Sample records for mrna da a-amilase

  1. Production of a-amylase in acid cheese whey culture media with automatic pH control Produção de a-amilase em soro ácido de queijo como meio de cultura, com controle automático do pH

    Rosana Ferreyra


    Full Text Available The influence of aeration and automatic pH control on the production of a-amylase by a strain of Bacillus subtilis NRRL 3411 from acid cheese whey was studied. Tests were carried out in a rotary shaker and in mechanically stirred fermenters. a-amylase was analysed according to DUN’s method. Oxygen absorption rate was determined by Cooper’s method. Cell oxygen demand was determined as oxygen consumption in a Warburg respirometer. The level of dissolved oxygen was measured by means of a galvanic silver-lead electrode. Results suggest the possibility of industrial use of acid cheese whey as a carbon source for a-amylase production, since the yield was similar to that produced with lactose. The highest a-amylase levels 10,000 DUN/ml units were not attained at higher aeration rates -431 mLO2/L.h-. The indicated value correspond to a 96 h process with automatic pH control at 7.5. These conditions resulted in double concentration of a-amylase. The enzyme production was directly related to growth in the form of cell aggregates.O presente artigo teve o objetivo de relatar a produção de a-amilase usando uma estirpe de Bacillus subtilis NRRL 3411, usando soro de queijo como fonte de carbono. Foi determinada a influência da aeração do meio de cultura bem como o controle automático do pH. As determinações de a-amilase foram realizadas pelo método de DUN e a taxa de absorção de oxigênio em diferentes condições de aeração pelo método de Cooper. A demanda celular de oxigênio foi estabelecida em um respirômetro de Warburg e o nível de oxigênio dissolvido por meio de eletrodos galvânicos prata-chumbo. Os resultados indicam a possibilidade de uso do soro de queijo como fonte de carbono pois foram similares aos obtidos com lactose. Melhores rendimentos, 10.000 unidade DUN/ml, foram obtidos em 96 h de processo com aeração média -431 mLO2/l.h- e com controle operativo do pH a 7.5, condições estas que permitiram dobrar a atividade de

  2. Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases Mutantes do inibidor-2 de alfa-amilase do feijão-comum para investigação da especificidade de ligação a alfa-amilases

    Maria Cristina Mattar da Silva


    Full Text Available Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2 in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.Apesar de possuir uma família de proteínas de defesa, o feijão-comum (Phaseolus vulgaris L. pode ser atacado por insetos bruquídeos causando sérios danos aos grãos armazenados. O P. vulgaris possui duas formas ativas de inibidores de a-amilases, denominadas a-AI1 e a-AI2, que apresentam diferentes especificidades em relação às a-amilases. A a-amilase de Zabrotes subfasciatus é inibida por a-AI2 mas não por a-AI1. Em contraste, a a-amilase pancreática de porco é inibida por a-AI1 mas não é por a-AI2. O objetivo deste trabalho foi entender as bases moleculares da especificidade desses inibidores em relação às a-amilases. Para tanto, foram construídos mutantes do a-AI2, os quais foram expressados em plantas de fumo

  3. Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-beta

    Issazadeh-Navikas, Shohreh; Lorentzen, J C; Mustafa, M I;


    ) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69-87 and 87-101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; interferon-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL......-1 beta and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CD8 and IL-2 receptor. The expression of m......RNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines, i.e. transforming growth factor-beta (TGF-beta), IL-10 and IL-4 were almost absent in DA rats, in both the CNS...

  4. RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are re-quired to produce the 56-kDa isoform

    Jia Chen; Ta-Yuan Chang; Bo-Liang Li; Xiao-Nan Zhao; Li Yang; Guang-Jing Hu; Ming Lu; Ying Xiong; Xin-Ying Yang; Catherine CY Chang; Bao-Liang Song


    We have previously reported that the human ACAT1 gene produces a chimeric mRNAthrough the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACATI and a novel enzymatically active 56-kDa isoform,respectively,with the latter being authentically present in human cells,including human monocyte derived macrophages. In this work,we report that RNA secondary structures located in the vicinity,of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268,1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type,deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to theORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome I were needed for production of the 56-kDa isoform,whereas the last stem-ioop1355-1384 from chromosome I was dispensable. The results of experi ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACATI transcript.

  5. Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells

    Marino, M.W.; Guidon, P.T. Jr.; Donner, D.B. (Cornell Univ. Graduate School of Medical Sciences, New York, NY (USA)); Pfeffer, L.M. (Rockefeller Univ., New York, NY (USA))


    Tumor necrosis factor {alpha} (TNF-{alpha}) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-{alpha} on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of {sup 125}I-labeled TNF-{alpha} to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-{alpha}, and the inhibition of cell proliferation by TNF-{alpha} occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-{alpha}-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5{prime}-triposphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-{alpha}-promoted phosphorylation, p28. Thus, TNF-{alpha} stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-{alpha}-receptor binding into cellular responses.

  6. Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells.

    Marino, M W; Pfeffer, L M; Guidon, P T; Donner, D B


    Tumor necrosis factor alpha (TNF-alpha) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-alpha on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of 125I-labeled TNF-alpha to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-alpha, and the inhibition of cell proliferation by TNF-alpha occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-alpha-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5'-triphosphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-alpha-promoted phosphorylation, p28. Thus, TNF-alpha stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-alpha-receptor binding into cellular responses.

  7. Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-beta.

    Issazadeh, S; Lorentzen, J C; Mustafa, M I; Höjeberg, B; Müssener, A; Olsson, T


    Experimental autoimmune encephalomyelitis (EAE) in rats is typically a brief and monophasic disease with sparse demyelination. However, inbred DA rats develop a demyelinating, prolonged and relapsing encephalomyelitis after immunization with rat spinal cord in incomplete Freund's adjuvant. This model enables studies of mechanisms related to chronicity and demyelination, two hallmarks of multiple sclerosis (MS). Here we have investigated, in situ, the dynamics of cytokine mRNA expression in the central nervous system (CNS) and peripheral lymphoid organs (lymph node cells and splenocytes) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69-87 and 87-101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; interferon-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL-1 beta and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CD8 and IL-2 receptor. The expression of mRNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines, i.e. transforming growth factor-beta (TGF-beta), IL-10 and IL-4 were almost absent in DA rats, in both the CNS and in vitro stimulated peripheral lymphoid cells, while their levels were elevated in the CNS of LEW rats during the recovery phase. We conclude that the MS-like prolonged and relapsing EAE in DA rats is associated with a prolonged production of proinflammatory cytokines and/or low or absent production of immunodownmodulatory cytokines.

  8. Expressão de TGFβ1 mRNA nas fases iniciais de expansão da sutura palatina mediana The expression of TGFβ1 mRNA in the early stage of the midpalatal suture cartilage expansion

    Emilia Teruko Kobayashi


    Full Text Available INTRODUÇÃO: a expansão da maxila induz a formação de novo osso na sutura palatina mediana por um processo de proliferação e diferenciação celular. A força de expansão pode estimular, nas células progenitoras, a produção de citocinas com atividade osteoindutiva, tais como o transforming growth factor β1(TGFβ1. OBJETIVOS: o principal objetivo deste estudo foi determinar a função dessa citocina nos estágios iniciais de expansão da sutura palatina mediana. MÉTODOS: um aparelho ortodôntico foi instalado entre os molares superiores direito e esquerdo de ratos com 4 semanas de idade. A força de expansão inicial foi de 50g. Os grupos controle e experimental foram sacrificados nos dias 0, 2 e 5. Cortes bucais de 6µm foram obtidos e sujeitos à técnica de hibridização in-situ. RESULTADOS: dois dias após a aplicação de força, as células osteocondroprogenitoras, distribuídas no lado interno do tecido cartilaginoso, exibiram altos níveis de transcrição de transforming growth factor β1. No dia 5, o nível de transcrição de TGFβ1 foi observado nos osteócitos e nas células osteoblásticas, na superfície do novo osso. A atividade osteoblástica foi confirmada por meio de um estudo imunohistoquímico utilizando-se Osteocalcina-Pro (OC-Pro. CONCLUSÕES: os dados sugerem que a expansão da sutura palatina induz a diferenciação de células osteocondroprogenitoras em osteoblastos, estimuladas pela produção de citocinasINTRODUCTION: The application of an orthodontic expansion force induces bone formation at the midpalatal suture because of cell proliferation and differentiation. Expansion forces may stimulate the production of osteoinductive cytokines, such as transforming growth factor β1 (TGFβ1, in the progenitor cells. OBJECTIVES: This study determined the role of TGFβ1 in the early stage of midpalatal suture cartilage expansion. METHODS: A rectangular orthodontic appliance was placed between the right and left

  9. Elementos regulatórios responsáveis pelo controle da estabilidade do mRNA de alfa tubulina de Trypanosoma cruzi

    Patricia Rosa de Araujo


    Um mecanismo de auto-regulação dos mRNAs de alfa e beta tubulina em Trypanosoma cruzi afeta a meia vida desses transcritos ao longo do ciclo de vida do parasita. Enquanto epimastigotas apresentam altos níveis dos mRNAs de tubulina, um excesso da proteína não polimerizada em amastigotas está relacionada com o decréscimo dos níveis dos seus mRNAs. Esta diminuição não é resultado de mudanças na transcrição e sim devido a redução da meia vida dos mRNAs de alfa e beta tubulina. Através do tratamen...

  10. Traditionally used plants in diabetes therapy: phytotherapeutics as inhibitors of alpha-amylase activity Plantas tradicionalmente utilizadas na terapia da diabetes: fitomedicamentos como inibidores da atividade alfa-amilase

    Ingrid Funke


    Full Text Available Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycaemia. There are many and diverse therapeutic strategies in the management of Type 2 diabetes. The inhibition of alpha-amylase activity is only one possibility to lower postprandial blood glucose levels. In our in-vitro studies we could demonstrate that different plants, mostly traditionally used in common diabetic therapy in Africa or Europe, are able to inhibit alpha-amylase, which is responsible for the breakdown of oligosaccharides into monosaccharides which are absorbed. An inhibition of alpha-amylase activity of 90% was seen with the extract of the leaves of Tamarindus indica. To quantify inhibtion rates, acarbose was used (IC50: 23.2 µM. Highest inhibition level of acarbose in our testmodel was about 85%. Additionally tests with pure polyphenolic compounds might explain the biological activity of the selected plants.Diabetes mellitus é uma desordem metabólica caracterizada pela hiperglicemia crônica. Existem diversas estratégias terapêuticas no tratamento da diabetes Tipo 2. A inibição da atividade da a-amilase é apenas uma possibilidade de reduzir os níveis de glicose posprandiais. Nos nossos estudos in vitro pudemos demonstrar que diferentes plantas, especialmente as tradicionalmente usadas em terapia comum de diabetes na África ou Europa, são capazes de inibir a a-amilase, a qual é responsável pela quebra dos oligossacarídeos em monossacarídeos, os quais são absorvidos. Uma inibição da atividade da a-amilase da ordem de 90% foi observada com o extrato das folhas de Tamarindus indica. Para quantificar os graus de inibição, acarbose foi usada (IC50: 23,2 mM. O maior grau de inibição de acarbose no nosso modelo de teste foi de cerca de 85%. Adicionalmente testes com compostos polifenólicos puros poderão explicar a atividade biológica das plantas selecionadas.

  11. Avaliação da expressão do mRNA do GLUT 4 em corpo lúteo de cadelas sadias ao longo do diestro


    O ciclo estral das cadelas difere das demais espécies domésticas. Estudos demonstraram que o aumento da concentração plasmática de P4 durante a fase luteínica das cadelas pode levar a alterações metabólicas como a resistência insulínica, acarretando complicações como Diabetes mellitus. A glicose é uma molécula transportada, na maioria das células, por proteínas transportadoras. O processo de instalação da resistência insulínica é caracterizado por alterações teciduais da expressão de algumas ...

  12. Transcrição reversa na determinação da expressão do mRNA para a enzima conversora de angiotensina testicular em animais tratados com zinco Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats

    Gilberto Simeone Henriques


    Full Text Available OBJETIVO: O objetivo deste trabalho foi otimizar as condições reacionais capazes de ocasionar variabilidade e de introduzir erros sistemáticos na reação em cadeia pela polimerase aplicada à análise da expressão gênica da isoforma testicular da enzima conversora de angiotensina. MÉTODOS: Avaliaram-se a concentração de cDNA, a concentração dos iniciadores, a temperatura de hibridização e o número de ciclos de desnaturação, hibridização e extensão. Para tanto, extraiu-se o RNA total por meio da reação com fenol-clorofórmio e isotiocianato de guanidina de amostras de testículos de ratos Wistar alimentados com uma ração contendo zinco. Em seguida, gerou-se o cDNA por transcrição reversa. Utilizando-se iniciadores específicos, amplificaram-se o gene de interesse (isoforma testicular da enzima conversora de angiotensina e o gene controle Gliceraldeído-3-Fosfato-Desidrogenase. As amostras foram então aplicadas em gel de agarose e submetidas à eletroforese, coradas em brometo de etídio e visualizadas sob luz ultravioleta. RESULTADOS: Demonstrou-se que a melhor condição reacional para a reação em cadeia pela polimerase da isoforma testicular da enzima conversora de angiotensina e do Gliceraldeído-3-Fosfato-Desidrogenase foi: (1 concentração inicial de cDNA de 2µg, (2 concentração de iniciadores de 200nM, (3 temperatura de hibridização entre 57,5ºC e 60,1ºC e (4 33 ciclos. CONCLUSÃO: Com essa otimização pôde-se minimizar as interferências sobre a técnica, contribuindo-se para a obtenção de dados comparativos a respeito da expressão gênica da enzima conversora de angiotensina testicular.OBJETIVE: The aim of the present work was to optimize the reaction conditions capable of generating variability and introducing systematic errors in the chain reaction of the polymerase used to analyze the gene expression for the testicular isoform of the angiotensin-converting enzyme. METHODS:The cDNA concentration

  13. Inhibition of carrageenan-induced expression of tissue and plasma prekallikreins mRNA by low level laser therapy in a rat paw edema model Inibição da expressão de RNA mensageiro de pré-calicreínas tecidual e plasmática pela laserterapia em modelo de edema de pata induzido pela carragenina em ratos

    Moisés P. Silva


    Full Text Available BACKGROND: Low level laser therapy (LLLT has been used clinically in order to treat inflammation, where tissue and plasma prekallikrein have crucial importance. Plasma prekallikrein (PPK is synthesized by the hepatocytes and secreted into the bloodstream, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Tissue prekallikrein is associated with important disease states (including cancer, inflammation, and neurodegeneration and has been utilized or proposed as clinically important biomarker or therapeutic target of interest. OBJECTIVE: To evaluate if LLLT modulates tissue and plasma prekallikreins mRNA expression in the carrageenan-induced rat paw edema. METHODS: Experimental groups were assigned as followed: A1 (Control-saline, A2 (Carrageenan-only, A3 (laser 660nm only and A4 (Carrageenan + laser 660nm. Edema was measured by a plethysmometer. Subplantar tissue was collected for the quantification of prekallikreins mRNA by Real time-Polymerase Chain Reaction. RESULTS: A significantly decrease in the edema was observed after laser irradiation. Expression of prekallikreins increased after carrageenan injection. Tissue and plasma prekallikrein mRNA expression significantly decreased after LLLT's 660nm wavelength. CONCLUSION: These results suggest that expression of tissue and plasma prekallikreins is modulated by LLLT, which can be used in clinical practice due to its anti-inflammatory effects.CONTEXTUALIZAÇÃO: A laserterapia de baixa potência tem sido usada para o tratamento de processos inflamatórios diversos em que a calicreína tecidual e a plasmática possuem participação ativa. A pré-calicreína plasmática (PPK é sintetizada pelos hepatócitos e secretada na corrente sanguínea, onde participa da ativação da coagulação, fibrinólise, geração de cininas e inflamação. A pré-calicreína tecidual está associada com importantes doenças (incluindo c

  14. Effect of glutamine on the mRNA level of key enzymes of malate-aspartate shuttle in the rat intestine subjected to ischemia reperfusion Efeito da glutamina sobre o nível de RNA Mensageiro das enzimas-chave do ciclo malato-aspartato no intestino de ratos submetidos à isquemia e reperfusão

    Paulo Roberto Cavalcante de Vasconcelos


    Full Text Available PURPOSE: To determine the effects of oral L-glutamine (L-Gln and the dipeptide l-alanyl-glutamine (L-Ala-Gln upon the activity of the malate-aspartate shuttle in the rat distal small intestine following ischemia and reperfusion. METHODS: Seventy-two Wistar rats (350-400g, were randomized in 2 groups (n = 36: group S (Sham and Group T (Treatment and divided into 12 subgroups (n = 6: A-A6, and B1-B6. The subgroups A1-A3 were subjected to sham procedures at 30 and 60 minutes. Thirty minutes before the study, rats were treated with calcium caseinate, 0.5g/Kg (subgroups A1, A4, B1, B4, L-Gln, 0.5g / kg (subgroups A2, A5, B2 and B5 or L-Ala-Gln, 0.75g/Kg (subgroups A3, A6, B3, B6, administered by gavage. Ischemia was achieved by clamping the mesenteric vessels, delimiting a segment of bowel 5 cm long and 5 cm apart from the ileocecal valve. Samples were collected 30 and 60 minutes after start of the study for real-time PCR assay of malate dehydrogenases (MDH1-2 and aspartate-aminotransferases (GOT1-2 enzymes. RESULTS: Tissue MDH and GOT mRNA expression in intestinal samples from rats preconditioned with either L-Gln or L-Ala-Gln showed no significant differences both during ischemia and early reperfusion. CONCLUSION: Activation of the malate-aspartate shuttle system appears not to be the mechanism of glutamine-mediated elevation of glucose oxidation in rat intestine during ischemia/reperfusion injury.OBJETIVO: Determinar os efeitos da administração oral de L-glutamina (L-Gln e do dipeptídeo L-alanil-glutamina (L-Ala-Gln sobre a atividade do ciclo malato-aspartato no intestino delgado distal de ratos após isquemia/reperfusão. MÉTODOS: Setenta e dois ratos Wistar (350-400g foram randomizados em 2 grupos (n = 36: T grupo S (Sham e grupo (Tratamento e distribuídos em 12 subgrupos (n = 6: A-A6, e B1-B6. Os subgrupos A1-A3 foram submetidos a procedimentos "sham" aos 30 e 60 minutos. Trinta minutos antes do estudo, os ratos foram tratados com


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

  16. Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and copper/zinc superoxide dismutase (SOD1) interact to modulate NFL mRNA stability. Implications for altered RNA processing in amyotrophic lateral sclerosis (ALS).

    Volkening, Kathryn; Leystra-Lantz, Cheryl; Yang, Wenchang; Jaffee, Howard; Strong, Michael J


    Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive motor neuron degeneration in association with neurofilament (NF) aggregate formation. This process is accompanied by an alteration in the stoichiometry of NF subunit protein expression such that the steady state levels of the low molecular weight NF (NFL) mRNA levels are selectively suppressed. We have previously shown that each of TDP-43, 14-3-3 and mutant SOD1 can function as NFL mRNA 3'UTR binding proteins that directly affect the stability of NFL transcripts. In this study, we demonstrate that the interaction of TDP-43 with the NFL mRNA 3' UTR involves ribonucleotide (UG) motifs present on stem loops of the 3'UTR as well as the RRM1 and RRM2 motifs of TDP-43. Ex vivo, TDP-43, 14-3-3 and SOD1 proteins interact to modulate NFL mRNA stability, although in vivo, only TDP-43 and either mutant or wild-type SOD1 co-localize in ALS motor neurons. TDP-43 was observed to co-localize to RNA transport granules (Staufen immunoreactive) in both control and ALS spinal motor neurons. In contrast, both stress granules (TIA-1 immunoreactive) and processing bodies (P-bodies; XRN-1 immunoreactive) were more prevalent in ALS motor neurons than in controls and demonstrated strong co-localization with TDP-43. Using RNA-IP-PCR, we further demonstrate that NFL mRNA is preferentially sequestered to both stress granules and P-bodies in ALS. These data suggest that NFL mRNA processing is fundamentally altered in ALS spinal motor neurons to favour compartmentalization within both stress granules and P-bodies, and that TDP-43 plays a fundamental role in this process.

  17. Reference: MRNA3ENDTAH3 [PLACE

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

  18. Systems perspectives on mRNA processing

    Adrienne E McKee; Pamela A Silver


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

  19. Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening.

    Dellapenna, D; Alexander, D C; Bennett, A B


    The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-alpha-D-galacturonide) glucanohydrolase, EC] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)(+) RNA isolated from ripe tomato fruit. The cDNA library was inserted into a lambda-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

  20. Serotonin 2A receptor mRNA levels in the neonatal dopamine-depleted rat striatum remain upregulated following suppression of serotonin hyperinnervation.

    Basura, G J; Walker, P D


    Sixty days after bilateral dopamine (DA) depletion (>98%) with 6-hydroxydopamine (6-OHDA) in neonatal rats, serotonin (5-HT) content doubled and 5-HT(2A) receptor mRNA expression rose 54% within the rostral striatum. To determine if striatal 5-HT(2A) receptor mRNA upregulation is dependent on increased 5-HT levels following DA depletion, neonatal rats received dual injections of 6-OHDA and 5,7-dihydroxytryptamine (5,7-DHT) which suppressed 5-HT content by approximately 90%. In these 6-OHDA/5,7-DHT-treated rats, striatal 5-HT(2A) receptor mRNA expression was still elevated (87% above vehicle controls). Comparative analysis of 5-HT(2C) receptor mRNA expression yielded no significant changes in any experimental group. These results demonstrate that upregulated 5-HT(2A) receptor biosynthesis in the DA-depleted rat is not dependent on subsequent 5-HT hyperinnervation.

  1. Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening

    DellaPenna, Dean; Alexander, Danny C.; Bennett, Alan B


    The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-α-D-galacturonide) glucanohydrolase, EC] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting...

  2. Problemas da sociologia da arte

    Bastide, Roger


    A partir de um balanço das realizações consideradas pelo autor como as mais relevantes no campo da sociologia da arte até o final dos anos de 1940, o texto propõe novos rumos para a disciplina, considerando o modo como a arte é socialmente produzida como linguagem e valor.

  3. Self-amplifying mRNA vaccines.

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


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

  4. Destabilization of survival factor MEF2D mRNA by neurotoxin in models of Parkinson's disease.

    Wang, Bao; Cai, Zhibiao; Lu, Fangfang; Li, Chen; Zhu, Xiaofei; Su, Linna; Gao, Guodong; Yang, Qian


    Progressive loss of dopaminergic (DA) neurons in the substantial nigra pars compacta (SNc) is an important pathological feature in Parkinson's disease (PD). Loss of transcription factor myocyte enhancer factor 2D (MEF2D), a key neuronal survival factor, has been shown to underlie the loss of DA neurons in SNc and the pathogenic process of PD. It is known that PD-associated neurotoxins reduce the level of MEF2D protein to trigger neuronal death. Although neurotoxins clearly destabilize MEF2D by post-translational mechanisms, it is not known whether regulation of MEF2D mRNA contributes to neurotoxin-induced decrease in MEF2D protein. In this work, we showed that MPP(+), the toxic metabolite of MPTP, caused a significant decrease in the half-life and total level of MEF2D mRNA in a DA neuronal cell line, SN4741 cells. Quantitative PCR analysis of the SNc DA neurons captured by immune-laser capture microdissection showed that exposure to MPTP led to a marked reduction in the level of MEF2D mRNA in SNc DA neurons compared to controls. Down-regulation of MEF2D mRNA alone reduced the viability of SN4741 cells and sensitized the cells to MPP(+)-induced toxicity. These results suggest that destabilization and reduction in MEF2D mRNA is in part responsible for neurotoxin-induced decrease in MEF2D protein and neuronal viability. Myocyte enhancer factor 2D (MEF2D) plays an important role in neuronal survival. How MEF2D mRNA is deregulated under toxic stress is unclear. We found that PD-associated neurotoxins destabilize MEF2D mRNA and reduce its level in vitro and in vivo. Reduction in MEF2D mRNA is sufficient to sensitize model cells to neurotoxin-induced toxicity, suggesting that destabilization of MEF2D mRNA is part of the mechanism by which neurotoxins trigger deregulation of neuronal survival.

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

    Krakauer, Martin; Sorensen, P; Khademi, M


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

  6. Gene regulation by mRNA editing

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


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

  7. Diagnostic value of cancer-testis antigen mRNA in peripheral blood from hepatocellular carcinoma patients


    AIM:To evaluate the diagnostic value of cancer-testis antigen(CTA) mRNA in peripheral blood samples from hepatocellular carcinoma(HCC) patients.METHODS:Peripheral blood samples were taken from 90 patients with HCC before operation.Expression of melanoma antigen-1(MAGE-1),synovial sarcoma X breakpoint-1(SSX-1),and cancer-testis-associated protein of 11 kDa(CTp11) mRNA in peripheral blood mononuclear cells(PBMC) was tested by nested reverse transcriptspolymerase chain reaction(RT-PCR).Serum α-fetoprotein(AFP)...

  8. Role of highly conserved pyrimidine-rich sequences in the 3' untranslated region of the GAP-43 mRNA in mRNA stability and RNA-protein interactions.

    Kohn, D T; Tsai, K C; Cansino, V V; Neve, R L; Perrone-Bizzozero, N I


    We have shown previously that the mRNA for the growth-associated protein GAP-43 is selectively stabilized during neuronal differentiation. In this study, we explored the role of its highly conserved 3' untranslated region (3'UTR) in mRNA stability and RNA-protein interactions. The 3'UTRs of the rat and chicken GAP-43 mRNAs show 78% sequence identity, which is equivalent to the conservation of their coding regions. In rat PC12 cells stably transfected with the full-length rat or chicken GAP-43 cDNAs, the transgene mRNAs decayed with same half-life of about 3 h. The GAP-43 3'UTR also caused the rabbit beta-globin mRNA to decay with a half-life of 4 h, indicating that the major determinants for GAP-43 mRNA stability are localized in its highly conserved 3'UTR. Three brain cytosolic RNA-binding proteins (molecular mass 40, 65 and 95 kDa) were found to interact with both the rat and chicken GAP-43 mRNAs. These RNA-protein interactions were specific and involved pyrimidine-rich sequences in the 3'UTR. Like the GAP-43 mRNA, the activity of these proteins was enriched in brain and increased during development. We propose that highly conserved pyrimidine-rich sequences in the 3'UTR of this mRNA regulate GAP-43 gene expression via interactions with specific RNA-binding proteins.

  9. Messenger RNA (mRNA) nanoparticle tumour vaccination

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


    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.

  10. Recent innovations in mRNA vaccines.

    Ulmer, Jeffrey B; Geall, Andrew J


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

  11. Alternative polyadenylation of mRNA precursors

    Tian, Bin; Manley, James L.


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

  12. 实时定量PCR检测耐异烟肼结核分枝杆菌Ag85B、38kDa及MPT64mRNA表达水平%The mRNA' s expression of Ag85B,38kDa and MPT64 in isoniazid resistant Mycobacterium tuberculosis by real-time PCR

    陈伟; 李永祥; 王远志; 米利古; 张辉; 张娟; 魏克娜; 袁俐


    The aim was to use real-time quantitative polymerase chain reaction for detecting the expression level of the coding gene of Ag85B, 38kDa and MPT64 (fbpB, pstsl ,and mpt64 ) in isoniazid-resistant and sensitive Mycobacterium tuberculosis and explore variability of their proteantigen in transcriptional level. The specific primers of fbpB, pstsl, rnpt64 and Siga were designed according to GenBank database. The fbpB, pstsl, mpt64 and Siga fragments were cloned to pMD18-T simple vector respectively, and recombinant plasmids were purified and gradient dilution. By establishing a method of detecting the expression of fbpB,pstsl and mpt64 by using SYBR Green I,it was employed to detect the expression of fbpB, pstsl, mpt64 in 22 isoniazid resistant Mycobacterium tuberculosis and 25 sensitive ones. The expression level of fbpB in isoniazid-resistant is 0. 65 ±0. 22 and the sensitive was 0. 36±0. 13, nhich was significantly different (t=5. 683, P<0. 01). The expression level of pstsl in isoniazid-resistant was 13. 63±4. 16 and the sensitive ones was 9. 86±2. 79, which was significantly different(2=3. 869, P <0. 01). The expression level of fbpB in isoniazid-resistant was 5. 89zb3. 22 and the sensitive ones was 7. 67±4. 52, nhich was not statisticaly significant. The method of detecting the expression level of fbpB, pstsl, and tnpt64 by real-time PCR has been estabished. This study find the expression level of fbpB andpstsl in isoniazid-resistant is significant high than that in the sensi-tive ones, providing the molecule identification for diagnosing the drug-resistant Mycobacterium tuberculosis.%目的 运用实时定量PCR(Real-time PCR)检测结核分枝杆菌耐异烟肼菌株和敏感株Ag85B、38kDa及MPT64编码基因(fbpB、psts1、mpt64)的mRNA表达水平,探讨结核分枝杆菌耐异烟肼株和敏感株的蛋白抗原在转录水平的差异性.方法 根据GenBank提供的序列,设计目的基因fbpB、psts1、mpt64及内参基

  13. Regional induction of c-fos and heat shock protein-72 mRNA following fluid-percussion brain injury in the rat

    Raghupathi, R.; Welsh, F.A.; Gennarelli, T.A. [Univ. of Pennsylvania, Philadelphia, PA (United States)] [and others


    To evaluate the cellular response to traumatic brain injury, the expression of mRNA for c-fos and the 72-kDa heat shock protein (hsp72) was determined using in situ hybridization following lateral fluid-percussion injury (2.2-2.4 atm) in rat brain. At 2 h after injury, induction of c-fos mRNA was restricted to regions of the cortex surrounding the contusion area. An increase in c-fos mRNA, but not hsp72 mRNA, was observed bilaterally in the CA{sub 3} subfield of the hippocampus and the granule cells of the dentate gyrus and in the thalamus ipsilateral to the impact site. By 6 h, increased expression of c-fos mRNA was observed only in the corpus callosum on the impact side; hsp72 mRNA persisted in the deep cortical layers and upper layers of the subcortical white matter below the site of maximal injury. By 24 h, both c-fos and hsp72 mRNA had returned to control levels in all regions of the brain. These results demonstrate that lateral fluid-percussion brain injury triggers regionally and temporally specific expression of c-fos and hsp72 mRNA, which may be suggestive of differential neurochemical alterations in neurons and glia following experimental brain injury. 33 refs., 3 figs., 1 tab.

  14. A Candida albicans gene expressed in Saccharomyces cerevisiae results in a distinct pattern of mRNA processing.

    Iborra, A; Sentandreu, R; Gozalbo, D


    Two plasmids (derived from YCplac22 and YEplac112) carrying a Candida albicans gene (including the 5' non-coding promoter sequences) coding for a 30 kDa membrane-bound protein, were used to transform Saccharomyces cerevisiae cells. A 30 kDa protein was immunodetected by Western blot in the membrane fraction of transformants. Northern analysis showed the presence of three mRNA species (of about 1.1, 0.7 and 0.5 kb) hybridizing with the C. albicans gene as a probe. The same result was obtained using the 5' and 3' regions of the gene as probes, whereas only a 1.1 kb mRNA was found in C. albicans and none was detected in S. cerevisiae control transformants. Thus, heterologous expression of this gene in S. cerevisiae results in a distinct pattern of mRNA processing, either due to the location on plasmid vectors and/or to differences in the mRNA processing systems in the two microorganisms.

  15. Differences in mRNA expression of calpains, calpastatin isoforms and calpain/calpastatin ratios among bovine skeletal muscles.

    Muroya, Susumu; Neath, Kate E; Nakajima, Ikuyo; Oe, Mika; Shibata, Masahiro; Ojima, Koichi; Chikuni, Koichi


    Messenger RNA (mRNA) expression of calpain-1 (µ-calpain), -2 (m-calpain), -3 (p94), small subunit (calpain-4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real-time and/or semi-quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P Calpain-1/CSTN I, calpain-2/CSTN I in LT and PM were higher than that in TN (P Calpain-3/CSTN-I and -III in LT and/or PM showed higher values than that in TN (P calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN-I ratios, especially 28 kDa/CSTN-I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles.

  16. mRNA pseudoknot structures can act as ribosomal roadblocks

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


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

  17. Functional Integration of mRNA Translational Control Programs

    Melanie C. MacNicol


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

  18. Probing dimensionality beyond the linear sequence of mRNA.

    Del Campo, Cristian; Ignatova, Zoya


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

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

    Gorospe Myriam


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

  20. Effects of DNA replication on mRNA noise.

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


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

  1. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring.

    Rossini, Kamila Fernanda; Oliveira, Camila Andrea de; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana


    The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de

  2. Substance P mRNA expression in the rat spinal cord following selective brachial plexus injury

    Na Liu; Longju Chen; Feng Li; Wutian Wu


    BACKGROUND: The neuropeptide, substance P, has various bioactivities and is widely distributed in the central nervous system. Substance P participates in neural transmission in the spinal cord and plays an important role in regeneration and repair of nerve injury.OBJECTIVE: To investigate substance P mRNA expression in the anterior horn of the spinal cord following brachial plexus injury.DESIGN, TIME AND SETTING: A molecular cell biology randomized controlled study was performed at the Department of Anatomy, Zhongshan Medical College, Sun Yat-sen University and the DaAn Gene Laboratory in May 2005.MATERIALS: A total of 29 adult male Sprague Dawley rats were randomly assigned to a control group (n=5) and an injury group (n = 24).METHODS: The injury group was divided into three subgroups. In subgroup A, the right seventh cervical vertebra (C7) anterior root was avulsed, and the residual nerve root at the distal end was removed. In subgroup B, the right C7 anterior root was avulsed, and the right C5 first thoracic vertebrae (TO posterior root was incised. Thus afferent pathways of the posterior root that connected with the anterior horn motor neurons were blocked. In subgroup C, the right C7 anterior root was avulsed, and a right C5-6 hemisection was performed. Thus the descending fiber pathways of the cortex that connected with anterior horn motor neurons were blocked. In the control group, the C5-T1 vertebral plate was opened, and then the skin was sutured.MAIN OUTCOME MEASURE: Substance P mRNA expression in the anterior horn of the spinal cord was quantified using fluorescent quantitative reverse transcription-polymerase chain reaction.RESULTS: Substance P mRNA expression was low in the anterior horn of the rat spinal cord in the control group. Substance P mRNA expression in the anterior horn of the spinal cord was upregulated and was significantly higher in the injury group compared with the control group (P < 0.01 ). Substance P mRNA expression was highest in

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

    John R. Sinnamon; Kevin Czaplinski


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

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

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


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

  5. Optimal Down Regulation of mRNA Translation

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir


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

  6. Optimal Down Regulation of mRNA Translation

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir


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

  7. Downregulation of VEGF mRNA expression by triamcinolone acetonide acetate-loaded chitosan derivative nanoparticles in human retinal pigment epithelial cells

    Zhou H


    Full Text Available Huaisheng Zhou,1 Liqun Yang,2,* Huajie Li,2 Haijun Gong,1 Liangzheng Cheng,2 Haisheng Zheng,1 Li-Ming Zhang,2 Yuqing Lan1,*1Department of Ophthalmology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 2Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-sen University, Guangzhou, China*Both corresponding authors contributed equally to this workBackground: The purpose of this study was to investigate the downregulation of mRNA expression of vascular endothelial growth factor (VEGF by triamcinolone acetonide acetate (TAA-loaded chitosan nanoparticles in human retinal pigment epithelial cells.Methods: TAA-loaded deoxycholic acid-modified chitosan (TAA/DA-Chit nanoparticles were prepared via a self-assembly mechanism, and their morphology and zeta potential were examined by transmission electron microscopy and zeta potential analysis, respectively. DA-Chit and TAA/DA-Chit nanoparticle toxicity was evaluated using a Cell Counting Kit-8 assay. The efficiency of cellular uptake was determined using fluorescein isothiocyanate-labeled DA-Chit nanoparticles, in place of TAA/DA-Chit nanoparticles, assessed by both inverted fluorescence microscopy and flow cytometry. Downregulation of VEGF mRNA expression by TAA/DA-Chit nanoparticles was further investigated by real-time reverse transcription polymerase chain reaction (RT-PCR assay of the treated human retinal pigment epithelial cells.Results: TAA/DA-Chit nanoparticles were prepared with a TAA-loading capacity in the range of 12%–82%, which increased the water solubility of TAA from 0.3 mg/mL to 2.1 mg/mL. These nanoparticles showed oblate shapes 100–550 nm in size in transmission electron microscopic images and had positive zeta potentials. The Cell Counting Kit-8 assay indicated that the DA-Chit and

  8. A Sintaxe da Janela

    Luís Antônio Jorge


    Full Text Available A preocupação fundamenta! do trabalho é de caráter ontológico: esboçar o problema das significações apriorísticas e autônomas dos elementos que compõem a sintaxe arquitetônica. O trabalho elege um desses elementos: a janela. Este artigo parte da aproximação histórica entre a janela e a secção da pirâmide visual, construção imaginária da teoria da perspectiva renascentista. A consideração da janela na linguagem arquitetônica implica reconhecer uma ambigüidade essencial: a janela deve inserir-se ordenadamente no plano da parede - raciocínio típico da modenatura - mas também relacionar-se com a imagem vista através dela, que extrapola o plano, ressaltando a tridimensionalidade - herança da perspectiva renascentista, fundamentada no espaço euclidiano. Ver janela ou ver através da janela são alternativas que se colocam tanto ao observador interno ao edifício, quanto ao externo. Texto de duplaface, a sintaxe da janela versa sobre o relacionamento desses dois lados

  9. Cloning and characterization of 37 kDa laminin receptor precursor in pearl oyster, Pinctada fucata

    Yaopeng Fu; Liping Xie; Rongqing Zhang


    A 1063 bp cDNA clone encoding a putative 37 kDa laminin receptor precursor (37 kDa LRP) is isolated from the mantle tissue of pearl oyster, Pinctadafucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molecular mass of 33.5 kDa. RT-PCR analysis shows that 37 kDa LRP mRNA is especially highly expressed in the mantle while widely expressed in several tissues. In situ hybridization analysis reveals that 37 kDa LRP is expressed in the outer epithelial cells of the mantle edge, suggesting its involvement in cell proliferation and secretion in P. Fucata. The identification and characterization of 37 kDa LRP in the pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissue formation.

