and Video Library

    2014-07-28

    MESSENGER Gets Closer to Mercury than Ever Before. This image is one of the highest resolution images taken by NASA MESSENGER spacecraft to date. It features a field of secondary craters in Mercury northern smooth plains.

  13. Evaluation of ribosomal RNA removal protocols for Salmonella RNA-Seq projects

    USDA-ARS?s Scientific Manuscript database

    Next generation sequencing is a powerful technology and its application to sequencing entire RNA populations of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is the massive abundance of ribosomal RNA. Unlike eukaryotic messenger RNA (mRNA), bacterial ...

  14. MESSENGER at Mercury: Early Orbital Operations

    NASA Technical Reports Server (NTRS)

    McNutt, Ralph L., Jr.; Solomon, Sean C.; Bedini, Peter D.; Anderson, Brian J.; Blewett, David T.; Evans, Larry G.; Gold, Robert E.; Krimigis, Stamatios M.; Murchie, Scott L.; Nittler, Larry R.; Slavin, James A.

    2012-01-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, launched in August 2004 under NASA's Discovery Program, was inserted into orbit about the planet Mercury in March 2011. MESSENGER's three flybys of Mercury in 2008-2009 marked the first spacecraft visits to the innermost planet since the Mariner 10 flybys in 1974-1975. The unprecedented orbital operations are yielding new insights into the nature and evolution of Mercury. The scientific questions that frame the MESSENGER mission led to the mission measurement objectives to be achieved by the seven payload instruments and the radio science experiment. Interweaving the full set of required orbital observations in a manner that maximizes the opportunity to satisfy all mission objectives and yet meet stringent spacecraft pointing and thermal constraints was a complex optimization problem that was solved with a software tool that simulates science observations and tracks progress toward meeting each objective. The final orbital observation plan, the outcome of that optimization process, meets all mission objectives. MESSENGER's Mercury Dual Imaging System is acquiring a global monochromatic image mosaic at better than 90%coverage and at least 250 m average resolution, a global color image mosaic at better than 90%coverage and at least 1 km average resolution, and global stereo imaging at better than 80%coverage and at least 250 m average resolution. Higher-resolution images are also being acquired of targeted areas. The elemental remote sensing instruments, including the Gamma-Ray and Neutron Spectrometer and the X-Ray Spectrometer, are being operated nearly continuously and will establish the average surface abundances of most major elements. The Visible and Infrared Spectrograph channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer is acquiring a global map of spectral reflectance from 300 to 1450 nm wavelength at a range of incidence and emission angles

  15. MESSENGER at Mercury: Early orbital operations

    NASA Astrophysics Data System (ADS)

    McNutt, Ralph L.; Solomon, Sean C.; Bedini, Peter D.; Anderson, Brian J.; Blewett, David T.; Evans, Larry G.; Gold, Robert E.; Krimigis, Stamatios M.; Murchie, Scott L.; Nittler, Larry R.; Phillips, Roger J.; Prockter, Louise M.; Slavin, James A.; Zuber, Maria T.; Finnegan, Eric J.; Grant, David G.; MESSENGER Team

    2014-01-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, launched in August 2004 under NASA's Discovery Program, was inserted into orbit about the planet Mercury in March 2011. MESSENGER's three flybys of Mercury in 2008-2009 marked the first spacecraft visits to the innermost planet since the Mariner 10 flybys in 1974-1975. The unprecedented orbital operations are yielding new insights into the nature and evolution of Mercury. The scientific questions that frame the MESSENGER mission led to the mission measurement objectives to be achieved by the seven payload instruments and the radio science experiment. Interweaving the full set of required orbital observations in a manner that maximizes the opportunity to satisfy all mission objectives and yet meet stringent spacecraft pointing and thermal constraints was a complex optimization problem that was solved with a software tool that simulates science observations and tracks progress toward meeting each objective. The final orbital observation plan, the outcome of that optimization process, meets all mission objectives. MESSENGER's Mercury Dual Imaging System is acquiring a global monochromatic image mosaic at better than 90% coverage and at least 250 m average resolution, a global color image mosaic at better than 90% coverage and at least 1 km average resolution, and global stereo imaging at better than 80% coverage and at least 250 m average resolution. Higher-resolution images are also being acquired of targeted areas. The elemental remote sensing instruments, including the Gamma-Ray and Neutron Spectrometer and the X-Ray Spectrometer, are being operated nearly continuously and will establish the average surface abundances of most major elements. The Visible and Infrared Spectrograph channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer is acquiring a global map of spectral reflectance from 300 to 1450 nm wavelength at a range of incidence and emission

  16. MESSENGER at Mercury: Early Orbital Operations

    NASA Technical Reports Server (NTRS)

    McNutt, Ralph L., Jr; Solomon, Sean C.; Bedini, Peter D.; Anderson, Brian J.; Blewett, David T.; Evans, Larry G.; Gold, Robert E.; Krimigis, Stamatios M.; Murchie, Scott L.; Nittler, Larry R.; hide

    2013-01-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, launched in August 2004 under NASA's Discovery Program, was inserted into orbit about the planet Mercury in March 2011. MESSENGER's three flybys of Mercury in 2008-2009 marked the first spacecraft visits to the innermost planet since the Mariner 10 flybys in 1974-1975. The unprecedented orbital operations are yielding new insights into the nature and evolution of Mercury. The scientific questions that frame the MESSENGER mission led to the mission measurement objectives to be achieved by the seven payload instruments and the radio science experiment. Interweaving the full set of required orbital observations in a manner that maximizes the opportunity to satisfy all mission objectives and yet meet stringent spacecraft pointing and thermal constraints was a complex optimization problem that was solved with a software tool that simulates science observations and tracks progress toward meeting each objective. The final orbital observation plan, the outcome of that optimization process, meets all mission objectives. MESSENGER's Mercury Dual Imaging System is acquiring a global monochromatic image mosaic at better than 90% coverage and at least 250 m average resolution, a global color image mosaic at better than 90% coverage and at least 1 km average resolution, and global stereo imaging at better than 80% coverage and at least 250 m average resolution. Higher-resolution images are also being acquired of targeted areas. The elemental remote sensing instruments, including the Gamma-Ray and Neutron Spectrometer and the X-Ray Spectrometer, are being operated nearly continuously and will establish the average surface abundances of most major elements. The Visible and Infrared Spectrograph channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer is acquiring a global map of spectral reflectance from 300 to 1450 nm wavelength at a range of incidence and emission

  17. Attitude Sensor and Gyro Calibration for Messenger

    NASA Technical Reports Server (NTRS)

    O'Shaughnessy, Daniel; Pittelkau, Mark E.

    2007-01-01

    The Redundant Inertial Measurement Unit Attitude Determination/Calibration (RADICAL(TM)) filter was used to estimate star tracker and gyro calibration parameters using MESSENGER telemetry data from three calibration events. We present an overview of the MESSENGER attitude sensors and their configuration is given, the calibration maneuvers are described, the results are compared with previous calibrations, and variations and trends in the estimated calibration parameters are examined. The warm restart and covariance bump features of the RADICAL(TM) filter were used to estimate calibration parameters from two disjoint telemetry streams. Results show that the calibration parameters converge faster with much less transient variation during convergence than when the filter is cold-started at the start of each telemetry stream.

  18. The Energy Messenger, Number 1, Volume 4

    SciTech Connect

    Stancil, J.

    1995-01-01

    `The Energy Messenger` is a Department of Energy publication on energy activities of interest to American Indians. The first issue of 1995 (in a magazine format) includes articles on: tribes winning grants to develop energy resources, recruiting of internships for DOE, information about Title XXVI-Indian Energy Resources, American Indian Heritage Month, tribal perspective on DOE actions, joint ventures between tribes and the DOE, and brief description of recent DOE activities.

  19. MESSENGER Observations of Substorm Activity at Mercury

    NASA Astrophysics Data System (ADS)

    Sun, W. J.; Slavin, J. A.; Fu, S.; Raines, J. M.; Zong, Q. G.; Poh, G.; Jia, X.; Sundberg, T.; Gershman, D. J.; Pu, Z.; Zurbuchen, T.; Shi, Q.

    2015-12-01

    MErcury Surface, Space ENviroment, GEochemistry, and Ranging (MESSENGER) magnetic field and plasma measurements taken during crossings of Mercury's magnetotail from 2011 to 2014 have been investigated for substorms. A number of events with clear Earth-like growth phase and expansion phase signatures were found. The thinning of the plasma sheet and the increase of magnetic field intensity in the lobe were observed during the growth phase and plasma sheet was observed to thicken during the expansion phase, which are similar to the observations at Earth. But the time scale of Mercury's substorm is only several minutes comparing with the several hours at Earth [Sun et al., 2015a]. Detailed analysis of magnetic field fluctuations during the substorm expansion phase have revealed low frequency plasma waves, e.g. Pi2-like pulsations. The By fluctuations accompanying substorm dipolarizations are consistent with pulses of field-aligned currents near the high latitude edge of the plasma sheet. Further study shows that they are near-circularly polarized electromagnetic waves, most likely Alfvén waves. Soon afterwards the plasma sheet thickened and MESSENGER detected a series of compressional waves. We have also discussed their possible sources [Sun et al., 2015b]. Sun, W.-J., J. A. Slavin, S. Y. Fu, et al. (2015a), MESSENGER observations of magnetospheric substorm activity in Mercury's near magnetotail. Geophys. Res. Lett., 42, 3692-3699. doi: 10.1002/2015GL064052.Sun, W.-J., J. A. Slavin, S. Y. Fu, et al. (2015b), MESSENGER observations of Alfvénic and compressional waves during Mercury's substorms. Geophys. Res. Lett., 42, in press. doi: 10.1002/ 2015GL065452.

  20. Mercury's Na Exosphere from MESSENGER data

    NASA Astrophysics Data System (ADS)

    Killen, Rosemary M.; Burger, M. H.; Cassidy, T. A.; Sarantos, M.; Vervack, R. J.; McClintock, W. E.; Merkel, A. W.; Sprague, A. L.; Solomon, S. C.

    2012-10-01

    MESSENGER entered orbit about Mercury on March 18, 2011. Since then, the Ultraviolet and Visible Spectrometer (UVVS) channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer (MASCS) has been observing Mercury's exosphere nearly continuously. Daily measurements of Na brightness were fitted with non-uniform exospheric models. With Monte Carlo sampling we traced the trajectories of a representative number of test particles, generally one million per run per source process, until photoionization, escape from the gravitational well, or permanent sticking at the surface removed the atom from the simulation. Atoms were assumed to partially thermally accommodate on each encounter with the surface with accommodation coefficient 0.25. Runs for different assumed source processes are run separately, scaled and co-added. Once these model results were saved onto a 3D grid, we ran lines of sight from the MESSENGER spacecraft to infinity using the SPICE kernels and we computed brightness integrals. Note that only particles that contribute to the measurement can be constrained with our method. Atoms and molecules produced on the nightside must escape the shadow in order to scatter light if the excitation process is resonant-light scattering, as assumed here. The aggregate distribution of Na atoms fits a 1200 K gas, with a PSD distribution, along with a hotter component. Our models constrain the hot component, assumed to be impact vaporization, to be emitted with a 2500 K Maxwellian. Most orbits show a dawnside enhancement in the hot component broadly spread over the leading hemisphere. However, on some dates there is no dawn/dusk asymmetry. The hot portion of the source appears to be highly variable. The authors acknowledge support from NASA through the MESSENGER Participating Scientist Program and Planetary Atmospheres research grants.

  1. Holographic gauge mediation via strongly coupled messengers

    SciTech Connect

    McGuirk, Paul; Shiu, Gary; Sumitomo, Yoske

    2010-01-15

    We consider a relative of semidirect gauge mediation where the hidden sector exists at large 't Hooft coupling. Such scenarios can be difficult to describe using perturbative field theory methods but may fall into the class of holographic gauge mediation scenarios, meaning that they are amenable to the techniques of gauge/gravity duality. We use a recently found gravity solution to examine one such case, where the hidden sector is a cascading gauge theory resulting in a confinement scale not much smaller than the messenger mass. In the original construction of holographic gauge mediation, as in other examples of semidirect gauge mediation at strong coupling, the primary contributions to visible sector soft terms come from weakly coupled messenger mesons. In contrast to these examples, we describe the dual of a gauge theory where there are significant contributions from scales in which the strongly coupled messenger quarks are the effective degrees of freedom. In this regime, the visible sector gaugino mass can be calculated entirely from holography.

  2. Occupational injuries among Boston bicycle messengers.

    PubMed

    Dennerlein, Jack Tigh; Meeker, John D

    2002-12-01

    Urban bicycle couriers may have a high incidence of injuries. Most messengers work as contractors and hence their injuries are not well documented. To quantify injury rates and severity among urban bicycle couriers a convenience sample of 113 couriers in the city of Boston completed a two-page self-administered survey. Most working couriers have suffered at least one injury resulting either in days lost from work (70%) and in visits to a health-care professional or hospital (55%). The annual incidence rate for injuries resulting in days away from work was 47/100-bike couriers. Bone fractures accounted for the most days lost from work, followed by dislocations, sprains, and strains. Collisions and avoiding collisions with motor vehicles, including being "doored," and collisions with pedestrians accounted for the majority (66%) of events leading to injury. Twenty-four percent of messengers reported wearing a helmet on a regular basis, and 32% have health insurance. Urban bicycle messengers are a poorly documented, largely unstudied workforce who suffer a very high rate of occupational injury. Copyright 2002 Wiley-Liss, Inc.

  3. (Dicer)phering roles of microRNA in platelets.

    PubMed

    Boilard, Eric; Belleannée, Clémence

    2016-04-07

    In this issue of Blood, Rowley et al report that noncoding RNAs precisely regulate the messenger RNA (mRNA) profile in platelets. Interfering in this process using genetically engineered mice affects hemostatic and thrombotic functions of platelets.

  4. MESSENGER: The Discovery Mission to Mercury

    NASA Astrophysics Data System (ADS)

    McNutt, R. L.; Solomon, S. C.; Gold, R. E.; Domingue, D. L.

    2004-12-01

    NASA's MErcury, Surface, Space ENvironment, GEochenistry, and Ranging (MESSENGER) spacecraft, launched on 3 August 2004, has begun its voyage to initiate a new era in our understanding of the terrestrial planets. The mission, spacecraft, and payload are designed to answer six fundamental questions regarding the innermost planet: What planetary formational processes led to Mercury's high metal/silicate ratio? What is the geological history of Mercury? What are the nature and origin of Mercury's magnetic field? What are the structure and state of Mercury's core? What are the radar-reflective materials at Mercury's poles? What are the important volatile species and their sources and sinks on and near Mercury? Planet formational hypotheses will be tested by measuring the surface abundances of major elements by X-ray and gamma-ray spectrometry. The geological history will be determined from high-resolution color imaging of the heavily cratered highlands, intercrater plains, and smooth plains. MESSENGER will provide detailed views of both the Caloris basin and its antipodal terrain. Topographic, mineralogical, and elemental abundance data will be used to seek evidence of volcanic features and units. Measurement of Mercury's magnetic field and its interaction with the solar wind will distinguish the intrinsic dipole and quadrupole components while separating these from the current systems driven by solar-wind-induced convection. The structure of the internal field will put constraints on dynamo models. Such models will also be constrained by measuring Mercury's libration to determine the extent of a fluid outer core. Both water ice and sulfur have been postulated as major constituents of the high-radar-backscatter polar deposits. MESSENGER will combine gamma-ray and neutron spectrometry of the surface with ultraviolet spectrometry and in situ particle measurements to detect both neutral and charged species originating from the surface. Such measurements will address the

  5. Metabolism of arginine-specific messenger ribonucleic acid in Escherichia coli K-12.

    PubMed Central

    Natter, W; Sens, D; James, E

    1977-01-01

    Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of the level of messenger RNA (mRNA) transcribed from seven of the nine genes of the arginine regulon of Escherichia coli K-12. The quantity of RNA complexing with each of the separated DNA strands of the argA, argF, argE, and argCBH operons carried on specialized transducing phages was measured. The derepressed:repressed ratio of mRNA formed in vivo was found to vary between about 3 and 4 when measured by hybridization to DNA isolated from specialized transducing phages carrying the argA, argE, argCBH, argF, and argI operons. PMID:326762

  6. Unsupervised Classification of MESSENGER MASC Data

    NASA Astrophysics Data System (ADS)

    de Sanctis, M. Cristina; Capaccioni, Fabrizio; Filacchione, Gianrico; Ammannito, Eleonora

    2010-05-01

    The MESSENGER spacecraft flew by Mercury as part of its journey to Mercury orbit insertion. The Mercury At-mospheric and Surface Composition Spectrometer (MASCS) observed Mercury during the first two flybys, includ-ing high-spatial-and spectral-resolution visible to near-infrared (IR) spectra of the Mercury surface. The Visible and InfraRed Spectrograph (VIRS) component of MASCS consists of two linear photodiode arrays covering a spectral range 320-1450 nm. We applied classification method to MASCS data in order to extract information on the mineralogy of Mercury. The classification of the Messenger data will permit to obtain maps of Mercury surface, giving us indication of the different mineralogy and maturity present on the Hermean surface. The data were pre-processed applying photometric correction and the VIS and NIR data were collected in a single spectrum. The data set show very similar featureless spectra. The main differences are in the reflectance levels and in the spectral slopes. To emphasize the spectral differences we have normalized the spectra to an average reflectance spectrum for each flyby. This allows to point out variation of different regions with respect to the aver-age spectral behaviour. Two different approaches have been used to analyze MASCS data of the two Messenger flybys: ISODATA unsupervised classification and a classification based on three different spectral slopes (in the wavelengths' ranges 0.3-0.55, 0.55-0.8 and 0.95-1.49 µm). The identified classes shows differences linked with slopes and reflectance's level: the proposed methods allows to correlate the most important classes with different morphological features on Mercury's surface which differ for weathering, maturity and composition. Our analysis is done in order to test and verify these classification methods that shall be necessary to analyze similar data harvested by SIMBIO-SYS/VIHI (Visible and Infrared Hyper-spectral Imager) aboard the future ESA's BepiColombo mission

  7. Mercury's Na Exosphere from MESSENGER Data

    NASA Technical Reports Server (NTRS)

    Killen, Rosemary M.; Burger, M. H.; Cassidy, T. A.; Sarantos, M.; Vervack, R. J.; McClintock, W. El; Merkel, A. W.; Sprague, A. L.; Solomon, S. C.

    2012-01-01

    MESSENGER entered orbit about Mercury on March 18, 2011. Since then, the Ultraviolet and Visible Spectrometer (UWS) channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer (MASCS) has been observing Mercury's exosphere nearly continuously. Daily measurements of Na brightness were fitted with non-uniform exospheric models. With Monte Carlo sampling we traced the trajectories of a representative number of test particles, generally one million per run per source process, until photoionization, escape from the gravitational well, or permanent sticking at the surface removed the atom from the simulation. Atoms were assumed to partially thermally accommodate on each encounter with the surface with accommodation coefficient 0.25. Runs for different assumed source processes are run separately, scaled and co-added. Once these model results were saved onto a 3D grid, we ran lines of sight from the MESSENGER spacecraft :0 infinity using the SPICE kernels and we computed brightness integrals. Note that only particles that contribute to the measurement can be constrained with our method. Atoms and molecules produced on the nightside must escape the shadow in order to scatter light if the excitation process is resonant-light scattering, as assumed here. The aggregate distribution of Na atoms fits a 1200 K gas, with a PSD distribution, along with a hotter component. Our models constrain the hot component, assumed to be impact vaporization, to be emitted with a 2500 K Maxwellian. Most orbits show a dawnside enhancement in the hot component broadly spread over the leading hemisphere. However, on some dates there is no dawn/dusk asymmetry. The portion of the hot/cold source appears to be highly variable.

  8. Mercury's Na Exosphere from MESSENGER Data

    NASA Technical Reports Server (NTRS)

    Killen, Rosemary M.; Burger, M. H.; Cassidy, T. A.; Sarantos, M.; Vervack, R. J.; McClintock, W. El; Merkel, A. W.; Sprague, A. L.; Solomon, S. C.