  10. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Carlo Cattani


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

  11. Expressão do mRNA de genes mitocondriais e desempenho produtivo de codornas alimentadas com glicerol

    Stefânia Caroline Claudino da Silva


    Full Text Available O objetivo deste trabalho foi avaliar o efeito de dietas com glicerol no desempenho produtivo de codornas japonesas de corte e na expressão do mRNA de genes mitocondriais da proteína adenina nucleotídeo translocase (ANT e da proteína desacopladora (UCP, envolvidas no metabolismo energético e na resposta ao estresse oxidativo. As codornas foram alimentadas com dietas contendo 0, 8 e 12% de glicerol, em substituição parcial ao milho. Aos 28 dias de idade, o RNA total foi extraído de amostras do músculo do peito e a síntese do cDNA foi feita por meio de qRT‑PCR com iniciadores específicos para genes da ANT e UCP, obtidos de Gallus gallus. A conversão alimentar e o consumo de ração foram avaliados para as três dietas testadas. A adição de 8% de glicerol não afetou o desempenho dos animais. No entanto, a adição de 12% aumentou o consumo de ração e piorou a conversão alimentar. O ganho de peso não foi afetado pela inclusão de glicerol na dieta. No grupo alimentado com 8% de glicerol, a expressão da UCP aumentou, mas a da ANT não variou, em comparação ao controle. A expressão da UCP foi menor e a da ANT foi maior no grupo alimentado com 12% de glicerol. A inclusão de 8% de glicerol na dieta não afeta o desempenho de codornas de corte, embora aumente a expressão da UCP. A inclusão de 12% de glicerol piora o desempenho e aumenta a expressão da ANT.

  12. Genealogias da alegria

    Bruno Vasconcelos de Almeid


    Full Text Available Genealogias da Alegria tem por objetivo rastrear conceitos e construir planos sobre os quais podemos pensar a alegria como campo problemático. Trata-se de traçar linhas e acoplar fragmentos históricos e literários com a finalidade de subsidiar a discussão contemporânea em torno da alegria. O procedimento genealógico vale-se de trechos das obras de Calímaco e Luciano de Samósata, da elegia erótica romana e da alegria cristã. O passo seguinte consiste em rastrear o pensamento da alegria na obra de Gilles Deleuze, especialmente em três momentos: uma passagem de Diferença e Repetição, um trecho dos Diálogos extraído de Da Superioridade da Literatura Anglo-americana e a comunicação Pensamento Nômade no Colóquio de Cerisy. A alegria não se define como sentimento ou emoção; ao contrário, está ligada à premissa de afirmação da vida e ao caráter trágico da existência.

  13. A sabedoria da democracia

    Gilson Matilde Diana


    Full Text Available a maior parte da história, a democracia foi desprezada como uma forma política inferior – a regra do povo, ou “dos muitos”, era logo identificada como a regra da ralé, da escória. Herdando do povo a volubilidade e a instabilidade, a democracia estava sempre perigosamente próxima do arbítrio e da anarquia, com toda a violência que se costuma associar a isso.

  14. MRNA-based skin identification for forensic applications

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


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

  15. Influenza virus mRNA trafficking through host nuclear speckles.

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


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

  16. Corporeidade da voz : estudo da vocalidade poetica

    Fernando Manoel Aleixo


    Resumo: Este material é o resultado da investigação prática da voz realizada a partir do desenvolvimento do princípio da sabedoria sensível do corpo. O texto expõe as etapas percorridas no levantamento de experiências e reflexões pedagógicas sobre a voz do ator, no estabelecimento de referências sobre o corpo e o aprendizado sensível e, também, no processo de composição poética do espetáculo teatral Voz Mercê Abstract: This material springs from a practical investigative work on voice base...

  17. Modelagem da laringe: da biologia ao computador

    Marcelo de Oliveira Rosa


    Full Text Available Neste trabalho apresentamos uma descrição daanatomia e fisiologia da laringe, enfatizando os aspectos relevantes à fonação. A partir destes aspectos, a modelagem da laringe é apresentada. O principal objetivo de modelara laringe é representar de uma maneira simples esse órgãoatravés de equações matemáticos intimamente relacionadoscom características fisiológicas. Equações aerodinâmicase viscoelásticas são simplificadas de acordo com as pesquisas,sempre buscando simplicidade e aplicabilidade computacional. Assim, pesquisadores podem compreender melhor a laringe e a fala humana em termos dos efeitos linguísticos, cirúrgicos e computacionais produzidos pelamovimentação desse órgão. Palavras-chave: Laringe, modelagem, sinal glotal,produção da fala.

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

    De Rubeis, Silvia; Bagni, Claudia


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

  19. A ciclovia da morte

    Gois, Ancelmo


    De acordo com levantamento da FGV/DAPP, houve mais de 36 mil menções ao desabamento no Twi er, até o fim da tarde de ontem, com palavras como "vergonha", "incompetência" e "culpa" em destaque, além de muitas criticas ao prefeito Eduardo Paes.

  20. Iptkalim inhibits cocaine challenge—induced enhancement of dopamine levels in nucleus accumbens and striatum of rats by up—regulating Kir6.1 and Kir6.2 mRNA expression

    HEHai-Rong; DINGJian-Hua; GUBing; WANGHai; HUGang; LIUYun


    AIM:To investigate the effect and mechanism of novel ATP-sensitive potassium channel opener (KCO) iptkalim (IPT) on acute and cocaine challenge-induced alterations in the levels of dopamine (DA) and glutamate (Glu) from nucleus accumbens (NAc), striatum, and prefrontal cortex (PFC) in rats. METHODS: The levels of DA and Glu were assayed using high performance liquid chromatography (HPLC) combined with amperometric and fluorescent detection, respectively. The mRNA levels of Kir6.1, Kir6.2, SUR1, and SUR2 were measured by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: IPT did not affect acute cocaine (30mg/kg,ip)-induced elevations in either DA levels from NAc and striatum or Glu levels from NAc and PFC. An acute cocaine challenge (30mg/kg,ip) on d 21 after withdrawal caused an elevation in DA levels in NAc and striatum. Moreover, the same treatment also increased Gluo levels in PFC and NAc of cocaine-pretreated rats. Repeated IPT injections reversed cocaine challenge-induced DA increase in NAc and striatum. Cocaine challenge increased Kir6.1 and Kir6.2 mRNA expression in striatum and NAc and only elevate Kir6.2 expression in PFC in both cocainepretreated rats and rats pretreated with IPT plus cocaine. Moreover, expression of Kir6.1 and Kir6.2 mRNA was augmented in rats pretreated with IPT plus cocaine compared to rats pretreated with cocaine alone. No significant change was found in the SUR1 and SUR2 expression of all four groups. CONCLUSION:IPT inhibited cocaine challenge-induced enhancement of DA levels in NAc and striatum by up-regulating Kir6.1 and Kir6.2 mRNA expression.

  1. A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol.

    Anji, Antje; Kumari, Meena


    Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half-life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis-acting region(s) within the NR1 3'-untranslated region (UTR) and identify NR1 3'-UTR binding trans-acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156-nt cis-acting region that binds to polysomal trans-acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis-trans interactions. Importantly, the 3'-UTR binding activity was significantly up-regulated in the presence of ethanol. UV cross-link analysis detected three NR1 3'-UTR binding proteins and their molecular mass calculated by Northwestern analysis was approximately 88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up-regulation of the 88-kDa protein after chronic ethanol treatment. The 88-kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIbeta). That GIIbeta is indeed a trans-acting protein and binds specifically to 3'-UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT-PCR. Western blotting data established a significant increase of GIIbeta polypeptide in chronic ethanol-exposed fetal cortical neurons. We hypothesize that the identified cis-acting region and the associated RNA-binding proteins are important regulators of NR1 subunit gene expression.

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

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


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

  3. Cartografia da paisagem alterada

    Simone Cortezão


    Full Text Available O presente trabalho de pesquisa surge da investigação de paisagens alteradas, áreas pós-industriais e da geografia urbana que é conformada pela indústria. A preocupação principal é revê-las como espaço de investigação crítica sob o ponto de vista particular, integradas às noções da peculiar natureza industrial com suas consequentes transformações na paisagem, além das relações do indivíduo com esse novo meio produzido. “Cartografia da paisagem alterada” é uma pesquisa que indicia a paisagem alterada em pelo menos dois estratos: um que é o da construção da geografia da cidade pela indústria, e o outro que é a paisagem alterada subjetivamente, o que configura uma crítica e uma reação à conformação sócio geográfica em outros parâmetros - da proximidade, da experiência vivida e da construção de um vocabulário próprio.  A pesquisa teórica sobre áreas pós-industriais, associada a referências nas artes e arquitetura, constitui o primeiro estrato. Teorias e procedimentos artísticos de Robert Smithson, Joseph Beuys e Rem Koolhaas fornecem material para o segundo, mediante a pesquisa do cotidiano no lugar, a região do Vale do Aço, que se configura em um guia - glossário

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

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


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

  5. Expressão do mRNA de genes mitocondriais e desempenho produtivo de codornas alimentadas com glicerol mRNA expression of mitochondrial genes and productive performance of quails fed with glycerol

    Stefânia Caroline Claudino da Silva


    Full Text Available O objetivo deste trabalho foi avaliar o efeito de dietas com glicerol no desempenho produtivo de codornas japonesas de corte e na expressão do mRNA de genes mitocondriais da proteína adenina nucleotídeo translocase (ANT e da proteína desacopladora (UCP, envolvidas no metabolismo energético e na resposta ao estresse oxidativo. As codornas foram alimentadas com dietas contendo 0, 8 e 12% de glicerol, em substituição parcial ao milho. Aos 28 dias de idade, o RNA total foi extraído de amostras do músculo do peito e a síntese do cDNA foi feita por meio de qRT‑PCR com iniciadores específicos para genes da ANT e UCP, obtidos de Gallus gallus. A conversão alimentar e o consumo de ração foram avaliados para as três dietas testadas. A adição de 8% de glicerol não afetou o desempenho dos animais. No entanto, a adição de 12% aumentou o consumo de ração e piorou a conversão alimentar. O ganho de peso não foi afetado pela inclusão de glicerol na dieta. No grupo alimentado com 8% de glicerol, a expressão da UCP aumentou, mas a da ANT não variou, em comparação ao controle. A expressão da UCP foi menor e a da ANT foi maior no grupo alimentado com 12% de glicerol. A inclusão de 8% de glicerol na dieta não afeta o desempenho de codornas de corte, embora aumente a expressão da UCP. A inclusão de 12% de glicerol piora o desempenho e aumenta a expressão da ANT.The objective of this work was to evaluate the effect of diets containing glycerol on the productive performance of Japanese meat quails and on the mRNA expression of the mitochondrial genes of the adenine nucleotide translocase (ANT and uncoupling protein (UCP, involved in energy metabolism and oxidative stress response. The quail were fed diets containing 0, 8, and 12% glycerol in partial substitution of corn. At 28 days of age, total RNA was extracted from samples of breast muscle and cDNA synthesis was done with qRT‑PCR using specific primers for ANT and UCP genes

  6. Translation with frameshifting of ribosome along mRNA transcript

    Li, Jingwei


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

  7. Interleukin-21 mRNA expression during virus infections

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


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

  8. Collagen mRNA levels changes during colorectal cancer carcinogenesis

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


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

  9. High lib mRNA expression in breast carcinomas.

    Satoh, Kazuki; Hata, Mitsumi; Yokota, Hiroshi


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

  10. Interleukin-21 mRNA expression during virus infections

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


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

  11. Membrane-Coupled mRNA Trafficking in Fungi.

    Haag, Carl; Steuten, Benedikt; Feldbrügge, Michael


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

  12. Interleukin-21 mRNA expression during virus infections

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


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

  13. Vibrational force alters mRNA expression in osteoblasts

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


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

  14. Protein Structure and the Sequential Structure of mRNA

    Brunak, Søren; Engelbrecht, Jacob


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

  15. Analysis of specific mRNA destabilization during Dictyostelium development.

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


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

  16. mRNA and DNA PCR tests in cutaneous tuberculosis

    Chandanmal Suthar


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


    Vera M.A.Tordino Brandão


    Full Text Available O Programa de Gerontologia da PUC desenvolve pesquisas focando o ser na perspectiva do envelhecimento. As atividades são interdisciplinares e reúnem docentes, alunos e ex-alunos. Nesta mesa redonda apresentamos um panorama sobre estes estudos. Na área da educação e comunicação o foco está centrado na longevidade e na preparação e representação social da velhice. São ressaltadas como temáticas: a formação continuada de profissionais e pesquisadores; o significado da religiosidade/espiritualidade ao longo da trajetória, e sua repercussão na qualidade de vida do idoso; os programas sócio-educacionais para idosos e concepções sobre educação; o monitoramento da mídia na cobertura da crescente longevidade e seus impactos. Em identidade e modos de morar reflete-se sobre soluções planejadas e sua adequação para a inclusão do envelhecimento populacional como questão fundamental, que implica a elaboração de novas políticas, investigações e, especialmente, apresenta-se como questão a ser analisada, refletida e vivenciada pela sociedade em geral. Quanto à saúde, o Programa desenvolve várias pesquisas numa abordagem interdisciplinar tendo como objetos de estudo: a terapia assistida com animais direcionada a idosos com diagnóstico de Alzheimer; o acompanhamento terapêutico e o atendimento psicoterapêutico em grupo, a aplicação da técnica de Calatonia em idosos moradores de ILPI, além da investigação sobre impasses clínicos no idoso frágil. Palavras-chave: gerontologia, longevidade, envelhecimento

  18. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    Hagedorn, C.H.


    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  19. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

    Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong


    In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.


    José Luiz Contador


    Full Text Available Este trabalho examina teoricamente os fatores determinantes da competitividade do produto e do negócio, imprescindíveis à elaboração de estratégias competitivas que visam à obtenção e sustentação de vantagens competitivas. Esse tema é objeto tanto da área de marketing estratégico quanto da de estratégia empresarial. Nas duas áreas, há um ponto de consenso que afirma que a empresa deve diferenciar sua oferta para conquistar e manter vantagem competitiva. Mas, os estudos conduzidos por meio do modelo de Campos e Armas da Competição revelaram que a empresa pode diferenciar seu produto e/ou serviço de modo mais amplo, levando em consideração, na formulação da sua estratégia competitiva, também o campo da competição, quer do produto, quer dela própria. Assim, o objetivo deste artigo é evidenciar que, para a empresa ser competitiva, além de diferenciar seu produto e/ou seu serviço no sentido tradicional, ela precisa: 1 diferenciar seu produto incluindo a definição em quais campos vai competir em cada segmento; e 2 escolher as armas da competição que irá utilizar e definir a intensidade de cada arma. Para atingir tal objetivo, foram necessários vários conceitos do modelo de Campos e Armas da Competição: campo da competição, arma da competição, tese do modelo, configuração dos campos da competição, campo coadjuvante, produto coadjuvante e par produto-mercado. Como uma proposta inovadora sempre desperta dúvida, é mostrado o processo de validação do modelo de Campos e Armas da Competição e são discutidas sua universalidade, consistência e completude.

  1. Molecular effects of leptin on peroxisome proliferator activated receptor gamma (PPAR-γ) mRNA expression in rat's adipose and liver tissue.

    Abbasi, A; Moghadam, A A; Kahrarian, Z; Abbsavaran, R; Yari, K; Alizadeh, E


    Leptin is a 16-kDa peptide hormone secreted by adipose tissue that participates in the regulation of energy homeostasis. The aim of this study was to determine the effect of leptin injection on mRNA expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) and comparison of PPAR-γ mRNA expression in rat's adipose and liver tissue. Twenty adult male rats were divided into the following groups: Group 1asa control (n=10) that did not receive any treatment. Group 2as a treatment (n=10) that received leptin (30 µg ⁄ kg BW) intraperitoneally (ip) for two successive days. Blood samples were taken before and one day after second leptin injection for triglyceride (TG), Free Fatty Acid (FFA), HLD-cholesterol, and LDL-cholesterol measurement. Total RNA was extractedfrom the adipose tissue and liver tissues of rats.  Adipose and liver tissue cells' cDNA was synthesized to characterize the expression of PPAR-γ. Gene expression of PPAR-γ mRNA was tested by RT- PCR technique. Results show leptin decreases expression of PPAR-γ on rat. Low levels of PPAR-γ mRNA were detected in adipose and liver tissues of treatment rats in comparison to control group. In treatment group, the level of PPAR-γ mRNA in liver tissue was very lower than the adipose tissue. The levels of HDL and FFA in treatment rats were increased whereas serum levels TG, VLDL and LDL were not changed. It is concluded that leptin signal with suppressing of PPAR-γ mRNA expression in rat's adipose and liver tissues can result in lipolysis instead of lipogenesis.

  2. Het Italiaanse voorzetsel da

    Boer, M.G. de


    SAMENVATTING In een voorzetselstudie in het kader van Jackendoffs semantische theorie wordt het sterk polyseme Italiaanse voorzetsel da geanalyseerd als een verzameling deelbetekenissen waarbij de meeste door een of meer semantische regels afgeleid kunnen worden van de basisbetekenis ruimtelijke

  3. Het Italiaanse voorzetsel da

    Boer, M.G. de


    SAMENVATTING In een voorzetselstudie in het kader van Jackendoffs semantische theorie wordt het sterk polyseme Italiaanse voorzetsel da geanalyseerd als een verzameling deelbetekenissen waarbij de meeste door een of meer semantische regels afgeleid kunnen worden van de basisbetekenis ruimtelijke He


    Kumiko K. Sakane


    Full Text Available São apresentados espectros infravermelhos da creatina, na faixa de 4000 a 400 cm−1. Os resultados são comparados com espectros observados e calculados, publicados na literatura.

  5. Impacto ambiental da apicultura


    A cultura da abelha doméstica Apis. mellifera L. ou apicultura é conhecida desde a antiguidade, sendo o mel uni alimento natural corrente entre os povos primitivos. Pinturas rupestres mostram que desde o neolítico o homem era atraído pelo mel, pois tentava recolher ninhos de abelhas selvagens e instalava-os em cavidades naturais das rochas, muito perto da sua habitação.

  6. The Pleiotropic Effects of Atorvastatin on Stable Angina Patients: Evidence by Analysis of High-Density Lipoprotein Size and Subclasses, and Plasma mRNA / Plejotropni Efekti Atorvastatina Kod Pacijenata Sa Stabilnom Anginom: Dokazi Dobijeni Analizom Veličine I Raspodele Subfrakcija Lipoproteina Velike Gustine I Plazmatske mRna

    Mirjanić-Azarić Bosa


    Full Text Available Uvod: Lipoproteini velike gustine (HDL imaju ateroprotektivne biološke osobine: antioksidativne, antiapoptotičke, antiinflamatorne kao i kapacitet da izvlače holesterol iz ćelija. Analiza plazmatske mRNA može da se koristi za ispitivanje plejotropnih efekata statina in vivo kao novo analitičko sredstvo za neinvazivnu procenu ekspresije gena u zidu krvnog suda. Cilj ove studije je bio da se procene plejotropni efekti atorvastatina kod pacijenata sa stabilnom anginom sa visokorizičnim vrednostima (grupa A u odnosu na pacijente sa graničnim i poželjnim vrednostima HDL holesterola (HDL- C (grupa B.

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

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


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

  8. Mechanisms of mRNA translation of interferon stimulated genes.

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


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

  9. mRNA analysis of single living cells

    Ikai Atsushi


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

  10. Control of Klebsiella pneumoniae nif mRNA synthesis.


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

  11. Transcriptional activators enhance polyadenylation of mRNA precursors

    Nagaike, Takashi; Manley, James L.


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

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

    Wang, Ke; Shi, Min; Cheng, Hong


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

  13. Clinical and biochemical evaluation of the saliva of patients with xerostomia induced by radiotherapy Avaliação clínica e bioquímica da saliva de pacientes com xerostomia induzida por radioterapia

    Cássio de Barros Pontes


    significativa na concentração de proteína. A atividade específica da a-amilase foi significativamente diminuída (p < 0,01 (856,6 ng/mg ± 88,0 antes e 567,0 ng/mg ± 120,6 após. No perfil eletroforético, foram observadas diferenças nas bandas protéicas, principalmente na faixa de peso molecular de 72.000 a 55.000 Da. Clinicamente, os pacientes com xerostomia induzida pela radioterapia apresentaram aumento de lesões na mucosa.

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

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


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

  15. The optional long 5'-untranslated region of human ACAT1 mRNAs impairs the production of ACAT1 protein by promoting its mRNA decay

    Xiaonan Zhao; Baoliang Song; Tayuan Chang; Boliang Li; Jia Chen; Lei Lei; Guangjing Hu; Ying Xiong; Jiajia Xu; Qin Li; Xinying Yang; Catherine C.Y.Chang


    We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG1397-1399 initiation codon,and also a minor 56 kDa isoform using the upstream in-frame GGC1274-1276initiation codon.The GGC1274-1276 codon is located at the optional long 5'-untranslated region(5'-UTR,nt 1-1396)of the mRNAs.The DNA sequences corresponding to this 5'-UTR are located in two different chromosomes,7 and 1.In the current work,we report that the optional long 5'-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG1397-1399 codon,mainly by promoting its mRNA decay.The western blot analyses indicated that the optional long 5'-UTR potently impaired the production of different proteins initiated from the AUG1397-1399codon,meaning that this impairing effect was not influenced by the 3'-UTR or the coding sequence of ACAT1 mRNA.The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5'-UTR dramatically reduced the contents of its linked mRNAs.Analyses of the protein to mRNA ratios showed that this 5'-UTR mainly decreased its mRNA stability rather than altering its translational efficiency.We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription.The results showed that this 5'-UTR promoted its mRNA decay.Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that,in both the cytoplasm and the nucleus of cells,the optional long 5'-UTR-linked mRNAs decayed faster than those without the link.Overall,our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.

  16. mRNA quality control pathways in Saccharomyces cerevisiae

    Satarupa Das; Biswadip Das


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

  17. Drosha mediates destabilization of Lin28 mRNA targets.

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


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

  18. Tracking single mRNA molecules in live cells

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


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

  19. Imaging mRNA and protein interactions within neurons

    Eliscovich, Carolina; Shenoy, Shailesh M.


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

  20. Myc Regulation of mRNA Cap Methylation

    Cowling, Victoria H.; Cole, Michael D.


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

  1. Cytokine mRNA quantification by real-time PCR.

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


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

  2. Em defesa da semiodiversidade

    Antônio Risério


    Full Text Available Semiodiversidade é um conceito que denomina a esfera da vida que se refere particularmente ao reino dos signos. Embora tenha sido criado no contexto das discussões sobre a biodiversidade, ele compreende um amplo campo que é quase desconhecido. Contrariamente à biodiversidade, não é um termo da moda, ainda que abarque um amploconjunto de problemas. As mais diferentes linguagens, signos e códigos culturais encontram lugar no domí­nio da semiodiversidade. Neste artigo a defesa da semiodiversidade é uma forma de preservar a vida humana neste momento da aventura do homem no planeta.Abstract: Semiodiversity is a concept to denominate the sphere of life that refers, particularly, to the realm of signs. Although it has been created in the context of biodiversity, it assigns a wider territory, which is almost unknown. Contrary to biodiversity, it is not a trendy' term, even though embeding a much wider scope. The most different languages, signs and cultural codes can be found in the domain of semiodiversity. In this article, the defense of semiodiversity is a way to preserve human life at this moment of humanadventure on Earth.

  3. Glucocorticoid receptor 1B and 1C mRNA transcript alterations in schizophrenia and bipolar disorder, and their possible regulation by GR gene variants.

    Duncan Sinclair

    Full Text Available Abnormal patterns of HPA axis activation, under basal conditions and in response to stress, are found in individuals with schizophrenia and bipolar disorder. Altered glucocorticoid receptor (GR mRNA and protein expression in the dorsolateral prefrontal cortex (DLPFC in psychiatric illness have also been reported, but the cause of these abnormalities is not known. We quantified expression of GR mRNA transcript variants which employ different 5' promoters, in 35 schizophrenia cases, 31 bipolar disorder cases and 34 controls. We also explored whether sequence variation within the NR3C1 (GR gene is related to GR mRNA variant expression. Total GR mRNA was decreased in the DLPFC in schizophrenia cases relative to controls (15.1%, p<0.0005 and also relative to bipolar disorder cases (8.9%, p<0.05. GR-1B mRNA was decreased in schizophrenia cases relative to controls (20.2%, p<0.05, while GR-1C mRNA was decreased in both schizophrenia and bipolar disorder cases relative to controls (16.1% and 17.2% respectively, both p<0.005. A dose-dependent effect of rs10052957 genotype on GR-1B mRNA expression was observed, where CC homozygotes displayed 18.4% lower expression than TC heterozygotes (p<0.05, and 31.8% lower expression than TT homozygotes (p<0.005. Similarly, a relationship between rs6190 (R23K genotype and GR-1C expression was seen, with 24.8% lower expression in GG homozygotes than GA heterozygotes (p<0.01. We also observed an effect of rs41423247 (Bcl1 SNP on expression of 67 kDa GRα isoform, the most abundant GRα isoform in the DLPFC. These findings suggest possible roles for the GR-1B and GR-1C promoter regions in mediating GR gene expression changes in psychotic illness, and highlight the potential importance of sequence variation within the NR3C1 gene in modulating GR mRNA expression in the DLPFC.

  4. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.


    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  5. da etiologia ao tratamento

    Soares, Tânia Alexandra Maia


    Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária A luxação da articulação temporomandibular (ATM) ocorre quando o côndilo mandibular se desloca para fora da fossa mandibular e não é capaz de retornar. As manifestações clinicas mais frequentes desta patologia são: dificuldade de fechar a boca, depressão pré-auricular, sialorréia, tensão dos músculos da mastigação e dor severa na reg...

  6. Fundamentos da psicologia social

    Kruger, Helmuth Ricardo


    O tema central deste estudo é o dos fundamentos da Psicologia Social. Neste sentido, foram examinados pressupostos ontológicos, epistemológicos, lógicos e axiológicos mas, também, as bases conceituais, teóricas e metodológicas deste setor da Psicologia contemporânea. A justificar este empreendimento, encontram-se duas suposições: a de que qualquer ciência é inexoravelmente construída sobre postulados metafísicos e lógicos, e a de que a falta de clareza em relação a estes impede a instalação d...


    Gustavo Bernardo


    Full Text Available Apresento uma concepção pessoal do ensino de literatura, condensando a discussão das principais questões que o afetam: a singularidade da disciplina entre as demais disciplinas escolares; a crítica do realismo como valor dominante; a importância do exemplo; os efeitos da desvalorização salarial e moral do professor; a praga das adaptações literárias para jovens. A partir dessas questões, apresento cinco modos de leitura e ensino do texto literário, a saber: ler como resposta; ler como pergunta; a leitura ingênua; a leitura crítica; a leitura teórica. Concluo pela necessidade de proteção radical à dúvida, fundadora da literatura e do próprio pensamento.

  8. Medidas de rentabilidade da empresa

    Mota, Haroldo Moura Vale


    Faz uma avaliação da medida de retorno contábil da corporação (return on investment - ROI) à luz da verdadeira medida de retorno econômico (taxa interna de retorno - TIR). Apresenta modelo para cálculo da taxa interna de retorno da empresa, o qual é submetido à testes para aferir o seu valor informativo. Utilizando o conceito de taxa interna de retorno da corporação, é criado um modelo denominado de Modelo de Avaliação Econômica Base Caixa, aplicável a uma empresa b...

  9. Da Vinci Linjen

    Nissen, Poul

    Afsluttende evalueringsrapport fra følgeforskningen gennem tre år i forbindelse med dety pædagogiske arbejde på Blåbjerggårdskolens Da Vinci Linje for skolemotiverede og underpræsterende elever på 7. til 9. klassetrin.......Afsluttende evalueringsrapport fra følgeforskningen gennem tre år i forbindelse med dety pædagogiske arbejde på Blåbjerggårdskolens Da Vinci Linje for skolemotiverede og underpræsterende elever på 7. til 9. klassetrin....



    Este artigo apresenta o conceito de agricultura urbana e sua contribuição para a integração da atividade agrícola com o ambiente urbano, incentivando o desenvolvimento de cidades sustentáveis. Analisa o grau de complexidade encontrado pelas autoridades ao criar e aplicar politicas pública onde os sistemas urbanos de suprimento de alimentos não são acessíveis a toda a população. Traz luz a algumas das questões diretamente relacionadas à implantação da agricultura em grandes centros urbanos, ta...

  11. Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

    Leick, Lotte; Lindegaard, Birgitte; Stensvold, Dorthe


    OBJECTIVES: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin......: AT IL-18 mRNA content and plasma IL-18 concentration were higher (p insulin resistance. While acute exercise did not affect IL-18 mRNA expression...... at the studied time-points, exercise training reduced AT IL-18 mRNA content by 20% in both sexes. DISCUSSION: Because obesity and insulin resistance were associated with elevated AT IL-18 mRNA and plasma IL-18 levels, the training-induced lowering of AT IL-18 mRNA content may contribute to the beneficial effects...

  12. Profilaxia da peste

    Renato dos Santos Araújo


    Full Text Available O autor parte da premissa de que a profilaxia da doença infecciosa decorre do encadeamento epidemiológico: fonte de infecção-veículo transmissor - receptivel, para explicar a da peste, baseada no mesmo princípio. Depois de citar os 4 principais complexos epidemiológicos da peste e afirmar que tôda a profilaxia da doença consiste em atingir os dois primeiros elos dessas cadeias e proteger o último, passa a classificar os vários métodos profiláticos empregados em 2 grandes grupos: o das medidas destrutivas ou provisórias e o das medidas supressivas ou definitivas. Entre as primeiras arrola a desratização e a despulização, às quais acrescenta o tratamento e isolamento do doente e do portaãor, e entre as segundas inclui a anti-ratização e a imunização. A seguir, passa a explicar em que consistem essas várias medidas profiláticas e quais os agentes de que se tem lançado mão para executá-las, expendendo ao curso dessa exposição o conceito que formula a respeito de cada uma delas, à guisa de apreciação do seu valor relativo. Enaltece sobretudo as medidas supressivas ou definitivas, às quais empresta a maior significação na luta antipestosa, chamando a atenção, em especial, para a necessidade de estudos imunológicos para aperfeiçoamento do poder imunitário das vacinas de germes vivos avirulentos, que considera um grande recurso para a profilaxia da doença, sobretudo para a proteção do rurícola, cujo labor e modo de vida o expõem inevitavelmente a se infectar, por ocasião da ocorrência de epizootias de origem silvestre. Concluída essa primeira parte, passa a fazer o histórico de como se desenvolveu a campanha contra a peste, no Brasil, desde a época da sua invasão em 1899 pelo pôrto de Santos até os nossos dias. Nesse histórico, detem-se um pouco para expôr a atuação do extinto Serviço Nacional de Peste, o qual, criado em 1941, após a reorganização do Departamento Nacional de Saúde, passou

  13. Development of a quantitative analysis method for mRNA from Mycobacterium leprae and slow-growing acid-fast bacteria

    Nakanaga, Kazue; Maeda Shinji; Matsuoka, Masanori; Kashiwabara, Yoshiko [National Inst. of Infectious Diseases, Tokyo (Japan)


    This study aimed to develop a specific method for detection and quantitative determination of mRNA that allows estimation of viable counts of M. leprae and other mycobacteria. Of heart-shock protein of 65 kDa (hsp65), mRNA was used as an indicator to discriminate the living cells and died ones. To compare mRNA detections by RNase protection assay (RPA) and Northern blot hybridization (NBH), labelled anti-sense RNA for hsp65 gene of M. leprae was synthesized using plasmid pUC8/N5. The anti-sense RNA synthesized from the template DNA containing about 580 bp (194 to 762) of hsp65 gene. When compared with NBH method, the amount of probe required for the detection by RPA method was 1/30 or less and the detection sensitivity of RPA was also 10 times higher. In addition, complicated procedures were needed to eliminate non-specific reactions in NBH method. These results indicated that RPA method is more convenient and superior for the mRNA detection. However, isotope degradation in the probe used for RPA method might affect the results. Therefore, {sup 33}P of {sup 35}P, of which degradation energy is less that {sup 32}P should be used for labelling. Total RNA was effectively extracted from M. chelonae, M. marinum by AGPC method, but not from M. leprae. In conclusion, RPA is a very effective detection method for these mRNA, but it seems necessary to further improve the sensitivity of detection for a small amount of test materials. (M.N.)

  14. Lipidoid mRNA Nanoparticles for Myocardial Delivery in Rodents.

    Turnbull, Irene C; Eltoukhy, Ahmed A; Anderson, Daniel G; Costa, Kevin D


    An area of active research in the field of cardiac gene therapy aims to achieve high transfection efficiency without eliciting immune or inflammatory reactions. Nanomedicine offers an attractive alternative to traditional viral delivery vehicles because nanoparticle technology can enable safer and more controlled delivery of therapeutic agents. Here we describe the use of lipidoid nanoparticles for delivery of modified mRNA (modRNA) to the myocardium in vivo, with a focus on rodent models that represent a first step toward preclinical studies. Three major procedures are discussed in this chapter: (1) preparation of lipid modRNA nanoparticles, (2) intramyocardial delivery of the lipid modRNA nanoparticles by direct injection with an open chest technique in rats, and (3) intracoronary delivery of the lipid modRNA nanoparticles with open chest and temporary aortic cross clamping in rats.

  15. Selenoprotein P mRNA expression in human hepatic tissues

    Chun-Li Li; Ke-Jun Nan; Tao Tian; Chen-Guang Sui; Yan-Fang Liu


    AIM: To investigate the expression of Selenoprotein P mRNA (SePmRNA) in tissues of normal liver, liver cirrhosis and hepatocellular carcinoma (HCC), and its relationship with HCC occurrence and development.METHODS: The expression of SePmRNA in tissues of normal liver, liver cirrhosis and HCC were detected by in situ hybridization using a cDNA probe.RESULTS: The enzyme digesting products of pBluescript-Human Selenoprotein P were evaluated by electrophoresis.The positive expression of SePmRNA was found in the tissues of normal liver,liver cirrhosis and HCC.The expression of SeP mRNA was found in hepatic interstitial substance,especially in endothelial cells and lymphocytes of vasculature.The positive rate of SePmRNA in normal liver tissue was 84.6% (11/13) and the positive signals appeared in the nucleus and cytoplasm,mostly in the nucleolus,and the staining granules were larger in the nucleolus and around the nucleus.The positive rate of SePmRNA in liver cirrhosis tissue was 45.O% (9/20) and the positive signals were mainly in the nucleolus and cytoplasm,being less around the nucleus and inner nucleus than that in normal liver tissue. The positive rate of SePmRNA in HCC tissue was 30.0% (9/30) and the positive signals were in the cytoplasm, but less in the nucleus.CONCLUSION: SePmRNA expression in the tissues of normal liver and HCC is significantly different (84.6% vs 30.0%, P = 0.003), suggesting that SeP might play a role in the occurrence and development of HCC.

  16. O livro da alma

    Oliver Tolle


    Full Text Available Trata-se de compreender os pressupostos da filosofia concreta pretendida por Johann Gottfried Herder (1744-1803 em uma série de textos redigidos durante as décadas de 1760-70. Se, por um lado, Herder demonstra afinidade com a mesma harmonia preestabelecida que está na base de leibnizianos como Gottsched e Baumgarten, por outro, ele aprofunda a discussão sobre a origem do conhecimento humano ao reduzir a importância de juízos de valor universal e acentuar a necessidade de uma aproximação constante das sensações que se agitam obscuramente no fundo da alma. Disso resulta uma concepção inusitada de sabedoria, a qual depende de uma renúncia à posse confortável de conhecimentos abstratos e da aceitação da natureza cambiante e apenas parcialmente perscrutável daquilo que nos move. Se com isso perde-se a certeza, abre-se em contrapartida uma dimensão essencialmente ativa e sensível de realização no mundo.