    2012-01-01

    MESSENGER entered orbit about Mercury on March 18, 2011. Since then, the Ultraviolet and Visible Spectrometer (UWS) channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer (MASCS) has been observing Mercury's exosphere nearly continuously. Daily measurements of Na brightness were fitted with non-uniform exospheric models. With Monte Carlo sampling we traced the trajectories of a representative number of test particles, generally one million per run per source process, until photoionization, escape from the gravitational well, or permanent sticking at the surface removed the atom from the simulation. Atoms were assumed to partially thermally accommodate on each encounter with the surface with accommodation coefficient 0.25. Runs for different assumed source processes are run separately, scaled and co-added. Once these model results were saved onto a 3D grid, we ran lines of sight from the MESSENGER spacecraft :0 infinity using the SPICE kernels and we computed brightness integrals. Note that only particles that contribute to the measurement can be constrained with our method. Atoms and molecules produced on the nightside must escape the shadow in order to scatter light if the excitation process is resonant-light scattering, as assumed here. The aggregate distribution of Na atoms fits a 1200 K gas, with a PSD distribution, along with a hotter component. Our models constrain the hot component, assumed to be impact vaporization, to be emitted with a 2500 K Maxwellian. Most orbits show a dawnside enhancement in the hot component broadly spread over the leading hemisphere. However, on some dates there is no dawn/dusk asymmetry. The portion of the hot/cold source appears to be highly variable.

  9. [Pathophysiological implications of the chemical messengers].

    PubMed

    Blázquez Fernández, Enrique

    2009-01-01

    To maintain a physical organization and a different composition of its surroundings environment, living beings use a great part of the energy that they produce. Vital processes require an elevated number of reactions which are regulated and integrated by chemical messengers. They use autocrine, paracrine, endocrine and synaptic signals through receptors of cell surface, nuclear or associated with ionic chanels, enzymes, trimeric G proteins and to intracellular kinases. Through these mechanisms pheromones play an important role in the relationships between different individuals, and hormones are able to regulate the integrative functions of our organism. In the nervous system, neurotransmitters, neuromodulators, sensors and receptors between other messengers, play functions of great relevance, while growth factors stimulate cell proliferation and cytokines have many effects but the most important is the ones related with the control of the immflamatory process. Alterations of these messengers permit us a better understanding of the diseases and possibly of its treatments in a near future. Modifications of the expression of genes from the nuclear and mitochondrial genomas are responsible of monogenic, polygenic and mitochondrial diseases, while alterations in the activities of dopamine and serotonin neurotransmitters are related with schizophrenia, Parkinson disease and depression, respectively. Other example is the hyperthyroidism of the Graves-Bassedow disease due to the competitive interference of the LATS immunoglobulin with TSH at the level of the folicular cells producing thyroid hormones Twenty five years ago in the reviews on the mechanisms of insulin action, there was presentations in which the insulin receptor was located in the plasma membrane of the target cells while in the cytoplasm only a big interrogative was observed, that at present is replaced by chemical mediators cascades responsible of the multiple effects of insulin. This finding is similar to

  10. Gravitational Waves and Multi-Messenger Astronomy

    NASA Technical Reports Server (NTRS)

    Centrella, Joan M.

    2010-01-01

    Gravitational waves are produced by a wide variety of sources throughout the cosmos, including the mergers of black hole and neutron star binaries/compact objects spiraling into central black holes in galactic nuclei, close compact binaries/and phase transitions and quantum fluctuations in the early universe. Observing these signals can bring new, and often very precise, information about their sources across vast stretches of cosmic time. In this talk we will focus on thee opening of this gravitational-wave window on the universe, highlighting new opportunities for discovery and multi-messenger astronomy.

  11. MESSENGER observations of Mercury's Plasma Mantle

    NASA Astrophysics Data System (ADS)

    Jasinski, J. M.; Slavin, J. A.; Raines, J. M.; DiBraccio, G. A.

    2016-12-01

    We present a survey of plasma mantle observations identified in particle and magnetic field data from four years of MESSENGER spacecraft measurements of Mercury's magnetosphere. The plasma mantle is a region of solar wind plasma entry into the nightside high-latitude magnetosphere. The two common observational signatures of this region are ion energy latitude dispersions as well as diamagnetic depressions. From these observations we estimate the contribution of plasma from the solar wind via the mantle and infer magnitude and variability in the cross-magnetospheric electric fields present at Mercury's dynamic magnetosphere.

  12. MicroRNA expression analysis using the Affymetrix Platform.

    PubMed

    Dee, Suzanne; Getts, Robert C

    2012-01-01

    Microarrays have been used extensively for messenger RNA expression monitoring. Recently, microarrays have been designed to interrogate expression levels of noncoding RNAs. Here, we describe methods for RNA labeling and the use of a miRNA array to identify and measure microRNA present in RNA samples.

  13. Multi-Messenger Astronomy and Dark Matter

    NASA Astrophysics Data System (ADS)

    Bergström, Lars

    This chapter presents the elaborated lecture notes on Multi-Messenger Astronomy and Dark Matter given by Lars Bergström at the 40th Saas-Fee Advanced Course on "Astrophysics at Very High Energies". One of the main problems of astrophysics and astro-particle physics is that the nature of dark matter remains unsolved. There are basically three complementary approaches to try to solve this problem. One is the detection of new particles with accelerators, the second is the observation of various types of messengers from radio waves to gamma-ray photons and neutrinos, and the third is the use of ingenious experiments for direct detection of dark matter particles. After giving an introduction to the particle universe, the author discusses the relic density of particles, basic cross sections for neutrinos and gamma-rays, supersymmetric dark matter, detection methods for neutralino dark matter, particular dark matter candidates, the status of dark matter detection, a detailled calculation on an hypothetical "Saas-Fee Wimp", primordial black holes, and gravitational waves.

  14. MESSENGER observations of magnetopause structure at Mercury

    NASA Astrophysics Data System (ADS)

    DiBraccio, G. A.; Slavin, J. A.; Boardsen, S. A.; Anderson, B. J.; Korth, H.; Zurbuchen, T.; Raines, J. M.; McNutt, R. L.; Solomon, S. C.

    2011-12-01

    On 18 March 2011, MESSENGER became the first spacecraft to orbit Mercury, providing a new opportunity to study the outer boundary of the innermost planet's magnetosphere - the magnetopause. The 12-hour orbital period yields a minimum of four magnetopause crossings per day, which facilitates the investigation of the effect of the interplanetary magnetic field (IMF) on the magnetopause structure. Here we use data from MESSENGER's Magnetometer (MAG) and Fast Imaging Plasma Spectrometer (FIPS) to characterize the magnetopause. A minimum variance analysis (MVA) is executed to transform the MAG data into current-sheet coordinates. In this new coordinate system we determine (1) the temporal duration and, with assumptions, the thickness of the magnetopause, (2) the magnetic shear angle across the boundary, and (3) the normal magnetic field across the current sheet, from which we infer the rate of reconnection. FIPS measurements provide a validation of the structure of the magnetopause determined from the MAG data, i.e., whether the magnetopause is magnetically open or closed, on the basis of its permeability to solar wind ions. The results of our analysis indicate that the structure of Mercury's magnetopause is highly responsive to IMF direction and, whenever the shear angle is greater than 90°, is generally open to the solar wind plasma under normal magnetic field components of order 1-10 nT.

  15. Multi-messenger aspects of cosmic neutrinos

    NASA Astrophysics Data System (ADS)

    Ahlers, Markus

    2016-04-01

    The recent observation of TeV-PeV neutrinos by IceCube has opened a new window to the high-energy Universe. I will discuss this signal in the context of multi-messenger astronomy. For extragalactic source scenarios the corresponding gamma-rays are not directly observable due to interactions with the cosmic radiation backgrounds. Nevertheless, the isotropic sub-TeV gamma ray background observed by Fermi-LAT contains indirect information from secondary emission produced in electromagnetic cascades. On the other hand, observation of PeV gamma rays would provide a smoking-gun signal for Galactic emission. Interestingly, the overall energy density of the observed neutrino flux is close to a theoretical limit for neutrino production in ultra-high energy cosmic ray sources and might indicate a common origin of these phenomena. I will highlight various multi-messenger relations and their implications for neutrino source scenarios. This article is an excerpt from an ICRC 2015 proceedings contribution [1].

  16. MESSENGER observations of Mercury's magnetic field structure

    NASA Astrophysics Data System (ADS)

    Johnson, Catherine L.; Purucker, Michael E.; Korth, Haje; Anderson, Brian J.; Winslow, Reka M.; Al Asad, Manar M. H.; Slavin, James A.; Alexeev, Igor. I.; Phillips, Roger J.; Zuber, Maria T.; Solomon, Sean C.

    2012-12-01

    We present a baseline, time-averaged model for Mercury's magnetosphere, derived from MESSENGER Magnetometer data from 24 March to 12 December 2011, comprising the spacecraft's first three Mercury years in orbit around the innermost planet. The model, constructed under the approximation that the magnetospheric shape can be represented as a paraboloid of revolution, includes two external (magnetopause and magnetotail) current systems and an internal (dipole) field and allows for reconnection. We take advantage of the geometry of the orbital Magnetometer data to estimate all but one of the model parameters, and their ranges, directly from the observations. These parameters are then used as a priori constraints in the paraboloid magnetospheric model, and the sole remaining parameter, the dipole moment, is estimated as 190 nT RM3 from a grid search. We verify that the best fit dipole moment is insensitive to changes in the other parameters within their determined ranges. The model provides an excellent first-order fit to the MESSENGER observations, with a root-mean-square misfit of less than 20 nT globally. The results show that the magnetopause field strength ranges from 10% to 50% of the dipole field strength at observation locations on the dayside and at nightside latitudes north of 60°N. Globally, the residual signatures observed to date are dominated by the results of magnetospheric processes, confirming the dynamic nature of Mercury's magnetosphere.