  17. O esvaziamento da cultura

    Andressa Paula de Andrade


    Full Text Available Trata-se da resenha da obra "A civilização do espetáculo: uma radiografia do nosso tempo e da nossa cultura" de autoria de Mario Vargas Llosa, ganhador do Nobel de Literatura em 2010. O trabalho busca compreender este tempo em que a diversão assume maior relevância em detrimento da cultura profunda e reflexiva. Destarte, a todo momento se olha para o passado com vistas a examinar este fenômeno que se tornou um verdadeiro "esvaziamento cultural". Outrossim, busca-se elucidar que embora ocorra um progresso científico, acesso a educação e a tecnologia de forma vertiginosa, isso não significa que a as artes avançaram na mesma velocidade, pelo contrário, acabou estagnando e já não visa mais a modificação de estruturas, mas apenas entreter.

  18. O labirinto da contemporaneidade

    Lang, Charles Elias


    Full Text Available O presente trabalho discute a modernidade e a produção da subjetividade contemporânea. Toma-se como modelo o labirinto de Eco (1985. Existem três tipos de labirintos: o labirinto clássico, o maneirístico, e a rede. O clássico é unicursal. Assim que se entra, a única coisa que se pode fazer é chegar ao centro e, do centro, só o que se pode fazer é encontrar uma saída. O maneirístico propõe escolhas alternativas. Todos os percursos levam a um ponto morto, com exceção de um que leva à saída. Na rede, cada ponto pode ter conexão com qualquer outro ponto. Se os labirintos anteriores têm um interior e um exterior, a rede, extensível ao infinito, não tem nem um dentro nem um fora. A partir deste modelo discute-se a produção de individualidades e subjetividades, do corpo como meio de comunicação e a produção de imagens do corpo através da mídia

  19. Dj Da Hai


    Miko Da Hai is DJ at a Xi’an disco bar. At work, in his cool attire, the pulsing disco beat punctuated by his distinctive vocal commentary, he makes a striking impression. The job having come his way by chance, Miko is now a skilled and experienced DJ of eight years’ standing. This may be

  20. Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

    Li, Long; Pintel, David J., E-mail:


    Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

  1. The mechanism of nonsense-mediated mRNA decay

    Costa, Nuno Filipe Teixeira


    Trabalho de investigação desenvolvido no Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge. [PT] A expressão genética representa uma das vias mais complexas e importantes para a célula, que culminam na síntese proteica. Como tal, quaisquer erros que possam surgir durante estes eventos podem ser rapidamente amplificados e terem um impacto severo na célula. Portanto surgiram diversos mecanismos de controlo de qualidade para assegurar a fidelidade da expr...

  2. qPCR based mRNA quality score show intact mRNA after heat stabilization

    Oskar Karlsson


    Full Text Available Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

  3. Transitoriedade ou o desterro da certeza

    Paulo José Carvalho da Silva


    Full Text Available O tema da transitoriedade era lugar-comum da medicina da alma da primeira modernidade. Este artigo analisa algumas dessas fontes para mostrar como a dor do tempo e os males da alma atinentes eram relacionados a um modo de encarar a fugacidade do tempo e a fragilidade da vida. Dá-se destaque, sobretudo, às construções em torno da condição humana enquanto desterro da certeza.

  4. Highest trkB mRNA expression in the entorhinal cortex among hippocampal subregions in the adult rat: contrasting pattern with BDNF mRNA expression.

    Tokuyama, W; Hashimoto, T; Li, Y X; Okuno, H; Miyashita, Y


    Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, regulate synaptic functions in the hippocampus of the adult rodent. In previous studies, in situ hybridization methods have been used to evaluate regional differences in BDNF and trkB mRNA expression levels in hippocampal subregions. However, these studies have failed to reach consensus regarding the regional differences in the mRNA expression levels. In the present study, we quantitated mRNA expression levels using two different methods, ribonuclease protection assays and a quantitative reverse-transcription polymerase chain reaction technique, in four hippocampal subregions: the entorhinal cortex, dentate gyrus (DG), CA3 and CA1. These two methods yielded the same results. We found that BDNF and trkB mRNA expression levels did not covary in the four subregions. BDNF and full length trkB (trkB FL) mRNA in the entorhinal cortex and the DG show contrasting expression patterns. The expression level of BDNF mRNA was highest in the DG among the hippocampal subregions and low in the entorhinal cortex and the CA1, whereas the trkB FL mRNA expression level was highest in the entorhinal cortex, low in the DG and lowest in the CA3. These results suggest regional differences in BDNF/TrkB signaling for maintenance and modifiability of neuronal connections in the hippocampal formation.

  5. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry


    concentrations of AMH, inhibin-B, progesterone and estradiol. Androgen Receptor mRNA expression in granulosa cells, and the FF content of androgens, both showed a highly significant positive association with to the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly...... with the expression of AMHR2, but did not correlate with any of the hormones in the FF. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the FF levels of androgen and FSHR expression...

  6. A FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA and its application



    The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response (FQ-RT-PCR) method for the quantitative determination of IL-2 mRNA and IL-4mRNA in Th cells, with which the Th cells status of the patients with gynaecological tumors and chronic renal failure (CRF) can be analyzed. IL-2 cDNA and IL-4 cDNA were prepared, and the plasmid pMD18 carrying IL-2 cDNA or IL-4 cDNA fragment was constructed and cloned as the template for quantitative determination. The primers and probes labelled with 6-carboxy-fluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) were prepared, and the experimental conditions were optimized to set up the FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA. Th cells enriched from peripheral blood mononuclear cells (PBMCs) of 20 healthy volunteers (HVs), 16 gynaecological benign (GB) cases, 18 gynaecological malignant (GM) tumor cases and 16 chronic renal failure (CRF) patients were tested for IL-2 mRNA and IL-4 mRNA by FQ-RT-PCR. The house-keeping gene 3-actin was used as the internal control gene of the experiment. The standard curve for log concentration of series of quantitative templates vs threshold cycle (CT) was established by linear regression, and the linear range was 102-107 copies/μl. The imprecision test showed the CV of inter-assay and intra-assay of a high content sample by FQ-RT-PCR were 7.8% and 12.5%, respectively. The CV of inter-assay and intra-assay of a low content sample were 10.8% and 19.5%, respectively. The IL-2 mRNA expressions in Th of the patients with gynaecological malignant tumor (compared with the HVs and the patients with gynaecological benign disease) and in Th of the CRF patients (compared with the HVs) were declined significantly and at the same time the IL-4 mRNA expression increased significantly ( P < 0. 001 ). A simple, sensitive and accurate FQ-RT-PCR method for the quantitative detection of IL-2 mRNA and IL-4 mRNA has

  7. Repeated stress increases catalytic TrkB mRNA in rat hippocampus.

    Nibuya, M; Takahashi, M; Russell, D S; Duman, R S


    Northern blot analysis was utilized to distinguish between catalytic and truncated TrkB mRNA on the basis of transcript size. Repeated (10 days), but not acute, immobilization stress significantly increased levels of catalytic TrkB mRNA, but did not influence expression of truncated TrkB transcripts in rat hippocampus. Exposure to another paradigm, a combination of different, unpredictable stressors, also increased levels of catalytic, but not truncated, TrkB mRNA. In situ hybridization analysis demonstrated that chronic stress up-regulated TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cells layers of hippocampus. As previously reported, both acute and chronic immobilization stress decreased expression of BDNF mRNA, suggesting that up-regulation of catalytic TrkB mRNA may be a compensatory adaptation to repeated stress.

  8. O Tema da Cidadania

    Adriano Moreira


    Full Text Available Neste artigo tentaremos evidenciar a natureza pelo menos tripartida da cidadania que responde ao incontroladoglobalismo: uma que se confunde com os direitos humanos, exigentes querem seja o Estado o seu interlocutor,quer não exista tal interlocutor; outra que se insere na fenomenologia dos grandes espaços em formação, de que aUnião Europeia é exemplo, e a que o projecto da Constituição Europeia, agora em recolhimento de meditação, jáoferece o apoio de uma definição específica de direitos; e finalmente, a cidadania que diz respeito à relação com oEstado soberano em crise de redefinição, sobretudo, pelo que nos interessa, no espaço europeu.

  9. Fisiopatologia da asma grave

    Todo-Bom, Ana; Mota-Pinto, Anabela


    Não tem resumo. Transcreve-se o primeiro parágrafo: A história natural da asma e os condicionalismos de uma evolução para formas moderadas ou graves não está completamente estabelecida. Contudo, quer os factores genéticos quer os factores ambientais, serão determinantes na fisiopatologia e no prognóstico da doença. A asma é, por definição, uma doença inflamatória crónica das vias aéreas caracterizada por uma obstrução brônquica generalizada mas variável que é, pelo menos parcialmente, reve...

  10. [da Vinci surgical system].

    Watanabe, Gou; Ishikawa, Norihiro


    The da Vinci surgical system was developed by Intuitive Surgical Inc. in the United States as an endoscopic surgical device to assist remote control surgeries. In 1998, the Da Vinci system was first used for cardiothoracic procedures. Currently a combination of robot-assisted internal thoracic artery harvest together with coronary artery bypass grafting (CABG) through a mini-incision (ThoraCAB) or totally endoscopic procedures including anastomoses under robotic assistance (TECAB) are being conducted for the treatment of coronary artery diseases. With the recent advances in catheter interventions, hybrid procedures combining catheter intervention with ThoraCAB or TECAB are anticipated in the future.On the other hand, with the decrease in number of coronary artery bypass surgeries, the share of valvular surgeries is expected to increase in the future. Among them, mitral valvuloplasty for mitral regurgitation is anticipated to be conducted mainly by low-invasive procedures, represented by minimally invasive cardiac surgery( MICS) and robot-assisted surgery. Apart from the intrinsic good surgical view, robotic-assisted systems offer additional advantages of the availability of an amplified view and the easy to observe the mitral valve in the physiological position. Thus, robotic surgical surgeries that make complicated procedures easier are expected to accomplish further developments in the future. Furthermore, while the number of surgeries for atrial septal defects has decreased dramatically following the widespread use of Amplatzer septal occluder, robotic surgery may become a good indication for cases in which the Amplatzer device is not indicated. In Japan, clinical trial of the da Vinci robotic system for heart surgeries has been completed. Statutory approval of the da Vinci system for mitral regurgitation and atrial septal defects is anticipated in the next few years.

  11. Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.

    Bondurant, M C; Koury, M J


    Regulation of the production of erythropoietin occurs in the kidney and liver largely through control of accumulation of erythropoietin mRNA. Erythropoietin mRNA was first detected in kidneys at 1.5 h postanemia and reached a plateau value at least 200-fold above the control value by 4 to 8 h. A 20-base sequence immediately upstream from the reported erythropoietin mRNA initiation site is complementary to a hypervariable sequence in 18S rRNA.

  12. Dynamics of translation by single ribosomes through mRNA secondary structures

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L.; Reiche, Michael; Farrell, Ian; Cooperman, Barry S.; Goldman, Yale E.


    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNA 2° structures. Downstream ...

  13. Regulation of mRNA translation during mitosis.

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D


    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  14. mRNA related to insulin family in human placenta

    Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.


    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  15. Nuclear imprisonment: viral strategies to arrest host mRNA nuclear export.

    Kuss, Sharon K; Mata, Miguel A; Zhang, Liang; Fontoura, Beatriz M A


    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  16. Correlation of mRNA and protein in complex biological samples.

    Maier, Tobias; Güell, Marc; Serrano, Luis


    The correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

  17. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.


    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  18. One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator

    Maria Letizia Di Martino


    Full Text Available VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF30 (30 kDa, and the shorter VirF21 (21 kDa, lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30 and VirF21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF21 is also translated from a leaderless mRNA (llmRNA whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30 is responsible for activation of the virulence system, VirF21 negatively autoregulates virF expression itself. Since VirF21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA.

  19. Elicitor induction of mRNA activity. Rapid effects of elicitor on phenylalanine ammonia-lyase and chalcone synthase mRNA activities in bean cells.

    Lawton, M A; Dixon, R A; Hahlbrock, K; Lamb, C J


    Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total

  20. BUB1 mRNA is significantly co-expressed with AURKA and AURKB mRNA in advanced-stage ovarian serous carcinoma.

    Davidson, Ben; Nymoen, Dag Andre; Elgaaen, Bente Vilming; Staff, Anne Cathrine; Tropé, Claes G; Kærn, Janne; Reich, Reuven; Falkenthal, Thea E Hetland


    The objective of this study was to investigate the expression and clinical role of the spindle checkpoint kinase budding uninhibited by benzimidazole 1 (Bub1) in primary and metastatic advanced-stage ovarian serous carcinoma. BUB1 mRNA expression was analyzed in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction (PCR). Bub1 protein expression by Western blotting was studied in 63 carcinomas (30 effusions and 33 solid lesions). BUB1 mRNA expression at different anatomic sites was studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. BUB1 mRNA was universally expressed in serous carcinomas, irrespective of anatomic site. BUB1 mRNA levels were uniformly low in six ovarian surface epithelium specimens analyzed for comparative purposes. Bub1 protein was expressed in 22/30 effusions and 28/33 solid lesions. BUB1 mRNA expression was significantly higher in chemo-naïve primary carcinomas and solid metastases compared to specimens obtained following neoadjuvant chemotherapy (p cancer. BUB1 mRNA levels are lower following chemotherapy exposure in solid lesions, though its presence is unrelated to clinical behavior including response to chemotherapy and survival. BUB1 is co-expressed with AURKA and AURKB suggesting biological relationship between these spindle cell components.

  1. Effect of Exercise on the Expression of Adiponectin mRNA and GLUT4 mRNA in Type 2 Diabetic Rats


    To investigate the impact of exercise on the expression of adiponectin and GLUT4 mR NA in type 2 diabetic rats, type 2 diabetic rat model was made. The diabetic rats were treated with swimming training for 8 weeks. The expression of adiponectin mRNA in perirenal fat and GLUT4mRNA in skeletal muscles were assessed by reverse transcription polymerase chain reaction (RT PCR) and the levels of blood glucose, serum insulin, and blood lipid were measured. Our results showed that the expression of adiponectin mRNA and GLUT4 mRNA in diabetic model group was decreased by 45 % (P<0.01), 43 % (P<0.01) respectively. The gene expression of adiponectin and GLUT4 was increased significantly in swimming group (P<0.05 and P<0.01, respectively).Compared with the model group, fasting insulin, TG, TC and FFA were decreased significantly in the training group (P<0.05 or P<0.01) as compared with model group. It is concluded that exercise can promote the expression of adiponectin mRNA and GLUT4 mRNA in type 2 diabetic rats,which may be one of the mechanisms responsible for the amelioration of insulin resistance in the rats.

  2. Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

    朱长虹; 田红


    In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT-PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

  3. O paradoxo da legitimidade a partir da legalidade segundo Habermas

    Aylton Barbieri Durão


    Full Text Available autonomia jurídica surge da conexão co-originária entre a autnomia privada dos indivíduos e a autonomia pública dos cidadãos, a qual, contudo, ficou velada ao longo da história da filosofia política e jurídica por causa da herança metafísica do direito natural racional e da filosofia da consciência que gerou uma relação de competição entre os direitos humanos e a vontade soberana do povo. Por causa desta conexão entre a autonomia privada e pública, o processo democrática de produção de normas jurídicas apresenta um aparente paradoxo porque os cidadãos tanto podem aprovar as normas motivados pela busca do êxito da racionalidade estratégica como orientados pelo reconhecimento de sua validade através da racionalidade comunicativa, o qual se dissolve, contudo, quando se considera a democracia como um processo aberto para a solidariedade dos cidadãos no mundo da vida.

  4. O Cognitivismo e o problema da Cientificidade da Psicologia

    Arja Castañon, Gustavo


    Este artigo trata do problema do estatuto científico da psicologia. Seu objetivo é avaliar em que medida o cognitivismo solucionou objeções históricas levantadas contra a possibilidade de uma psicologia científica. Com base em avaliação de textos clássicos da “revolução cognitiva”, se conclui que esta abordagem apresentou um projeto filosófico de ciência psicológica que resolveu a maior parte dos problemas ontológicos e epistemológicos endêmicos da disciplina, como o da natureza inquantificáv...

  5. Stage and strain specific expression of the tandemly repeated 90 kDa surface antigen gene family in Trypanosoma cruzi.

    Beard, C A; Wrightsman, R A; Manning, J E


    A recombinant cDNA library constructed in the expression vector lambda gtll using mRNA from the trypomastigote stage of Trypanosoma cruzi was screened with two monoclonal antibodies that have been shown to react with a 105 kDa and a 90 kDa surface antigen in trypomastigotes of the Peru and Y strains of T. cruzi. One recombinant lambda phage, designated Tcc-20, was reactive to both monoclonals. The beta-galactosidase/T. cruzi hybrid protein encoded in Tcc-20 is recognized by the monoclonal antibodies and by serum antibodies from mice infected with strains of T. cruzi which contain the 90 kDa antigen. Antibodies immunoselected from serum of mice infected with the Peru strain by adsorption to Tcc-20 fusion protein react specifically with a 90 kDa polypeptide in trypomastigote but not epimastigote lysates of T. cruzi. The mRNA complementary to the DNA insert in Tcc-20 is present only in those stages and strains of T. cruzi which express the 90 kDa surface antigen. These characteristics are strong evidence that the T. cruzi DNA fragment cloned into Tcc-20 encodes a portion of the 90 kDa surface antigen. The gene(s) which encodes this polypeptide is shown to be present in approximately 20 copies per haploid genome and most, and possibly all, of the copies are found in a tandemly linked multigene family.

  6. Active Fe-Containing Superoxide Dismutase and Abundant sodF mRNA in Nostoc commune (Cyanobacteria) after Years of Desiccation

    Shirkey, Breanne; Kovarcik, Don Paul; Wright, Deborah J.; Wilmoth, Gabriel; Prickett, Todd F.; Helm, Richard F.; Gregory, Eugene M.; Potts, Malcolm


    Active Fe-superoxide dismutase (SodF) was the third most abundant soluble protein in cells of Nostoc commune CHEN/1986 after prolonged (13 years) storage in the desiccated state. Upon rehydration, Fe-containing superoxide disumutase (Fe-SOD) was released and the activity was distributed between rehydrating cells and the extracellular fluid. The 21-kDa Fe-SOD polypeptide was purified, the N terminus was sequenced, and the data were used to isolate sodF from the clonal isolate N. commune DRH1. sodF encodes an open reading frame of 200 codons and is expressed as a monocistronic transcript (of approximately 750 bases) from a region of the genome which includes genes involved in nucleic acid synthesis and repair, including dipyrimidine photolyase (phr) and cytidylate monophosphate kinase (panC). sodF mRNA was abundant and stable in cells after long-term desiccation. Upon rehydration of desiccated cells, there was a turnover of sodF mRNA within 15 min and then a rise in the mRNA pool to control levels (quantity of sodF mRNA in cells in late logarithmic phase of growth) over approximately 24 h. The extensive extracellular polysaccharide (glycan) of N. commune DRH1 generated superoxide radicals upon exposure to UV-A or -B irradiation, and these were scavenged by SOD. Despite demonstrated roles for the glycan in the desiccation tolerance of N. commune, it may in fact be a significant source of damaging free radicals in vivo. It is proposed that the high levels of SodF in N. commune, and release of the enzyme from dried cells upon rehydration, counter the effects of oxidative stress imposed by multiple cycles of desiccation and rehydration during UV-A or -B irradiation in situ. PMID:10613879

  7. A Utopia da Paz Universal

    Daniel Serrão


    Full Text Available Como o Homem atingiu a fase da cultura exterior simbólica em que se encontra actualmente. Apresentaçãosucinta da teoria dos patamares evolutivos de Merlin Donald. O paradigma "popperiano" da investigaçãocientífica moderna: verificar ou falsificar hipóteses. O caso típico da teoria darwiniana da evolução das espéciesvivas. A Bioética como diálogo de saberes em ordem à criação de uma nova sabedoria (wisdom. Apresentação doconceito de Van Rensselaer Potter. O "credo" de Potter e a utopia da paz universal.

  8. Custos da qualidade

    Gilberto Caetano


    Full Text Available Este artigo pretende demonstrar através de uma síntese histórico-conceitual , a evolução da importância do sistema de qualidade, bem como a sua mensuração no processo de tomada de decisão das organizações frente à competitividade estabelecida no contexto da globalização. Assim, procura dar ênfase à ação planejada no sentido de reduzir ou eliminar falhas e defeitos nos vários processos, com vistas a agregar valor ao cliente e ao mesmo tempo reduzir os custos da não-conformidade. Palavras-chave: Custo, qualidade, satisfação, inspeção, controle, requisito, conformidade, não-conformidade, mensuração, processo, prevenção, problema e avaliação. Abstract This article intends to demonstrate through a historical-conceptual synthesis, the evolution of the importance of the quality system, as well as your measurement the process of decision making of the organizations front to the established competitiveness in the context of the globalization. Like this, it tries to give emphasis to the action drifted in the sense of to reduce or to eliminate flaws and defects in the several processes, with views to join value to the customer and at the same time to reduce the costs of the no-conformity. Key-words: Cost, quality, satisfaction, inspection, control, requirement, conformity, no-conformity, measurement, process, prevention, problem and evaluation.

  9. Pioneirismo da associacao brasileira de enfermagem no desenvolvimento da pesquisa: da revista ao centro de pesquisa

    Lais de Araujo Pereira


    Full Text Available O presente estudo tem como objetivo analisar as primeiras iniciativas da Associação Brasileira de Enfermagem para o desenvolvimento da pesquisa em enfermagem. Com um recorte temporal que engloba o período de 1932 a 1971, ano de criação da Revista Brasileira de Enfermagem à criação do Centro de Estudos e Pesquisas, da Associação Brasileira de Enfermagem. As fontes utilizadas foram documentos escritos localizados no Centro de Documentação da Escola de Enfermagem Anna Nery da Universidade Federal do Rio de Janeiro e no Centro de Memória da Associação Brasileira de Enfermagem, além de edições da revista localizadas na Biblioteca Setorial da Pós-Graduação da Escola de Enfermagem Anna Nery. Percebe-se, nesta análise, que os empreendimentos realizados por essa entidade representaram um verdadeiro espaço de investigação e de formação de enfermeiros pesquisadores no Brasil.

  10. Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

    Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.


    A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

  11. Pensamento da America


    Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Filosofia e Ciências Humanas, Programa de Pós-Graduação em História, Florianópolis, 2013. Esta dissertação objetivou investigar a atividade editorial de Rui Ribeiro Couto e Renato Costa Almeida enquanto estes intelectuais estiveram à frente do Pensamento da America, uma publicação mensal vinculada ao A Manhã, jornal porta voz do Estado Novo. Este suplemento panamericano veio a público entre 1941 e 1949, no entanto a...

  12. Propriedades antioxidantes da melatonina



    Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Neurociências. Foram investigadas as ações da melatonina (MEL) contra a lipoperoxidação (LPO) induzida por hidroxil (OH), ascorbil (Asc) e peroxinitrito (ONOO-) em diversos modelos de membranas como lipossomas de fosfatidilcolina (LipPC) e asolecitina (LipASO), microssomas de cérebro (MicC) e fígado (MicF) e homogeneizado de cérebro (HC); e seu efeito contra enzimas ...

  13. A moral da infidelidade

    Bundt, Roger


    Full Text Available Através das representações da infidelidade conjugal encontradas em três filmes do diretor Adrian Lyne realizados nas décadas de 1980, 1990 e 2000 (Atração Fatal, Proposta Indecente e Infidelidade, busca- se entender as idéias trabalhadas em cada obra, as maneiras de representar a mulher, a família, a relação amorosa e a monogamia, a partir das noções de dever e moral trabalhadas por Gilles Lipovetsky

  14. A moralidade da nanotecnologia


    A nanotecnologia é um conjunto formado por saberes, técnicas e práticas que estudam e exploram as novas propriedades dos materiais, quando manipulados em níveis atômicos e moleculares. A possibilidade técnica de organizar e controlar a matéria, desde suas menores dimensões e unidades, pode implicar profundas transformações no processo industrial de produção e ter consequências, moralmente significativas, sobre as inter-relações humanas, a organização da conjuntura social vigente e o próprio f...

  15. Elevated neurofilament light chain (NFL) mRNA levels in prediabetic peripheral neuropathy.

    Celikbilek, Asuman; Tanik, Nermin; Sabah, Seda; Borekci, Elif; Akyol, Lutfi; Ak, Hakan; Adam, Mehmet; Suher, Murat; Yilmaz, Neziha


    Evidence suggests that peripheral nerve injury occurs during the early stages of disease with mild glycemic dysregulation. Two proteins, neuron-specific enolase (NSE) and neurofilament light chain (NFL), have been examined previously as possible markers of neuronal damage in the pathophysiology of neuropathies. Herein, we aimed to determine the potential value of circulatory NSE and NFL mRNA levels in prediabetic patients and in those with peripheral neuropathy. This prospective clinical study included 45 prediabetic patients and 30 age- and sex-matched controls. All prediabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy and nephropathy. mRNA levels of NSE and NFL were determined in the blood by real-time polymerase chain reaction. NSE mRNA levels were similar between prediabetic and control groups (p > 0.05), whereas NFL mRNA levels were significantly higher in prediabetics than in controls (p 0.05), while NFL mRNA levels were significantly higher in prediabetics with peripheral neuropathy than in those without (p = 0.038). According to correlation analysis, NFL mRNA levels were positively correlated with the Douleur Neuropathique 4 questionnaire score in prediabetic patients (r = 0.302, p = 0.044). This is the first study to suggest blood NFL mRNA as a surrogate marker for early prediction of prediabetic peripheral neuropathy, while NSE mRNA levels may be of no diagnostic value in prediabetic patients.

  16. Cotranscriptional assembly of mRNP complexes that determine the cytoplasmic fate of mRNA

    Forget, Amélie; Chartrand, Pascal


    Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence has revealed that the full picture is more complex and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA.

  17. Impaired expression of glycogen synthase mRNA in skeletal muscle of NIDDM patients

    Vestergaard, H; Bjørbaek, C; Andersen, P H


    with NIDDM were accompanied by a 39% reduction (P less than 0.02) in the steady state level of GS mRNA per microgram DNA of muscle. In both diabetic and control subjects, the mRNA expression of GS was unaffected after euglycemic-hyperinsulinemic clamp for 4 h. With single-stranded conformation polymorphism...

  18. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Cristian Del Campo


    Full Text Available Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  19. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya


    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  20. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.


    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in

  1. Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

    Michaud, Morgane; Ubrig, Elodie; Filleur, Sophie; Erhardt, Mathieu; Ephritikhine, Geneviève; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie


    Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

  2. Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

    Arae, Toshihiro


    Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

  3. Distribution of the mRNA for protein phosphatase T in rat brain

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG


    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  4. Impaired expression of glycogen synthase mRNA in skeletal muscle of NIDDM patients

    Vestergaard, H; Bjørbaek, C; Andersen, P H


    with NIDDM were accompanied by a 39% reduction (P less than 0.02) in the steady state level of GS mRNA per microgram DNA of muscle. In both diabetic and control subjects, the mRNA expression of GS was unaffected after euglycemic-hyperinsulinemic clamp for 4 h. With single-stranded conformation polymorphism...

  5. Neurotrophin-3 mRNA expression in rat intrafusal muscle fibres after denervation and reinnervation

    Copray, JCVM; Brouwer, N


    We have studied the regulation of the expression of neurotrophin-3 (NT-3) mRNA in neonatal and adult rat muscle spindles after denervation and after denervation followed by reinnervation. Denervation of the intrafusal fibres did not result in an upregulation of the NT-3 mRNA expression but decreased

  6. The intragraft cytokine mRNA pattern reflects the efficacy of steroid anti rejection therapy

    Baan, C C; Niesters, H G; Balk, A H; Mochtar, B; Zondervan, P E; Weimar, W


    BACKGROUND: We studied the effect of antirejection therapy on intragraft cytokine mRNA expression. METHODS: Therapy consisted of three doses of 1 gm of intravenous methylprednisolone. We determined its effect on intragraft mRNA expression of immunoregulatory (interleukin-2, interleukin-4) and inflam

  7. Distribution of the mRNA for protein phosphatase T in rat brain

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG


    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  8. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.


    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in ter

  9. Simultaneous isolation of mRNA and native protein from minute samples of cells

    Petersen, Tonny Studsgaard; Andersen, Claus Yding


    Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native pr...

  10. Relative workload determines exercise-induced increases in PGC-1alpha mRNA

    Nordsborg, Nikolai Baastrup; Lundby, Carsten; Leick, Lotte;


    trial. No change in HIF1alpha, PFK, CS, LDH-A or LDH-B mRNA expression was detected after any of the exercise trials. CONCLUSION:: The relative intensity of brief intermittent exercise is of major importance for the exercise induced increase of several mRNA's, including PGC-1alpha....

  11. Aspectos moleculares da anemia falciforme

    Galiza Neto Gentil Claudino de


    Full Text Available No presente artigo abordaram-se vários aspectos relacionados à natureza molecular da anemia falciforme, desordem hematológica de caráter hereditário que acomete expressivo número de indivíduos em várias regiões do mundo. As pesquisas realizadas em torno desta patologia da hemácia, ao longo de quase um século, a partir de 1910, cooperaram para a criação de um novo e importante segmento da ciência, denominado biologia molecular. A descoberta dos polimorfismos da mutação (GAT->GTG no gene que codifica a cadeia beta da hemoglobina, originando diferentes haplótipos da doença, permitiu um melhor e mais amplo conhecimento em torno da heterogeneidade clínica nos pacientes falcêmicos. Analisando a hemoglobina na sua estrutura normal e mutante, sua produção e evolução, pode-se ter um entendimento mais completo da fisiopatologia desta doença e da sua complexidade clínica.

  12. Fractais : da geometria à videoarte


    A descoberta da geometria fractal é datada de meados do séc. XX. É conhecida como a geometria da natureza já que permite descrever muitos fenómenos naturais que apresentam irregularidades que são impossíveis de descrever através dos princípios da geometria tradicional. O aparecimento de novas tecnologias digitais veio alterar o modo como a arte é encarada. Este trabalho pretende demonstrar possibilidades de integração da geometria fractal e a Teoria do Caos na criação de videoarte, b...

  13. Fisiopatogênese da dor

    Miguel R. Covian


    Full Text Available O estudo da dor é um capítulo do estudo das bases neurofisiológicas da sensação, pois a dor nada mais é que uma sensação de conteúdo desagradável originada por estímulos nocivos. Enquanto a função das outras modalidades sensoriais é informativa ou gnósica, a dor é de proteção. O estudo da fisiología da dor compreende o estudo dos receptores, dos estímulos, das vias, das estruturas do sistema nervoso central que partici- pam da fisiología da dor, da percepção dolorosa e das reações motoras e neurovegetativas. São analisados os receptores e as vias e discutidas sua especificidade, assim como a transmissão da dor rápida e lenta por vias nervosas diferentes até o córtex cerebral. É comentada a influência da atenção na intensidade da percepção dolorosa e, com base em estudos experimentais, é considerado que o circuito retículo-córtico-reticular seja indispensável para a percepção da dor. No tocante à patologia da dor é examinada a insensibilidade congênita à dor, sendo admitida a hipótese de que em algumas sinapses é modificado o esquema de impulsos que darão lugar à sensação dolorosa. É destacado o papel do lobo frontal como parte de um mecanismo pòtenciador que condiciona o sofrimento geral do paciente. A dor referida é explicada pela teoria da "convergência-projeção" de fibras viscerais com fibras cutâneas dolorosas sôbre o mesmo neurônio em algun ponto da via sensitiva. O resultado da hipofisectomia trazendo uma diminuição ou anulação da dor no câncer do seio abre debates em tôrno do papel dos hormônios na percepção dolorosa.

  14. Correlation between indoleamine 2,3 dioxygenase mRNA and CDKN2A/p16 mRNA: a combined strategy to cervical cancer diagnosis.

    Saffi Junior, Mario Cezar; Duarte, Ivone da Silva; Brito, Rodrigo Barbosa de Oliveira; Prado, Giovana Garcia; Makabe, Sergio; Dellê, Humberto; Camacho, Cleber P


    Cervical cancer (CC) is one of the most common cancers among women worldwide. The relation of the human papillomavirus (HPV) with CC and its precursor lesions was first suspected for over 40 years. The indoleamine 2,3 dioxygenase (IDO) is an immune modulator enzyme responsible for the immune system tissue protection mechanism, which may be the key to the tumoural persistence. HPV oncoprotein E7 promotes the increase in cyclin-dependent kinase inhibitor p16 (CDKN2A/p16). The isolated and combined analysis of CDKN2A/p16 mRNA to CC diagnosis was done with promising results. The aim of this study is to evaluate the correlation between IDO mRNA and CDKN2A/p16 mRNA. We will explore the potential of both as diagnostic tools. RNA was extracted from tissue samples. cDNA was generated with High Capacity RNA-to-cDNA kit. The real-time PCR results were analysed using nonlinear curve estimation, ROC curve, Chi-squared test, the proportion of variance explained and Galen and Gambino formulas. From 270 patients attended, colposcopy examination was performed in 110 and the biopsy in 75 patients. We found a positive correlation in patients older than 28 years old with low-risk lesions, but the correlation is lost in high-risk lesions. Although cytology, IDO mRNA and CDKN2A/p16 mRNA could not differentiate the risk groups, IDO combined with CDKN2A/p16 mRNA results could (p = 0.028). The best diagnostic result was achieved by IDO coupled with CDKN2A/p16 mRNA, which may considerably increase the sensitivity of screening for CC.

  15. mRNA expression of adipocytokines and glucocorticoid-related genes are associated with downregulation of E-cadherin mRNA in colorectal adenocarcinomas.

    Størkson, Ragnhild H; Aamodt, Rolf; Vetvik, Katja Kannisto; Pietilainen, Kirsi; Bukholm, Geir; Jonsdottir, Kristin; Vollan, Hilde S; Sonerud, Tonje; Lüders, Torben; Jacobsen, Morten B; Bukholm, Ida R K


    There is a consistently reported relationship between the incidence of colon cancer and obesity. It is thought that adipose tissue, particularly visceral fat, which secretes systemic factors that alter immunological, metabolic and endocrine milieu and promotes insulin resistance by producing adipocytokines, is important in cancer progression. Systemic high concentrations of adipocytokines, such as tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and glucocorticoid metabolism-related genes have been associated with gastrointestinal cancer. However, limited information exists about the expression of these cytokines within tumour tissue. mRNA expression of TNF-α, IL-6,IL-8, IL-10, IL-1RN, glucocorticoid receptor alpha (GR-α), 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), plasminogen activator inhibitor-1 (PAI-1), Slug, vimentin, Snail and E-cadherin was analysed in paired samples of tumour tissue and normal mucosa in 60 surgical patients for Dukes B and C colorectal adenocarcinomas using quantitative reverse transcription PCR and microarray technology. The mRNA expression level of analysed genes was compared between tumour tissue and normal mucosa from the same patients, and a correlation to mRNA expression of E-cadherin in the same tissue samples was also performed. A highly significant difference in mRNA expression level of several of the analysed genes was observed between tumour tissue and the normal intestinal mucosa. Inverse correlation between mRNA expression of 11βHSD1, IL-6, GR-α and PAI-1 on one hand and mRNA expression of E-cadherin on the other hand was observed. Results show that the adipocytokines and glucocorticoid metabolism-related genes are overexpressed in colorectal adenocarcinomas, and expression of these genes is associated with the downregulation of E-cadherin mRNA, connecting these genes to carcinogenesis and progression of colorectal cancer.

  16. Relationship Between Heart Damages and HSPs mRNA in Persistent Heat Stressed Broilers

    SUN Pei-ming; LIU Yu-tian; ZHAO Yong-gang; BAO En-dong; WANG Zhi-liang


    The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activities of creatine kinase (CK) and glutamic-pyruvic transaninase (GPT) were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC70) mRNA was changeable. The transcription of heat shock protein 70 (HSP70) mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.

  17. The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy

    匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民


    Objective To evaluate the potential role of insulin-like growth factor-1 receptor mRNA(IGF-IR mRNA) in the onset and development of retinopathy in diabetic rats.Methods A diabetic model was duplicated in Wistar rats. The early changes in the retina were examined using light and transmission electron microscopy. Expression of IGF-IR mRNA was analyzed using in situ hybridization.Results Weak expression of IGF-IR mRNA(5%) was found in retinas of normal rats, but was significantly increased (15% and 18%) in the retinas of diabetic rats after 3 and 6 months of diabetes (P<0.01). In situ hybridization and morphological study demonstrated that there was a positive correlation between IGF-IR mRNA expression and retinal changes at various stages.Conclusion Increased IGF-IR mRNA might play an important role in the onset and development of diabetic retinopathy.