  17. Imaging During MESSENGER's Second Flyby of Mercury

    NASA Astrophysics Data System (ADS)

    Chabot, N. L.; Prockter, L. M.; Murchie, S. L.; Robinson, M. S.; Laslo, N. R.; Kang, H. K.; Hawkins, S. E.; Vaughan, R. M.; Head, J. W.; Solomon, S. C.; MESSENGER Team

    2008-12-01

    During MESSENGER's second flyby of Mercury on October 6, 2008, the Mercury Dual Imaging System (MDIS) will acquire 1287 images. The images will include coverage of about 30% of Mercury's surface not previously seen by spacecraft. A portion of the newly imaged terrain will be viewed during the inbound portion of the flyby. On the outbound leg, MDIS will image additional previously unseen terrain as well as regions imaged under different illumination geometry by Mariner 10. These new images, when combined with images from Mariner 10 and from MESSENGER's first Mercury flyby, will enable the first regional- resolution global view of Mercury constituting a combined total coverage of about 96% of the planet's surface. MDIS consists of both a Wide Angle Camera (WAC) and a Narrow Angle Camera (NAC). During MESSENGER's second Mercury flyby, the following imaging activities are planned: about 86 minutes before the spacecraft's closest pass by the planet, the WAC will acquire images through 11 different narrow-band color filters of the approaching crescent planet at a resolution of about 5 km/pixel. At slightly less than 1 hour to closest approach, the NAC will acquire a 4-column x 11-row mosaic with an approximate resolution of 450 m/pixel. At 8 minutes after closest approach, the WAC will obtain the highest-resolution multispectral images to date of Mercury's surface, imaging a portion of the surface through 11 color filters at resolutions of about 250-600 m/pixel. A strip of high-resolution NAC images, with a resolution of approximately 100 m/pixel, will follow these WAC observations. The NAC will next acquire a 15-column x 13- row high-resolution mosaic of the northern hemisphere of the departing planet, beginning approximately 21 minutes after closest approach, with resolutions of 140-300 m/pixel; this mosaic will fill a large gore in the Mariner 10 data. At about 42 minutes following closest approach, the WAC will acquire a 3x3, 11-filter, full- planet mosaic with an

  18. The cosmic mult-messenger background field

    NASA Astrophysics Data System (ADS)

    Hartmann, Dieter

    2016-04-01

    The cosmic star formation history associated with baryon flows within the large scale structure of the expanding Universe has many important consequences, such as cosmic chemical- and galaxy evolution. Stars and accreting compact objects subsequently produce light, from the radio band to the highest photon energies, and dust within galaxies reprocesses a significant fraction of this light into the IR region. The Universe creates a radiation background that adds to the relic field from the big bang, the CMB. In addition, Cosmic Rays are created on variouys scales, and interact with this diffuse radiation field, and neutrinos are added as well. A multi-messenger field is created whose evolution with redshift contains a tremendous amount of cosmological information. We discuss several aspects of this story, emphasizing the background in the HE regime and the neutrino sector, and disccus the use of gamma-ray sources as probes.

  19. Gauge mediation models with adjoint messengers

    NASA Astrophysics Data System (ADS)

    Gogoladze, Ilia; Mustafayev, Azar; Shafi, Qaisar; Ün, Cem Salih

    2016-10-01

    We present a class of models in the framework of gauge mediation supersymmetry breaking where the messenger fields transform in the adjoint representation of the standard model gauge symmetry. To avoid unacceptably light right-handed sleptons in the spectrum we introduce a nonzero U (1 )B-L D-term. This leads to an additional contribution to the soft supersymmetry breaking mass terms which makes the right-handed slepton masses compatible with the current experimental bounds. We show that in this framework the observed 125 GeV Higgs boson mass can be accommodated with the sleptons accessible at the LHC, while the squarks and gluinos lie in the multi-TeV range. We also discuss the issue of the fine-tuning and show that the desired relic dark matter abundance can also be accommodated.

  20. Mercury's magnetosphere after MESSENGER's first flyby.

    PubMed

    Slavin, James A; Acuña, Mario H; Anderson, Brian J; Baker, Daniel N; Benna, Mehdi; Gloeckler, George; Gold, Robert E; Ho, George C; Killen, Rosemary M; Korth, Haje; Krimigis, Stamatios M; McNutt, Ralph L; Nittler, Larry R; Raines, Jim M; Schriver, David; Solomon, Sean C; Starr, Richard D; Trávnícek, Pavel; Zurbuchen, Thomas H

    2008-07-04

    Observations by MESSENGER show that Mercury's magnetosphere is immersed in a comet-like cloud of planetary ions. The most abundant, Na+, is broadly distributed but exhibits flux maxima in the magnetosheath, where the local plasma flow speed is high, and near the spacecraft's closest approach, where atmospheric density should peak. The magnetic field showed reconnection signatures in the form of flux transfer events, azimuthal rotations consistent with Kelvin-Helmholtz waves along the magnetopause, and extensive ultralow-frequency wave activity. Two outbound current sheet boundaries were observed, across which the magnetic field decreased in a manner suggestive of a double magnetopause. The separation of these current layers, comparable to the gyro-radius of a Na+ pickup ion entering the magnetosphere after being accelerated in the magnetosheath, may indicate a planetary ion boundary layer.

  1. Cosmic muons, as messengers from the Universe

    SciTech Connect

    Brancus, I. M.; Rebel, H.

    2015-02-24

    Penetrating from the outer space into the Earth atmosphere, primary cosmic rays are producing secondary radiation by the collisions with the air target subsequently decaying in hadrons, pions, muons, electrons and photons, phenomenon called Extensive air Shower (EAS). The muons, considered as the “penetrating” component, survive the propagation to the Earth and even they are no direct messenger of the Universe, they reflect the features of the primary particles. The talk gives a description of the development of the extensive air showers generating the secondary particles, especially the muon component. Results of the muon flux and of the muon charge ratio, (the ratio between the positive and the negative muons), obtained in different laboratories and in WILLI experiment, are shown. At the end, the contribution of the muons measured in EAS to the investigation of the nature of the primary cosmic rays is emphasized in KASCADE and WILLI-EAS experiments.

  2. [Irisin: a messenger from the gods?].

    PubMed

    Moreno, María; Moreno-Navarrete, José María; Fernández-Real, José Manuel

    2014-01-01

    Due to the need to understand the basis of the metabolic benefits of exercise, irisin was discovered a few years ago. This cytokine, secreted by skeletal muscle due to exercise, should have positive effects on energetic metabolism. In particular, it could act as a messenger on white adipose tissue, modifying its phenotype into the beige adipocyte, and increasing its thermogenic capacity. Since it was described, there have been numerous studies led to depict its function, with the aim of determining if irisin could become a therapeutic target in the context of diseases associated with a caloric excess, such as obesity and diabetes. In this review, the irisin discovery is summarized, along with its in vitro and in vivo effects described up until now.

  3. MESSENGER Observations of Mercury's Dynamic Magnetosphere

    NASA Technical Reports Server (NTRS)

    Slavin, James A.

    2009-01-01

    MESSENGER's 14 January and 6 October 2008 encounters with Mercury have provided new measurements dynamic variations in the coupled atmosphere magnetosphere system. The two flybys took place under very different interplanetary magnetic field (IMF) conditions. The northward IMF during the first encounter produced a very quiet, stable magnetosphere. Neutral sodium atoms and photo-ions were observed to high altitudes ; > 2000 km, even in the subsolar region demonstrating the important role played by more energetic neutral atom production processes such as sputtering. Consistent with predictions of magnetospheric models for northward IMF, the neutral atmosphere was observed to have its strongest sources in the high latitude northern hemisphere for the first flyby. The southward IMF for the second encounter revealed a highly dynamic magnetosphere. Reconnection between the interplanetary and planetary magnetic fields is known to control the rate of energy transfer from the solar wind and to drive magnetospheric convection. The MESSENGER magnetic field measurements revealed that the rate at which interplanetary magnetic fields were reconnecting to planetary fields was a factor of 10 greater than is usually observed at Earth. This extremely high reconnection results in a large magnetic field component normal to the magnetopause and the formation of flux transfer events that are much larger relative to the size of the forward magnetosphere than is observed at Earth. The resulting magnetospheric configuration allows the solar wind access to much of the dayside surface of the Mercury. This widespread impingement of the solar wind on Mercury's surface is a likely source of the less structured sodium exosphere imaged during the second flyby and quite possibly the high degree of exospheric temporal variability observed by ground-based telescopes.

  4. Current Understanding of Mercury's Magnetosphere before MESSENGER

    NASA Astrophysics Data System (ADS)

    Krimigis, S. M.

    The MESSENGER spacecraft is scheduled to be launched mid-May, 2004 on a trajectory that includes two flybys (October 07, July 08) and eventual orbit insertion in July 2009 around the planet Mercury. Embedded in its payload are instruments to examine the basic properties of the planet's magnetosphere, including magnetometer, plasma, and energetic particle measurements (Gold et al, 2001). Our present knowledge of Mercury's magnetosphere is derived from two nightside Mariner 10 flybys in 1974, 1975 that established the presence of an intrinsic magnetic field and some energetic particles. Unfortunately not even the magnetic dipole term was well-resolved, and the fluxes and identity of energetic particles have been a subject of extensive discussion and varying interpretations (e.g. Armstrong et al, 1975, Christon, 1989). There has been evidence of field-aligned currents (e.g. Slavin et al, 1997), but alternative interpretations of magnetic signatures suggest that the magnetosphere may be driven by changing external boundary conditions (Luhman et al, 1998). These uncertainties, coupled with the observed presence of volatiles (H, He, O, Na, K, Ca) raise obvious questions on current closure, hot plasma injection and acceleration, the frequency with which the planetary surface is exposed to the solar wind, and potential sputtering of material due to particle impingement on the regolith. The talk will review our current knowledge and describe the measurements expected from MESSENGER that will address some of the key science questions. Armstrong et al, JGR, 80, 4015, 1975 Gold et al, Planet and Space Sci, 49, 1467, 2001 Christon, S.P., JGR, 94, 6481, 1989 Slavin et al, Planet and Space Sci, 45, 133, 1997 Luhman et al, JGR, 103, 9113, 1998

  5. Endogenous Arabidopsis messenger RNAs transported to distant tissues.

    PubMed

    Thieme, Christoph J; Rojas-Triana, Monica; Stecyk, Ewelina; Schudoma, Christian; Zhang, Wenna; Yang, Lei; Miñambres, Miguel; Walther, Dirk; Schulze, Waltraud X; Paz-Ares, Javier; Scheible, Wolf-Rüdiger; Kragler, Friedrich

    2015-03-23

    The concept that proteins and small RNAs can move to and function in distant body parts is well established. However, non-cell-autonomy of small RNA molecules raises the question: To what extent are protein-coding messenger RNAs (mRNAs) exchanged between tissues in plants? Here we report the comprehensive identification of 2,006 genes producing mobile RNAs in Arabidopsis thaliana. The analysis of variant ecotype transcripts that were present in heterografted plants allowed the identification of mRNAs moving between various organs under normal or nutrient-limiting conditions. Most of these mobile transcripts seem to follow the phloem-dependent allocation pathway transporting sugars from photosynthetic tissues to roots via the vasculature. Notably, a high number of transcripts also move in the opposite, root-to-shoot direction and are transported to specific tissues including flowers. Proteomic data on grafted plants indicate the presence of proteins from mobile RNAs, allowing the possibility that they may be translated at their destination site. The mobility of a high number of mRNAs suggests that a postulated tissue-specific gene expression profile might not be predictive for the actual plant body part in which a transcript exerts its function.