  18. Expression of hippocampal adrenergic receptor mRNA in a rat model of depression

    Jianbin Zhang; Lingling Wang; Xinjun Wang; Jingfeng Jiang; Xiaoren Xiang; Tianjun Wang


    Adrenergic receptor dysfunction is suggested as a potential cause of hippocampal vulnerability to stress-related pathology. We examined mRNA expression of adrenergic receptor (AR) subtypes α1-AR, α2-AR, and β1-AR in hippocampal subregions (CA1, CA3, dentate gyrus) using in situ hybridization in a depression model induced by chronic unpredictable mild stress and social isolation. α1-AR mRNA expression was significantly increased in the CA3 and dentate gyrus, β1-AR mRNA was significantly increased in the CA1, and α2-AR mRNA remained unchanged in all regions of depression rats compared with controls. Thus, different AR subtypes exhibit a differing pattern of mRNA expression in various hippocampal subregions following depression.

  19. Quantification of mRNA Levels Using Real-Time Polymerase Chain Reaction (PCR).

    Li, Yiyi; Wang, Kai; Chen, Longhua; Zhu, Xiaoxia; Zhou, Jie


    Real-time quantitative reverse transcription PCR technique has advanced greatly over the past 20 years. Messenger RNA (mRNA) levels in cells or tissues can be quantified by this approach. It is well known that changes in mRNA expression in disease, and correlation of mRNA expression profiles with clinical parameters, serve as clinically relevant biomarkers. Hence, accurate determination of the mRNA levels is critically important in describing the biological, pathological, and clinical roles of genes in health and disease. This chapter describes a real-time PCR approach to detect and quantify mRNA expression levels, which can be used for both laboratorial and clinical studies in breast cancer research.

  20. Challenges and advances towards the rational design of mRNA vaccines.

    Pollard, Charlotte; De Koker, Stefaan; Saelens, Xavier; Vanham, Guido; Grooten, Johan


    In recent years, mRNA vaccines have emerged as a safe and potent approach for the induction of cellular immune responses. Whereas initial studies were limited to the ex vivo loading of dendritic cells (DCs) with antigen-encoding mRNA, recent progress has led to the development of improved mRNA vaccines that enable direct in vivo targeting of DCs. Although preclinical studies demonstrated their potency in inducing antitumor immunity, several bottlenecks hinder the broader application of mRNA vaccines. In this review, we discuss the challenges associated with mRNA-based vaccination strategies, the technological advances that have been made to overcome these limitations, and the hurdles that remain to be tackled for the development of an optimal mRNA vaccine.

  1. Glucocorticoids modulate BDNF mRNA expression in the rat hippocampus after traumatic brain injury.

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M


    Brain-derived neurotrophic factor (BDNF) expression in rat hippocampus is increased after experimental traumatic brain injury (TBI) and may be neuroprotective. Glucocorticoids are important regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) on the expression of BDNF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury (FPI) and in situ hybridization to evaluate the expression of BDNF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomized rats (with or without corticosterone replacement). FPI and ADX independently increased expression of BDNF mRNA. In animals undergoing FPI, prior ADX caused further elevation of BDNF mRNA and this upregulation was prevented by corticosterone replacement in ADX rats. These findings suggest that glucocorticoids are involved in the modulation of the BDNF mRNA response to TBI.

  2. Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury.

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M


    Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.

  3. Ontogeny of pituitary growth hormone and growth hormone mRNA in the chicken.

    McCann-Levorse, L M; Radecki, S V; Donoghue, D J; Malamed, S; Foster, D N; Scanes, C G


    The changes in pituitary growth hormone (GH) mRNA levels have been determined by Northern blot analysis and laser densitometry during embryonic development and posthatch growth of white Leghorn cockerels. Pituitary GH mRNA levels were observed to progressively increase between 18 days of embryonic development to a maximum at 4 weeks of age (posthatch). Subsequently, pituitary GH mRNA levels declined between 4 and 8 weeks of age, and between 12 weeks of age and adulthood. Pituitary GH contents showed increases during embryonic development and posthatch growth that paralleled the rise in GH mRNA. The decline in pituitary GH mRNA levels between 4 weeks of age and adulthood occurs when GH secretion has been observed previously to decline.

  4. Tumores cartilaginosos da laringe

    Thomé Robert


    Full Text Available Introdução: Os tumores cartilaginosos da laringe são raros, sendo a cartilagem cricóide a mais acometida. A raridade desses tumores tem como conseqüência experiência limitada e, portanto, o conhecimento a respeito destas neoplasias apresenta pontos obscuros. Objetivo: Apresentar os resultados cirúrgicos bem como o seguimento a longo prazo de 6 pacientes com tumores cartilaginosos da laringe, 4 condrossarcomas de baixa malignidade e 2 condromas. Forma de estudo: Retrospectivo não randomizado. Material e método: Cinco dos tumores acometiam a cartilagem cricóide e 1 a tireóide. Os pacientes foram submetidos a laringectomia parcial (4 e total (2, com seguimento de 6 a 30 anos (média-19,5 anos. Resultados: Nenhum dos pacientes apresentou metástase ou morte relacionada ao tumor. A sobrevida alcançada por 5 anos foi de 100% e a por 20 anos, 67%, sendo a comorbidade responsável por tal decréscimo. Conclusões: Nossos resultados reforçam o conceito de que a laringectomia conservadora seja a modalidade ideal de tratamento, reservando a ressecção total para tumores extensos ou casos de recorrência. Há indícios de que 5 anos pode não ser tempo suficiente para observar recorrência no condrossarcoma de baixo grau de malignidade.

  5. Dos Males da Medida

    Schwarcz Lilia Moritz


    Full Text Available Esse artigo tem como objeto a difícil percepção da "diferença". Tema polêmico, o debate sobre as variedades culturais foi transformado, em meados do século XIX, em uma questão racial e, nesse sentido, vinculado às regularidades da biologia. Tecendo comparações entre os teóricos raciais brasileiros e os impasses mais contemporâneos, sobre a aferição de inteligência e capacidade, esse ensaio pretende provocar. Em pauta estarão as contradições que têm se destacado, mais recentemente, quando se opõem de um lado as etnicidades locais, de outro a tão propalada globalização. Tal qual dois lados de uma mesma moeda, a afirmação de identidades tem se mostrado tão evidente como o próprio estreitamento das relações internacionais. Mudam os termos do debate - de raça, para etnia -, mas, de alguma maneira, continuamos presos a uma lógica positiva de estabelecimento de hierarquias ontológicas.

  6. Os usos da diversidade

    Clifford Geertz


    Full Text Available Resumo Ao discorrer sobre o “Futuro do Etnocentrismo”, este artigo parte da premissa de que a globalização, apesar de ter – em muitos casos – diminuído as diferenças entre povos, não tem amenizado os preconceitos e as formas de discriminação que ocorrem em nome dessas diferenças. Ele resgata a importância do antropólogo neste cenário lembrando que a diversidade cultural faz parte da sociedade complexa, remetendo-se não apenas a grupos étnicos ou nacionais bem delimitados, mas também a diferenças de geração, gênero, sexo e classe, entre outros. Neste contexto, a tolerância passiva de modos distantes de vida assim como a aceitação pragmática de nosso próprio paroquialismo são atitudes não somente intelectualmente desonestas mas também moralmente repreensíveis. É no encontro incômodo de subjetividades variantes na sua própria sociedade que o antropólogo define seu lugar.

  7. A morte da bailarina

    Nancy Mangabeira Unger


    Full Text Available A história do Ocidente pode ser lida de acordo com o sentido da palavra grega hybris: transgressão, presunção, desmesura. Hoje, vivemos o ápice de um percurso marcado pela desejo de tudo dominar. Por sua extrema gravidade, a crise contemporânea nos coloca diante da responsabilidade de questionar a própria dimensão na qual pensamos, e a maneira em que compreendemos nossa identidade enquanto humanos e nosso lugar na totalidade do real.Palavras-chave: civilização ocidental; crise; hybris; questionamento. AbstractThe history of the West can be read according to the meaning of the greek word hybris: transgression, arrogance, loss of measure. We presently live the pinnacle of a course marked by the desire to dominate all beings.  The extreme gravity of  contemporary crisis confronts us with the responsibility of questioning the very dimension in which we think, as well as the manner in which we understand our identity as human beings and our place in the totality of being.Key words: western civilization; crisis; hybris; questioning. 

  8. Cellular stress increases RGS2 mRNA and decreases RGS4 mRNA levels in SH-SY5Y cells.

    Song, Ling; Jope, Richard S


    Modulation of the expression of regulator of G-protein signaling (RGS) proteins is a major mechanism used to modulate their actions. Besides control by second messengers, the expression of RGS proteins, particularly RGS2, can be regulated by cell stress. Because RGS2 and RGS4 expression can be regulated by the cell cycle, we examined if cell cycle signals are involved in their regulation following stress. Treatment of SH-SY5Y cells with camptothecin increased RGS2 mRNA and decreased RGS4 mRNA levels. This effect on RGS2 mRNA was blocked by the cyclin-dependent kinase-2 (cdk2) inhibitors roscovitine and purvalanol. Cell cycle arrest was further implicated in regulating RGS mRNA levels because geldanamycin, which causes cell cycle arrest by inhibiting the actions of heat shock protein 90, caused changes in the mRNA levels of RGS2 and RGS4 similar to, and additive with, the effects of camptothecin. Overall, these results indicate that cell cycle arrest regulates the expression of RGS2 and RGS4, and that the expression of these two RGS family members is oppositely regulated by stress that causes cell cycle arrest.

  9. Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples

    Negi S


    Full Text Available Purpose: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR amplification of M . tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85 B protein from different clinical samples of pulmonary and extrapulmonary tuberculosis. Methods: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system and PCR. PCR was performed with specific primers for the targets: IS6110, 65kDa, 38kDa and 85B. Results: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture ( P < 0.05. Smear positive samples (56 and the samples (36 showing positive results by conventional methods (smear and LJ medium culture and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols ( P >0.05. The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%, followed by 65kDa, 38kDa and 85B RT-PCR in descending order. Conclusions: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.

  10. The alteration of mRNA expression of SOD and GPX genes, and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles.

    Alharby, Hesham F; Metwali, Ehab M R; Fuller, Michael P; Aldhebiani, Amal Y


    Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L(-1)) and ZnO-NPs (0, 15 and 30 mg L(-1)). Treatments with NaCl at both 3 and 6 g L(-1) suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS-PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.

  11. Short hairpin RNA targeting NP mRNA inhibiting Newcastle disease virus production and other viral structural mRNA transcription.

    Yue, Hua; Deng, Shu; Yang, Fa-Long; Li, Ding-Fei; Fu, An-Jing; Yang, Fan; Tang, Cheng


    Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiological agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry. Better understanding of the molecular basis of viral structural genes involved with production should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here we show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding mRNA but also P, HN, F, M gene mRNA. The findings reveal that newly synthesized NP mRNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

  12. Heat shock response in yeast involves changes in both transcription rates and mRNA stabilities.

    Laia Castells-Roca

    Full Text Available We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25 °C to 37 °C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins.

  13. Circulating hTERT mRNA as a tumor marker in cholangiocarcinoma patients

    Kawin Leelawat; Surang Leelawat; Thawee Ratanachu-Ek; Somboon Trubwongchareon; Jerasak Wannaprasert; Saad Tripongkaruna; Suchart Chantawibul; Panadda Tepaksorn


    AIM: To investigate human telomerase reverse transcriptase (hTERT) mRNA in the serum of cholangiocarcinoma patients.METHODS: The serum of thirty three cholangiocarcinoma patients, forty one benign biliary tract disease patients and ten healthy volunteers were collected and analyzed for the expression of hTERT mRNA by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).We then examined the correlation between values of serum hTERT mRNA and the pathological staging of cholangiocarcinoma.RESULTS: hTERT mRNA was detected in 28 of 33(84.85%) of serum obtained from cholangiocarcinoma patients and 9 of 41 (21.9%) of serum obtained from benign biliary tract disease patients. hTERT mRNA was not detected in any serum obtained from healthy volunteers. on the other hand the common tumor marker, CA19-9 was detected in 20 of 33 (60.6%) of serum obtained from cholangiocarcinoma patients and 8 of 41 (19.5%) of serum obtained from benign biliary tract disease patients. However, no correlation was found between the present of serum hTERT mRNA and tumor staging.CONCLUSION: These results indicate that the detection of circulating hTERT mRNA was identified in almost all cholangiocarcinoma patients. It offers anovel tumor marker, which can be used as a complementary study for diagnosis of cholangiocarcinoma.

  14. Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation.

    Mezquita, Pau; Parghi, Sean S; Brandvold, Kimberly A; Ruddell, Alanna


    Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

  15. Fisiologia da caquexia

    Gonçalves, Catarina Vicente


    Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz A caquexia é uma síndrome de desnutrição multifatorial que se caracteriza por uma perda de peso e perda de massa muscular. Esta síndrome parte de uma doença subjacente e pode estar presente em várias doenças crónicas como por exemplo HIV, doença renal crónica, DPOC, doença hepática crónica, insuficiência cardíaca congénita e cancro. Com este trabalho, pretendeu-se entender a definição de ca...

  16. Em favor da talassografia

    Jean-Louis Boudou


    Full Text Available A Talassografia (“descrição do mar” interessa-sepelos impactos físicos, biológicos, ecológicos... culturais da violenta antropização dos ambientes costeiros (oceânicos e continentais, caracterizados pelaexigüidade, vulnerabilidade, fragilidade e plasticidade. Como o Brasil é um “país marítimo”, os geó-grafos (os talassógrafos brasileiros são convidadosa intensificar suas pesquisas nas áreas costeiras e acriar novas estruturas para divulgá-las: Revista, Encontros, Associação, Pós-Graduação... tudo em prolda talassografia.

  17. Fisiologia da caquexia

    Gonçalves, Catarina Vicente


    Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz A caquexia é uma síndrome de desnutrição multifatorial que se caracteriza por uma perda de peso e perda de massa muscular. Esta síndrome parte de uma doença subjacente e pode estar presente em várias doenças crónicas como por exemplo HIV, doença renal crónica, DPOC, doença hepática crónica, insuficiência cardíaca congénita e cancro. Com este trabalho, pretendeu-se entender a definição de ca...

  18. Berengario da Carpi.

    De Santo, N G; Bisaccia, C; De Santo, L S; De Santo, R M; Di Leo, V A; Papalia, T; Cirillo, M; Touwaide, A


    Berengario da Carpi was magister of anatomy and surgery at the University of Bologna from 1502 to 1527. Eustachio and Falloppia defined him as 'the restaurator of anatomy'. He was a great surgeon, anatomist and physician of illustrious patients including Lorenzo II dei Medici, Giovanni dalle Bande Nere, Galeazzo Pallavicini, Cardinal Colonna, and Alessandro Soderini. He had strong links to the intellectuals of his time (Forni, Bonamici, Manuzio, Pomponazzi) as well as with the Medici family. He was respected by the Popes Julius II, Leo X and Clement VII. His main contributions are the Isogogae Breves, De Fractura calvae sive cranei, and the illustrated Commentaria on the Anatomy of Mondino de Liucci, a textbook utilized for more than 200 years, which Berengario aimed to restore to its initial text. The Commentaria constitutes the material for the last part of this paper which concludes with a personal translation of some passages on 'The kidney', where the author gives poignant examples of experimental ingenuity.

  19. Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Fleischer, Jennifer G; Woster, Patrick M; Casero, Robert A


    The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

  20. Myxovirus Resistance Protein A mRNA Expression Kinetics in Multiple Sclerosis Patients Treated with IFNβ

    Libertinova, Jana; Meluzinova, Eva; Tomek, Ales; Horakova, Dana; Kovarova, Ivana; Matoska, Vaclav; Kumstyrova, Simona; Zajac, Miroslav; Hyncicova, Eva; Liskova, Petra; Houzvickova, Eva; Martinkovic, Lukas; Bojar, Martin; Havrdova, Eva; Marusic, Petr


    Introduction Interferon-β (IFNß) is the first-line treatment for relapsing-remitting multiple sclerosis. Myxovirus resistance protein A (MxA) is a marker of IFNß bioactivity, which may be reduced by neutralizing antibodies (NAbs) against IFNß. The aim of the study was to analyze the kinetics of MxA mRNA expression during long-term IFNβ treatment and assess its predictive value. Methods A prospective, observational, open-label, non-randomized study was designed in multiple sclerosis patients starting IFNß treatment. MxA mRNA was assessed prior to initiation of IFNß therapy and every three months subsequently. NAbs were assessed every six months. Assessment of relapses was scheduled every three months during 24 months of follow up. The disease activity was correlated to the pretreatment baseline MxA mRNA value. In NAb negative patients, clinical status was correlated to MxA mRNA values. Results 119 patients were consecutively enrolled and 107 were included in the final analysis. There was no correlation of MxA mRNA expression levels between baseline and month three. Using survival analysis, none of the selected baseline MxA mRNA cut off points allowed prediction of time to first relapse on the treatment. In NAb negative patients, mean MxA mRNA levels did not significantly differ in patients irrespective of relapse status. Conclusion Baseline MxA mRNA does not predict the response to IFNß treatment or the clinical status of the disease and the level of MxA mRNA does not correlate with disease activity in NAb negative patients. PMID:28081207

  1. Isolation and amplification of mRNA within a simple microfluidic lab on a chip.

    Reinholt, Sarah J; Behrent, Arne; Greene, Cassandra; Kalfe, Ayten; Baeumner, Antje J


    The major modules for realizing molecular biological assays in a micro-total analysis system (μTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 10(15) fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.

  2. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

    Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)


    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

  3. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    Sayaka Aizawa

    Full Text Available The pars tuberalis (PT is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD, such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2 mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

  4. COX-2 mRNA expression in esophageal squamous cell carcinoma (ESCC) and effect by NSAID.

    Liu, X; Li, P; Zhang, S-T; You, H; Jia, J-D; Yu, Z-L


    To investigate cyclooxygenase-2 (COX-2) mRNA expression in human esophageal squamous cell carcinoma and the effect of a non-steroidal anti-inflammatory drug (NSAID) on it, in order to explore the mechanism of COX-2 in esophageal squamous cell carcinoma (ESCC) carcinogenesis and the ability of NSAID to prevent or treat ESCC. Frozen specimens of human ESCC and adjacent normal esophageal squamous epithelium pairs (n = 22) were examined for COX-2 mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). After incubation with aspirin (a non-selective COX inhibitor) or Nimesulide (a selective COX-2 inhibitor), the proliferation status of two human esophageal squamous cancer cell lines, EC-9706 and EC-109, was quantified by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide assay. The expression of COX-2 mRNA in these cells was detected by RT-PCR. COX-2 mRNA was expressed in 12 of 22 (54.5%) ESCC tissue samples, but it was undetectable in all the specimens of adjacent normal esophageal squamous epithelium COX-2 mRNA expression. Both aspirin (5-20 mmol/L) and Nimesulide (0.1-0.8 mmol/L) inhibited EC-9706 cell line proliferation and suppressed its COX-2 mRNA expression dose-dependently. However, only aspirin (5-20 mmol/L) could inhibit proliferation in the EC-109 cell line and suppress COX-2 mRNA expression. Nimesulide (0.1-0.8 mmol/L) could neither inhibit EC-109 cell growth nor suppress COX-2 mRNA expression. COX-2 mRNA expression is a frequent phenomenon in human ESCC tissue samples and plays an important role in the carcinogenesis of ESCC. NSAID may be useful in the chemoprevention and therapy of human ESCC and its effects are likely to be mediated by modulating COX-2 activity.

  5. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

    Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina


    between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed...

  6. Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

    Sartor, Francesca; Anderson, Jihan; McCaig, Colin; Miedzybrodzka, Zosia; Müller, Berndt


    Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention.

  7. An Experimental Study on the Expression of Heme Oxygenase-2 mRNA in Hirschsprung's Disease

    朱珉; 魏明发; 刘芳


    Summary: In order to investigate the relationship between the expression of heme oxygenase-2 (HO-2) mRNA and the pathogenesis of Hirschsprung's disease (HD), total ribonucleic acid (RNA) was extracted in the aganglionic and ganglionic segments of colon respectively from 15 cases of HD. The single-stranded cDNA of HO-2 was synthesized and further amplified by reverse transcription-poly merase chain reaction (RT-PCR). The expression of HO-2 mRNA was normal in ganglionic seg ments, but absent in aganglionic segments. It is concluded that the absence of HO-2 mRNA expres sion may be an important mechanism responsible for HD.

  8. Assessing mRNA nuclear export in mammalian cells by microinjection.

    Lee, Eliza S; Palazzo, Alexander F


    The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Overexpression of protease nexin-1 mRNA and protein in oral squamous cell carcinomas

    Gao, Shan; Krogdahl, Annelise; Sørensen, Jens Ahm;


    -1 has been almost totally neglected. We have now compared the level of PN-1 mRNA in 20 cases of oral squamous cell carcinomas and in matched samples of the corresponding normal oral tissues. We found that the average PN-1 mRNA level in tumours and normal tissues was significantly different, being...... increased up to 13 fold in tumour samples compared with the average level in normal tissues. The PN-1 mRNA level was significantly higher in tumours from patients with lymph node metastasis than in tumours from patients without. We could conclude that PN-1 is frequently overexpressed in oral squamous cell...

  10. Stable RNA hairpins in 88 coding regions of human mRNA

    PAN Min; WANG Chuanming; LIU Ciquan


    RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R=G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

  11. The topological configuration and conformational analysis of mRNA in translation


    The theoretical model construction of mRNA hairpin structure and single-stranded structure as well as the simulation studies on RNA structure determined by the X-ray crystal diffraction and nuclear magnetic resonance revealed that in translation, after mRNA being unfolded into single-stranded structure, its topological configuration was closely correlative with the original hairpin structure. The conformational features of single-stranded mRNA appeared as helical regions alternating with curly regions to different extents, which might exert the influence on the folding of nascent polypeptide by various regulating effects including different translational rates.

  12. A influência da Internet no processo ensino-aprendizagem da leitura e da escrita

    Evanice Ramos Lima Barreto


    Full Text Available

    A Internet constitui um dos grandes avanços da comunicação humana. Através dela, é possível o contato com uma infinidade de informações, veiculadas pelos mais diversos gêneros de texto. Por isso, sendo um dos meios de comunicação que mais se expandiu no mundo, a Internet se apresenta como uma ferramenta indispensável no ensino-aprendizagem da leitura e da escrita. Partindo desse pressuposto, o presente trabalho pretende apresentar os resultados de um estudo que tem por objetivo investigar, à luz dos pressupostos empreendidos por Soares (1998 e Dionísio (2005, qual o “lugar” da Internet no processo de ensino-aprendizagem da leitura e da escrita, bem como até que ponto ela interfere de forma positiva e/ou negativa nesse processo. Para tanto, foram aplicados questionários a professores e alunos do Ensino Médio. A partir da análise dos dados, pretende-se verificar como a sobrecarga de informações, bem como o uso inadequado da Internet podem trazer implicações para o desenvolvimento dessas atividades.

  13. Dimensão da maturidade à luz da logoterapia

    Libardi, Tadeu Antonio


    Full Text Available O artigo aborda o tema da maturidade humana à luz da logoterapia, fundada por Viktor Emil Frankl. Nos dias de hoje, existe a frustração existencial, não mais a sexual, como analisada por Freud. Cada ser humano, como pessoa livre e responsável e tendo como meta a busca da maturidade, vai percorrendo seu próprio caminho, que o leva à ceitação de si, de seus limites e potencialidades e ao relacionamento social e religioso, capacitando-o ao confronto com o sofrimento humano

  14. Mito da guerra nos Estados Unidos

    Spini, Ana Paula; UFU


    Este artigo tem como objetivo compreender aspectos do mito da guerra nos Estados Unidos, em uma abordagem que prioriza as dissensões internas acerca da legitimidade de uma guerra e os dispositivos midiáticos de criação do consenso nacional. Procura-se, assim, demonstrar que o mito da guerra é constantemente reformulado à luz da conjuntura internacional, da prática da guerra, da propaganda e da resistência interna às intervenções militares. Palavras-Chave: Guerra; Estados Unidos; Mídia....

  15. Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of Saccharomyces cerevisiae.

    Chhetri, Gaurav; Pandey, Tripti; Kumar, Bijay; Akhtar, Md Sohail; Tripathi, Timir


    The nuclear export of macromolecules is facilitated by the nuclear pore complexes (NPCs), embedded in the nuclear envelope and consists of multi-protein complexes. MEX67 is one of the nuclear export factor responsible for the transport of the majority of cellular mRNAs from the nucleus to the cytoplasm. The mechanism of mRNA transport through NPCs is unclear due to the unavailability of structures and the known interacting partners of MEX67. The mex67 gene was cloned in pQE30A and was expressed in Escherichia coli. A strategy has been developed to purify the insoluble MEX67 using a nickel affinity column with chelating Sepharose fast flow media, after solubilizing with sodium lauroyl sarcosinate (Sarkosyl). The IMAC purified recombinant MEX67 was further purified using SEC to apparent homogeneity (∼8 mg/L). Following SEC, MEX67 was stable and observed to be a 67 kDa monomeric protein as determined by PAGE and the size exclusion chromatography. The availability of large quantities of the protein will help in its biochemical and biophysical characterization, which may lead to the identification of new interaction partners of MEX67 or MEX67 complex.

  16. Expression of NK1 receptor at the protein and mRNA level in the porcine female reproductive system.

    Bukowski, R


    The presence and distribution of substance P (SP) receptor NK1 was studied in the ovary, the oviduct and the uterus (uterine horn and cervix) of the domestic pig using the methods of molecular biology (RT-PCR and immunoblot) and immunohistochemistry. The expression of NK1 receptor at mRNA level was confirmed with RT-PCR in all the studied parts of the porcine female reproductive system by the presence of 525 bp PCR product and at the protein level by the detection of 46 kDa protein band in immunoblot. Immunohistochemical staining revealed the cellular distribution of NK1 receptor protein. In the ovary NKI receptor was present in the wall of arterial blood vessels, as well as in ovarian follicles of different stages of development. In the tubular organs the NK1 receptor immunohistochemical stainings were observed in the wall of the arterial blood vessels, in the muscular membrane, as well as in the mucosal epithelium. The study confirmed the presence of NK1 receptor in the tissues of the porcine female reproductive tract which clearly points to the possibility that SP can influence porcine ovary, oviduct and uterus.

  17. Rice black-streaked dwarf virus genome segment S5 is a bicistronic mRNA in infected plants.

    Yang, Jian; Zhang, Heng-Mu; Ying, Lu; Li, Jing; Lv, Ming-Fang; Xie, Li; Li, Pei-Pei; Liu, Xiao-Ya; Liang-Ying, Dai; Chen, Jian-Ping


    Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus, family Reoviridae. Genome segment S5 has a putative second ORF partially overlapping the major ORF but in a different reading frame. This putative ORF is present in a published sequence and in two Chinese isolates now sequenced. Antibodies were raised against purified P5-1 and P5-2 fusion proteins expressed in a prokaryotic system. In western blots, these antibodies reacted with proteins of about 106 and 27 kDa, respectively, as predicted by sequence analysis. In immunoelectron microscopy, antibodies to P5-1 reacted with viroplasms, indicating that P5-1 is a component of viroplasms, but no labeling was observed with P5-2 antisera. Northern blot assays showed that the genome segment S5 was transcribed as a single mRNA with no subgenomic RNA. These results show that S5 is functionally bicistronic in infected plants. Possible translational mechanisms for P5-2 are discussed.

  18. Controle da ferrugem e da broca-dos-ramos da figueira com diferentes fungicidas e inseticidas

    Éder Júnior Mezzalira


    Full Text Available Temperaturas e precipitações elevadas favorecem a incidência da ferrugem (Cerotelium fici(Cast. e da broca-dos-ramos (Azochis gripusalis (Walker, 1859 (Lepidoptera: Pyralidae, limitando a produção comercial de figos. O objetivo deste trabalho foi comparar a eficiência de fungicidas e inseticidas alternativos em relação à de produtos convencionais registrados para a cultura. Foram realizados dois experimentos, no delineamento de blocos inteiramente casualizados, com quatro repetições, no setor de fruticultura da Universidade Tecnológica Federal do Paraná, Campus Dois Vizinhos. Para controle da ferrugem, foram utilizados, em 100 L de água, azoxistrobin (10 g, calda bordalesa (1.500 g de cal virgem + 1.500 g de sulfato de cobre e testemunha (água. No controle da broca-dos-ramos utilizaram-se, em 100 L de água, azadiractina (1.000 mL P.C., alho (Allium sativumL. (100 mL P.C., cinza (20.000 g, extrato de fumo (nicotina (10.000 mL do preparado, deltametrina (50 mL P.C., Bacillus thuringiensisBerliner (100 g P.C., rotenona (1.000 mL P.C., sabão de coco (1.000 g e testemunha (água. A calda bordalesa foi o tratamento mais efetivo no controle da ferrugem, promovendo aumento da produtividade e da qualidade dos frutos. A deltametrina promoveu o melhor controle da broca-dos-ramos da figueira. Entre os produtos alternativos testados, o alho foi o mais efetivo no controle dessa praga.

  19. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick


    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent;


    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation......-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor....

  1. Dificuldades da aprendizagem na leitura

    Solange Oliveira Nascimento


    Full Text Available Este presente artigo propôs verificar os fatores que influenciam no desenvolvimento da aprendizagem na leitura dos alunos da 4º ano no Ensino Fundamental na Escola Municipal Armando Dias no município de Sinop/MT, cujo tema é dificuldade da aprendizagem na leitura. O objetivo do trabalho foi investigar as relações interpessoais em sala de aula e de que forma a professora introduz a leitura nos conteúdos dados aos alunos. Como fundamentação teórica, recorremos a vários autores, entre eles: Enrique Cártula Fita, Elisabete da Assunção Jóse, Jésus Alonso Tapia, Maria Tereza Coelho e Ruth Caribe da Rocha Drouet. A metodologia usada consistiu na pesquisa qualitativa, no estudo de caso e questionário com perguntas abertas e fechadas aplicadas à professora e três alunos que estão com dificuldades na leitura. Através dos relatos dos sujeitos realizamos um estudo analítico confrontando os dados da pesquisa com os autores principais que fundamentam a mesma. O presente trabalho pretende contribuir e mostrar ao professor em sala de aula a importância de incentivar os alunos a aprenderem a ler com prazer motivando-os a despertar o interesse e gosto pela leitura. Os resultados da investigação revelam que a professora pesquisada está ciente de seu papel e procura meios para enfrentar os problemas existentes em sala de aula, em especial às dificuldades da aprendizagem na leitura e os alunos percebem a importância da leitura e tem vontade em avançar nesse aprendizado.Palavras-chave: educação; dificuldades na aprendizagem. professor; aluno; sala de aula.


    Mayra Ferreira


    Full Text Available O presente trabalho é fruto de uma pesquisa em desenvolvimento em parceria com a TV Unesp, emissora universitária vinculada à Universidade Estadual Paulista Júlio de Mesquita, campus de Bauru/SP e que tem por objetivo identificar os pontos convergentes e divergentes na produção de conteúdo audiovisual informativo para a TV aberta e demais plataformas digitais e móveis. O texto em questão é a conclusão da primeira etapa da pesquisa que se pautou por identificar os pressupostos básicos que deverão nortear os conteúdos que serão produzidos posteriormente e veiculados pela emissora no decorrer da pesquisa. Foram analisadas as características do jornalismo audiovisual online e o modelo de negócios da TV aberta em contraste com o modelo de negócios presente na Internet.

  3. Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

    Glahder, Jacob-Andreas Harald; Norrild, Bodil


    Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR......-based poly(A) test and in vitro poly(A) assay. Taken together, our results suggest that hCPEB1 and hGLD-2 are antagonizing factors regulating p53 mRNA stability....

  4. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))


    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  5. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing


    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  6. c-myc mRNA in cytoskeletal-bound polysomes in fibroblasts.

    Hesketh, J E; Campbell, G P; Whitelaw, P F


    3T3 fibroblasts were treated sequentially with 25 mM-KCl/0.05% Nonidet P40, 130 mM-KCl/0.05% Nonidet P40 and finally with 1% Nonidet P40/1% deoxycholate in order to release free, cytoskeletal-bound and membrane-bound polysomes respectively. The membrane-bound fraction was enriched in the mRNA for the membrane protein beta 2-microglobulin, whereas the cytoskeletal-bound polysomes were enriched in c-myc mRNA. Actin mRNA was present in both free and cytoskeletal-bound polysomes. The results suggest that cytoskeletal-bound polysomes are involved in the translation of specific mRNA species.

  7. Lipoprotein Lipase mRNA expression in different tissues of farm ...

    Lipoprotein Lipase mRNA expression in different tissues of farm animals. ... Lipoprotein lipase (LPL) controls triacylglycerol partitioning between adipose tissues and muscles, so it is important enzyme for ... Article Metrics. Metrics Loading .

  8. Control of mRNA stability during development of Dictyostelium discoideum.

    Mangiarotti, G


    A large group of mRNA species (which are mainly pre-spore specific) accumulate only after the formation of multicellular aggregates. They are transcribed at a constant rate from the beginning of development and their accumulation is controlled by a 10-20-fold increase in their stability. This mRNA stabilization is dependent upon multicellularity. When aggregates are dispersed, the mRNAs are destabilized; if cells are allowed to reaggregate, the destabilization is reversed. Destabilization is not due to a selective exclusion of mRNA from polyribosomes, but is a primary control event. It does not require synthesis of new RNA or protein, but it may require an interaction between ribosome and the 5'-end of mRNA molecules.

  9. The potential lipolysis function of musclin and its mRNA expression ...



    Nov 16, 2009 ... Musclin mRNA levels in adipose, muscle tissues and primary adipocytes were examined by quantitative PCR. The ... reduced fat mass in mice. Here, we ..... increased, suggesting that musclin might be involved in adipocyte ...

  10. Mapping interactions between mRNA export factors in living cells.

    I-Fang Teng

    Full Text Available The TREX complex couples nuclear mRNA processing events with subsequent export to the cytoplasm. TREX also acts as a binding platform for the mRNA export receptor Nxf1. The sites of mRNA transcription and processing within the nucleus have been studied extensively. However, little is known about where TREX assembly takes place and where Nxf1 is recruited to TREX to form the export competent mRNP. Here we have used sensitized emission Förster resonance energy transfer (FRET and fluorescence lifetime imaging (FLIM-FRET, to produce a spatial map in living cells of the sites for the interaction of two TREX subunits, Alyref and Chtop, with Nxf1. Prominent assembly sites for export factors are found in the vicinity of nuclear speckles in regions known to be involved in transcription, splicing and exon junction complex formation highlighting the close coupling of mRNA export with mRNP biogenesis.

  11. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Beatriz M. A. Fontoura


    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  12. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P


    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol.

  13. Dificuldades da aprendizagem na leitura

    Nascimento, Solange Oliveira; Trugillo, Edneuza Alves


    Este presente artigo propôs verificar os fatores que influenciam no desenvolvimento da aprendizagem na leitura dos alunos da 4º ano no Ensino Fundamental na Escola Municipal Armando Dias no município de Sinop/MT, cujo tema é dificuldade da aprendizagem na leitura. O objetivo do trabalho foi investigar as relações interpessoais em sala de aula e de que forma a professora introduz a leitura nos conteúdos dados aos alunos. Como fundamentação teórica, recorremos a vários autores, entre eles: Enri...

  14. Controlo da Qualidade em Hemoglobinopatias

    Miranda, Armandina


    Experiência do Controlo da qualidade em Hemoglobinopatias, pelo Grupo de Trabalho de Hematologia do Programa Nacional de Avaliação Externa da Qualidade (PNAEQ), INSA. As Hemoglobinopatias são doenças monogénicas hereditárias de transmissão autossómica recessiva resultantes de mutações que afetam os genes responsáveis pela síntese das cadeias de globina da hemoglobina, ou as suas regiões regulatórias.




    Full Text Available

    Estudou-se a pasteurização da polpa de graviola (Annona muricata L., visando estabelecer condições simples e eficientes para sua conservação. Foram realizadas avaliações microbiológica, enzimática e de cor da polpa, para identificar parâmetros a serem utilizados no acompanhamento da pasteurização. O produto foi submetido a tratamento térmico, em copos de inox, em diferentes tempos (1 a 300 seg e temperaturas (70°C a 90°C, para identificar o efeito destas variáveis sobre parâmetros de qualidade do produto. Constatou-se que os tratamentos não provocaram variação na atividade da peroxidase, contagem de bactérias, bolores e leveduras e cor da polpa. Em seguida foram realizadas pasteurizações, em escala piloto, a 80°C e 90°C; sendo os produtos estocados a 25°C e avaliados durante 120 dias. A redução da atividade da peroxidase e da concentração de bactérias, bolores e leveduras, a níveis aceitáveis, e a manutenção da cor, demonstraram a eficiência dos processos térmicos. A manutenção destes parâmetros, nos níveis aceitáveis, durante a estocagem, confirmou a aplicabilidade dos processos, sem a necessidade da adição de conservante na polpa. A pasteurização da polpa de graviola a 80°C ou 90°C, mostrou ser um tratamento térmico adequado para a conservação do produto, proporcionando 120 dias de vidade- prateleira à temperatura ambiente ( 25°C.

  16. Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

    Bire, Solenne; Gosset, David; Jégot, Gwenhael; Midoux, Patrick; Pichon, Chantal; Rouleux-Bonnin, Florence


    Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration

  17. TGF beta 2 mRNA expression and pregnancy failure in mice.

    Gorivodsky, M; Torchinsky, A; Zemliak, I; Savion, S; Fein, A; Toder, V


    We describe here a pattern of transforming growth factor (TGF) beta2 mRNA expression at the fetomaternal interface in mice with high rate of resorptions as well as its expression following maternal immunopotentiation. TGF beta 2 mRNA expression was evaluated in the uteroplacental units of mice with spontaneous (CBA/J x DBA/2J mouse combination) or cyclophosphamide (CP)-induced pregnancy loss. The effect of immunopotentiation on TGF beta 2 mRNA expression was determined in CP-treated females who underwent nonspecific immunostimulation with xenogeneic (rat) leukocytes. A quantitative analysis of TGF beta 2 mRNA level was performed using RNase protection assay. Distribution of TGF beta 2 mRNA transcripts at the fetomaternal interface was studied by in situ hybridization analysis. RNase protection analysis revealed four TGF beta 2 specific mRNA forms (330, 270, 230, and 170 bp) in the uteroplacental units of mice with either normal or decreased reproductive performance. A significant decrease (about 50%) in the level of TGF beta 2 mRNA was registered in the uteroplacental unit of mice with pregnancy loss as compared to the control mice. TGF beta 2 transcripts were abundant in the uterine epithelium and stroma. A specific hybridization signal was detected also in metrial gland cells and it was found to be substantially lower in CP-treated as compared to intact mice. In the resorbing uteroplacental unit, the expression of TGF beta 2 mRNA was completely lost in the uterine epithelium, and the number of TGF beta 2 mRNA-positive metrial gland cells was lower as compared to the control. Immunopotentiation decreased the resorption rate in mice with CP-induced pregnancy loss and caused a dramatic increase in TGF beta 2 mRNA expression: the level of TGF beta 2 mRNA was found to be higher by 2.0-3.2 fold in the uteroplacental unit of immunized as compared to nonimmunized CP-treated mice. These data suggest that distortion of TGF beta 2 expression at the fetomaternal interface

  18. OX40 mRNA in peripheral blood as a biomarker of acute renal allograft rejection

    WANG Yu-liang; FU Ying-xin; ZHU Zhi-jun; WANG Hui; SHEN Zhong-yang


    Background Acute rejection remains an important cause of renal allograft dysfunction and the need for accurate diagnosis is essential to successfully treat transplant recipients.The purpose of this study was to determine the costimulatory molecules OX40 and OX40L messenger RNA (mRNA) levels in peripheral blood mononuclear cells (PBMCs) to predict acute renal transplant rejection.Methods The whole blood samples from 20 recipients with biopsy-confirmed acute rejection (rejection group),20 recipients with stable graft function and normal biopsy results (stable group) after kidney transplantation,and 20 healthy volunteers (control group) were collected.The mRNA levels of OX40 and OX40L were analyzed with TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR).The association of OX40 and OX40L mRNA levels with disease severity was investigated.Results There was no significant difference of OX40,OX40L mRNA levels in PBMCs between the stable group and control group (P>0.05).The levels of OX40 and OX40L mRNA were significantly higher in the rejection group than in the control group (P<0.01 and P<0.05,respectively).Non-significantly higher OX40L mRNA and significantly higher OX40 mRNA in PBMCs were observed in subjects in the rejection group compared with the stable group (P >0.05 and P <0.01,respectively).Receiver operating characteristic (ROC) curve analysis demonstrated that OX40 mRNA levels could discriminate recipients who subsequently suffered acute allograft rejection (area under the curve,0.908).OX40 and OX40L mRNA levels did not significantly correlate with serum creatinine levels in the rejection group (P >0.05).Levels of OX40 mRNA after anti-rejection therapy were lower than those at the time of protocol biopsy in the rejection group (P<0.05).Conclusion Our data suggest that measurement of OX40 mRNA levels after transplant might offer a noninvasive means for recognizing recipients at risk of acute renal allograft rejection.

  19. Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA.

    Sharon, Ronit; Abramovitz, Rene; Miskin, Ruth


    Plasminogen (Plg), which can be converted to the active protease plasmin by plasminogen activators, has been previously implicated in brain plasticity and in toxicity inflicted in hippocampal pyramidal neurons by kainate. Here we have localized Plg. mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate injection (i.p.). The results indicated that Plg mRNA was undetectable in the normal brain, but after kainate injection it was induced in neuronal cells in multiple, but specific areas, including layers II-III of the neocortex; the olfactory bulb, anterior olfactory nucleus, and the piriform cortex; the caudate/putamen and accumbens nucleus shell; throughout the amygdaloid complex; and in the CAI/CA3 subfields of the hippocampus. Interestingly, this distribution pattern coincided with what we have recently described for the plasminogen activator inhibitor-2 (PAI-2) mRNA, however differing from that of the plasminogen activator inhibitor-1 (PAI-1) mRNA, as also shown here. These results suggest that enhanced Plg gene expression could be involved in events associated with olfactory, striatal, and limbic structures. Furthermore, because PAI-2 is thought to intracellularly counteract cytotoxic events, our results raise the possibility that PAI-2 can act in the brain as an intracellular neuroprotector against potential plasmin-mediated toxicity.


    Péricles Pereira de Sousa


    Full Text Available O texto tem o objetivo de apresentar como o filósofo francês Gilles Deleuze, a partir da obra Nietzsche e a Filosofia, interpreta a Genealogia da Moral, obra escrita pelo filósofo alemão Friedrich Nietzsche, levando em consideração as formas mais gerais do niilismo: ressentimento, má consciência e ideal ascético.

  1. Involvement of the p53 and HPV-16 early 3'UTRs in mRNA regulation

    Glahder, Jacob-Andreas Harald

    tilstede. hCPEB1 nedsætter i øvrigt marginelt halveringtiden på mRNA. hGLD-2 kan delvis fjerne den hCPEB1 inducerede repression. Andre proteiner regulerer også p53 mRNA stabiliteten så som Wig-1, der ogå induceres af p53, hvilket medfører en positiv feed-back loop i p53 reguleringen. Som konklusion lader...

  2. Methylation Inactivation Mechanism of Parkin Gene mRNA Expression in Nasopharyngeal Carcinoma

    Ni Haifeng; Jiang Bo; Zhou Zhen; Li Yong; Huang Guangwu


    Objective:To investigate the methylation inactivation and the clinical signiifcance of Parkin gene mRNA expression in nasopharyngeal carcinoma (NPC). Methods: The methylation-speciifc PCR (MSP) and semi-quantitative reverse transcription PCR (RT-PCR) were used to detect methylation and the mRNA expression level of Parkin gene in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues.The mRNA expression of Parkin gene in two NPC cell lines (CNE1 and CNE2) were detected before and after treatment with the methyltransferase inhibitor 5-aza-2-deoxycytidine so as to analyze the effects of Parkin gene methylation and demethylation on Parkin gene mRNA expression and the relationship between Parkin gene mRNA expression and clinical factors. Results: The methylation frequency of Parkin gene in human NPC tissues was 62.96% (34/54), but didn’t happen in any of 16 cases of NNE tissues. The mRNA expression level was (0.3430±0.4947) in 54 cases of NPC tissues and (1.0052±0.4911) in NNE tissues, showing that the mRNA expression level of NPC tissues was significantly down-regulated (P0.05), but was closely related to lymph node metastasis (P<0.05). Conclusion:Parkin gene mRNA expression, serving as a cancer suppressor gene in the occurrence and development of NPC, is inactivated and regulated by methylation, which also has a negative correlation with lymph node metastasis and could be considered as the judgment of predictive index of clinical prognosis of NPC.

  3. Rapid changes in ovarian mRNA induced by brief photostimulation in Siberian hamsters (Phodopus sungorus).

    Shahed, Asha; McMichael, Carling F; Young, Kelly A


    This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2 (PT day-2), 4 (PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone β-subunit (FSHβ-subunit), luteinizing hormone β-subunit (LHβ-subunit), FSH and LH receptors, estrogen receptors α and β (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Müllerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHβ- and LHβ-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation.

  4. mRNA levels of TLR4 and TLR5 are independent of H pylori

    Elvira Garza-González; Virgilio Bocanegra-García; Francisco Javier Bosques-Padilla; Juan Pablo Flores-Gutiérrez; Francisco Moreno; Guillermo Ignacio Perez-Perez


    AIM:To determine if the presence Hpylori or its virulence affect toll-like receptor 4 (TLR4) and TLR5 mRNA expression levels.METHODS:For the in vivo assays,gastric biopsies were obtained from 40 patients and H pylori status was determined.For the in vitro assays,human gastric adenocarcinoma mucosal cells (AGS) were cultured in the presence or absence of twelve selected H pylori strains.H pylori strains isolated from culture-positive patients and selected strains were genotyped for cagA and vacA.The cDNA was obtained from mRNA extracted from biopsies and from infected AGS cells.TLR4 and TLR5 mRNA levels were examined by real-time PCR.RESULTS:The presence of Hpylori did not affect the mRNA levels of TLR4 or TLR5 in gastric biopsies.The mRNA levels of both receptors were not influenced by the vacA status (P>0.05 for both receptors) and there were no differences in TLR4 or TLR5 mRNA levels among the different clinical presentations/histological findings (P>0.05).In the in vitro assay,the mRNA levels of TLR4 or TLR5 in AGS cells were not influenced by the vacAsl status or the clinical condition associated with the strains (P>0.05 for both TLR4 and TLR5).CONCLUSION:The results of this study show that the mRNA levels of TLR4 and TLR5 in gastric cells,both in vivo and in vitro,are independent of H pylori colonization and suggest that vacA may not be a significant player in the first step of innate immune recognition mediated by TLR4 or TLR5.

  5. Changes in mRNA for metabotropic glutamate receptors after transient cerebral ischaemia

    Rosdahl, D; Seitzberg, D A; Christensen, Thomas;


    Using a rat 4-vessel occlusion model of cerebral ischaemia we studied the changes in the mRNA level for the metabotropic receptor subtypes mGluR1 alpha, mGluR1 beta, mGluR2, mGluR3, mGluR4, and mGluR5 by means of in situ hybridization with oligonucleotides. After 24 hours of reperfusion the mRNA ...

  6. Expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patient

    Li Zhang; Zhen Yang; Bao-Min Shi; Da-Peng Li; Chong-Yun Fang; Fa-Zu Qiu


    AIM: To investigate the expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patients.METHODS: The expression of local renin and angiotensinogen mRNA in the liver, splenic artery and vein of PH patients was detected by RT-PCR analysis.RESULTS: Expression of local renin mRNA in the liver of control group was (0.19±0.11), significantly lower than that in splenic artery(0.45±0.17)or splenic vein(0.39±0.12)respectively, (P<0.05). Expression of local angiotensinogen mRNA in the liver was (0.64±0.21), significantly higher than that in splenic artery(0.41±0.15) or in splenic vein (0.35±0.18)respectively, (P<0.05). Expression of local renin mRNA in the liver, splenic artery and vein of PH group was (0.78±0.28),(0.86±0.35) and (0.81±0.22) respectively, significantly higher than that in the control group, (P<0.05). Expression of local angiotensinogen mRNA in the liver, splenic artery and vein of PH group was (0.96±0.25), (0.83±0.18) and (0.79±0.23)respectively, significantly higher than that in the control group,(P<0.05). There was no significant difference between the liver, splenic artery and vein in the expression of local renin or local angiotensinogen mRNA in PH group, (P<0.05).CONCLUSION: In normal subjects the expression of local renin and angiotensinogen mRNA was organ specific, but with increase of the expression of LRAS, the organ-specificity became lost in cirrhotic patients. LRAS may contribute to increased resistance of portal vein with liver and formation of splanchnic vasculopathy.

  7. Maternally inherited npm2 mRNA is crucial for egg developmental competence in zebrafish.

    Bouleau, Aurélien; Desvignes, Thomas; Traverso, Juan Martin; Nguyen, Thaovi; Chesnel, Franck; Fauvel, Christian; Bobe, Julien


    The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates.

  8. O sentido da espiritualidade na transitoriedade da vida

    Isabel Cristina de Oliveira Arrieira

    Full Text Available Resumo Objetivo: Compreender o sentido da espiritualidade para a pessoa em cuidados paliativos. Métodos: Abordagem qualitativa, fundamentada na fenomenologia existencialista e referencial de Viktor Frankl. Os participantes foram nove pessoas em cuidados paliativos atendidas no domicílio. Informações coletadas por meio da observação e de entrevista realizadas no período de junho a outubro de 2014, gravadas, transcritas e transformadas em texto interpretado com a abordagem fenomenológica hermenêutica. Resultados: Surgiram as seguintes categorias: sentido de continuidade da vida; sentido de alívio do sofrimento; sentido de naturalidade da morte e sentido de valorização do viver. Conclusão: Para a integralidade da atenção faz-se necessário a inclusão da espiritualidade na prática do cuidado em saúde. A espiritualidade proporciona o encontro existencial entre a pessoa em cuidados paliativos e os profissionais que a cuidam em sua integralidade.


    Rafael Koehler; Rosana Jardim Candeloro


    Full Text Available O objetivo deste artigo consiste em analisar como Nietzsche, baseado na filosofia schopenhauriana, resolveu o problema do surgimento da tragédia grega através da elevação dos elementos Apolo e Dionísio a um estatuto ontológico, e como os gregos fizeram isso em seu tempo através da Música, além de revelar como, em Nietzsche, o pensamento de Sócrates surge como o “perverso algoz da arte”.O estudo foi realizado através de uma pesquisa bibliográfica com base nas seguintes obras do filósofo: A visão Dionisíaca de Mundo (uma compilação de duas conferências e um texto não publicado e O nascimento da tragédia no espírito da Música. Na segunda parte, sugere-se como Nietzsche faz esse resgate do trágico em sua época e qual a recepção no século XIX de seu trabalho.

  10. O uso da aromaterapia na melhora da autoestima

    Juliana Rizzo Gnatta


    Full Text Available Os objetivos deste estudo foram verificar se a inalação dos óleos essenciais de rosa e de ylang-ylang alteram a percepção da autoestima e comparar a eficácia dos mesmos. O projeto foi aprovado pelo Comitê de Ética em Pesquisa do Hospital da Universidade de São Paulo. Participaram 43 funcionários dos setores de Higienização e da Central de Materiais e Esterilização durante os meses de maio a julho de 2009. Os sujeitos foram randomizados em três grupos: dois que receberem os óleos essenciais e outro que recebeu placebo (essência de rosa. A avaliação da autoestima foi feita através de uma escala já validada no Brasil, sendo aplicada antes do uso dos aromas, depois de 30 dias de uso e ao completar 60 dias. Dentre os resultados, verificou-se que a amostra era constituída por 88,6% de indivíduos com média e alta autoestima e que os óleos essenciais em questão não alteraram de forma significativa a percepção da autoestima.


    Alfram Roberto Rodrigues de Albuquerque


    Full Text Available Expõe-se o estado atual da disciplina Arquitetura da Informação quanto a problemática terminológica da definição do campo de estudo, apresentando-se argumentos para uma definição de Arquitetura da Informação de uma forma ampla na qual os usos correntes dessa expressão sejam vistos como especializações. Defende-se uma definição com a utilização de um conjunto de propriedades mínimas cujas interações levam a uma terminologia formal e cuja abstração leva a uma definição formal do conceito que emprega a linguagem de morfismos da Teoria das Categorias. Trata-se de um recorte de alguns dos resultados obtidos na tese de doutoramento de um dos autores, defendida em setembro de 2010. O contexto da pesquisa é exposto e alguns dos resultados obtidos são delineados.

  12. Carboxylesterase 1 gene duplication and mRNA expression in adipose tissue are linked to obesity and metabolic function

    Friedrichsen, Martin; Poulsen, Pernille; Wojtaszewski, Jørgen


    involved in the control of mRNA expression. Here, we investigated mRNA expression level in adipose tissue and its association with measures of adiposity and metabolic function in a population of elderly twins. Furthermore, the heritability of mRNA expression level in adipose tissue and the effect of gene...

  13. Chamber-dependent expression of brain natriuretic peptide and its mRNA in normal and diabetic pig heart

    Christoffersen, Christina; Goetze, Jens P; Bartels, Emil D;


    -induced diabetes doubled the expression of BNP mRNA in porcine atrial myocardium (P=0.03), but was without effect on BNP mRNA in the ventricular myocardium. The data suggest that BNP mRNA processing and proBNP storage differ between the atrial and ventricular myocardium. The results also imply that diabetes...

  14. Prognostic relevance of circulating CK19 mRNA in advanced malignant biliary tract diseases

    Kawin Leelawat; Siriluck Narong; Wandee Udomchaiprasertkul; Jerasak Wannaprasert; Sa-ard Treepongkaruna; Somboon Subwongcharoen; Tawee Ratanashu-ek


    AIM: To determine the role of circulating tumor cells (CTCs) in prediction of the overall survival of patients with advanced malignant biliary tract obstruction. METHODS: We investigated the prognostic value of CTCs by examining two markers, cytokeratin (CK) 19 and human telomerase reverse transcriptase (hTERT) mRNA, in 40 patients diagnosed with advanced malignant biliary tract diseases. Quantitative real-time reverse transcription polymerase chain reaction was used to detect CK19 and hTERT mRNA in the peripheral blood of these patients. Overall survival was analyzed using the Kaplan-Meier method and Cox regression modeling. RESULTS: Positive CK19 and hTERT mRNA expression was detected in 45% and 60%, respectively, of the 40 patients. Univariable analysis indicated that positive CK19 mRNA expression was significantly associated with worse overall survival (P = 0.009). Multivariable analysis determined that positive CK19 mRNA expression, patient's age and serum bilirubin were each independently associated with overall survival. CONCLUSION: CK19 mRNA expression levels in peripheral blood appear to provide a valuable marker to predict the overall survival of patients with advanced malignant biliary tract obstruction.

  15. Foxg1 mRNA overexpression in neonatal rats following hypoxic brain injury

    Luquan Li; Yi Zheng; Guoliang Mo; Fang Li; Jialin Yu


    Forkhead box G1 (Foxg1) is expressed during the embryonic stage and in postnatal brain regions sensitive to hypoxia/ischemia injury,such as the hippocampus and cerebral cortex.To date,very little is known about Foxg 1 expression changes in the brain following hypoxia injury (HI).The present study measured Foxg 1 mRNA expression using reverse-transcription polymerase chain reaction on days 3,7,14,28,and 56 following HI to determine self-restorative features in the injured brain.In addition,mRNA expression of other related layer markers,such as Reelin,RORB,Foxp1,Foxp2,ER81,and Otx-1,was detected following HI.Results revealed significantly decreased Foxg1 mRNA expression at 3 days after HI,which significantly increased by 56 days.Reelin and Foxp2 mRNA expression were upregulated until 56 days after HI,but Foxp1 and ER81 mRNA expression decreased from day 14 to 56 following HI.In addition,Otx-1 and RORB mRNA expression decreased from day 3 to 28 after HI.These findings revealed Fxog1 mRNA overexpression and varying degrees of restoration in the neonatal rat brain following HI.

  16. mRNA transfection of mouse and human neural stem cell cultures.

    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  17. Drosophila glutamate receptor mRNA expression and mRNP particles.

    Ganesan, Subhashree; Karr, Julie E; Featherstone, David E


    The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.

  18. [Interrelationship between protein synthesis and mRNA metabolism in rat liver cells].

    Arbuzov, V A


    It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.

  19. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

    Steven J. Schnell


    Full Text Available The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE. Plenty of nuclear pore complexes (NPCs embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

  20. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    林茂芳; 魏国庆; 黄河; 蔡真


    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  1. RNase III controls the degradation of corA mRNA in Escherichia coli.

    Lim, Boram; Sim, Se-Hoon; Sim, Minji; Kim, Kyungsub; Jeon, Che Ok; Lee, Younghoon; Ha, Nam-Chul; Lee, Kangseok


    In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.

  2. Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA.

    Thomas J Stoddard

    Full Text Available Plant genome engineering using sequence-specific nucleases (SSNs promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA encoding TAL effector nucleases (TALENs. mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were 10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5' and 3' untranslated regions (UTRs from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740 enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing.

  3. HDAC3 regulates stability of estrogen receptor α mRNA

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Murayama, Akiko; Yanagisawa, Junn, E-mail:


    Highlights: ► HDAC inhibitors decrease the stability of ERα mRNA in MCF-7 cells. ► HDAC3 is involved in maintaining ERα mRNA stability in MCF-7 cells. ► ERα mRNA instability by knockdown of HDAC3 reduces the estrogen-dependent proliferation of ERα-positive MCF-7 cells. ► HDAC3 specific inhibitor will be one of new drugs for ERα-positive breast cancers. -- Abstract: Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.

  4. Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

    Giraldo-Vela, Juan P; Kang, Wonmo; McNaughton, Rebecca L; Zhang, Xuemei; Wile, Brian M; Tsourkas, Andrew; Bao, Gang; Espinosa, Horacio D


    New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

  5. A utilização da teoria da aprendizagem significativa no ensino da Enfermagem

    Alana Tamar Oliveira de Sousa


    Full Text Available RESUMOObjetivo:sintetizar a produção científica acerca da Teoria da Aprendizagem Significativa no processo de ensino-aprendizagem em Enfermagem.Método:revisão integrativa realizada nas bases de dados MEDLINE, LILACS, SciELO, BDENF e CINAHL, com artigos que abordaram a temática ou aspectos da teoria da aprendizagem significativa de David Ausubel. Fizeram parte da amostra dez artigos, sendo seis escritos no idioma português e quatro no inglês, publicados de 1998 a 2013.Resultados:cinco artigos fizeram alusão à Teoria da Aprendizagem Significativa, quatro citaram Ausubel como autor dessa teoria e apenas dois seguiram a estrutura para a aplicação da teoria completamente. Há escassez de estudos sobre o tema e os que o abordam não o correlacionam com a teoria de Ausubel.Conclusão:faz-se necessário romper com a dicotomia entre teoria e prática e promover a articulação dos conteúdos com a ação, introduzindo o aluno como autor de seu próprio conhecimento.


    Michelle de Freitas Bissoli


    Full Text Available Este trabalho, de cunho teórico-bibliográfico, tem por objetivo trazer à discussão a relação que se estabelece entre a prática pedagógica desenvolvida na Educação Infantil e a formação da personalidade da criança. Tem por fundamentos os pressupostos da Teoria Histórico-Cultural e busca responder, fundamentalmente, às seguintes questões: O que é a personalidade?; Quais são as forças motrizes do seu processo de desenvolvimento nos primeiros anos de vida da criança?; Quais as especificidades do trabalho dos professores da Educação Infantil e como a atividade pedagógica pode contribuir para o desenvolvimento da personalidade infantil? Para tanto, pretendemos refletir sobre alguns princípios essenciais de um trabalho docente capaz de impulsionar o processo formativo integral da criança na Educação Infantil. Consideramos que, com a consolidação de práticas pedagógicas que medeiem as relações entre as crianças e a cultura, é possível organizar atividades que focalizem as diferentes dimensões do desenvolvimento humano e a personalidade.

  7. Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

    Soetanto, R; Hynes, C J; Patel, H R; Humphreys, D T; Evers, M; Duan, G; Parker, B J; Archer, S K; Clancy, J L; Graham, R M; Beilharz, T H; Smith, N J; Preiss, T


    miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Atlas nacional da Espanha, demografia

    Hervé Théry


    Full Text Available Calvo Palacios J.L., Pueyo Campos Á. (2008, Atlas nacional de España, Demografía. Madrid: Centro Nacional de Información Geográfica, 386 p. ISBN: 978-8-4416-0685-2.O volume, que faz parte da série Monografias do Atlas Nacional de Espanha, inclui 420 mapas, 91 dos quais apresentam informações por município que dão uma visão global, concisa e muito completa da realidade da população espanhola e da sua evolução (em alguns casos analisada desde 1857. Seus diretores científicos são José Luis Cal...

  9. A subjetividade fora da mente

    Alexandre Simões


    Full Text Available O artigo contextualiza uma tradição que se apresenta de maneira recorrente nas práticas clínicas da psicologia e da saúde mental: o Mentalismo. O Mentalismo pressupõe uma concepção interiorizada do sujeito em prol de modelos instrumentais de clínica. A partir da crítica ao Mentalismo, os autores examinam outras possibilidades de se lidar não mais com o sujeito essencializado, porém, com os complexos processos de subjetivação. Esta é uma perspectiva clínica que recorrendo a Deleuze, Guattari e à Filosofia da Diferença oferece estratégias mais condizentes com a complexidade do mundo contemporâneo, introduzindo debates éticos, políticos e estéticos na dimensão psíquica.

  10. Reduced brain-derived neurotrophic factor (BDNF) mRNA expression and presence of BDNF-immunoreactive granules in the spinocerebellar ataxia type 6 (SCA6) cerebellum.

    Takahashi, Makoto; Ishikawa, Kinya; Sato, Nozomu; Obayashi, Masato; Niimi, Yusuke; Ishiguro, Taro; Yamada, Mitsunori; Toyoshima, Yasuko; Takahashi, Hitoshi; Kato, Takeo; Takao, Masaki; Murayama, Shigeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro


    Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder caused by a small expansion of tri-nucleotide (CAG) repeat encoding polyglutamine (polyQ) in the gene for α(1A) voltage-dependent calcium channel (Ca(v) 2.1). Thus, this disease is one of the nine neurodegenerative disorders called polyQ diseases. The Purkinje cell predominant neuronal loss is the characteristic neuropathology of SCA6, and a 75-kDa carboxy-terminal fragment (CTF) of Ca(v) 2.1 containing polyQ, which remains soluble in normal brains, becomes insoluble in the cytoplasm of SCA6 Purkinje cells. Because the suppression of the brain-derived neurotrophic factor (BDNF) expression is a potentially momentous phenomenon in many other polyQ diseases, we implemented BDNF expression analysis in SCA6 human cerebellum using quantitative RT-PCR for the BDNF mRNA, and by immunohistochemistry for the BDNF protein. We observed significantly reduced BDNF mRNA levels in SCA6 cerebellum (n = 3) compared to controls (n = 6) (Mann-Whitney U-test, P = 0.0201). On immunohistochemistry, BDNF protein was only weakly stained in control cerebellum. On the other hand, we found numerous BDNF-immunoreactive granules in dendrites of SCA6 Purkinje cells. We did not observe similar BDNF-immunoreactive granules in other polyQ diseases, such as Huntington's disease or SCA2. As we often observed that the 1C2-positive Ca(v) 2.1 aggregates existed more proximally than the BDNF-positive granules in the dendrites, we speculated that the BDNF protein trafficking in dendrites may be disturbed by Ca(v) 2.1 aggregates in SCA6 Purkinje cells. We conclude that the SCA6 pathogenic mechanism associates with the BDNF mRNA expression reduction and abnormal localization of BDNF protein.

  11. Effects of chronic fluoride exposure on object recognition memory and mRNA expression of SNARE complex in hippocampus of male mice.

    Han, Haijun; Du, Wenjuan; Zhou, Bingrui; Zhang, Wen; Xu, Guoli; Niu, Ruiyan; Sun, Zilong


    This study aimed to investigate the effects of long-term fluoride exposure on object recognition memory and mRNA expression of soluble N-ethylmaleimidesensitive fusion protein attachment protein receptors (SNARE) complex (synaptosome-associated protein of 25 kDa (SNAP-25), vesicle-associated membrane protein 2 (VAMP-2), and syntaxin 1A) in the hippocampus of male mice. Sixty sexually matured male Kunming mice were randomly divided into four groups: control group (given distilled water), low F group (25 mg/L NaF, corresponding to 11 mg/L F(-)), medium F group (50 mg/L NaF, corresponding to 22 mg/L F(-)), and high F group (100 mg/L NaF, corresponding to 45 mg/L F(-)). After 180 days, the spontaneous locomotor behavior and object recognition memory were detected by open field test and novel object recognition (NOR) test. Results showed that compared with the control group, frequency in each zone, total distance, and line crosses were significantly increased in low F and medium F groups, suggesting fluoride enhanced excitement of mice, while there were no marked changes in high F group. Twenty-four hours after training, a deficit of long-term memory (LTM) occurred only in high F group (P recognition memory, and upregulate VAMP-2 mRNA expression, which are involved in the adverse effects of fluoride on the object recognition memory of nervous system.

  12. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    Ris-Stalpers, C.; Kuiper, G.G.J.M.; Faber, P.W.; van Rooij, H.C.J.; Degenhart, H.J.; Trapman, J.; Brinkmann, A.O. (Erasmus Univ., Rotterdam (Netherlands)); Schweikert, H.U. (Univ. of Bonn (Germany)); Zegers, N.D. (Medical Biological Laboratory-Organization for Applied Scientific Research, Rijswijk (Netherlands)); Hodgins, M.B. (Glasgow Univ. (United Kingdom))


    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G {r arrow} T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein {approx}5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

  13. Induction of aromatic L-amino acid decarboxylase mRNA by interleukin-1 beta and prostaglandin E2 in PC12 cells.

    Li, X M; Juorio, A V; Boulton, A A


    Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1-beta and prostaglandin (PG) E2 in PC12 cells. The cells were treated with different doses of IL 1-beta and PGE2 for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1-beta and PGE2 produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE2 formation, its effects may be occurring by increasing level of PGE2.

  14. Dioscorea Extract (DA-9801) Modulates Markers of Peripheral Neuropathy in Type 2 Diabetic db/db Mice.

    Moon, Eunjung; Lee, Sung Ok; Kang, Tong Ho; Kim, Hye Ju; Choi, Sang Zin; Son, Mi-Won; Kim, Sun Yeou


    The purpose of this study was to investigate the therapeutic effects of DA-9801, an optimized extract of Dioscorea species, on diabetic peripheral neuropathy in a type 2 diabetic animal model. In this study, db/db mice were treated with DA-9801 (30 and 100 mg/kg, daily, p.o.) for 12 weeks. DA-9801 reduced the blood glucose levels and increased the withdrawal latencies in hot plate tests. Moreover, it prevented nerve damage based on increased nerve conduction velocity and ultrastructural changes. Decrease of nerve growth factor (NGF) may have a detrimental effect on diabetic neuropathy. We previously reported NGF regulatory properties of the Dioscorea genus. In this study, DA-9801 induced NGF production in rat primary astrocytes. In addition, it increased NGF levels in the sciatic nerve and the plasma of type 2 diabetic animals. DA-9801 also increased neurite outgrowth and mRNA expression of Tieg1/Klf10, an NGF target gene, in PC12 cells. These results demonstrated the attenuation of diabetic peripheral neuropathy by oral treatment with DA-9801 via NGF regulation. DA-9801 is currently being evaluated in a phase II clinical study.

  15. Histone gene expression and histone mRNA 3' end structure in Caenorhabditis elegans

    Pettitt Jonathan


    Full Text Available Abstract Background Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development. Results Sequence analysis of replication-dependent histone genes revealed the presence of several highly conserved sequence elements in the 3' untranslated region of histone pre-mRNAs, including an RNA hairpin element and a polyadenylation signal. To determine whether in C. elegans histone mRNA 3' end formation occurs at this polyadenylation signal and results in polyadenylated histone mRNA, we investigated the mRNA 3' end structure of histone mRNA. Using poly(A selection, RNAse protection and sequencing of histone mRNA ends, we determined that a majority of C. elegans histone mRNAs lack a poly(A tail and end three to six nucleotides after the hairpin structure, after an A or a U, and have a 3' OH group. RNAi knock down of CDL-1, the C. elegans HBP/SLBP, does not significantly affect histone mRNA levels but severely depletes histone protein levels. Histone gene expression varies during development and is reduced in L3 animals compared to L1 animals and adults. In adults, histone gene expression is restricted to the germ line, where cell division occurs. Conclusion Our findings indicate that the expression of C. elegans histone genes is subject to control mechanisms similar to the ones in other

  16. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)


    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

  17. A teoria da gastrea de Ernst Haeckel

    Guilherme Francisco Santos


    O objetivo principal de nosso trabalho é descrever e analisar criticamente o núcleo da teoria da gastrea de Ernst Haeckel. Ele gira em torno de duas noções principais: forma gastrular e metazoário. A teoria da gastrea é um conjunto de formulações que visa estabelecer uma definição de metazoário a partir da noção de forma gastrular. O argumento central da teoria da gastrea articula essas duas noções para organizar a partir de estudos de embriologia comparativa uma visão geral da história evolu...

  18. Desafios da leitura e da escrita em meio digital

    Adair de Aguiar Neitzel


    Full Text Available Este artigo trata das estratégias de ensino no campo da leitura literária que os licenciandos de Letras promoveram no decorrer do Programa Docência na Educação Básica/PIBID/UNIVALI. Foram analisadas as concepções que subjazem essas estratégias e como elas colaboram para a formação de leitores no ensino médio. Esta investigação, de caráter qualitativo, utilizou como instrumentos de coleta de dados os portfólios redigidos pelos licenciandos de Letras, bolsistas do PIBID, disponíveis no ambiente virtual da instituição, e entrevistas com os alunos do ensino médio que participaram da pesquisa. Os resultados apontam que, por meio da leitura e da produção de contos multimídias, os alunos foram capazes de fazer relações com outros textos; ampliar o diálogo sobre a obra lida; colocar a pesquisa no centro do processo; fazer uso da autonomia na escolha do material; participar de um processo criativo; trabalhar em equipe; aprender a importância da revisão na produção de textos; negociar saberes. Ao trabalhar-se com a literatura de forma a resguardar sua função estética, os estudantes mudaram sua visão sobre o texto literário, passando a compartilhá-lo com os pares e a reconhecer as literariedades do texto e as sutilezas das construções literárias.

  19. Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex.

    Worch, Remigiusz; Stepinski, Janusz; Niedzwiecka, Anna; Jankowska-Anyszka, Marzena; Mazza, Catherine; Cusack, Stephen; Stolarski, Ryszard; Darzynkiewicz, Edward


    Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.


    Renata Longo


    gerencial (PCDA foi o Action que apresentou maior grau de aderência. Há uma concordância acentuada às características fundamentais do ciclo gerencial na Gestão da Responsabilidade Socioambiental.

  1. A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

    Mandy Muller


    Full Text Available During lytic Kaposi's sarcoma-associated herpesvirus (KSHV infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA. However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6, a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

  2. Maternal mRNA expression levels of H19 are inversely associated with risk of macrosomia.

    Jiang, Hua; Yu, Yang; Xun, Pengcheng; Zhang, Jun; Luo, Guanghua; Wang, Qiuwei


    To investigate the associations between the mRNA levels of H19 in term placenta and risk of macrosomia. Term placentas were collected from 37 macrosomia and 37 matched neonates with normal birth weight (controls) born in Changzhou Women and Children Health Hospital, Jiangsu province, P. R. China from March 1 to June 30, 2008. The mRNA levels of H19 in those placentas were measured by real-time polymerase chain reaction (PCR). Simple and multiple logistic regression models were used to explore the risk factors in the development of macrosomia. All analyses were performed using Stata 10.0 (StataCorp, College Station, Texas, USA). The average H19 mRNA level of the macrosomia group was 1.450 ±0.456 while in the control group it was 2.080 ±1.296. Based on the result of Student's t test, there was a significant difference in H19 mRNA level between the macrosomia group and the control group (p = 0.008). After controlling for potential confounders, the multivariable adjusted odds ratio (OR) of macrosomia for those in the highest tertile of H19 mRNA level was 0.12 (95% CI: 0.02-0.59) when compared to those in the lowest tertile (p for linear trend = 0.009). The term placental H19 mRNA levels were inversely related to the occurrence of macrosomia. Our findings suggest that the low expression of H19 mRNA may contribute to the development of macrosomia.

  3. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    Guidon, P T; Salvatori, R; Bockman, R S


    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  4. The relationship between MRNA levels and the locomotor response to novelty.