  6. Addition of Polyadenylate Sequences to Virus-Specific RNA during Adenovirus Replication

    PubMed Central

    Philipson, L.; Wall, R.; Glickman, G.; Darnell, J. E.

    1971-01-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA. PMID:5315962

  7. Addition of polyadenylate sequences to virus-specific RNA during adenovirus replication.

    PubMed

    Philipson, L; Wall, R; Glickman, G; Darnell, J E

    1971-11-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA.

  8. RNA Processing and Export

    PubMed Central

    Hocine, Sami; Singer, Robert H.; Grünwald, David

    2010-01-01

    Messenger RNAs undergo 5' capping, splicing, 3'-end processing, and export before translation in the cytoplasm. It has become clear that these mRNA processing events are tightly coupled and have a profound effect on the fate of the resulting transcript. This processing is represented by modifications of the pre-mRNA and loading of various protein factors. The sum of protein factors that stay with the mRNA as a result of processing is modified over the life of the transcript, conferring significant regulation to its expression. PMID:20961978

  9. Mercury's interior from MESSENGER geodetic measurements

    NASA Astrophysics Data System (ADS)

    Genova, Antonio; Mazarico, Erwan; Goossens, Sander; Lemoine, Frank G.; Neumann, Gregory A.; Smith, David E.; Zuber, Maria T.; Solomon, Sean C.

    2016-04-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft completed more than 4 years of operations in orbit about Mercury. One of the main mission goals was the determination of the interior structure of Mercury enabled by geodetic observations of the topography, gravity field, rotation, and tides by the Mercury Laser Altimeter (MLA) and radio science system. MLA acquired over 25 million individual measurements of Mercury's shape that are mostly limited to the northern hemisphere because of MESSENGER's eccentric orbit. However, the lack of laser altimetry in the southern hemisphere has been partly compensated by ˜400 occultations of spacecraft radio signals. X-band radio tracking data collected by the NASA Deep Space Network (DSN) allowed the determination of Mercury's gravity field to spherical harmonic degree and order 100, the planet's obliquity, and the Love number k2. The combination of altimetry and radio measurements provides a powerful tool for the investigation of Mercury's orientation and tides, which enable a better understanding of the interior structure of the planet. The MLA measurements have been assembled into a digital elevation model (DEM) of the northern hemisphere. We then used individual altimetric measurements from the spacecraft for orbit determination, together with the radio tracking, over a continuous span of time using a batch least-squares filter. All observations were combined to recover directly the gravity field coefficients, obliquity, librations, and tides by minimizing the discrepancies between the computed observables and actual measurements. We will present the estimated 100×100 gravity field model, the obliquity, the Love number k2, and, for the first time, the tidal phase lag φ and the amplitude of the longitudinal libration from radio and altimetry data. The k2 phase provides information on Mercury's dissipation and mantle viscosity and allows a determination of the Q factor. A refinement of

  10. Calcium in Mercury's Exosphere: Modeling MESSENGER Data

    NASA Technical Reports Server (NTRS)

    Burger, Matthew H.; Killen, Rosemary M.; McClintock, William E.; Merkel, Aimee; Vervack, Ronald J.; Sarantos, Menelaos; Sprague, Ann L.

    2011-01-01

    Mercury is surrounded by a surface-bounded exosphere comprised of atomic species including hydrogen, sodium, potassium, calcium, magnesium, and likely oxygen. Because it is collisionless. the exosphere's composition represents a balance of the active source and loss processes. The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) on the MErcury Surface. Space ENvironment. GEochemistry. and Ranging (MESSENGER) spacecraft has made high spatial-resolution observations of sodium, calcium, and magnesium near Mercury's surface and in the extended, anti-sunward direction. The most striking feature of these data has been the substantial differences in the spatial distribution of each species, Our modeling demonstrates that these differences cannot be due to post-ejection dynamics such as differences in photo-ionization rate and radiation pressure. but instead point to differences in the source mechanisms and regions on the surface from which each is ejected. The observations of calcium have revealed a strong dawn/dusk asymmetry. with the abundance over the dawn hemisphere significantly greater than over the dusk. To understand this asymmetry, we use a Monte Carlo model of Mercury's exosphere that we developed to track the motions of exospheric neutrals under the influence of gravity and radiation pressure. Ca atoms can be ejected directly from the surface or produced in a molecular exosphere (e.g., one consisting of CaO). Particles are removed from the system if they stick to the surface or escape from the model region of interest (within 15 Mercury radii). Photoionization reduces the final weighting given to each particle when simulating the Ca radiance. Preliminary results suggest a high temperature ( I-2x 10(exp 4) K) source of atomic Ca concentrated over the dawn hemisphere. The high temperature is consistent with the dissociation of CaO in a near-surface exosphere with scale height <= 100 km, which imparts 2 eV to the freshly produced Ca atom. This

  11. Calcium in Mercury's Exosphere: Modeling MESSENGER Data

    NASA Astrophysics Data System (ADS)

    Burger, M. H.; Killen, R. M.; McClintock, W. E.; Merkel, A. W.; Vervack, R. J.; Sarantos, M.; Sprague, A. L.

    2011-12-01

    Mercury is surrounded by a surface-bounded exosphere known to contain hydrogen, sodium, potassium, calcium, and magnesium. Because the exosphere is collisionless, its composition represents a balance of active source and loss processes. The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft has made high-spatial-resolution observations of sodium, calcium, and magnesium near Mercury's surface and in the extended, anti-sunward direction. The most striking feature of these data is the substantial differences among species, which was detected during three close flybys of the planet and has been persistantly present during MESSENGER's orbital phase. Our modeling demonstrates that these differences are not because of post-ejection dynamics such as differences in photo-ionization rate and radiation pressure, but rather result from differences in the source mechanisms and regions on the surface from which each species is ejected. The observations of calcium have revealed a strong dawn/dusk asymmetry, with the abundance over the dawn hemisphere substantially greater than that on the dusk side. To understand this asymmetry, we use a Monte Carlo model of Mercury's exosphere that we developed to track the motions of exospheric neutrals under the influence of gravity and radiation pressure. In this model, Ca atoms can be ejected directly from the surface or produced by ejection of CaO followed by dissociation to produce Ca and O. Particles are removed from the system if they stick to the surface or escape from the model region of interest (within 15 Mercury radii). Photoionization reduces the final weighting given to each particle when simulating the Ca radiance. Data from the flybys are consistent with a high temperature (~1-2 x 104 K) source of atomic Ca concentrated over the dawn hemisphere. Such a high temperature resutls from dissociation of CaO in a near

  12. Active transport of messenger ribonucleoprotein particles in a reconstituted cell-free system.

    PubMed

    French, B T; Schumm, D E; Webb, T E

    1987-08-01

    The ability of a reconstituted cell-free system to transport mRNA as a ribonucleoprotein particle has been examined. Poly(A) messenger ribonucleoproteins (mRNPs), UV cross-linked after release from isolated liver nuclei in a cell-free system, exhibited a buoyant density of 1.33 g/cm3 in cesium sulfate and 1.47 g/cm3 in cesium chloride, values identical to those of poly(A) mRNP isolated directly from liver polysomes. Furthermore, the in vivo and in vitro transported mRNP showed a similar degree of resistance to RNase digestion and had sedimentation coefficients approximately 2.5 times that of the isolated mRNA. Release of both total mRNA and alpha 2 mu-globulin mRNA was proportional to the concentration of a specific cytoplasmic protein. Removal of the transport proteins from the cytosol with streptomycin sulfate provided a basal system incapable of supporting the active transport of alpha 2 mu-globulin mRNA. Hybridization of released RNA with a recombinant probe specific for intron 6 of alpha 2 mu-globulin showed that intron sequences were retained within the nucleus under optimal alpha 2 mu-globulin mRNA transport conditions and that the transported alpha 2 mu-globulin mRNA was of mature size.

  13. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.

    PubMed

    Wilbert, Melissa L; Huelga, Stephanie C; Kapeli, Katannya; Stark, Thomas J; Liang, Tiffany Y; Chen, Stella X; Yan, Bernice Y; Nathanson, Jason L; Hutt, Kasey R; Lovci, Michael T; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B; Morris, Quaid; Hoon, Shawn; Yeo, Gene W

    2012-10-26

    LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

  14. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance

    PubMed Central

    Wilbert, Melissa L.; Huelga, Stephanie C.; Kapeli, Katannya; Stark, Thomas J.; Liang, Tiffany Y.; Chen, Stella X.; Yan, Bernice Y.; Nathanson, Jason L.; Hutt, Kasey R.; Lovci, Michael T.; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B.; Morris, Quaid; Hoon, Shawn; Yeo, Gene W.

    2012-01-01

    SUMMARY LIN28 is a conserved RNA binding protein implicated in pluripotency, reprogramming and oncogenesis. Previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through cross-linking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28 binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions. PMID:22959275

  15. Evidence of direct complementary interactions between messenger RNAs and their cognate proteins

    PubMed Central

    Polyansky, Anton A.; Zagrovic, Bojan

    2013-01-01

    Recently, the ability to interact with messenger RNA (mRNA) has been reported for a number of known RNA-binding proteins, but surprisingly also for different proteins without recognizable RNA binding domains including several transcription factors and metabolic enzymes. Moreover, direct binding to cognate mRNAs has been detected for multiple proteins, thus creating a strong impetus to search for functional significance and basic physico-chemical principles behind such interactions. Here, we derive interaction preferences between amino acids and RNA bases by analyzing binding interfaces in the known 3D structures of protein–RNA complexes. By applying this tool to human proteome, we reveal statistically significant matching between the composition of mRNA sequences and base-binding preferences of protein sequences they code for. For example, purine density profiles of mRNA sequences mirror guanine affinity profiles of cognate protein sequences with quantitative accuracy (median Pearson correlation coefficient R = −0.80 across the entire human proteome). Notably, statistically significant anti-matching is seen only in the case of adenine. Our results provide strong evidence for the stereo-chemical foundation of the genetic code and suggest that mRNAs and cognate proteins may in general be directly complementary to each other and associate, especially if unstructured. PMID:23868089

  16. Identification of messenger RNAs and microRNAs associated with fragile X mental retardation protein.

    PubMed

    Duan, Ranhui; Jin, Peng

    2006-01-01

    Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of the Fragile X mental retardation protein (FMRP). FMRP, which may regulate translation in neurons, not only associates with specific messenger RNAs (mRNAs) and with microRNAs (miRNAs), but also associates with the components of the miRNA pathway, including the Dicer and Argonaute proteins. It has been proposed that FMRP regulates the translation of its mRNA targets through miRNAs. In this chapter, we describe the protocol to identify the mRNAs and miRNAs associated with FMRP in vivo. The same method could also be applied to other RNA-binding proteins interacting with specific mRNAs or miRNAs.