    Hooks, M S; Sorg, B A; Kalivas, P W


    Differences in behavioral and neurochemical responses to drugs of abuse and environmental stress have been observed between rats that have a greater locomotor response in a novel environment (high responders: HR) compared to those that have a low response to novelty (low responders: LR). This study examined nuclei associated with the nigrostriatal and mesolimbic systems for differences in mRNA content between HR and LR using Northern blot analysis. These brain regions were chosen because of their role in both drug abuse and stress responses. The mRNAs examined code for either peptide transmitters that interact with the dopaminergic system or components of the dopaminergic system that have not been previously examined for differences between HR and LR. HR rats had approximately 50% lower levels of mRNA for beta-preprotachykinin (PPT) in the core of the nucleus accumbens (NACC) compared to LR. No differences between HR and LR in mRNA levels for dynorphin (DYN), preproenkephalin (PPE), glutamic acid decarboxylase (GAD) or neurotensin (NT) were observed in the core of the NACC. In the shell region of the NACC, HR exhibited a 25% reduction in the level of mRNA for NT compared to LR. No differences between HR and LR in mRNA levels for PPT, DYN, PPE or GAD were observed in the shell of the NACC. In the medial frontal cortex and the dorsal striatum, no differences between HR and LR in mRNA levels for PPT, DYN, PPE, GAD or NT were found. In the substantia nigra and ventral tegmental area no differences between HR and LR in mRNA levels for tyrosine hydroxylase, GAD, cholecystokinin, or NT were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    Kropp, Jenna; Khatib, Hasan


    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  6. Developmental expression of parvalbumin mRNA in the cerebral cortex and hippocampus of the rat.

    de Lecea, L; del Río, J A; Soriano, E


    Parvalbumin (PARV) belongs to the family of calcium-binding proteins bearing the EF hand domain. Immunocytochemical studies in the cerebral cortex have demonstrated that neurons containing PARV include two types of GABAergic interneurons, namely, basket and axo-axonic chandelier cells. The present study examines the onset and pattern of PARV mRNA expression during the development of rat neocortex and hippocampus by means of 'in situ' hybridization with an oligonucleotide probe corresponding to rat PARV cDNA. In animals aged P0-P6 no signal was detected above background in neocortex or hippocampus. At P8, a few cortical cells displayed a number of silver grains just above background levels. By P10 PARV mRNA-expressing cells in the neocortex were detected almost exclusively in layer V of somatosensory, frontal and cingulate cortices. At P12 PARV mRNA was mainly detected in layers IV, V and VIa. By P14 there was a marked overall increase in the entire neocortex, including layer II-III, both in the number of cells and in their intensity of labelling. Further maturation in the pattern of PARV mRNA concentration was observed between P16 and P21. In the hippocampus low hybridization was observed at P10-P12. In subsequent stages both the number of positive cells and the intensity of labelling increased steadily. No clear-cut radial gradients for the expression of PARV mRNA were observed in the hippocampal region. Our results show that the developmental radial gradient followed by PARV mRNA expression in the neocortex does not follow an 'inside-out' gradient, consistent with previous immunocytochemical findings. Taken together, these data indicate that the developmental sequence followed by the PARV protein directly reflects mRNA abundance and suggest that PARV mRNA expression correlates with the functional maturation of cortical interneurons.


    Xin-yun Wang; Xing-ye Wu; Zhi Yao; Yan Li; Ting Liu; Hai-yan Zheng; Cong-zhong Zhu; Cui-yun Sun; Ai-xiang Wang; Min Zhao


    Objective To investigate the expression of Survivin mRNA in lung cancer tissue microarray (TMA) by fluorescence in situ hybridization (FISH) method, and determine the role and significance of it in lung cancer genesis and progress. Methods The expression of Survivin mRNA was detected by FISH method and TMA technology. Fifty-four cases of lung cancer and 10 cases of normal lung tissue were examined. Results Survivin mRNA was expressed in 66.7% (36/54) of lung cancer; the positive ratio of lung cancer was significantly higher than that of normal lung tissue (0/10; x2= 15.238, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in poor differentiated cancer (20/24, 83.3%) than moderate and well differentiated cancer (16/30, 53.3%; x2= 5.40, P <0.05). The positive ratio of Survivin mRNA was significantly higher in group with lymph node metastasis (27/32, 84.4%) than without lymph node metastasis (9/22, 40.9%; x2= 11.084, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in stage Ⅲ-Ⅳ(12/13, 92.3%) than stage Ⅰ - Ⅱ (24/41, 58.5%; x2= 5.066, P < 0.05). Conclusion Survivin mRNA highly expresses in lung cancer, which is related to the progress and malignant behavior. Survivin may play a promoting role in lung cancer genesis and progress and provide a basis for estimating prognosis and treatment.

  8. [The expression of human telomerase reverse transcriptase mRNA and its significance in acute leukemia].

    Meng, Xiao-Li; Lin, Mao-Fang; Jin, Jie


    To investigate the expression of hTERT mRNA in bone marrow mononuclear cells (MNCs) from acute leukemia patients, the method of semi-quntitative RT-PCR was used to examine the expression of hTERT mRNA in marrow MNCs, and the telomerase activity of marrow MNCs was determined with the method of TRAP-PCR-ELISA by using a commercial kit. The results indicated that the expression of hTERT mRNA of marrow MNCs in 30 untreated AL patients was markedly higher than that in 12 CR cases (0.71 +/- 0.34 vs 0.43 +/- 0.25, P < 0.05) and 6 normal volunteers (0.71 +/- 0.34 vs 0.22 +/- 0.21, P < 0.01), respectively. Telomerase activity of marrow MNCs in 30 untreated AL patients was significantly higher than that in 12 CR cases (0.235 +/- 0.395 vs 0.012 +/- 0.015, P = 0.007). Moreover, there was a positive correlation between the hTERT mRNA synthesis and telomerase activity in AL cells (r = 0.421, P < 0.01). The pencentage of blast cells in marrow smear of the untreated AL patients was positively correlated with both the expression of hTERT mRNA and the telomerase activity of bone marrow MNCs (r = 0.457, P < 0.05 and r = 0.411, P < 0.05), respectively. It is concluded that the expression of hTERT mRNA in bone marrow MNCs from untreated AL patients was correlated with their telomerase activity. It is suggested that the expression of hTERT mRNA leukemic cells indicates their higher proliferation ability.

  9. Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system

    DeRosa, F; Guild, B; Karve, S; Smith, L; Love, K; Dorkin, J R; Kauffman, K J; Zhang, J; Yahalom, B; Anderson, D G; Heartlein, M W


    DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA. Nanoparticles formulated with lipids and lipid-like materials were developed for delivery of two separate mRNA transcripts encoding either human erythropoietin (hEPO) or factor IX (hFIX) protein. Dose-dependent protein production was observed for each mRNA construct. Upon delivery of hEPO mRNA in mice, serum EPO protein levels reached several orders of magnitude (>125 000-fold) over normal physiological values. Further, an increase in hematocrit (Hct) was established, demonstrating that the exogenous mRNA-derived protein maintained normal activity. The capacity of producing EPO in non-human primates via delivery of formulated mRNA was also demonstrated as elevated EPO protein levels were observed over a 72-h time course. Exemplifying the possible broad utility of mRNA drugs, therapeutically relevant amounts of human FIX (hFIX) protein were achieved upon a single intravenous dose of hFIX mRNA-loaded lipid nanoparticles in mice. In addition, therapeutic value was established within a hemophilia B (FIX knockout (KO)) mouse model by demonstrating a marked reduction in Hct loss following injury (incision) to FIX KO mice. PMID:27356951

  10. Analysis of novel NEFL mRNA targeting microRNAs in amyotrophic lateral sclerosis.

    Muhammad Ishtiaq

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal disease characterized by progressive motor neuron degeneration and neurofilament aggregate formation. Spinal motor neurons in ALS also show a selective suppression in the levels of low molecular weight neurofilament (NEFL mRNA. We have been interested in investigating the role of microRNAs (miRNAs in NEFL transcript stability. MiRNAs are small, 20-25 nucleotide, non-coding RNAs that act as post-transcriptional gene regulators by targeting the 3' untranslated region (3'UTR of mRNA resulting in mRNA decay or translational silencing. In this study, we characterized putative novel miRNAs from a small RNA library derived from control and sporadic ALS (sALS spinal cords. We detected 80 putative novel miRNAs, 24 of which have miRNA response elements (MREs within the NEFL mRNA 3'UTR. From this group, we determined by real-time PCR that 10 miRNAs were differentially expressed in sALS compared to controls. Functional analysis by reporter gene assay and relative quantitative RT-PCR showed that two novel miRNAs, miR-b1336 and miR-b2403, were downregulated in ALS spinal cord and that both stabilize NEFL mRNA. We confirmed the direct effect of these latter miRNAs using anit-miR-b1336 and anti-miR-b2403. These results demonstrate that the expression of two miRNAs (miRNAs miR-b1336 and miR-b2403 whose effect is to stabilize NEFL mRNA are down regulated in ALS, the net effect of which is predicted to contribute directly to the loss of NEFL steady state mRNA which is pathognomic of spinal motor neurons in ALS.

  11. Enhanced alpha 1(I) mRNA expression in frozen shoulder and dupuytren tissue.

    Kilian, Olaf; Pfeil, U; Wenisch, S; Heiss, C; Kraus, R; Schnettler, R


    The purpose of this study has been to investigate collagen I and III synthesis during the fibrosing stage of frozen shoulder and Dupuytren samples in comparison to normal capsule tissue. - By using the quantitative PCR significantly increased levels of alpha 1(I) mRNA transcription in samples of frozen shoulder (p = 0.016) and Duypuytren (p = 0.041) could be demonstrated, whereas alpha 2(I) and alpha 1(III) chains have shown the same mRNA levels as in normal capsule tissue. - Despite an enhancement of alpha 1(I) mRNA transcription in frozen shoulder and Dupuytren samples the intracellular precursor procollagen I and extracellular mature collagen I was detected immunohistochemically in reduced levels. - The structural alteration of collagen I assembly might be caused by disturbed post-translation from the polypeptide chains into the triple helices procollagen I though alpha 1(I) mRNA transcription was significantly increased and alpha 2(I) mRNA transcription was in normal range. Fibroblasts might release high quantities of free alpha 1(I) polypeptide chains or (alpha 1(I)) 3 homotrimer into the extracellular space during the fibrosing stage of frozen shoulder and Dupuytren disease. - In all samples neither differences of alpha 1(III) mRNA transcription nor differences of immunohistochemical staining intensity of collagen III could be seen. This might result from apoptosis of myofibroblasts in the final phase of the fibrosing processes. - The stimulating effect of insulin-like growth factor type I (IGF-I) to induce fibrosis in connective tissue such as scarlet is known. In all patients suffering from frozen shoulder and Dupuytren disease the serum IGF-I level was in a normal range and the IGF-I receptor - (IGFR-I) mRNA transcription in the samples was also in the same level compared with normal capsule tissue.

  12. Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

    Vanesa Garcia

    Full Text Available BACKGROUND: We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC and favorable outcome (LMO2, BCL6, FN1 in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS: mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE: Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

  13. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Małgorzata; Przybyszewska, Wiesława; Kaczmarek, Elżbieta; Koczorowska, Maria; Kościński, Tomasz; Zabel, Maciej; Drews, Michał


    The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 mRNA

  14. mRNA expression of Rho GTPase-related signaling molecules during rat hippocampal development

    Guoqing Guo; Jifeng Zhang; Li Xin; Jing Chen; Weizai Shen; Lin Yuan; Shizhen Zhong


    BACKGROUND:Rho GTPase family members have been shown to participate in neurite growth by regulating the neuronal cytoskeleton.However,there are very few reports of developmental roles of signaling molecules related to Rho GTPases.OBJECTIVE:To investigate messenger ribonucleic acid mRNA expression of signaling molecules associated with Rho GTPases,including Rho-A,Rac-1,collapsin response mediator protein 1(CRMP-1),and tubulin β3 (Tub β3) during rat hippocampus development.DESIGN,TIME AND SETTING:A non-randomized,controlled,animal experiment,based on different developmental stages of the rat hippocampus,was performed at the Guangdong Key Laboratory of Tissue Construction and Detection,Institute of Clinical Anatomy,Southern Medical University between December 2005 and July 2007.MATERIALS:Trizol reagent was purchased from Invitrogen,USA.RNA PCR kit (AMV) Ver 3.0 and 150 bp DNA Ladder Marker were purchased from TaKaRa,Japan.Unless otherwise specified,all other reagents were purchased from Sigma-Aldrich,USA.METHODS:Twenty-five Sprague Dawley rats were assigned to five groups (n=5) according to developmental stages:embryonic (embryonic 15 days),neonatal (postnatal 5 days),juvenile (postnatal 1 month),adult (postnatal 3 months),and senile (postnatal 18 months).MAIN OUTCOME MEASURES:Detection of mRNA expression of Rho-A,Rac-1,CRMP-1,and Tub β3 during various hippocampal developmental stages by reverse-transcription polymerase chain reaction.RESULTS:Hippocampal mRNA expression of Rho-A,as well as Rac-1,reached peak levels at embryonic,juvenile,and senile stages,and was relatively less during neonatal and adult stages.mRNA expression of Rac-1 was greater than Rho-A during each hippocampal developmental stage.CRMP-1 mRNA expression levels were as follows:embryonic>neonatal>juvenile>adult<senile,while Tub β3 mRNA expression was embryonic>neonatal>juvenile>adult=senile.CONCLUSION:Rho-A and Rac-1 shared similar expression profiles,which demonstrated similar

  15. Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells.

    Gondo, Yusuke; Satsu, Hideo; Ishimoto, Yoko; Iwamoto, Taku; Shimizu, Makoto


    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. β-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. β-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by β-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions.

    Kalynchuk, Lisa E; Meaney, Michael J


    Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear.

  17. HSF1-TPR interaction facilitates export of stress-induced HSP70 mRNA.

    Skaggs, Hollie S; Xing, Hongyan; Wilkerson, Donald C; Murphy, Lynea A; Hong, Yiling; Mayhew, Christopher N; Sarge, Kevin D


    Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear pore-associating TPR protein in a stress-responsive manner. TPR is brought into proximity of the HSP70 promoter after stress and preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the export of mRNAs expressed from the HSP70 promoter, both endogenous HSP70 mRNA and a luciferase reporter mRNA. These results suggest that HSP mRNA export escapes stress inhibition via HSF1-mediated recruitment of the nuclear pore-associating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression pathway, transcription and mRNA export.

  18. Detection of histidine decarboxylase mRNA in human vascular smooth muscle and endothelial cells.

    Tippens, A S; Gruetter, C A


    The objective of this study was to investigate histamine synthesis capability of human vascular smooth muscle and endothelial cells by detecting histidine decarboxylase (HDC) mRNA. HDC catalyzes exclusively the formation of histamine in mammalian cells. Experiments utilizing nested reverse transcription-polymerase chain reaction (nRT-PCR) were conducted to detect the presence of HDC mRNA. Human aortic smooth muscle cells (HAoSMC) and human aortic endothelial cells (HAEC) were cultured and RNA was extracted and amplified using two sets of HDC-specific primers. Rat liver and kidney RNA were isolated and amplified to serve as positive and negative controls, respectively. Gel electrophoresis of HAoSMC, HAEC and liver mRNA revealed bands coinciding with an expected product size of 440 base pairs. Sequence analysis revealed that the observed bands were the appropriate HDC amplicons. These findings are the first to indicate the presence of HDC mRNA in vascular smooth muscle cells and confirm the presence of HDC mRNA in endothelial cells which is consistent with an ability of these cell types to synthesize histamine in the vascular wall.

  19. Translation by polysome: theory of ribosome profile on a single mRNA transcript

    Sharma, Ajeet K


    The process of polymerizing a protein by a ribosome, using a messenger RNA (mRNA) as the corresponding template, is called {\\it translation}. Ribosome may be regarded as a molecular motor for which the mRNA template serves also as the track. Often several ribosomes may translate the same (mRNA) simultaneously. The ribosomes bound simultaneously to a single mRNA transcript are the members of a polyribosome (or, simply, {\\it polysome}). Experimentally measured {\\it polysome profile} gives the distribution of polysome {\\it sizes}. Recently a breakthrough in determining the instantaneous {\\it positions} of the ribosomes on a given mRNA track has been achieved and the technique is called {\\it ribosome profiling} \\cite{ingolia10,guo10}. Motivated by the success of these techniques, we have studied the spatio-temporal organization of ribosomes by extending a theoretical model that we have reported elsewhere \\cite{sharma11}. This extended version of our model incorporates not only (i) mechano-chemical cycle of indivi...

  20. Kinetin in familial dysautonomia carriers: implications for a new therapeutic strategy targeting mRNA splicing.

    Gold-von Simson, Gabrielle; Goldberg, Judith D; Rolnitzky, Linda M; Mull, James; Leyne, Maire; Voustianiouk, Andrei; Slaugenhaupt, Susan A; Axelrod, Felicia B


    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IkappaB kinase-associated protein gene (IKBKAP) that leads to partial skipping of exon 20 and tissue-specific reduction of IkappaB kinase-associated protein/elongator protein 1 (IKAP/ELP-1 protein). Kinetin increases IKBKAP mRNA and protein expression in FD cell lines. To determine whether oral kinetin alters IKBKAP splicing in vivo, we administered kinetin to 29 healthy carriers of the major FD mutation for 8 d. Adverse effects, kinetin, and IKBKAP mRNA levels were monitored. In the highest dosing cohorts (23.5 mg/kg/d), the target plasma kinetin level was achieved in 91% of subjects at 2 h. After 8 d, IKBKAP mRNA expression in leukocytes increased as kinetin levels increased. There is a linear association between log plasma kinetin level and corresponding log change from baseline in IKBKAP mRNA expression that allows estimation of IKBKAP mRNA levels because of kinetin ingestion. Adverse effects were transient and mild. This is the first report of in vivo IKBKAP splicing modification and strongly suggests kinetin's therapeutic potential in FD and perhaps in other splicing disorders. Furthermore, our findings support our hypothesis that treatments, which target a particular splicing mutation, can be successfully developed.

  1. The life of an mRNA in space and time.

    Ben-Ari, Ya'ara; Brody, Yehuda; Kinor, Noa; Mor, Amir; Tsukamoto, Toshiro; Spector, David L; Singer, Robert H; Shav-Tal, Yaron


    Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed beta-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic beta-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the beta-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell.

  2. Probabilistic Boolean Network Modelling and Analysis Framework for mRNA Translation.

    Zhao, Yun-Bo; Krishnan, J


    mRNA translation is a complex process involving the progression of ribosomes on the mRNA, resulting in the synthesis of proteins, and is subject to multiple layers of regulation. This process has been modelled using different formalisms, both stochastic and deterministic. Recently, we introduced a Probabilistic Boolean modelling framework for mRNA translation, which possesses the advantage of tools for numerically exact computation of steady state probability distribution, without requiring simulation. Here, we extend this model to incorporate both random sequential and parallel update rules, and demonstrate its effectiveness in various settings, including its flexibility in accommodating additional static and dynamic biological complexities and its role in parameter sensitivity analysis. In these applications, the results from the model analysis match those of TASEP model simulations. Importantly, the proposed modelling framework maintains the stochastic aspects of mRNA translation and provides a way to exactly calculate probability distributions, providing additional tools of analysis in this context. Finally, the proposed modelling methodology provides an alternative approach to the understanding of the mRNA translation process, by bridging the gap between existing approaches, providing new analysis tools, and contributing to a more robust platform for modelling and understanding translation.

  3. Dynein regulates epithelial polarity and the apical localization of stardust A mRNA.

    Horne-Badovinac, Sally; Bilder, David


    Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

  4. Dynein regulates epithelial polarity and the apical localization of stardust A mRNA.

    Sally Horne-Badovinac


    Full Text Available Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

  5. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter


    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems.

    Siwaszek, Aleksandra; Ukleja, Marta; Dziembowski, Andrzej


    The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5'-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3'>5'direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals.

  7. Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

    Michaud, Morgane; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie


    Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex.

  8. Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

    YU Duowei; SUN Yun; WU Jianqing; YUAN Sheng


    By inducing and amplifying dendritic cells(DCs)derived from the bone marrow of asthma murine in vitro,cytokines mRNA were expressed,and the functions of DCS were investigated.Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4,and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates.Large numbers of DCS have been obtained from bone marrow,and they expressed interleukin-13(IL-13),interleukin-9(IL-9),and interleukin-3(IL-3)mRNA.Moreover.the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups(P<0.05).But,the level of IL-3 mRNA showed no discrepancy between the two groups(P>0.05).Des are very important in the forming and developing of asthma,which implies that the therapy targeted at DCs will possibly become a new goal.

  9. Selective translation of the measles virus nucleocapsid mRNA by La protein

    Yoshihisa eInoue


    Full Text Available Measles, caused by measles virus (MeV infection, is the leading cause of death in children because of secondary infections attributable to MeV-induced immune suppression. Recently, we have shown that wild-type MeVs induce the suppression of protein synthesis in host cells (referred to as "shutoff" and that viral mRNAs are preferentially translated under shutoff conditions in infected cells. To determine the mechanism behind the preferential translation of viral mRNA, we focused on the 5 untranslated region (UTR of nucleocapsid (N mRNA. The La/SSB autoantigen (La was found to specifically bind to an N-5UTR probe. Recombinant La enhanced the translation of luciferase mRNA containing the N-5UTR (N-fLuc, and RNA interference of La suppressed N-fLuc translation. Furthermore, recombinant MeV lacking the La-binding motif in the N-5UTR displayed delayed viral protein synthesis and growth kinetics at an early phase of infection. These results suggest that La induced predominant translation of N mRNA via binding to its 5UTR under shutoff conditions. This is the first report on a cellular factor that specifically regulates paramyxovirus mRNA translation.

  10. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)


    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  11. The use of Molecular Beacons to Directly Measure Bacterial mRNA Abundances and Transcript Degradation

    Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Morrison, John M.; Dunman, Paul M.


    The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript. PMID:18992285

  12. Acute stress increases neuropsin mRNA expression in the mouse hippocampus through the glucocorticoid pathway.

    Harada, Akiko; Shiosaka, Sadao; Ishikawa, Yasuyuki; Komai, Shoji


    Stress affects synaptic plasticity and may alter various types of behaviour, including anxiety or memory formation. In the present study, we examined the effects of acute stress (1 h restraint with or without tail-shock) on mRNA levels of a plasticity-related serine protease neuropsin (NP) in the hippocampus using semiquantitative RT-PCR and in situ hybridization. We found that NP mRNA expression was dramatically increased shortly after exposure to the acute restraint tail-shock stress and remained at high level for at least 24 h. The level of NP mRNA would be correlated to the elevated plasma concentration of the glucocorticoid corticosterone (CORT) and to the stress intensity. Application of CORT either onto primary cultured hippocampal neurons (5 nM) or in vivo to adrenalectomized (ADX) mice (10 mg/kg B.W., s.c.) mimicked the effect of stress and significantly elevated NP mRNA. These results suggest that the upregulation of NP mRNA after stress is CORT-dependent and point to a role for neuropsin in stress-induced neuronal plasticity.

  13. Influenza virus targets the mRNA export machinery and the nuclear pore complex.

    Satterly, Neal; Tsai, Pei-Ling; van Deursen, Jan; Nussenzveig, Daniel R; Wang, Yaming; Faria, Paula A; Levay, Agata; Levy, David E; Fontoura, Beatriz M A


    The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral-host interactions and provide insights into potential molecular therapies that may interfere with influenza infection.

  14. MOS11: a new component in the mRNA export pathway.

    Hugo Germain


    Full Text Available Nucleocytoplasmic trafficking is emerging as an important aspect of plant immunity. The three related pathways affecting plant immunity include Nuclear Localization Signal (NLS-mediated nuclear protein import, Nuclear Export Signal (NES-dependent nuclear protein export, and mRNA export relying on MOS3, a nucleoporin belonging to the Nup107-160 complex. Here we report the characterization, identification, and detailed analysis of Arabidopsis modifier of snc1, 11 (mos11. Mutations in MOS11 can partially suppress the dwarfism and enhanced disease resistance phenotypes of snc1, which carries a gain-of-function mutation in a TIR-NB-LRR type Resistance gene. MOS11 encodes a conserved eukaryotic protein with homology to the human RNA binding protein CIP29. Further functional analysis shows that MOS11 localizes to the nucleus and that the mos11 mutants accumulate more poly(A mRNAs in the nucleus, likely resulting from reduced mRNA export activity. Epistasis analysis between mos3-1 and mos11-1 revealed that MOS11 probably functions in the same mRNA export pathway as MOS3, in a partially overlapping fashion, before the mRNA molecules pass through the nuclear pores. Taken together, MOS11 is identified as a new protein contributing to the transfer of mature mRNA from the nucleus to the cytosol.

  15. Detection of MDR1 mRNA expression with optimized gold nanoparticle beacon

    Zhou, Qiumei; Qian, Zhiyu; Gu, Yueqing


    MDR1 (multidrug resistance gene) mRNA expression is a promising biomarker for the prediction of doxorubicin resistance in clinic. However, the traditional technical process in clinic is complicated and cannot perform the real-time detection mRNA in living single cells. In this study, the expression of MDR1 mRNA was analyzed based on optimized gold nanoparticle beacon in tumor cells. Firstly, gold nanoparticle (AuNP) was modified by thiol-PEG, and the MDR1 beacon sequence was screened and optimized using a BLAST bioinformatics strategy. Then, optimized MDR1 molecular beacons were characterized by transmission electron microscope, UV-vis and fluorescence spectroscopies. The cytotoxicity of MDR1 molecular beacon on L-02, K562 and K562/Adr cells were investigated by MTT assay, suggesting that MDR1 molecular beacon was low inherent cytotoxicity. Dark field microscope was used to investigate the cellular uptake of hDAuNP beacon assisted with ultrasound. Finally, laser scanning confocal microscope images showed that there was a significant difference in MDR1 mRNA expression in K562 and K562/Adr cells, which was consistent with the results of q-PCR measurement. In summary, optimized MDR1 molecular beacon designed in this study is a reliable strategy for detection MDR1 mRNA expression in living tumor cells, and will be a promising strategy for in guiding patient treatment and management in individualized medication.

  16. Major role for mRNA stability in shaping the kinetics of gene induction

    Zeller Karen I


    Full Text Available Abstract Background mRNA levels in cells are determined by the relative rates of RNA production and degradation. Yet, to date, most analyses of gene expression profiles were focused on mechanisms which regulate transcription, while the role of mRNA stability in modulating transcriptional networks was to a large extent overlooked. In particular, kinetic waves in transcriptional responses are usually interpreted as resulting from sequential activation of transcription factors. Results In this study, we examined on a global scale the role of mRNA stability in shaping the kinetics of gene response. Analyzing numerous expression datasets we revealed a striking global anti-correlation between rapidity of induction and mRNA stability, fitting the prediction of a kinetic mathematical model. In contrast, the relationship between kinetics and stability was less significant when gene suppression was analyzed. Frequently, mRNAs that are stable under standard conditions were very rapidly down-regulated following stimulation. Such effect cannot be explained even by a complete shut-off of transcription, and therefore indicates intense modulation of RNA stability. Conclusion Taken together, our results demonstrate the key role of mRNA stability in determining induction kinetics in mammalian transcriptional networks.

  17. Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons.

    Jakobson, M; Jakobson, M; Llano, O; Palgi, J; Arumäe, U


    N-Bak is a neuron-specific BH3-only splice variant of pro-apoptotic Bcl-2 family member Bak. We have shown that its mRNA is stable in the neurons, whereas the protein cannot be detected by antibodies, suggesting a strong translational arrest of the mRNA. Here we identify two regulatory elements in the N-Bak mRNA that significantly repress translation in the luciferase reporter assay: an upstream open reading frame in the 5'-untranslated region (UTR) and naturally spliced exon-exon junction downstream of the premature translation termination codon in the 3'UTR. We also show that N-Bak mRNA is stored in granular structures in the sympathetic neurons and stays in these granules during intrinsic apoptosis. Finally, we confirm the absence of N-Bak protein by quantitative mass spectrometry analysis in the healthy, apoptotic or stressed sympathetic and cortical neurons. We conclude that N-Bak mRNA is translationally repressed by multiple mechanisms, and the protein does not participate in the classical apoptosis or cellular stress response.

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

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


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

  19. GLUT3 protein and mRNA in autopsy muscle specimens

    Stuart, C. A.; Wen, G.; Jiang, J.


    GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

  20. Effect of Interventional Therapy on the Expression of Survivin mRNA in Cervical Cancer.

    Wang, Shao-Guang; Mu, Nan; Sun, Hai-Yan


    To examine the expression of survivin mRNA in cervical squamous cancer at different time points during interventional therapy in order to find the optimal time point for continual treatment. Fifty patients with stage IB2-IIB cervical squamous cancer receiving transarterial infusion chemotherapy and chemoembolization were included in the present study. The expression of survivin mRNA in cancer samples before and after interventional therapy (on days 7, 14 and 21) were examined by reverse transcription-polymerase chain reaction. The expression of survivin mRNA in cancer samples before the interventional therapy was significantly higher than that of any time point after the interventional therapy (ptreatment, the expression of survivin mRNA decreased until day 14. The expression of survivin mRNA was inhibited by the interventional therapy. It seems that day 14 after interventional treatment is the right time point for continuation of treatment. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. Mobile FT mRNA contributes to the systemic florigen signalling in floral induction.

    Li, Chunyang; Gu, Mei; Shi, Nongnong; Zhang, Hang; Yang, Xin; Osman, Toba; Liu, Yule; Wang, Huizhong; Vatish, Manu; Jackson, Stephen; Hong, Yiguo


    In inducing photoperiodic conditions, plants produce a signal dubbed "florigen" in leaves. Florigen moves through the phloem to the shoot apical meristem (SAM) where it induces flowering. In Arabidopsis, the FLOWERING LOCUS T (FT) protein acts as a component of this phloem-mobile signal. However whether the transportable FT mRNA also contributes to systemic florigen signalling remains to be elucidated. Using non-conventional approaches that exploit virus-induced RNA silencing and meristem exclusion of virus infection, we demonstrated that the ArabidopsisFT mRNA, independent of the FT protein, can move into the SAM. Viral ectopic expression of a non-translatable FT mRNA promoted earlier flowering in the short-day (SD) Nicotiana tabacum Maryland Mammoth tobacco in SD. These data suggest a possible role for FT mRNA in systemic floral signalling, and also demonstrate that cis-transportation of cellular mRNA into SAM and meristem exclusion of pathogenic RNAs are two mechanistically distinct processes.

  2. Systemic movement of FT mRNA and a possible role in floral induction

    Stephen eJackson


    Full Text Available FLOWERING LOCUS T (FT protein is known to be part of the mobile flowering inducing ‘florigen’ signal in plants, but it may not be acting alone. This article reviews the data that FT mRNA can also move systemically throughout the plant and into the shoot apical meristem (SAM independently of the FT protein. There is a promotion of flowering when increased levels of virally expressed FT mRNA are present together with endogenously produced FT protein in inducing conditions, even if the additional FT mRNA is non-translatable and thus not increasing the overall levels of FT protein. A specific sequence, or ‘zip code’ of the FT mRNA is required for systemic movement and this sequence binds a specific protein(s in plant extracts. This raises the possibility the FT mRNA may be moving systemically through the plant and into the SAM as an RNA-protein complex, whether FT protein is also a component of this mobile complex remains to be determined.

  3. Integrating mRNA Decay Information into Co-Regulation Study

    Liang Chen; Hong-Yu Zhao


    Absolute or relative transcript amounts measured through high-throughput technologies (e.g., microarrays)are now commonly used in bioinformatics analysis, such as gene clustering and DNA binding motif finding. However,transcription rates that represent mRNA synthesis may be more relevant in these analyses. Because transcription rates are not equivalent to transcript amounts unless the mRNA degradation rates as well as other factors that affect transcript amount are identical across different genes, the use of transcription rates in bioinformatics analysis may lead to a better description of the relationships among genes and better identification of genomic signals. In this article, we propose to use experimentally measured mRNA decay rates and mRNA transcript amounts to jointly infer transcription rates, and then use the inferred transcription rates in downstream analyses. For gene expression similarity analysis, we find that there tends to be higher correlations among co-regulated genes when transcription-rate-based correlations are used compared to those based on transcript amounts. In the context of identifying DNA binding motifs, using inferred transcription rates leads to more significant findings than those based on transcript amounts. These analyses suggest that the incorporation of mRNA decay rates and the use of the inferred transcription rates can facilitate the study of gene regulations and the reconstruction of transcriptional regulatory networks.


    COLI, Katiane Cristie


    Full Text Available To present researches “The pleasure of the learning through of the poetry” has for objective to emphasize the importance of pleased poetries in classroom of the fundamental teaching, proposing to the students one moment ludic, tends in view the exercise of the imagination, of the fantasy and of the creativity, developing like this a form teaching more cheerful, more poetic and with larger freedom to build the knowledge. The research also has for purpose to call the teachers’ attention for the importance of the reading of poetries in the classroom and to develop in the students the taste for her. For the accomplishment of this research works of renowned national prominence were used, among other materials. It was also accomplished a field research, in way to still give larger credibility to the study. A presente pesquisa “O prazer da aprendizagem através da poesia” teve por objetivo ressaltar a importância de poesias prazerosas em sala de aula do ensino fundamental, propondo aos alunos um momento lúdico, tendo em vista o exercício da imaginação, da fantasia e da criatividade, desenvolvendo assim um ensino de forma mais alegre, mais poético e com maior liberdade para construir o conhecimento. A pesquisa também tem por propósito chamar a atenção dos professores para a importância da leitura de poesias na sala de aula e desenvolver nos alunos o gosto por ela. Para a realização desta pesquisa foram utilizadas obras de renomado destaque nacional, entre outros materiais. Foi realizada também uma pesquisa de campo, de forma a dar maior credibilidade ao estudo.

  5. Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system.

    Schröder, H C; Rottmann, M; Bachmann, M; Müller, W E; McDonald, A R; Agutter, P S


    Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.

  6. Publicidade e ética: um estudo da construção da imagem da mulher

    Elizabeth Moraes Gonçalves


    Full Text Available O texto propõe uma reflexão sobre a ética da responsabilidade na publicidade veiculada nas revistas Claudia e Nova. Trata-se de uma pesquisa descritiva, resultante da leitura dos anúncios selecionados, subsidiada por teóricos da Análise do Discurso da linha francesa, que busca averiguar como a mulher é representada. Constatou-se que no contexto da sociedade contemporânea o retrato da mulher como sedutora ainda está presente, mesmo que em vários momentos ela apareça como protagonista de sua própria vida.

  7. Efeitos da acupuntura no tratamento da ansiedade: revisao integrativa

    Sueli Leiko Takamatsu Goyatá; Carolina Costa Valcanti Avelino; Sérgio Valverde Marques dos Santos; Deusdete Inácio de Souza Junior; Maria Dorise Simão Lopes Gurgel; Fábio de Souza Terra


    RESUMO Objetivo: avaliar as evidências científicas disponíveis na literatura sobre os efeitos da acupuntura no tratamento da ansiedade e a qualidade desses estudos. Método: revisão integrativa, realizada nas bases/bancos de dados CINAHL, LILACS, PUBMEDPICO, SciELO, The Cochrane Library, no período entre 2001 a 2014. Os descritores anxiety, acupuncture therapy, acupuncture e anxiety disorders foram combinados entre si para garantir a ampla busca de estudos primários. Resultados: dos 514 art...

  8. O apagamento da oralidade na historiografia da literatura brasileira

    Schneider, Sabrina; Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)


    Este artigo tem, por objetivo, mostrar como a oralidade tem sido abordada – quando é abordada – pelos historiadores da literatura brasileira. Trabalha com a hipótese de que a historiografia, por meio de estratégias discursivas, tem marginalizado a oralidade, bem como a contribuição das tradições ágrafas africana e indígena. Para verificá-la, perfaz um trajeto que tem início em Ferdinand Denis, cujo "Resumo da história literária do Brasil" foi publicado em 1826, e termina em Alexei Bueno, que ...


    João Paulo Rodrigues


    Full Text Available O projeto acadêmico de Habermas busca contribuir para a realização das metas emancipatórias da modernidade, através da teoria da ação comunicativa. Portanto, será examinado aqui se o processo de racionalização da sociedade moderna, presente na teoria da ação comunicativa, realmente representa um potencial para a emancipação humana. Primeiramente, pretende-se abordar no respectivo trabalho os principais conceitos que Habermas utiliza em sua teoria da ação comunicativa. Em um segundo momento, serão explorados os elementos fundamentais da teoria da sociedade. Após isso, será analisada a teoria da modernidade e a sua relação com a teoria da sociedade. Tal caminho terá o objetivo de investigar a possibilidade de resgate do projeto da modernidade, ao explicar a possibilidade da teoria da ação comunicativa ser considerada o potenciador emancipatório da sociedade moderna, formando tanto o cidadão como um indivíduo autônomo, preservando a sua identidade pessoal, quanto o cidadão como participante de uma sociedade política, possuidor de uma identidade cultural, étnica, entre outras, que abrange variados grupos sociais.

  10. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    Köster, Tino


    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  11. Expression of SART-1 mRNA in canine squamous cell carcinomas.

    Takaishi, Yumi; Yoshida, Yukari; Nakagaki, Kazuhide; Fujita, Michio; Taniguchi, Akiko; Orima, Hiromitsu


    SART-1, a squamous cell carcinoma (SCC) antigen recognized by cytotoxic T lymphocytes, has been useful in human cancer therapy. The SART-1(259) peptide is a potential candidate for vaccine. The present study examined an orthologue of the mRNA coding this peptide in canine SCCs. Specimens were obtained from seven canine patients with SCC, and the mRNA was isolated from the samples. The SART-1 and beta-actin genes were amplified by reverse-transcription polymerase chain reaction, using the isolated mRNA as a template. Canine SART-1 was amplified in six of the seven specimens, while beta-actin was detected in all the samples. In dogs, carcinomas expressing SART-1 could be a target for cytotoxic T lymphocyte mediated immunotherapy.

  12. A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1.

    Mozdziak, Paul E; Giamario, Carol; Dibner, Julia J; McCoy, Darell W


    Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.

  13. Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

    Frydelund-Larsen, Lone; Åkerström, Thorbjörn; Nielsen, Søren


    Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression...... in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4.......5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied...

  14. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    Elgart, Vlad; Fenley, Andrew T; Kulkarni, Rahul V


    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distributio...

  15. Choreography of molecular movements during ribosome progression along mRNA.

    Belardinelli, Riccardo; Sharma, Heena; Caliskan, Neva; Cunha, Carlos E; Peske, Frank; Wintermeyer, Wolfgang; Rodnina, Marina V


    During translation elongation, ribosome translocation along an mRNA entails rotations of the ribosomal subunits, swiveling motions of the small subunit (SSU) head and stepwise movements of the tRNAs together with the mRNA. Here, we reconstructed the choreography of the collective motions of the Escherichia coli ribosome during translocation promoted by elongation factor EF-G, by recording the fluorescence signatures of nine different reporters placed on both ribosomal subunits, tRNA and mRNA. We captured an early forward swiveling of the SSU head taking place while the SSU body rotates in the opposite, clockwise direction. Backward swiveling of the SSU head starts upon tRNA translocation and continues until the post-translocation state is reached. This work places structures of translocation intermediates along a time axis and unravels principles of the motions of macromolecular machines.

  16. Class II antigen-associated invariant chain mRNA in mouse small intestine.

    Ouellette, A J; Frederick, D; Hagen, S J; Katz, J D


    MHC class II antigen-associated invariant (Ii) chain mRNA appears in mouse small intestine during postnatal development. Ii chain cDNA hybridizes to RNA from epithelial sheets dissociated from the lamina propria with EDTA. Of several mouse organs tested, only bone marrow and spleen contain higher levels of Ii chain mRNA than small bowel. Ii chain mRNA is not detected in stomach, colon, duodenum, testis, liver, submandibular gland, or L-cell RNA; brain contains a cross-reactive but uncharacterized sequence. cDNA amplification using primers specific for both Ii31 and Ii41 chain mRNAs showed that both forms occur in small intestine. These results support the conclusion that regulation of the class II Ii chain gene is associated with the ontogeny of intestinal immunity.

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

    Natalizio, Barbara J.; Wente, Susan R.


    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

  18. The expression of TRPA1 mRNA in the rat brain

    Peng Du; Shua Li; Jinyu Zheng; Zhi-yuan Yu; Minjie Xie; Wei Wang


    Objective: To investigate the distribution of TRPA1 (one kind of the TRP-like ion channel family) channel in the hippocampus and cerebral cortex of rat. Methods: RT-PCR was used to amplify the fragment of TRPA1 in the DRG (dorsal root ganglion), hippocampus and cerebral cortex of adult SD rat. In situ hybridization staining was used to show the distribution of TRPA1 mRNA in the hippocampus and cerebral cortex of adult rat brain. Results: Both RT-PCR and in situ hybridization staining showed that TRPA1 mRNA was expressed in hippocampus and cerebral cortex of the adult rat brain. Conclusion: Ourresults suggest that there is expression of TRPA1 mRNA both in the hippocampus and cerebral cortex of the adult rat brain.

  19. CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China.

    An, Lihui; Hu, Jianying; Yang, Min; Zheng, Binghui; Wei, An; Shang, Jingjing; Zhao, Xingru


    Induction of cytochrome P4501A (CYP1A) has been used as a biomarker in fish for monitoring aromatic and organic contaminants. In this study, a partial of CYP1A gene in redeye mullet (Liza haematocheila) was isolated and sequenced, and then a real-time quantitative reverse-transcription polymerase chain reaction assay was developed for quantification of CYP1A mRNA normalized to β-actin. The developed method was applied to detect CYP1A mRNA expression in redeye mullets collected from Nandaihe (reference site) and Dashentang (impacted site) in Bohai Bay, China. CYP1A mRNA expression values were significantly elevated in redeye mullets from Dashentang compared to a reference site--Nandaihe, which was correlated with the contents of different environmentally relevant pollutants in tissues, particularly with PCBs and PBDEs.

  20. Expression of TLR9 and Its mRNA in the Lesions of Lichen Planus

    LI Jiawen; CHEN Jing; TAN Zhijian; LIU Houjun; LIU Zhixiang


    To investigate the role of toll-like receptor 9 (TLR9) in the pathogenesis of lichen planus,the expressions of TLR9 and its mRNA in the lesional skin of lichen planus were detected by immunohistochemical technique (SP) and RT-PCR. As control, normal skin of healthy volunteers was also tested. The immunohistochemical study showed that the expression of TLR9 in the lesional skin of lichen planus was significantly higher than that in the normal controls. The results of RT-PCR showed that both skin lesions and normal controls had TLR9 expression. In skin lesions, the expression level of TLR9 mRNA was 1.6075±0.0930, which was significantly higher than that in normal controls (P<0.001). These findings indicated that up-regulated expression of TLR9 and its mRNA might be involved in the pathogenesis of lichen planus.

  1. Codon optimality is a major determinant of mRNA stability.

    Presnyak, Vladimir; Alhusaini, Najwa; Chen, Ying-Hsin; Martin, Sophie; Morris, Nathan; Kline, Nicholas; Olson, Sara; Weinberg, David; Baker, Kristian E; Graveley, Brenton R; Coller, Jeff


    mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.

  2. mRNA vaccination as a safe approach for specific protection from type I allergy.

    Weiss, Richard; Scheiblhofer, Sandra; Roesler, Elisabeth; Weinberger, Esther; Thalhamer, Josef


    Allergic diseases are on the increase and current therapies are lacking in efficacy and patient compliance. In recent years, the idea of prophylactic measures, especially for children at high risk for allergy, has become increasingly popular. This review summarizes the available preclinical data for protective allergy vaccines, with a focus on one of the most promising vaccine candidates; mRNA vaccines. Recently, mRNA vaccines have been rediscovered as an alternative to their more prominent counterparts, the DNA vaccines. Allergen-encoding mRNA vaccines elicit long-lasting protection from sensitization, and induce a type of immunity similar to the natural protective response that is acquired in the presence of microbial burden early in life. Owing to their excellent safety profile, they represent the ideal candidates for a vaccine that aims to protect at-risk children who have not yet been sensitized to allergens.

  3. Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome.

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D; Jaffrey, Samie R


    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide-repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5' untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA.

  4. Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

    Weischenfeldt, Joachim Lütken; Waage, Johannes Eichler; Tian, Geng;


    a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes......ABSTRACT: BACKGROUND: Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation...... of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. RESULTS: We developed...

  5. Adaptações bioquimicas em fibras musculares esqueleticas de equinos treinados para enduro : correlação entre tipagem muscular e expressão da isoforma neuronal da oxido nitrico sintase

    Fernando Jose Gondim Peixoto


    Resumo: Em várias espécies de mamíferos o músculo esquelético expressa "splicings" variantes do mRNA da isoforma neuronal da enzima Óxido Nítrico Sintase (nNOS). O músculo esquelético é composto por uma população de fibras musculares metabolicamente heterogêneas. A distribuição destas fibras no músculo esquelético de eqüinos está correlacionada com aptidão atlética destes animais. Investigamos a expressão da nNOS em músculo esquelético eqüino, e analisamos se esta expressão está relacionada à...

  6. Carencia de dados e desafios metodologicos para o desenvolvimento dos estudos da industria da musica

    Herschmann, Micael


    Balanco das dificuldades metodologicas enfrentadas pelos pesquisadores que vem trabalhando com a industria da musica no contexto atual marcado pela crise e reestruturacao deste setor da producao cultural...

  7. Translational control of maskin mRNA by its 3' untranslated region

    Meijer, Hedda A.; Radford, Helois E.; Wilson, Lolita S.; Lissenden, Sarah; de Moor, Cornelia H.


    Background information. Maskin is a member of the acidic transforming coiled-coil (TACC) domain proteins found in Xenopus leavis oocytes and embryos. It is implicated in the coordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. Results We report here that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3' UTR of maskin can confer the translational regulation to a reporter mRNA, and so can the 3' UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV crosslinking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3' UTRs. As previously reported, CPEB binds to the cyclin B1 3' UTR, but its binding to the maskin 3' UTR is minimal. By RNA affinity chromatography and mass spectrometry, we identified the embryonic deadenylation element binding protein (EDEN-BP) as one of the proteins binding to both the maskin and the cyclin B1 3' UTRs. Conclusion Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3' UTRs, indicating it may be involved in the deadenylation of these mRNAs. PMID:17241108

  8. Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples.

    Hui Zhang

    Full Text Available There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA tubes and PAXgene Blood RNA Tubes (PAXgene tubes. These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.

  9. Adaptive and maladaptive expression of the mRNA regulatory protein HuR

    Suman; Govindaraju; Beth; S; Lee


    The RNA-binding proteins involved in regulation of mRNA post-transcriptional processing and translation control the fates of thousands of mRNA transcripts and basic cellular processes. The best studied of these, HuR, is well characterized as a mediator of mRNA stability and translation, and more recently, as a factor in nuclear functions such as pre-mRNA splicing. Due to HuR’s role in regulating thousands of mRNA transcripts, including those for other RNA-binding proteins, HuR can act as a master regulator of cell survival and proliferation. HuR itself is subject to multiple post-translationa modifications including regulation of its nucleocytoplasmic distribution. However, the mechanisms that govern HuR levels in the cell have only recently begun to be defined. These mechanisms are critical to cell health, as it has become clear in recent years that aberrant expression of HuR can lead alternately to decreased cell viability or to promotion of pathological proliferation and invasiveness. HuR is expressed as alternate mRNAs that vary in their untranslated regions, leading to differences in transcript stability and translatability. Multiple transcription factors and modulators of mRNA stability that regulate HuR mRNA expression have been identified. In addition, translation of HuR is regulated by numerous microRNAs, several of which have been demonstrated to have anti-tumor properties due to their suppression of HuR expression. This review summarizes the current state of knowledge of the factors that regulate HuR expression, along with the circumstances under which these factors contribute to cancer and inflammation.

  10. Functional characterization of the HuR:CD83 mRNA interaction.

    Dorothea Pieper

    Full Text Available Maturation of dendritic cells (DC is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE, which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1-3 in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein:RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway.

  11. Characterization of a major late herpes simplex virus type 1 mRNA.

    Costa, R H; Devi, B G; Anderson, K P; Gaylord, B H; Wagner, E K


    A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

  12. Quantitation of HDAC1 mRNA expression in invasive carcinoma of the breast

    Zhenhuan Zhang; Hirotaka Iwase; Hiroko Yamashita; Tatsuya Toyama; Hiroshi Sugiura; Yoshiaki Ando; Keiko Mita; Maho Hamaguchi; Yasuo Hara; Shunzo Kobayashi


    Estrogen is well-established as a mitogenic factor implicated in the tumorigenesis and progression of breast cancer via its binding to the estrogen receptor a(ERα). Recent data indicate that chromatin inactivation mediated by histone deacetylation(HDAC) and DNA methylation is a critical component of ERα silencing in human breast cancer cells. The aim of this study was to determine the expression of the HDAC1 gene in malignant human breast tissue and to correlate our observations with available clinical information. In the present study, the level of expression of HDAC1 mRNA was assessed by LightCycler-based quantitative real-time reverse transcriptase (RT)-PCR analvsis in 162 cases of invasive carcinoma of the breast. Associations between HDAC1 mRNA expression and different clinicopathological factors were sought. It was found that HDAC1 mRNA was expressed at significantly higher levels in tumors from patients over 50 years of age and in those tumors without axillary lymph node involvement, that are less than 2 cm, that are of a non-high histological grade, that are HER2 negative and that are ERα/PgR positive. Patients with tumors displaying high levels of HDAC1 mRNA expression tended to have a better prognosis in terms of both disease-free and overall survival. However, univariate and multivariate analysis did not show HDAC1 mRNA expression level to be an independent prognostic factor for either disease-free or overall survival. These results imply that HDAC1 mRNA expression could have potential as an endocrine response marker and may have prognostic implications for breast cancer progression.

  13. Expression of Npas4 mRNA in telencephalic areas of adult and postnatal mouse brain

    Joanne C Damborsky


    Full Text Available The transcription factor neuronal PAS domain-containing protein 4 (Npas4 is an inducible immediate early gene which regulates the formation of inhibitory synapses, and could have a significant regulatory role during cortical circuit formation. However, little is known about basal Npas4 mRNA expression during postnatal development. Here, postnatal and adult mouse brain sections were processed for isotopic in situ hybridization using an Npas4 specific cRNA antisense probe. In adults, Npas4 mRNA was found in the telencephalon with very restricted or no expression in diencephalon or mesencephalon. In most telencephalic areas, including the anterior olfactory nucleus (AON, piriform cortex, neocortex, hippocampus, dorsal caudate putamen (CPu, septum and basolateral amygdala nucleus (BLA, basal Npas4 expression was detected in scattered cells which exhibited strong hybridization signal. In embryonic and neonatal brain sections, Npas4 mRNA expression signals were very low. Starting at postnatal day 5 (P5, transcripts for Npas4 were detected in the AON, CPu and piriform cortex. At P8, additional Npas4 hybridization was found in CA1 and CA3 pyramidal layer, and in primary motor cortex. By P13, robust mRNA expression was located in layers IV and VI of all sensory cortices, frontal cortex and cingulate cortex. After onset of expression, postnatal spatial mRNA distribution was similar to that in adults, with the exception of the CPu, where Npas4 transcripts became gradually restricted to the most dorsal part. In conclusion, the spatial distribution of Npas4 mRNA is mostly restricted to telencephalic areas, and the temporal expression increases with developmental age during postnatal development, which seem to correlate with the onset of activity-driven excitatory transmission.

  14. Nuclear accumulation of CDH1 mRNA in hepatocellular carcinoma cells

    Ghafoory, S; Mehrabi, A; Hafezi, M; Cheng, X; Breitkopf-Heinlein, K; Hick, M; Huichalaf, M; Herbel, V; Saffari, A; Wölfl, S


    Expression of E-cadherin has a central role in maintaining epithelial morphology. In solid tumors, reduction of E-cadherin results in disruption of intercellular contacts. Consequently, cells lose adhesive properties and gain more invasive mesenchymal properties. Nevertheless, the mechanism of E-cadherin regulation is not completely elucidated. Here we analyzed the distribution of E-cadherin expression at the cell level in human hepatocellular carcinoma, in which human liver paraffin blocks from 25 hepatocellular carcinoma patients were prepared from cancerous (CA) and noncancerous areas (NCA). In situ hybridization (ISH) was performed to detect E-cadherin and hypoxia-induced factor-1α (HIF1α) mRNAs and immunohistochemistry to stain E-cadherin protein. In parallel, RNA was extracted from CA and NCA, and E-cadherin and HIF1α were quantified by quantitative reverse transcription PCR. ISH revealed abundant E-cadherin mRNA in nuclei of hepatocellular carcinoma cells (HCCs), whereas immunohistochemistry showed depletion of E-cadherin protein from these areas. In sections of NCA, E-cadherin mRNA was also found in the cytosol, and E-cadherin protein was detected on the membrane of cells. Experiments in cell lines confirmed E-cadherin mRNA in nuclei of cells negative for E-cadherin protein. HIF1α expression is elevated in CAs, which is associated with a clear cytosolic staining for this mRNA. Our results demonstrate that E-caderhin mRNA is selectively retained in nuclei of HCCs, whereas other mRNAs are still exported, suggesting that translocation of E-cadherin mRNA from nuclei to cytoplasm has a role in regulating E-cadherin protein levels during epithelial mesenchymal transition. PMID:26029826

  15. Heparanase mRNA expression and point mutation in hepatocellular carcinoma

    Xiao-Peng Chen; Yin-Bib Liu; Jing Rui; Shu-You Peng; Cheng-Hong Peng; Zi-Yan Zhou; Liang-Hui Shi; Hong-Wei Shen; Bin Xu


    AIM: To explore the expression of heparanase mRNA and point mutation in hepatocellular carcinoma (HCC).METHODS: Reverse transcription polymerase chain reaction was used to measure the expression of heparanase mRNA in the primary tumor tissues and surrounding liver tissues of 33 HCC patients. T-A cloning and sequencing were used to detect whether there was any mutation in the amplified PCR products.RESULTS: The expression of heparanase mRNA was positive in 16 primary tumor tissues of HCC, and the positive rate was 48.5%, which was significantly higher than that in the surrounding liver parenchyma (P<0.01). The positive rate for heparanase gene in high-tendency to metastatic recurrence group (71.4%, 10/14) was obviously higher than that in low-tendency to metastatic recurrence group(31.6%, 6/19) (P= 0.023). The positive rate for heparanase gene in patients with metastatic recurrence during postoperative follow-up (78.6%, 11/14) was also significantly higher than that in those without metastatic recurrence (21.4%, 3/14)(P = 0.003). Sequence analysis of the HPA PCR products was made in 7 patients, and 2-point mutations were found in 4 patients, one of which was sense mutation, neither base insertion nor deletion was detected. The mutation rate was 57.1% (4/7).CONCLUSION: The expression rate of heparanase mRNA increases in HCC, and HPA mRNA may be one of the reliable markers for the metastatic activity gained by the liver tumor cells and could be used clinically in predicting metastatic recurrence of HCC. Point mutation may be one of the causes for enhanced heparanase mRNA expression.

  16. Real-time determination of human telomerase reverse transcriptase mRNA in gastric cancer

    Li-Hua Hu; Feng-Hua Chen; Yi-Rong Li; Lin Wang


    AIM: To set up a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay,to detect human telomerase reverse transcriptase (hTERT)messenger RNA in gastric carcinomas, and to evaluate quantitative determination of hTERT mRNA in the diagnostic value of gastric carcinomas, and to analyze the correlation between the expression level of hTERT mRNA and dinicopathological parameters in patients with gastric cancer.METHODS: A real-time quantitative RT-PCR (RQ-PCR)based on TaqMan fluorescence methodoloogy and the LightCyder system was used to quantify the full range of hTERT mRNA copy numbers in 35 samples of gastric carcinomas and corresponding adjacent non-cancerous tissues. The normalized hTERT (NhTERT) was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100× (hTERT/GAPDH) ratio. Variables were analyzed by the Student's t-test, χ2 test and Fisher's exact test.RESULTS: NhTERT from gastric carcinomas and corresponding adjacent non-cancerous tissues was 6.27±0.89 and 0.93±0.18,respectively (t= 12.76, P<0.001). There was no significant association between gastric cancer hTERT mRNA expression level and patient's age, gender, tumor size, location and stage (pTNM), but a significant correlation was found between hTERT mRNA expression level in gastric carcinomas and the degree of differentiation.CONCLUSION: Quantitative determination of hTERT mRNA by RQ-PCR is a rapid and sensitive method. hTERT might be a potential biomarker for the early detection of gastric cancer.

  17. A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells.

    Haider, Michaela; Ji, Bozhi; Haselgrübler, Thomas; Sonnleitner, Alois; Aberger, Fritz; Hesse, Jan


    Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties - e.g. low sample dilution, low contamination - that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.


    C. T. R. M. BASTOS


    Full Text Available

    O taperebá (Spondias mombim é uma pequena drupa elipsóide, de cor amarelo alaranjado, com casca fi na que pode ser facilmente rompida, tornando o fruto susceptível à contaminação por ação de agentes externos. Estudou-se a pasteurização da polpa de taperebá, visando estabelecer condições simples e efi cientes de conservação a temperatura ambiente. Parâmetros microbiológicos (enumeração de microrganismos mesófílos aeróbios e pesquisa de coliformes a 45°C, químicos (teor de vitamina C, enzimáticos (atividade da peroxidase – POD e colorimétricos (colorimetria tristimulus foram utilizados como variáveis de resposta. Em bancada, foram testados diferentes tempos (1 a 300 s e temperaturas (70ºC a 90ºC de tratamento térmico, não tendo sido observado efeito estatisticamente signifi cativo (p < 0,05 sobre as contagens de microrganismos mesófi los aeróbios, coliformes a 45°C e sobre a cor da polpa. Em escala piloto, realizou-se a pasteurização da polpa a 85°C por 3 minutos, sendo avaliados parâmetros de qualidade do produto durante 14 dias de manutenção a 40°C, sob iluminação artifi cial, e realizados testes sensoriais. A redução da contagem de microrganismos mesófi los aeróbios e coliformes a 45°C, para níveis aceitáveis pela legislação brasileira, a manutenção da cor e os índices de aceitação da polpa confi rmaram a efi ciência do tratamento térmico. Não se observou atividade da POD na polpa pasteurizada, devendo o controle de qualidade do produto ser feito com base nos parâmetros microbiológicos e de cor da polpa.

  19. Subchronic exposure to arsenic disturbed the biogenic amine neurotransmitter level and the mRNA expression of synthetase in mice brains.

    Zhang, J; Liu, X; Zhao, L; Hu, S; Li, S; Piao, F


    Little is known about the influence of arsenic (As) exposure on monoamine neurotransmitters and the underlying mechanisms, although arsenic toxicity on the central nervous system has been well documented. In the present study, the levels of norepinephrine (NE), dopamine (DA), and 5-HT were determined by high performance liquid chromatography in the cerebrum and cerebellum of mice exposed to 1, 2 and 4 ppm As2O3 through drinking water for 60 days. The ultra-structural change of vesicles in the synapses of mice brains was observed by transmission electron microscopy; the mRNA expressions of dopamine beta hydroxylase (DBH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) as NE, DA and 5-HT synthetases were quantitatively assessed by real time reverse transcription-polymerase chain reaction. It was shown that the concentrations of NE, DA and 5-HT in the cerebrum or cerebellum of mice exposed to As were significantly lower than those in the control group. The number of synaptic vesicles significantly decreased in the brain of mice exposed to As. Moreover, the expressions of TH, TPH and DBH genes were significantly lower in the brains of mice exposed to As than those in the controls. These results suggested that subchronic exposure to As might decrease the concentrations of the three monoamine neurotransmitters in the mouse brain and downregulate TH, TPH and DBH gene expressions. It was also indicated that the decreased concentrations of the three monoamine neurotransmitters in the brain might be related to the down-regulated gene expressions of these synthetases by As.

  20. O panorama das Drogas no México: da margem da sociedade ao centro da cultura

    Diana Palaversich


    Full Text Available O artigo destaca o percurso das manifestações culturais ligadas ao tráfico de drogas no México, de um lugar marginal, de subcultura, na sociedade mexicana das décadas de 1970 e 1980, expressas principalmente através dos narcocorridos e do narcocine, para um lugar no mainstream cultural mexicano, desde o início do século 21, com destaque especial para a narcoliteratura. O aumento da violência, provocado pela guerra ao narcotráfico, produz uma proliferação da temática nos meios de comunicação e se reflete em áreas da cultura como as artes plásticas e a literatura, produzindo um novo tipo de expressão artística ou narrativa, que tanto pode ser oportunista e contribuir para a banalização da violência do narcotráfico como ser crítica e narrar o impacto individual e coletivo do tráfico de drogas, narrar a desenfreada e violenta realidade mexicana sem cair no estilo da imprensa marrom, do folclore ou dos lugares comuns dos romances de ação.

  1. MRN1 implicates chromatin remodeling complexes and architectural factors in mRNA maturation

    Düring, Louis; Thorsen, Michael; Petersen, Darima;


    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6¿¿ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin....... Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309¿, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing....

  2. An in vitro system from Plasmodium falciparum active in endogenous mRNA translation

    Ferreras Ana


    Full Text Available An in vitro translation system has been prepared from Plasmodium falciparum by saponin lysis of infected-erythrocytes to free parasites which were homogeneized with glass beads, centrifuged to obtain a S-30 fraction followed by Sephadex G-25 gel filtration. This treatment produced a system with very low contamination of host proteins (<1%. The system, optimized for Mg2+ and K+, translates endogenous mRNA and is active for 80 min which suggests that their protein factors and mRNA are quite stable.

  3. Role of the C terminus of Lassa virus L protein in viral mRNA synthesis.

    Lehmann, Maria; Pahlmann, Meike; Jérôme, Hanna; Busch, Carola; Lelke, Michaela; Günther, Stephan


    The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.

  4. Effects of Ginseng and Echinacea on Cytokine mRNA Expression in Rats

    Deniz Uluışık


    Full Text Available The aim of the study was to determine the effect of ginseng and echinacea on the mRNA expression of IL-10, TNF-α, and TGF-β1 in healthy rats. Six-week-old male Fischer 344 rats (n=48 were used. The animals were divided into three equal groups, as follows: control (C; ginseng (G; echinacea (E. While the C group was fed a standard rat diet (Purina ad libitum for a period of 40 days, the G and E groups animals received the same diet containing 0.5 g/kg of Panax ginseng root powder and 0.75 g/kg of Echinacea purpurea root powder, respectively. Blood samples were obtained from 8 rats in each group after 20 and 40 days of treatment, and the mRNA expression of IL-10, TNF-α, and TGF-β1 was determined. After 20 days of treatment, the expression of IL-10 mRNA in the G group was different from the C group (P<0.05; however, after 40 days of treatment, there was no difference between the groups. There was no difference after 20 and 40 days of treatment between the groups with respect to the expression of TGF-β1 mRNA. After 20 days of treatment, the expression of TNF-α mRNA in the E group was higher (P<0.05 than the C group. After 40 days of treatment, the expression of TNF-α mRNA was similar in all of the groups. Based on the current study, the increase in expression of IL-10 mRNA in the G group and the increase in expression of TNF-α mRNA in the E group support the use of these plants for purposes of modulating the immune system. However, a more detailed study regarding the effects of ginseng and echinacea on these cytokines and other cytokines is needed.

  5. Increased thymidylate synthase mRNA concentration in blood leukocytes following an experimental stressor

    Ehrnrooth, Eva; Zacharia, Robert; Svendsen, Gunner


    to a computerized mental stressor; (2) relaxation, and (3) control. Measurements included TS mRNA levels, total leukocyte number, leukocyte subtypes, and serum cortisol before (baseline), immediately after, and 1 h after each experimental condition. RESULTS: While no significant differences were found between...... in percentage of neutrophil cells after stress. CONCLUSION: The results suggest that TS mRNA levels in peripheral leukocytes may be sensitive to mental stress and confirm previous findings indicating that subjects scoring high on the personality trait of absorption exhibit greater physiological stress...

  6. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas


    In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce...... intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...... and hence is important to recombinant and, most certainly, endogenous gene expression....

  7. Optimization of Differential Display of Prokaryotic mRNA: Application to Pure Culture and Soil Microcosms

    Fleming, James T.; Yao, Wen-Hsiang; Sayler, Gary S.


    The differential display (DD) technique, which is widely used almost exclusively for eukaryotic gene discovery, was optimized to detect differential mRNA transcription from both pure-culture and soil-derived bacterial RNA. A model system which included toluene induction of todC1 in Pseudomonas putida F1 was used to optimize the procedure. At 24-h tod induction was determined to be approximately 8 × 107 transcripts/μg or 0.08% of the total mRNA. The primer concentration, primer length, anneali...

  8. Differential Gene Expression in Retina of Myopic Chicken Eyes Using mRNA Differential Display

    ShenHX; ZhangQJ


    Purpose:To study differentially expressed genes in retina of experimental myopic chicken.Methods:Experimental myopia in chicken was induced by form-deprivatin.The mRNA in chicen retina was analyzed by using differential display.Results:Experimental myopia was successfully induced in chicken through form-deprivation.Differentially expressed gene fragments were detected in retina of chicken with myopic evelopment and recovery as compared with normal controld.Conclusion:The differential display of mRNA may be a useful way in cloning myopic-related genes.

  9. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L


    Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

  10. Signatures of Host mRNA 5′ Terminus for Efficient Hantavirus Cap Snatching

    Cheng, Erdong


    Hantaviruses, similarly to other negative-strand segmented RNA viruses, initiate the synthesis of translation-competent capped mRNAs by a unique cap-snatching mechanism. Hantavirus nucleocapsid protein (N) binds to host mRNA caps and requires four nucleotides adjacent to the 5′ cap for high-affinity binding. N protects the 5′ caps of cellular transcripts from degradation by the cellular decapping machinery. The rescued 5′ capped mRNA fragments are stored in cellular P bodies by N, which are later efficiently used as primers by the hantaviral RNA-dependent RNA polymerase (RdRp) for transcription initiation. We showed that N also protects the host mRNA caps in P-body-deficient cells. However, the rescued caps were not effectively used by the hantavirus RdRp during transcription initiation, suggesting that caps stored in cellular P bodies by N are preferred for cap snatching. We examined the characteristics of the 5′ terminus of a capped test mRNA to delineate the minimum requirements for a capped transcript to serve as an efficient cap donor during hantavirus cap snatching. We showed that hantavirus RdRp preferentially snatches caps from the nonsense mRNAs compared to mRNAs engaged in translation. Hantavirus RdRp preferentially cleaves the cap donor mRNA at a G residue located 14 nucleotides downstream of the 5′ cap. The sequence complementarity between the 3′ terminus of viral genomic RNA and the nucleotides located in the vicinity of the cleavage site of the cap donor mRNA favors cap snatching. Our results show that hantavirus RdRp snatches caps from viral mRNAs. However, the negligible cap-donating efficiency of wild-type mRNAs in comparison to nonsense mRNAs suggests that viral mRNAs will not be efficiently used for cap snatching during viral infection due to their continuous engagement in protein synthesis. Our results suggest that efficiency of an mRNA to donate caps for viral mRNA synthesis is primarily regulated at the translational level. PMID

  11. Programmed death 1 mRNA in peripheral blood as biomarker of acute renal allograft rejection

    WANG Ya-wen; WANG Zhen; SHI Bing-yi


    Background Invasive kidney biopsy is a priority diagnostic method for the acute rejection after renal transplantation for the past decades. However, no effective and noninvasive assay for predicting the severity of acute rejection is in wide use at present. This study was designed to investigate the predictive value of programmed death 1 (PD-1) mRNA for acute rejection after renal transplantation with real-time reverse transcriptase polymerase chain reaction (RT-PCR). A noninvasive diagnostic method has been expected to replace the tranditional kidney biopsy for the diagnosis of acute rejection and prediction of the outcome after kidney transplantation.Methods The whole blood samples from 19 subjects with acute rejection, 20 subjects with delayed graft function (DGF)and 21 subjects with stable recipients after kidney transplantation in a single kidney transplantation center between 2006 and 2009 were collected. The messenger RNA (mRNA) of PD-1 was analyzed with real-time RT-PCR. The associations of PD-1 mRNA levels with acute rejection and disease severity were investigated.Results The log-transformed ratio of PD-1 mRNA to GAPDH mRNA was higher in peripheral blood mononuclear cell (PBMC) from the group with acute rejection (4.52±1.1) than that from the group with DGF (1.12±0.6) or the group with normal biopsy results (0.7±0.4) (P <0.01, by the Kruskal-Wallis test). PD-1 mRNA levels were correlated with serum creatinine levels measured at the time of biopsy in the acute rejection group (Spearman's correlation coefficient, r=0.81,P=0.03), but not in the group with DGF or the group with normal biopsy results. PD-1 mRNA levels identified subjects at risk for graft failure within six months after the incident episode of acute rejection.Conclusions Our data suggest that PD-1 status may be a new predictor of acute rejection and the levels of PD-1mRNA in whole blood cells may positively correlate with the severity of acute rejection after renal transplantation

  12. Signatures of host mRNA 5' terminus for efficient hantavirus cap snatching.

    Cheng, Erdong; Mir, Mohammad A


    Hantaviruses, similarly to other negative-strand segmented RNA viruses, initiate the synthesis of translation-competent capped mRNAs by a unique cap-snatching mechanism. Hantavirus nucleocapsid protein (N) binds to host mRNA caps and requires four nucleotides adjacent to the 5' cap for high-affinity binding. N protects the 5' caps of cellular transcripts from degradation by the cellular decapping machinery. The rescued 5' capped mRNA fragments are stored in cellular P bodies by N, which are later efficiently used as primers by the hantaviral RNA-dependent RNA polymerase (RdRp) for transcription initiation. We showed that N also protects the host mRNA caps in P-body-deficient cells. However, the rescued caps were not effectively used by the hantavirus RdRp during transcription initiation, suggesting that caps stored in cellular P bodies by N are preferred for cap snatching. We examined the characteristics of the 5' terminus of a capped test mRNA to delineate the minimum requirements for a capped transcript to serve as an efficient cap donor during hantavirus cap snatching. We showed that hantavirus RdRp preferentially snatches caps from the nonsense mRNAs compared to mRNAs engaged in translation. Hantavirus RdRp preferentially cleaves the cap donor mRNA at a G residue located 14 nucleotides downstream of the 5' cap. The sequence complementarity between the 3' terminus of viral genomic RNA and the nucleotides located in the vicinity of the cleavage site of the cap donor mRNA favors cap snatching. Our results show that hantavirus RdRp snatches caps from viral mRNAs. However, the negligible cap-donating efficiency of wild-type mRNAs in comparison to nonsense mRNAs suggests that viral mRNAs will not be efficiently used for cap snatching during viral infection due to their continuous engagement in protein synthesis. Our results suggest that efficiency of an mRNA to donate caps for viral mRNA synthesis is primarily regulated at the translational level.

  13. Angiotensin II receptor mRNA expression and vasoconstriction in human coronary arteries

    Wackenfors, Angelica; Pantev, Emil; Emilson, Malin;


    Angiotensin II is a potent vasoconstrictor that is implicated in the pathogenesis of hypertension, heart failure and atherosclerosis. In the present study, angiotensin II receptor mRNA expression levels were quantified by real-time polymerase chain reaction and the vasocontractile responses...... to angiotensin II were characterised by in vitro pharmacology in endothelium-denuded human coronary arteries. Angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptor mRNA expression levels were significantly down-regulated in arteries from patients with heart failure as compared to controls. The angiotensin II...

  14. Ischemic heart disease induces upregulation of endothelin receptor mRNA in human coronary arteries

    Wackenfors, Angelica; Emilson, Malin; Ingemansson, Richard;


    and controls using real-time polymerase chain reaction (real-time PCR). In addition, the suitability of organ culture as a model mimicking endothelin receptor changes in cardiovascular disease was evaluated by in vitro pharmacology and real-time PCR. Endothelin ETA and ETB receptor mRNA levels were......Endothelin has been implicated in the pathogenesis of ischemic heart disease and congestive heart failure. The aims were to quantify endothelin type A (ETA) and type B (ETB) receptor mRNA levels in human coronary arteries from patients with ischemic heart disease, congestive heart failure...

  15. [Comparative study of nuclear RNA and polysomal mRNA in sunflower seedlings].

    Kuntsevich, V I; Tishchenko, E N; Lobov, V P; Zhil'ko, T D


    A comparative study of nRNA and polysomal mRNA complexity in the sunflower seedlings by molecular DNA:RNA hybridization method was carried out. It is established that nRNA complexity 4 times exceeds that of mRNA and is equal to 4.85.10(8) bp. Thus the nuclear RNA is expressed from at least 50.47% of single-copy DNA or 10.40% of genome. This data allow the presence of regulation of sunflower genome expression on the posttranscriptional level to be assumed.

  16. mRNA detection in living cell using phosphorothioate-modified molecular beacon

    TANG HongXing; YANG XiaoHai; WANG KeMin; TAN WeiHong; LI Wei


    In this study, GFP mRNA in COS-7 cell and GFP-transfected COS-7 cell was detected in real time using phosphorothioate-modified molecular beacon based on living cell imaging method. Results showed that phosphorothioate-modified molecular beacon still kept the advantages of molecular beacon, such as, excellent selectivity, high sensitivity, and no separation detection. In addition, this modification could significantly increase the nuclease resistance of molecular beacon. Phosphorothioate-modified molecular beacon can efficiently reduce the false positive signal and improve the accuracy of living cell mRNA detection.

  17. Promoter-Bound Trinucleotide Repeat mRNA Drives Epigenetic Silencing in Fragile X Syndrome

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S.; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D.; Jaffrey, Samie R.


    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide–repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5′ untranslated region, which hybridizes to the complementary CGG-repeat ...

  18. Investigation of HSP60 gene expression in mRNA level in heart at dilated cardiomyopathy

    Riabenko D. V.


    Full Text Available The expression of HSP60 in the mRNA level in human hearts at the end-stage of dilated cardiomyopathy (DCM as well as in the hearts of mice with disease model similar to human DCM was investigated. We observed a significant increase in the Hsp60 mRNA level at the beginning of the disease and decrease to a normal level at the end stage. As the Hsp60 level was increased during the disease up to the end stage we can presume some changes in the regulation of Hsp60 synthesis or its degradation at DCM progression


    Antônio Flávio Pierucci


    Full Text Available Este artigo procura analisar os efeitos perversos da apropriação pela esquerda de um tema definidor das tradições de direita, a saber, a diferença. Com base no caso Sears (EUA e no caso Le Pen (França, o autor aponta para as armadilhas racistas e sexistas presentes nos discursos que focalizam e enfatizam a diferença, sobretudo num período histórico de reemergência dos conservadorismos que, por sua vez, se apropriam do argumento da diferença, dirigindo-o contra os próprios movimentos de esquerda.