  17. Expression of P450c17 messenger ribonucleic acid in postmenopausal human ovary tissues.

    PubMed

    José, M; Puche, C; Cabero, A; Cabero, L; Meseguer, A

    1999-03-01

    To investigate the expression of the P450c17 gene in postmenopausal human ovaries compared with normal cycling ovaries. Prospective nonrandomized clinical research study. Servei de Medicina Reproductiva and Centre d'Investigacions en Bioquimica i Biologia Molecular, Hospitals Vall d'Hebron, Barcelona, Spain. Six premenopausal women and four postmenopausal women undergoing bilateral oophorectomy for nonovarian gynecologic disease. Extraction of 10 mL of peripheral venous blood for hormone measurements. Extraction of RNA from surgically removed ovaries for Northern blot, ribonuclease protection, and reverse transcriptase polymerase chain reaction Southern blot assays. Definition of the reproductive cycle state of each patient and determination of the level of P450c17 gene expression in all samples with the use of the semiquantitative reverse transcriptase polymerase chain reaction Southern blot assay. P450c17 messenger RNA levels in postmenopausal ovaries varied considerably between samples. Although the levels were similar to those detected in the early follicular phase, one of the samples had levels as high as those observed in the late follicular phase. Although the degree varied from one sample to another, all the postmenopausal ovaries studied expressed the P450c17 gene at the messenger RNA level. In a sample from a patient with endometrial adenocarcinoma, the level was as high as the levels observed in the late follicular phase.

  18. Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

    PubMed Central

    Braicu, C; Tomuleasa, C; Monroig, P; Cucuianu, A; Berindan-Neagoe, I; Calin, G A

    2015-01-01

    Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by ‘trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics. PMID:25236394

  19. Messenger Observations of Mercury's Bow Shock and Magnetopause

    NASA Technical Reports Server (NTRS)

    Slavin J. A.; Acuna, M. H.; Anderson, B. J.; Benna, M.; Gloeckler, G.; Krimigis, S. M.; Raines, M.; Schriver, D.; Travnicek, P.; Zurbuchen, T. H.

    2008-01-01

    The MESSENGER spacecraft made the first of three flybys of Mercury on January 14.2008 (1). New observations of solar wind interaction with Mercury were made with MESSENGER'S Magnetometer (MAG) (2.3) and Energetic Particle and Plasma Spectrometer (EPPS) - composed of the Energetic Particle Spectrometer (EPS) and Fast Imaging Plasma Spectrometer (FIPS) (3,4). These MESSENGER observations show that Mercury's magnetosphere has a large-scale structure that is distinctly Earth-like, but it is immersed in a comet-like cloud of planetary ions [5]. Fig. 1 provides a schematic view of the coupled solar wind - magnetosphere - neutral atmosphere - solid planet system at Mercury.

  20. Neutrinos as the messengers of CPT violation

    NASA Astrophysics Data System (ADS)

    Borissov, Liubomir Anguelov

    CPT violation has the potential to explain all three existing neutrino oscillation signals without enlarging the neutrino sector. CPT violation in the Dirac mass terms of the three neutrino flavors preserves Lorentz invariance, but generates in dependent masses for neutrinos and antineutrinos. This specific signature can be motivated by braneworld scenarios with extra dimensions, where neutrinos are the natural messengers for Standard Model physics of CPT violation in the bulk. A simple model of maximal CPT violation is sufficient to explain the exisiting neutrino data, while accommodating the recent results from the KamLAND experiment and making dramatic predictions for the ongoing MiniBooNE experiment. In addition, the model fits the existing SuperKamiokande data, at least as well as the standard atmospheric neutrino oscillation models. Another attractive feature of the presented model is that it provides a new promising mechanism for baryogenesis, which obviates two of the three Sakharov conditions necessary to generate the baryon asymmetry of the universe. CPT-violating scenarios can give new insights about the possible nature of neutrinos. Majorana neutrino masses are still allowed, but in general, there are no longer Majorana neutrinos in the conventional sense. However, CPT-violating models still have interesting consequences for neutrinoless double beta decay. Compared to the usual case, while the larger mass scale (from LSND) may appear, a greater degree of suppression can also occur.

  1. Mercury's Seasonal Sodium Exosphere: MESSENGER Orbital Observations

    NASA Technical Reports Server (NTRS)

    Cassidy, Timothy A.; Merkel, Aimee W.; Burger, Matthew H.; Killen, Rosemary M.; McClintock, William E.; Vervack, Ronald J., Jr.; Sarantos, Menelaos

    2014-01-01

    The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) Ultraviolet and Visible Spectrometer (UVVS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft now orbiting Mercury provides the first close-up look at the planet's sodium exosphere. UVVS has observed the exosphere from orbit almost daily for over 10 Mercury years. In this paper we describe and analyze a subset of these data: altitude profiles taken above the low-latitude dayside and south pole. The observations show spatial and temporal variations, but there are no obvious year-to-year variations in most of the observations. We do not see the episodic variability reported by some ground-based observers. We used these altitude profiles to make estimates of sodium density and temperature. The bulk of the exosphere, at about 1200 K, is much warmer than Mercury's surface. This value is consistent with some ground-based measurements and suggests that photon-stimulated desorption is the primary ejection process. We also observe a tenuous energetic component but do not see evidence of the predicted thermalized (or partially thermalized) sodium near Mercury's surface temperature. Overall we do not see the variable mixture of temperatures predicted by most Monte Carlo models of the exosphere.

  2. Mercury's Seasonal Sodium Exosphere: MESSENGER Orbital Observations

    NASA Technical Reports Server (NTRS)

    Cassidy, Timothy A.; Merkel, Aimee W.; Burger, Matthew H.; Sarantos, Menelaos; Killen, Rosemary M.; McClintock, William E.; Vervack, Ronald J., Jr.

    2014-01-01

    The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) Ultraviolet and Visible Spectrometer (UVVS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft now orbiting Mercury provides the first close-up look at the planet's sodium exosphere. UVVS has observed the exosphere from orbit almost daily for over 10 Mercury years. In this paper we describe and analyze a subset of these data: altitude profiles taken above the low-latitude dayside and south pole. The observations show spatial and temporal variation but there is little or no year-to-year variation; we do not see the episodic variability reported by ground-based observers. We used these altitude profiles to make estimates of sodium density and temperature. The bulk of the exosphere is about 1200 K, much warmer than Mercury's surface. This value is consistent with some ground-based measurements and suggests that photon-stimulated desorption is the primary ejection process. We also observe a tenuous energetic component but do not see evidence of the predicted thermalized (or partially thermalized) sodium near Mercury's surface temperature. Overall we do not see the variable mixture of temperatures predicted by most Monte Carlo models of the exosphere.

  3. Mercury's Seasonal Sodium Exosphere: MESSENGER Orbital Observations

    NASA Technical Reports Server (NTRS)

    Cassidy, Timothy A.; Merkel, Aimee W.; Burger, Matthew H.; Sarantos, Menelaos; Killen, Rosemary M.; McClintock, William E.; Vervack, Ronald J., Jr.

    2014-01-01

    The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) Ultraviolet and Visible Spectrometer (UVVS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft now orbiting Mercury provides the first close-up look at the planet's sodium exosphere. UVVS has observed the exosphere from orbit almost daily for over 10 Mercury years. In this paper we describe and analyze a subset of these data: altitude profiles taken above the low-latitude dayside and south pole. The observations show spatial and temporal variation but there is little or no year-to-year variation; we do not see the episodic variability reported by ground-based observers. We used these altitude profiles to make estimates of sodium density and temperature. The bulk of the exosphere is about 1200 K, much warmer than Mercury's surface. This value is consistent with some ground-based measurements and suggests that photon-stimulated desorption is the primary ejection process. We also observe a tenuous energetic component but do not see evidence of the predicted thermalized (or partially thermalized) sodium near Mercury's surface temperature. Overall we do not see the variable mixture of temperatures predicted by most Monte Carlo models of the exosphere.

  4. Extracellular RNA in aging.

    PubMed

    Dluzen, Douglas F; Noren Hooten, Nicole; Evans, Michele K

    2017-03-01

    Since the discovery of extracellular RNA (exRNA) in circulation and other bodily fluids, there has been considerable effort to catalog and assess whether exRNAs can be used as markers for health and disease. A variety of exRNA species have been identified including messenger RNA and noncoding RNA such as microRNA (miRNA), small nucleolar RNA, transfer RNA, and long noncoding RNA. Age-related changes in exRNA abundance have been observed, and it is likely that some of these transcripts play a role in aging. In this review, we summarize the current state of exRNA profiling in various body fluids and discuss age-related changes in exRNA abundance that have been identified in humans and other model organisms. miRNAs, in particular, are a major focus of current research and we will highlight and discuss the potential role that specific miRNAs might play in age-related phenotypes and disease. We will also review challenges facing this emerging field and various strategies that can be used for the validation and future use of exRNAs as markers of aging and age-related disease. WIREs RNA 2017, 8:e1385. doi: 10.1002/wrna.1385 For further resources related to this article, please visit the WIREs website.

  5. Differential regulation of catalytic and non-catalytic trkB messenger RNAs in the rat hippocampus following seizures induced by systemic administration of kainate.

    PubMed

    Dugich-Djordjevic, M M; Ohsawa, F; Okazaki, T; Mori, N; Day, J R; Beck, K D; Hefti, F

    1995-06-01

    Ribonuclease protection analysis and quantitative in situ hybridization histochemistry were used to investigate the coordination and regional expression of catalytic and non-catalytic trkB messenger RNAs in the adult rat hippocampus following systemic kainate-induced seizures. Changes in trkB expression were compared with the messenger RNA expression of its neurotrophic ligands, brain-derived neurotrophic factor and neurotrophin-3. TrkB messenger RNA expression was increased in the dentate granule cells at 1-4 h following the onset of seizures, and returned to control levels 16-24 h thereafter. In addition, seizures also induced expression of trkB messenger RNA in putative non-neuronal cells at four to seven days in the molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1 region. Hybridization with probes specific for the non-catalytic trkB receptor and the catalytic trkB receptor revealed that the increases at four and seven days in the molecular layers of the hippocampus reflected an up-regulation of only the non-catalytic form of the receptor. Furthermore, the neuronal increases observed 1-4 h were due to an up-regulation of both trkB TK- and trkB TK+ messenger RNAs. It was established that systemic administration of kainate increased brain-derived neurotrophic factor messenger RNA levels in the pyramidal and granule cell regions of the hippocampus 1-4 h following the onset of behaviorally manifested seizure activity. Early changes in neuronal expression of trkB TK- and trkB TK+ messenger RNA paralleled changes in brain-derived neurotrophic factor messenger RNA in the dentate granule cell and CA1 pyramidal cell layers, but not in the CA3 subregion. These data suggest that concomitant regulation of brain-derived neurotrophic factor and its cognate receptor may play a role in the selective vulnerability of hippocampal subregions to kainate-induced neuropathology. Furthermore, these data suggest a dual function for trkB receptor

  6. 3. Photocopy of photograph (from Fort Dodge Messenger, no issue ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Photocopy of photograph (from Fort Dodge Messenger, no issue or date known) Photographer and date unknown INTERIOR, STAIRWAY - Swain-Vincent House, 824 Third Avenue, South, Fort Dodge, Webster County, IA

  7. 5. Photocopy of photograph (from Fort Dodge Messenger, no issue ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. Photocopy of photograph (from Fort Dodge Messenger, no issue or date known) Photographer and date unknown INTERIOR, FIRST FLOOR, SITTING ROOM, DETAIL OF FIREPLACE - Swain-Vincent House, 824 Third Avenue, South, Fort Dodge, Webster County, IA

  8. MESSENGER Observations of Mercury's Bow Shock and Magnetopause

    NASA Technical Reports Server (NTRS)

    Slavin, James A.; Boardsen, S. A.; Sarantos, M.; Acuna, M. H.; Anderson, B. J.; Baker, D. N.; Benna, M.; Gloeckler, G.; Gold, R. E.; Ho, G. C.; Korth, H.; Krimigis, S. M.; Livi, S. A.; McNutt, R. L., Jr.; Raines, J. M.; Schriver, D.; Solomon, S. C.; Travnicek, P.; Zurbuchen, T. H.

    2008-01-01

    MESSENGER'S 14 January 2008 encounter with Mercury will provide the first new observations of the solar wind interaction with this planet since the Mariner 10 flybys that took place over 30 years ago. The closest approach distance for this first MESSENGER flyby is targeted for an altitude of 200 km as compared with the 707 km and 327 km attained by Mariner 10 on 29 March 1974 and 16 March 1975, respectively. The locations of the bow shock and magnetopause boundaries observed by MESSENGER will be examined and compared against those found in the earlier Mariner 10 measurements and the predictions of theoretical models and numerical simulations. The structure of the magnetopause will be investigated for the presence of flux transfer events or other evidence of magnetic reconnection as will the more general implications of these new MESSENGER bow shock and magnetopause observations for the global solar wind interaction with Mercury.

  9. Biogenesis, assembly, and export of viral messenger ribonucleoproteins in the influenza A virus infected cell.

    PubMed

    York, Ashley; Fodor, Ervin

    2013-08-01

    The flow of genetic information from sites of transcription within the nucleus to the cytoplasmic translational machinery of eukaryotic cells is obstructed by a physical blockade, the nuclear double membrane, which must be overcome in order to adhere to the central dogma of molecular biology, DNA makes RNA makes protein. Advancement in the field of cellular and molecular biology has painted a detailed picture of the molecular mechanisms from transcription of genes to mRNAs and their processing that is closely coupled to export from the nucleus. The rules that govern delivering messenger transcripts from the nucleus must be obeyed by influenza A virus, a member of the Orthomyxoviridae that has adopted a nuclear replication cycle. The negative-sense genome of influenza A virus is segmented into eight individual viral ribonucleoprotein (vRNP) complexes containing the viral RNA-dependent RNA polymerase and single-stranded RNA encapsidated in viral nucleoprotein. Influenza A virus mRNAs fall into three major categories, intronless, intron-containing unspliced and spliced. During evolutionary history, influenza A virus has conceived a way of negotiating the passage of viral transcripts from the nucleus to cytoplasmic sites of protein synthesis. The major mRNA nuclear export NXF1 pathway is increasingly implicated in viral mRNA export and this review considers and discusses the current understanding of how influenza A virus exploits the host mRNA export pathway for replication.

  10. Biogenesis, assembly, and export of viral messenger ribonucleoproteins in the influenza A virus infected cell

    PubMed Central

    York, Ashley; Fodor, Ervin

    2013-01-01

    The flow of genetic information from sites of transcription within the nucleus to the cytoplasmic translational machinery of eukaryotic cells is obstructed by a physical blockade, the nuclear double membrane, which must be overcome in order to adhere to the central dogma of molecular biology, DNA makes RNA makes protein. Advancement in the field of cellular and molecular biology has painted a detailed picture of the molecular mechanisms from transcription of genes to mRNAs and their processing that is closely coupled to export from the nucleus. The rules that govern delivering messenger transcripts from the nucleus must be obeyed by influenza A virus, a member of the Orthomyxoviridae that has adopted a nuclear replication cycle. The negative-sense genome of influenza A virus is segmented into eight individual viral ribonucleoprotein (vRNP) complexes containing the viral RNA-dependent RNA polymerase and single-stranded RNA encapsidated in viral nucleoprotein. Influenza A virus mRNAs fall into three major categories, intronless, intron-containing unspliced and spliced. During evolutionary history, influenza A virus has conceived a way of negotiating the passage of viral transcripts from the nucleus to cytoplasmic sites of protein synthesis. The major mRNA nuclear export NXF1 pathway is increasingly implicated in viral mRNA export and this review considers and discusses the current understanding of how influenza A virus exploits the host mRNA export pathway for replication. PMID:23807439

  11. Mercury's global evolution: New views from MESSENGER

    NASA Astrophysics Data System (ADS)

    Hauck, S. A., II; Byrne, P. K.; Denevi, B. W.; Grott, M.; McCoy, T.; Stanley, S.

    2015-12-01

    MESSENGER's exploration of Mercury has revealed the planet's rich and dynamic history and provided new constraints on the processes that control its internal evolution. Mercury's surface records evidence of an extensive geological history. This evidence includes resurfacing by impacts and volcanism prior to the end of the late heavy bombardment (LHB) and a subsequent rapid waning of effusive volcanism. Volcanism is an important indicator of the history of melt production. Thousands of globally distributed, contractional tectonic landforms collectively have accommodated a decrease in Mercury's radius of 5-7 km since the end of the LHB. Such contraction results from planetary cooling and crystallization within Mercury's metallic core. Measurements of surface chemistry have provided constraints on internal radiogenic heat production necessary to understand more fully Mercury's thermal evolution. Elemental abundances also reveal that Mercury is strongly chemically reduced, suggesting that the core's iron is alloyed with silicon as well as sulfur, which constrains the dynamics and crystallization of the metallic core. Magnetometer observations show that Mercury's dynamo-generated, dominantly dipolar field is displaced ~500 km northward along the rotation axis. Low-altitude magnetic field observations late in the mission led to the discovery of crustal magnetization in Mercury's ancient crust, dating to at least 3.7 Ga, which places a new constraint on the timing of the dynamo. Monte Carlo parameterized mantle convection models, constrained by these observations, indicate that for global contraction of 7 km or less, mantle convection persists to the present ~40% of the time, with the likelihood of modern convection decreasing with less global contraction. Slow present cooling in these models indicates that dynamo generation is strongly influenced by both a static layer at the top of the core and convective motions within the core driven by compositional buoyancy.

  12. MESSENGER observations of magnetopause structure at Mercury

    NASA Astrophysics Data System (ADS)

    DiBraccio, G. A.; Slavin, J. A.; Boardsen, S. A.; Anderson, B. J.; Korth, H.; Zurbuchen, T. H.; Raines, J. M.; McNutt, R. L., Jr.; Solomon, S. C.

    2012-04-01

    MESSENGER Magnetometer (MAG) data from the first series of "hot-season" orbits at Mercury, when periapsis was positioned over the subsolar region, have been used to augment our initial studies to characterize the magnetopause structure as a function of local time. Minimum variance analysis (MVA) was applied to transform the MAG data into boundary-normal coordinates in order to determine (1) the thickness of the magnetopause, (2) the magnetic shear angle across the boundary, and (3) the normal magnetic field, BN, across the current sheet and, by inference, the rate of reconnection and the magnetosphere electric potential. We applied the MVA to all distinct magnetopause crossings within the subsolar region between 0800 and 1600 local time and within ± 25° latitude. A well-defined normal direction, specified by a ratio of the eigenvalue for intermediate variance to that for minimum variance that is greater than 8, was determined for 72 crossings. For this data set, 72.2% of the magnetopause traversals had a substantial normal component (i.e., BN > 4 nT). For a mean boundary motion velocity of 10 km s-1, the average current sheet thickness was 29 km, which is comparable to 2 gyroradii for solar wind protons. The mean ratio of the normal magnetic field component to the total field magnitude, a measure of the reconnection rate, was 0.2 and is independent of magnetic field shear angle across the magnetopause. We conclude that Mercury's magnetopause structure is generally open to the solar wind plasma under a wide range of interplanetary magnetic field shear angles.

  13. Star Messenger: Galileo at the Millennium

    NASA Astrophysics Data System (ADS)

    White, R. E.