  20. O mito fascista da romanidade

    Giardina, Andrea


    Este ensaio procura analisar como o mito de Roma foi utilizado por Mussolini para criar uma "especificidade" fascista que coincidisse com uma "especificidade" da nação italiana. Práticas rituais, gestos, valores, símbolos foram retomados, de forma variamente arbitrária, para construir um mito que gerava novas ambigüidades, e que constituiu, à diferença do nazismo, um empecilho para a própria sobrevivência da "romanidade" após Mussolini.

  1. Cinco discursos da digitalidade audiovisual

    Gerbase, Carlos


    Full Text Available Michel Foucault ensina que toda fala sistemática - inclusive aquela que se afirma “neutra” ou “uma desinteressada visão objetiva do que acontece” - é, na verdade, mecanismo de articulação do saber e, na seqüência, de formação de poder. O aparecimento de novas tecnologias, especialmente as digitais, no campo da produção audiovisual, provoca uma avalanche de declarações de cineastas, ensaios de acadêmicos e previsões de demiurgos da mídia

  2. Cinco discursos da digitalidade audiovisual

    Gerbase, Carlos


    Full Text Available Michel Foucault ensina que toda fala sistemática - inclusive aquela que se afirma “neutra” ou “uma desinteressada visão objetiva do que acontece” - é, na verdade, mecanismo de articulação do saber e, na seqüência, de formação de poder. O aparecimento de novas tecnologias, especialmente as digitais, no campo da produção audiovisual, provoca uma avalanche de declarações de cineastas, ensaios de acadêmicos e previsões de demiurgos da mídia.

  3. Escola Vila : pedagogia da sustentabilidade

    Rocha, Ilka Maria Zoza


    Esta pesquisa teve como objetivo conhecer de que forma a educação ambiental é colocada em prática na Escola VILA de Fortaleza, buscando compreender o seu significado como conteúdo transversal e as relações que as crianças e as demais pessoas da comunidade escolar estabelecem com o ambiente, por meio da observação de como atuam e se comportam no cotidiano. Inicia situando o leitor no contexto educacional: leis e exigências, seguido dos conceitos: relação homem x meio ambiente...

  4. da Vinci decoded: does da Vinci stereopsis rely on disparity?

    Tsirlin, Inna; Wilcox, Laurie M; Allison, Robert S


    In conventional stereopsis, the depth between two objects is computed based on the retinal disparity in the position of matching points in the two eyes. When an object is occluded by another object in the scene, so that it is visible only in one eye, its retinal disparity cannot be computed. Nakayama and Shimojo (1990) found that a precept of quantitative depth between the two objects could still be established for such stimuli and proposed that this precept is based on the constraints imposed by occlusion geometry. They named this and other occlusion-based depth phenomena "da Vinci stereopsis." Subsequent research found quantitative depth based on occlusion geometry in several other classes of stimuli grouped under the term da Vinci stereopsis. However, Nakayama and Shimojo's findings were later brought into question by Gillam, Cook, and Blackburn (2003), who suggested that quantitative depth in their stimuli was perceived based on conventional disparity. In order to understand whether da Vinci stereopsis relies on one type of mechanism or whether its function is stimulus dependent we examine the nature and source of depth in the class of stimuli used by Nakayama and Shimojo (1990). We use three different psychophysical and computational methods to show that the most likely source for depth in these stimuli is occlusion geometry. Based on these experiments and previous data we discuss the potential mechanisms responsible for processing depth from monocular features in da Vinci stereopsis.

  5. Efeitos da acupuntura no tratamento da ansiedade: revisao integrativa

    Sueli Leiko Takamatsu Goyatá


    Full Text Available RESUMO Objetivo: avaliar as evidências científicas disponíveis na literatura sobre os efeitos da acupuntura no tratamento da ansiedade e a qualidade desses estudos. Método: revisão integrativa, realizada nas bases/bancos de dados CINAHL, LILACS, PUBMEDPICO, SciELO, The Cochrane Library, no período entre 2001 a 2014. Os descritores anxiety, acupuncture therapy, acupuncture e anxiety disorders foram combinados entre si para garantir a ampla busca de estudos primários. Resultados: dos 514 artigos, 67 foram selecionados para leitura na íntegra e 19 incluídos. Desses, 11 apresentaram forte nível de evidência. Dos seis artigos de estudos clínicos randomizados, cinco apresentaram qualidade classificada como razoável. Dois estudos utilizaram acupunturistas enfermeiros para a aplicação da intervenção. Os resultados mostram efeitos positivos e estatisticamente significativos do uso da acupuntura para tratamento de indivíduos com ansiedade. Conclusão: a acupuntura parece ser um tratamento promissor para a ansiedade, no entanto, há necessidade de melhorar a qualidade metodológica das pesquisas nessa temática.

  6. Efeito da urina de vaca no estado nutricional da alface

    Nelson Licínio C. de Oliveira


    Full Text Available Existem diversos relatos da utilização da urina de vaca em hortaliças, todavia sua eficácia carece de comprovação. Objetivou-se avaliar o efeito da urina de vaca no estado nutricional da alface. O experimento foi constituído de 12 tratamentos, esquema de parcelas subdivididas, em blocos ao acaso, com quatro repetições. Nas parcelas foram alocadas as vias de aplicação (solo ou foliar e nas subparcelas as concentrações das soluções (0,00; 0,25; 0,50; 0,75; 1,00 e 1,25% v/v. Aplicou-se 60 mL de solução/planta, divididos em cinco aplicações de 5; 5; 10; 20 e 20 mL/planta, aos 7, 14, 21, 28 e 35 após o transplante, respectivamente. Durante o ciclo avaliou-se o índice SPAD e na colheita a massa da matéria seca de cabeça (MSCA e os teores de N, P, K, Ca, Mg, S, Na, Zn, Fe, Mn, Cu e B na matéria seca das folhas (MSF, caule (MSC e raízes (MSR. Em ambas as vias de aplicação o índice SPAD apresentou incremento linear às concentrações e resposta quadrática ao longo do tempo. A MSCA teve comportamento linear às concentrações, com aumento de 25,9 e 35,4% nas aplicações via foliar e solo, respectivamente. Não houve efeito de concentrações sobre teores de nutrientes na MSF e MSC. Na MSR, via solo, os teores de P e K apresentaram pontos de máximo enquanto Fe e Mn de mínimo; o Na apresentou incremento linear às concentrações via foliar. Os efeitos da urina sobre o crescimento da alface provavelmente são devidos a fatores outros que não somente a quantidade de nutrientes veiculados nas soluções.

  7. Efeito agudo da suplementacao da cafeina no desempenho da forca muscular e alteracoes cardiovasculares durante o treino de forca

    Materko, W; Santos, E.L


    A proposta do presente estudo foi avaliar o efeito agudo da cafeina no desempenho da forca muscular, paralelamente a possiveis alteracoes hemodinamicas ao longo de uma sessao de treino de forca muscular...

  8. Laser de diodo no tratamento da retinopatia da prematuridade

    Liarth Josilene de Carvalho Soares


    Full Text Available Objetivo: Determinar a eficácia do laser de diodo e suas complicações no tratamento da retinopatia da prematuridade, estágio 3 limiar. Métodos: De 348 crianças pré-termo examinadas na Clínica Oftalmológica do Hospital Getúlio Vargas e Instituto de Olhos do Piauí, em Teresina, Piauí, no período de julho/89 a março/99, 152 (43,7% apresentaram retinopatia da prematuridade. As crianças com retinopatia da prematuridade no estágio 3 limiar foram submetidas a laserablação retiniana com laser de diodo indireto, no centro cirúrgico, sob anestesia geral. Resultados: Vinte crianças (38 olhos foram submetidas a laserablação retiniana. Doze pacientes (80,0% tiveram regressão da retinopatia e três (20,0% evoluíram para o estágio 5. Cinco crianças não retornaram para controle, sendo excluídas do estudo quanto aos resultados do laser. Não foram observadas complicações oculares. Cinco crianças apresentaram apnéia relacionada à anestesia. Conclusões: Neste grupo de crianças, o laser de diodo foi eficaz em 80,0% dos pacientes tratados. As complicações encontradas (cinco casos de apnéia se relacionaram possivelmente à anestesia geral.

  9. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent


    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation...

  10. A identidade à flor da pele: etnografia da prática da tatuagem na contemporaneidade

    Andrea Lissett Pérez


    Full Text Available No presente artigo, realiza-se uma descrição etnográfica da prática da tatuagem na contemporaneidade, a partir de uma perspectiva que prioriza as noções da prática, das interações e das experiências vividas durante o processo de ser tatuado. A reflexão tenta reconstruir uma visão total da prática da tatuagem que abrange o mundo subjetivo e as dinâmicas sociais como partes constitutivas desse ato. Entre as idéias conclusivas estão, de um lado, o surgimento de uma nova normalidade estética e vivencial no seio da sociedade ocidental e, de outro, a configuração de uma nova subjetividade, a dos "tatuados", como um processo interativo, inovador, emotivo e reflexivo, em que o corpo se converte em uma forma de expressão e construção do sujeito.This article provides a descriptive account of contemporary tattooing, based on a perspective that prioritizes the notion of practice, as well as the interactions and experiences involved in the process of being tattooed. The analysis attempts to rebuild a holistic view of tattooing, combining subjective processes and social dynamics as equally constitutive parts of this activity. Among the conclusions reached are: (a the emergence of tattooing as a new aesthetic norm and a lived experience at the heart of the western society; and (b the configuration of a new subjectivity, the ‘tattooed,’ as an interactive, innovative, emotional and reflexive process in which the body is converted into a form of expressing and constructing the subject.

  11. O panorama das Drogas no México: da margem da sociedade ao centro da cultura

    Diana Palaversich


    O artigo destaca o percurso das manifestações culturais ligadas ao tráfico de drogas no México, de um lugar marginal, de subcultura, na sociedade mexicana das décadas de 1970 e 1980, expressas principalmente através dos narcocorridos e do narcocine, para um lugar no mainstream cultural mexicano, desde o início do século 21, com destaque especial para a narcoliteratura. O aumento da violência, provocado pela guerra ao narcotráfico, produz uma proliferação da temática nos meios de comunicação e...

  12. O panorama das Drogas no México: da margem da sociedade ao centro da cultura

    Diana Palaversich


    O artigo destaca o percurso das manifestações culturais ligadas ao tráfico de drogas no México, de um lugar marginal, de subcultura, na sociedade mexicana das décadas de 1970 e 1980, expressas principalmente através dos narcocorridos e do narcocine, para um lugar no mainstream cultural mexicano, desde o início do século 21, com destaque especial para a narcoliteratura. O aumento da violência, provocado pela guerra ao narcotráfico, produz uma proliferação da temática nos meios de comunicação e...

  13. Novas e velhas abordagens da teoria da escolha e da utilidade

    Wagner, Sergio Artur Luz


    Até pouco tempo havia apenas um posicionamento frente o dilema da escolha envolvendo o risco, que era baseado no processo do indivíduo racional. Atualmente, com novas técnicas de compreensão do risco e principalmente do complexo instituto da incerteza, já é possível de se delimitar o que leva as pessoas a decidirem e a se equivocarem em alguns processos de decisão. Neste trabalho apresentaremos um pequeno resumo histórico dos processos, visando certa linha de pensamento, no que tange aos meca...

  14. IMP3 RNP safe houses prevent miRNA-directed HMGA2 mRNA decay in cancer and development

    Jønson, Lars; Christiansen, Jan; Hansen, Thomas van Overeem;


    The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated...... that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression....

  15. Mulheres da Norô

    Flavia Liberman

    Full Text Available O projeto Cartografias femininas: ações territoriais junto às mulheres da região noroeste de Santos realiza diferentes ações em uma região que apresenta vulnerabilidade social e ambiental na cidade de Santos. Envolvendo docentes e estudantes de diferentes cursos de graduação da área da saúde da Universidade Federal de São Paulo, campus Baixada Santista, teve como resultado uma Exposição Fotográfica itinerante cujas imagens explicitam um processo de construção de vínculos com as participantes. A fotógrafa Nice Gonçalvez, do Instituto Arte no Dique, acompanhou e fotografou momentos desta experiência, oferecendo-nos um material delicado que retrata seus rostos, corpos, detalhes e expressividades. Esta exposição ofereceu, às mulheres, uma oportunidade de reconhecerem-se como protagonistas ativas e fortes em todo o processo, tornando-as visíveis para além das fronteiras de suas casas, de seu bairro ou, mesmo, de sua região.

  16. Tratamento da dor em queimados

    Rodrigo José Alencar de Castro


    Full Text Available JUSTIFICATIVA E OBJETIVOS: Apesar dos avanços, ainda é observado manejo analgésico inadequado dos pacientes com queimaduras. O objetivo desta revisão foi coletar dados sobre tratamento da dor em queimados. CONTEÚDO: Foi feita revisão sobre os mecanismos de dor, avaliação do paciente com queimadura e o tratamento farmacológico e não farmacológico. CONCLUSÕES: O manejo da dor em pacientes vítimas de queimaduras ainda é um desafio por parte da equipe multiprofissional. A avaliação frequente e contínua da resposta apresentada pelo paciente é muito importante, tendo em vista os vários momentos por que passa o paciente internado em decorrência de uma queimadura, além de uma terapêutica combinada com medicações analgésicas e medidas não farmacológicas. Entender a complexidade de alterações fisiopatológicas, psicológicas e bioquímicas apresentadas por um paciente em tratamento de queimadura é o primeiro passo para alcançar o sucesso no seu manejo analgésico.

  17. Leonardo da Vinci and the Downburst.

    Gedzelman, Stanley David


    Evidence from the drawings, experiments, and writings of Leonardo da Vinci are presented to demonstrate that da Vinci recognized and, possibly, discovered the downburst and understood its associated airflow. Other early references to vortex flows resembling downbursts are mentioned.

  18. Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae.

    Harigaya, Yuriko; Parker, Roy


    The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3' to 5' degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5' monophosphate ends on mRNAs in wild-type and dcp2 xrn1 yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low-level endonucleolytic cleavage were observed, suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5' monophosphates on some mRNAs, suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested, Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential step to regulate gene expression.

  19. Interleukin-6 modifies mRNA expression in mouse skeletal muscle

    Hassing, Helle Adser; Wojtaszewski, Jørgen; Jakobsen, Anne Hviid;


    Aim: The aim of the present study was to test the hypothesis that interleukin-6 plays a role in exercise-induced PGC-1a and TNFa mRNA responses in skeletal muscle and to examine the potential IL-6 mediated AMPK regulation in these responses. Methods: Whole body IL-6 knockout and wildtype (WT) male...

  20. Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy

    Fisker, Niels; Westergaard, Majken; Hansen, Henrik Frydenlund;


    synergistic effect when combining the mRNA antagonists against Survivin with the chemotherapeutic Taxol. This effect was demonstrated at concentrations of antagonists far lower than any previously demonstrated, indicating the high potential of locked nucleic acid for therapeutic use. Further characterisations...

  1. Internal Initiation of Translation of mRNA in the Methylotrophic Yeast Hansenula polymorpha.

    Mardanova, E S; Beletsky, A V; Ravin, N V


    Besides regular cap-dependent translation of mRNA, eukaryotes exploit internal initiation of translation driven by internal ribosome entry sites (IRESs). It is supposed that internal initiation provides translation of cellular mRNAs under stress conditions where the cap-dependent initiation is reduced. A number of IRESs have been characterized in mammalian mRNAs, but only a few examples are known in lower eukaryotes, particularly in yeasts. Here we identified two IRESs in the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1. These sites are located in 5'-untranslated regions of genes HPODL_02249 and HPODL_04025 encoding a hypothetical membrane protein and actin-binding protein, respectively. In Saccharomyces cerevisiae cells, both IRESs drive expression of a second gene of a bicistronic mRNA, as well as translation of hairpin-containing monocistronic mRNA. The possibility of spurious splicing or presence of a cryptic promoter in the IRES sequences was ruled out, indicating that expression of a second gene of a bicistronic mRNA was IRES-dependent. We evaluated IRES activity of both elements and found that under normal physiological conditions its contribution to the overall translation of the respective mRNAs in yeast cells is about 0.3-0.4%. Therefore, these results suggest that the IRES-dependent translation initiation mechanism exists in Hansenula polymorpha.

  2. Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation.

    Queenie Wing-Lei Wong

    Full Text Available The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs and in ESC derived Neural Precursor Cells (NPCs using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.

  3. Proprotein convertase 1 mRNA and protein expression in ischemic rat cortex after reperfusion

    Shuqin Zhan; An Zhou; Jingquan Lan; Tao Yang


    Proprotein convertase 1 (PC1) is a member of the family of proprotein convertases (PCs), which are the processing enzymes of neuropeptides. Previous studies have addressed PC1 effects with regard to the neuroendocrine system. In this study, the developing changes of PC1 mRNA and PC1 protein in rat cortices after transient focal cerebral ischemia were investigated by fluorescent double labeling (both in situ hybridization and immunocytochemistry) using a transient focal cerebral ischemia model in rats. The results were compared with those of sham-operated rat cortices. Both the mRNA and protein levels of PC1 in ischemic cortices decreased gradually at 4, 8, and 16 hours of reperfusion after 100 minutes of middle cerebral artery occlusion. After 24 hours of reperfusion, enhanced intensities of signals for PC1 protein were observed, while signals for PC1 mRNA remained low. These results suggest that transient focal cerebral ischemia influences PC1 mRNA and protein expression in cortices of ischemic rats. Thus, PC1 is regulated by ischemic stress.

  4. Eosinophil cationic protein mRNA expression in children with bronchial asthma.

    Yu, H Y; Li, X Y; Cai, Z F; Li, L; Shi, X Z; Song, H X; Liu, X J


    Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P children with bronchial asthma.

  5. Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

    Min HONG; Yan-chun CHE; Gui-zhen TANG; Wei CUN; Xue-mei ZHANG; Long-ding LIU; Qi-han LI


    HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.

  6. MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

    Xu, Kai; Lin, Jing; Zandi, Roza; Roth, Jack A; Ji, Lin


    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

  7. Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA

    Roberto Plebani


    Full Text Available mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (≈2 x 10-5 of all mRNA. Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.

  8. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok


    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  9. Quantification of mRNA by RT-competitive-PCR and high performance liquid chromatography


    The use of RT-competitive-PCR with high performance liquid chromatography (HPLC) detection to quantify the absolute number of mRNA copies in mammalian cells is reported. As an example, the glutathione transferase (GST)-α mRNA in human hepatoma Hep G2 cells has been estimated. A PCR-generated internal standard was used as a competitor, co-amplified with the GST-α target sequence. The RT-competitive-PCR method was improved by designing target and competitor molecules which differed in only 30 base pairs. This allowed the two sequences to be co-amplified with the same efficiency. This improvement also facilitated a wider ratio to be used than previous methods (target:competitor ratio between 0.2 and 5). Products were baseline separated by HPLC using an ion-exchange column readily quantified at 260 nm. To validate the improved methodology, the effect of a known GST-α inducer, the anticancer drug oltipraz, was shown to induce GST-α mRNA up to 3-fold in Hep G2 cells. The RT-competitive PCR-HPLC method provides a reliable and sensitive way to quantify the amount of specific mRNA with 0.1 ng of total RNA.

  10. The influence of eccentric exercise on mRNA expression of skeletal muscle regulators.

    Jensky, Nicole E; Sims, Jennifer K; Rice, Judd C; Dreyer, Hans C; Schroeder, E Todd


    To evaluate change in myostatin, follistatin, MyoD and SGT mRNA gene expression using eccentric exercise to study mechanisms of skeletal muscle hypertrophy. Young (28+/-5 years) and older (68+/-6 years) men participated in a bout of maximal single-leg eccentric knee extension on an isokinetic dynamometer at 60 degrees /s: six sets, 12-16 maximal eccentric repetitions. Muscle biopsies of the vastus lateralis were obtained from the dominant leg before exercise and 24 h after exercise. Paired t tests were used to compare change (pre versus post-exercise) for normalized gene expression in all variables. Independent t tests were performed to test group differences (young vs. older). A probability level of Peccentric exercise. Similarly, we did not observe significant change in myostatin (-3.83+/-8.8; P=0.23), follistatin (-2.66+/-5.2; P=0.17), MyoD (-0.13+/-3.1; P=0.90), or SGT (-1.6+/-3.5; P=0.19) mRNA expression in older subjects. Furthermore, the non-significant changes in mRNA expression were not different between young and older subjects, P>0.23 for all variables. Our data suggests that a single bout of maximal eccentric exercise does not alter myostatin, follistatin, MyoD or SGT mRNA gene expression in young or older subjects.

  11. c-Myc co-ordinates mRNA cap methylation and ribosomal RNA production.

    Dunn, Sianadh; Lombardi, Olivia; Cowling, Victoria H


    The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)-RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT-RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT-RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT-RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT-RAM is suppressed, indicating that RNMT-RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT-RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts. © 2017 The Author(s).

  12. mRNA profiling for the identification of blood--Results of a collaborative EDNAP exercise

    Haas, C.; Hanson, E.; Bär, W.;


    A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, usi...

  13. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia.

    Axelrod, Felicia B; Liebes, Leonard; Gold-Von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A


    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex-associated protein/elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase WT IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine whether oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/d for 28 d. An increase in WT IKBKAP mRNA expression in leukocytes was noted after 8 d in six of eight individuals; after 28 d, the mean increase compared with baseline was significant (p = 0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients but also that effect seems to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine whether kinetin will prove therapeutic in FD patients.

  14. Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation.

    Johannes H Urban


    Full Text Available Small noncoding RNAs (sRNA can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3' end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms.

  15. Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A Polymorphism

    Yoshihiko Inaoka


    Full Text Available Alpha 2-HS glycoprotein (AHSG, also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG∗1 and AHSG∗2. The serum level in the AHSG∗1 homozygote is significantly higher than that of the AHSG∗2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG∗1 to AHSG∗2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

  16. Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A) Polymorphism.

    Inaoka, Yoshihiko; Osawa, Motoki; Mukasa, Nahoko; Miyashita, Keiko; Satoh, Fumiko; Kakimoto, Yu


    Alpha 2-HS glycoprotein (AHSG), also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG(∗) 1 and AHSG(∗) 2. The serum level in the AHSG(∗) 1 homozygote is significantly higher than that of the AHSG(∗) 2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG(∗) 1 to AHSG(∗) 2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

  17. Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution.

    Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander


    Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the 'long-term evolution experiment'. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins.

  18. A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis

    Guchte, Maarten van de; Lende, Ted van der; Kok, Jan; Venema, Gerard


    Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression. Muta

  19. Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

    Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W


    Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

  20. Expression of Nogo-A mRNA after injury of the rat central nervous system

    Xigao Guo; Yang Guo; Tao Huang


    BACKGROUND: Nogo protein has been identified as an inhibitor of axonal growth, which was highly expressed in central nervous system; however, there are only a few studies on changes of Nogo-A expression following central nervous system injury.OBJECTIVE: To investigate the dynamic expression of Nogo-A mRNA after rat central nervous system injury.DESIGN: Randomized controlled animal study.MATERIALS: Thirty-five rats were randomly divided into two groups, normal animal group (n = 5) and model group (n = 30). The model group was then divided into six subgroups at six time points: 12, 24 hours and 3, 9, 15, and 21 days post-injury, with five rats in each subgroup.METHODS: The left parietal lobe of rats was contused by free-fall strike, and total RNA was extracted from the entire brain tissue. Semi-quantitative RT-PCR was used to detect Nogo-A mRNA expression, and the ratio between expression of the target gene and glyceraldehyde phosphate dehydrogenase was used to determine the relative expression level.MAIN OUTCOME MEASURES: To determine whether Nogo-A mRNA expression was higher than usual following brain injury.RESULTS: The level of Nogo-A mRNA started to increase 12 hours after injury (P 0.05).CONCLUSION: After injury of the central nervous system, Nogo-A may play a pivotal role in obstructing regeneration of the nerve.

  1. Differential distribution of calcineurin Aα isoenzyme mRNA's in rat brain

    Buttini, M.; Limonta, S.; Luyten, M.; Boddeke, H.


    Specific antisense oligonucleotide probes for the α isoforms of the catalytic subunit (A-subunit) of calcineurin were prepared and the distribution of Aα1 and Aα2 mRNA's has been studied in rat brain using in situ hybridization histochemistry. Clear regional differences have been observed for the Aα

  2. VDR mRNA overexpression is associated with worse prognostic factors in papillary thyroid carcinoma

    June Young Choi


    Full Text Available The purpose of this study was to assess the relationship between vitamin D receptor gene (VDR expression and prognostic factors in papillary thyroid cancer (PTC. mRNA sequencing and somatic mutation data from The Cancer Genome Atlas (TCGA were analyzed. VDR mRNA expression was compared to clinicopathologic variables by linear regression. Tree-based classification was applied to find cutoff and patients were split into low and high VDR group. Logistic regression, Kaplan–Meier analysis, differentially expressed gene (DEG test and pathway analysis were performed to assess the differences between two VDR groups. VDR mRNA expression was elevated in PTC than that in normal thyroid tissue. VDR expressions were high in classic and tall-cell variant PTC and lateral neck node metastasis was present. High VDR group was also associated with classic and tall cell subtype, AJCC stage IV and lower recurrence-free survival. DEG test reveals that 545 genes were upregulated in high VDR group. Thyroid cancer-related pathways were enriched in high VDR group in pathway analyses. VDR mRNA overexpression was correlated with worse prognostic factors such as subtypes of papillary thyroid carcinoma that are known to be worse prognosis, lateral neck node metastasis, advanced stage and recurrence-free survival.

  3. CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

    Eira Valeria Barrón

    Full Text Available The cyclin-dependent kinase inhibitor 3 (CDKN3 gene, involved in mitosis, is upregulated in cervical cancer (CC. We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa. CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney. A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17 died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression. In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

  4. CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

    Barrón, Eira Valeria; Roman-Bassaure, Edgar; Sánchez-Sandoval, Ana Laura; Espinosa, Ana María; Guardado-Estrada, Mariano; Medina, Ingrid; Juárez, Eligia; Alfaro, Ana; Bermúdez, Miriam; Zamora, Rubén; García-Ruiz, Carlos; Gomora, Juan Carlos; Kofman, Susana; Pérez-Armendariz, E Martha; Berumen, Jaime


    The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

  5. Codon Usage and 3' UTR Length Determine Maternal mRNA Stability in Zebrafish.

    Mishima, Yuichiro; Tomari, Yukihide


    The control of mRNA stability plays a central role in regulating gene expression. In metazoans, the earliest stages of development are driven by maternally supplied mRNAs. The degradation of these maternal mRNAs is critical for promoting the maternal-to-zygotic transition of developmental programs, although the underlying mechanisms are poorly understood in vertebrates. Here, we characterized maternal mRNA degradation pathways in zebrafish using a transcriptome analysis and systematic reporter assays. Our data demonstrate that ORFs enriched with uncommon codons promote deadenylation by the CCR4-NOT complex in a translation-dependent manner. This codon-mediated mRNA decay is conditional on the context of the 3' UTR, with long 3' UTRs conferring resistance to deadenylation. These results indicate that the combined effect of codon usage and 3' UTR length determines the stability of maternal mRNAs in zebrafish embryos. Our study thus highlights the codon-mediated mRNA decay as a conserved regulatory mechanism in eukaryotes.

  6. Reduced fructosamine-3-kinase activity and its mRNA in human distal colorectal carcinoma.

    Notarnicola, M; Caruso, Maria G; Tutino, V; Guerra, V; Frisullo, S; Altomare, D F; Misciagna, G


    Fructosamine-3-Kinase (FN3K) is an enzyme phosphorilating fructoselysine (FL) residues on glycated proteins, resulting in the production of protein-bound FL-3-phosphate. The pathological role of the non-enzymatic modification of proteins by reducing sugars has become increasingly evident in various types of disorders, including the cancer. In this study, our aim was to study FN3K enzyme activity, as well as its mRNA in human colorectal cancer (CRC). Thirty consecutive CRC patients undergoing surgery of the colon were enrolled in the study. FN3K enzymatic activity and gene expression were analyzed using a radiometric assay and quantitative RT-PCR, respectively. FN3K is a functionally active enzyme in human colon tissue, without significant differences between normal mucosa and cancer. The mean level of FN3K mRNA was significantly lower in cancer than in the corresponding normal colorectal mucosa The colorectal tumors located on the left side showed lower levels of both enzymatic activity and mRNA FN3K than tumors located in the right side of colon. This paper is the first studying FN3K enzyme activity in human CRC, showing a significant relationship between enzymatic activity, its mRNA and tumor side.

  7. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V


    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  8. Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi.

    Tanaka, Mizuki; Tokuoka, Masafumi; Gomi, Katsuya


    Filamentous fungi, particularly Aspergillus species, have recently attracted attention as host organisms for recombinant protein production. Because the secretory yields of heterologous proteins are generally low compared with those of homologous proteins or proteins from closely related fungal species, several strategies to produce substantial amounts of recombinant proteins have been conducted. Codon optimization is a powerful tool for improving the production levels of heterologous proteins. Although codon optimization is generally believed to improve the translation efficiency of heterologous genes without affecting their mRNA levels, several studies have indicated that codon optimization causes an increase in the steady-state mRNA levels of heterologous genes in filamentous fungi. However, the mechanism that determines the low mRNA levels when native heterologous genes are expressed was poorly understood. We recently showed that the transcripts of heterologous genes are polyadenylated prematurely within the coding region and that the heterologous gene transcripts can be stabilized significantly by codon optimization, which is probably attributable to the prevention of premature polyadenylation in Aspergillus oryzae. In this review, we describe the detailed mechanism of premature polyadenylation and the rapid degradation of mRNA transcripts derived from heterologous genes in filamentous fungi.

  9. Cellular differentiation state modulates the mRNA export activity of SR proteins.

    Botti, Valentina; McNicoll, François; Steiner, Michaela C; Richter, Florian M; Solovyeva, Anfisa; Wegener, Marius; Schwich, Oliver D; Poser, Ina; Zarnack, Kathi; Wittig, Ilka; Neugebauer, Karla M; Müller-McNicoll, Michaela


    SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells. © 2017 Botti et al.

  10. Model of EF4-induced ribosomal state transitions and mRNA translocation

    Xie, Ping


    EF4, a highly conserved protein present in bacteria, mitochondria and chloroplasts, can bind to both the posttranslocation and pretranslocation ribosomal complexes. When binding to the posttranslocation state, it catalyzes backward translocation to a pretranslocation state. When binding to the pretranslocation state, it catalyzes transition to another pretranslocation state that is similar and possibly identical to that resulting from the posttranslocation state bound by EF4, and competes with EF-G to regulate the elongation cycle. However, the molecular mechanism on how EF4 induces state transitions and mRNA translocation remains unclear. Here, we present both the model for state transitions induced by EF4 binding to the posttranslocation state and that by EF4 binding to the pretranslocation state, based on which we study the kinetics of EF4-induced state transitions and mRNA translocation, giving quantitative explanations of the available experimental data. Moreover, we present some predicted results on state transitions and mRNA translocation induced by EF4 binding to the pretranslocation state complexed with the mRNA containing a duplex region.

  11. Methods to analyze microRNA-mediated control of mRNA translation.

    Clancy, Jennifer L; Nousch, Marco; Humphreys, David T; Westman, Belinda J; Beilharz, Traude H; Preiss, Thomas


    MicroRNAs (miRs) are an important class of gene regulators that affect a wide range of biological processes. Despite the early recognition of miRs as translational regulators and intense interest in studying this phenomenon, it has so far not been possible to derive a consensus model for the underlying molecular mechanism(s). The potential of miRs to act in a combinatorial manner and to also promote mRNA decay creates conceptual and technical challenges in their study. Here, we discuss critical parameters in design and analysis of experiments used to study miR function including creation of synthetic miR and mRNA partners for assay of translational inhibition using luciferase reporters; measurement of mRNA stability after miR action; defining poly(A) tail length in miR target mRNA; determining the distribution of miRs and their target mRNAs in polysome profiles; and visualization of P-body components. We describe protocols for each of these procedures.

  12. Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation.

    Wong, Queenie Wing-Lei; Vaz, Candida; Lee, Qian Yi; Zhao, Tian Yun; Luo, Raymond; Archer, Stuart K; Preiss, Thomas; Tanavde, Vivek; Vardy, Leah A


    The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.

  13. poolMC: Smart pooling of mRNA samples in microarray experiments

    Schiefelbein John


    Full Text Available Abstract Background Typically, pooling of mRNA samples in microarray experiments implies mixing mRNA from several biological-replicate samples before hybridization onto a microarray chip. Here we describe an alternative smart pooling strategy in which different samples, not necessarily biological replicates, are pooled in an information theoretic efficient way. Further, each sample is tested on multiple chips, but always in pools made up of different samples. The end goal is to exploit the compressibility of microarray data to reduce the number of chips used and increase the robustness to noise in measurements. Results A theoretical framework to perform smart pooling of mRNA samples in microarray experiments was established and the software implementation of the pooling and decoding algorithms was developed in MATLAB. A proof-of-concept smart pooled experiment was performed using validated biological samples on commercially available gene chips. Differential-expression analysis of the smart pooled data was performed and compared against the unpooled control experiment. Conclusions The theoretical developments and experimental demonstration in this paper provide a useful starting point to investigate smart pooling of mRNA samples in microarray experiments. Although the smart pooled experiment did not compare favorably with the control, the experiment highlighted important conditions for the successful implementation of smart pooling - linearity of measurements, sparsity in data, and large experiment size.

  14. Induction of interleukin-1β mRNA after focal cerebral ischaemia in the rat

    Buttini, M.; Sauter, A.; Boddeke, H.W.G.M.


    The expression of interleukin-1β (IL-1β) mRNA in the brain in response to cerebral ischaemia in rats was examined using in situ hybridization histochemistry. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent occlusion of the left middle cerebral artery (MCAO). Wher

  15. Light-dark condition regulates sirtuin mRNA levels in the retina.

    Ban, Norimitsu; Ozawa, Yoko; Inaba, Takaaki; Miyake, Seiji; Watanabe, Mitsuhiro; Shinmura, Ken; Tsubota, Kazuo


    Sirtuins (Sirt1-7) are nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases/ADP-ribosyltransferases that modulate many metabolic responses affecting aging. Sirtuins expressed in tissues and organs involved in systemic metabolism have been extensively studied. However, the characteristics of sirtuins in the retina, where local energy expenditure changes dynamically in response to light stimuli, are largely unknown. Here we analyzed sirtuin mRNA levels by real-time PCR, and found that all seven sirtuins are highly expressed in the retina compared with other tissues, such as liver. We then analyzed the sirtuin mRNA profiles in the retina over time, under a 12-h light/12-h dark cycle (LD condition) and in constant darkness (DD condition). All seven sirtuins showed significant daily variation under the LD condition, with all except Sirt6 being increased in the dark phase. The expression patterns were different under the DD condition, suggesting that sirtuin mRNA levels except Sirt6 are affected by light-dark condition. These findings were not obtained in the brain and liver. In addition, the mRNA expression patterns of Nicotinamide phosphoribosyltransferase (Nampt), peroxisome proliferator-activated receptor gamma coactivator (PGC1α), and transcription factor A, mitochondrial (Tfam) in the retina, were similar to those of the sirtuins except Sirt6. Our observations provide new insights into the metabolic mechanisms of the retina and the sirtuins' regulatory systems.

  16. Effect of dietary betaine supplementation on mRNA level of ...



    Mar 22, 2012 ... age, the significant difference in egg production was observed between the control and 0.08% ... Key words: Betaine, CpG methylation, gene expression, laying hen, lipogenesis genes. ... as the FAS mRNA level in abdominal adipose tissue in finishing pigs with less fat deposition (Huang et al., 2008).

  17. Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

    Birgisdottir, Asa B; Nielsen, Henrik; Beckert, Bertrand;


    processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor...

  18. c-Myc deregulation induces mRNA capping enzyme dependency

    Lombardi, Olivia; Varshney, Dhaval; Phillips, Nicola M.; Cowling, Victoria H.


    c-Myc is a potent driver of many human cancers. Since strategies for directly targeting c-Myc protein have had limited success, upstream regulators and downstream effectors of c-Myc are being investigated as alternatives for therapeutic intervention. c-Myc regulates transcription and formation of the mRNA cap, which is important for transcript maturation and translation. However, the direct mechanism by which c-Myc upregulates mRNA capping is unclear. mRNA cap formation initiates with the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide, catalysed by mRNA capping enzyme (CE/RNGTT). Here we report that c-Myc increases the recruitment of catalytically active CE to RNA polymerase II and to its target genes. c-Myc-induced target gene expression, cell proliferation and cell transformation is highly dependent on CE, but only when c-Myc is deregulated. Cells retaining normal control of c-Myc