    1999-05-01

    Smith College has recently established the Louise B. and Edmund J. Kahn Liberal Arts Institute to foster interdisciplinary scholarship among the faculty. In the 1999-2000 academic year, the Kahn Institute is sponsoring a project entitled "Star Messenger: Galileo at the Millennium." The project will explore the impact of the astronomical discoveries of Galileo and his contemporaries on the Renaissance world-view and also use Galileo's experience as a lens for examining scientific and cultural developments at the symbolic juncture represented by the year 2000. Seven faculty fellows and 10-12 student fellows will participate in a year-long colloquium pursuing these themes, aided by the participation of some five Visiting Fellows. The inaugural public event will be a symposium on the historical Galileo, with presentation by three noted scholars, each of whom will return to campus for a second meeting with the Kahn colloquium. Additional events will include an exhibit of prints, artifacts, and rare books related to Galileo and his time, an early music concert featuring music composed by Galileo's father, and a series of other events sponsored by diverse departments and programs, all related to the broad themes of the Galileo project. The culminating events will be the premiere of a new music theater work, which will encapsulate the insights of the colloquium about human reactions to novel insights about the world, and a symposium presenting the research results of faculty and student fellows. The symposium will feature a capstone lecture by an visionary scholar projecting the implication of historical and contemporary trends into the future.

  14. Neutrino-Gamma Multi-Messenger Source Detection via the Astrophysical Multi-Messenger Observatory Network

    NASA Astrophysics Data System (ADS)

    Fixelle, Josh; Miles, S.; AMON

    2014-01-01

    The idea of multi-messenger event detection has long been explored in the context of above-threshold analysis performed by the IceCube collaboration using Swift BAT and by the Amanda collaboration using BATSE. While these investigations produced null results, they left the event space of sub-threshold events untouched. This untapped event space, combined with the addition of new observatories for various bands and messenger types, provides the obvious niche for a GBN style network to exist: AMON. We consider Monte-carlo models of pair-wise detection between sub-threshold IceCube neutrino doublets, sub-threshold neutrino-gamma doublets with Swift BAT, and with sub-threshold higher multiplicity neutrino-gamma coincidences with Fermi LAT. Several detection methods were considered and compared to the status quo analyses of neutrino doublets by IceCube, demonstrating significant sensitivity gain. The MC model analysis was followed by an archival doublet analysis between IceCube-40 and Fermi LAT data within their co-temporal window of observation. Several methods for detecting statistical signal excess in the archival analysis were considered, providing an upper limit on source population parameters for the given analysis sensitivity.

  15. The pivotal regulatory landscape of RNA modifications.

    PubMed

    Li, Sheng; Mason, Christopher E

    2014-01-01

    Posttranscriptionally modified nucleosides in RNA play integral roles in the cellular control of biological information that is encoded in DNA. The modifications of RNA span all three phylogenetic domains (Archaea, Bacteria, and Eukarya) and are pervasive across RNA types, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and (less frequently) small nuclear RNA (snRNA) and microRNA (miRNA). Nucleotide modifications are also one of the most evolutionarily conserved properties of RNAs, and the sites of modification are under strong selective pressure. However, many of these modifications, as well as their prevalence and impact, have only recently been discovered. Here, we examine both labile and permanent modifications, from simple methylation to complex transcript alteration (RNA editing and intron retention); detail the models for their processing; and highlight remaining questions in the field of the epitranscriptome.

  16. Identification and sequencing of remnant messenger RNAs found in domestic swine (Sus scrofa) fresh ejaculated spermatozoa.

    PubMed

    Yang, C C; Lin, Y S; Hsu, C C; Wu, S C; Lin, E C; Cheng, W T K

    2009-07-01

    The existence of specific messenger RNA remnants contained within freshly ejaculated spermatozoa was described in several species. Those investigations, using high-throughput techniques to screen the population of transcripts in ejaculated spermatozoa, were limited to the probes which mostly derived from nucleic acids of testicular tissues of either human or mice. The objective of this study was to investigate mRNA remnants from ejaculated spermatozoa of the domestic swine (Sus scrofa), a valuable model for biomedical research. A non-redundant 5'-end complementary DNA library was generated from swine ejaculated spermatozoa. After sequence quality verification, 4562 clones remained. These clones were then clustered and assembled into 514 unique sequences including 188 contigs (36.58%) and 326 singletons (63.42%), representing those clusters containing at least two clones and those clusters without having enough similarity with other clones. These unique gene sequences were annotated in Gene Ontology (GO) hierarchy; they included biological processes (38.7%), molecular functions (39.1%) and cellular components (40.3%). Based on the analysis, a broad spectrum of messenger RNAs existed in swine ejaculated spermatozoa and was closely correlated with nucleic acid binding, structural modifications, and transcriptional regulation. All of these categories are considered to have profound effects on the male reproductive system. Therefore, our work provides initial results on potential spermatozoal gene expression for future studies regarding the tightly regulated spermiogenic processes and later fertilization events.

  17. A Spectral Map Of Mercury From MESSENGER

    NASA Astrophysics Data System (ADS)

    Izenberg, N. R.; Pahsai, P.; Klima, R. L.; Blewett, D. T.; Goudge, T. A.; Solomon, S. C.

    2013-12-01

    We use orbital data from the Mercury Surface and Atmospheric Composition Spectrometer (MASCS) Visible and Near Infrared Spectrograph (VIRS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft to study subtle compositional variations across the surface of Mercury. VIRS reflectance spectra obtained from orbit allow identification and classification of spectral units, many of which collocate with geologic features such as pyroclastic deposits; low-reflectance material (LRM); bright, fresh-appearing impact craters; and hollows. The vast majority of the surface is composed of plains units with brightness and spectral reflectance ratios (e.g., 415 nm / 750 nm and 310 nm / 390 nm) that vary within a small range about mean values for the planet. Analysis of VIRS reflectance data in the context of Mercury Dual Imaging System (MDIS) color and high-resolution images enables identification of large regions with similar spectral properties. Our spectral map of Mercury covers approximately 70% of the planet (excluding polar regions and two regions for which calibration refinement is pending). On the basis of brightness, spectral ratio variations, and superposition relationships in the image data, we define four large-scale spectral units in Mercury plains, as well as six additional spectral units of smaller area. The four large-scale spectral units cover (1) 48.7% (brightness and spectral ratio parameters within a few percent of planetary mean values) (2) 31.6% (higher reflectance, higher 310 nm / 390 nm values than mean), (3) 12.9% (higher reflectance, lower 415 nm / 750 nm values than mean), and (4) 6.8% (lower reflectance and higher 310 nm / 390 nm values than mean) of the mapped area. Spectrally defined plains units correspond broadly to plains units defined by morphology and color imaging; e.g., unit 2 corresponds to the previously defined high-reflectance red plains (HRP), unit 3 to the northern smooth plains and the smooth plains

  18. microRNA Decay: Refining microRNA Regulatory Activity.

    PubMed

    Pepin, Genevieve; Gantier, Michael P

    2016-01-01

    MicroRNAs (miRNAs) are short 19-25 nucleotide RNA molecules that impact on most biological processes by regulating the efficiency of messenger RNA (mRNA) translation. To date, most research activities have been focused on the control of miRNA expression and its functional consequences. Nonetheless, much remains unknown about the mechanisms affecting the level of specific miRNAs in the cell, a critical feature impacting their regulatory activity. This review focuses on the factors that regulate the abundance of miRNAs, including synthesis, post-transcriptional modifications, nucleases, target binding, and secretion.

  19. Thermal evolution of Mercury as constrained by MESSENGER observations

    NASA Astrophysics Data System (ADS)

    Michel, Nathalie C.; Hauck, Steven A.; Solomon, Sean C.; Phillips, Roger J.; Roberts, James H.; Zuber, Maria T.

    2013-05-01

    observations of Mercury by the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft provide new constraints on that planet's thermal and interior evolution. Specifically, MESSENGER observations have constrained the rate of radiogenic heat production via measurement of uranium, thorium, and potassium at the surface, and identified a range of surface compositions consistent with high-temperature, high-degree partial melts of the mantle. Additionally, MESSENGER data have placed new limits on the spatial and temporal variation in volcanic and tectonic activity and enabled determination that the planet's core is larger than previously estimated. Because Mercury's mantle layer is also thinner than previously thought, this result gives greater likelihood to the possibility that mantle convection is marginally supercritical or even that the mantle is not convecting. We simulate mantle convection and magma generation within Mercury's mantle under two-dimensional axisymmetry and a broad range of conditions to understand the implications of MESSENGER observations for the thermal evolution of the planet. These models demonstrate that mantle convection can persist in such a thin mantle for a substantial portion of Mercury's history, and often to the present, as long as the mantle is thicker than ~300 km. We also find that magma generation in Mercury's convecting mantle is capable of producing widespread magmas by large-degree partial melting, consistent with MESSENGER observations of the planet's surface chemistry and geology.

  20. MESSENGER Measurements of Mercury's Magnetic Field during the First Flyby

    NASA Technical Reports Server (NTRS)

    Slavin, James A.; Boardsen, S. A.; Acuna, M. H.; Anderson, B. J.; Johnson, C. L.; Korth, H.; Krimigis, S. M.; McNutt, R. L., Jr.; Purucker, M. E.; Solomon, S. C.

    2008-01-01

    On 14 January 2008 the MESSENGER spacecraft will encounter Mercury for the first time. Depending upon the solar wind conditions, this initial flyby will return Magnetometer measurements of Mercury's magnetic field over a time interval lasting between - 30 md 60 min. Closest approach for MESSENGER is targeted for an altitude of 200 km as compared with the 707 krn and 327 km attained by Mariner 10 on 29 March 1974 and 16 March 1975, respectively. Furthermore, the differences in the MESSENGER and Mariner 10 encounter trajectories, with respect both to magnetospheric and body-fixed coordinates are highly complementary and expected to lead to significant improvements in our knowledge of Mercury's magnetic field. We present an overview of the MESSENGER magnetic field observations, an initial subtraction of the magnetic fields attributable to magnetospheric current systems from the total measured magnetic field, and an improved model of Mercury's intrinsic magnetic field. We also discuss the expected advances afforded by the two additional MESSENGER flybys, which occur in October 2008 and September 2009, as well as the orbital phase that will begin in March 201 1.

  1. Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

    1994-01-01

    The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

  2. Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

    1994-01-01

    The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase m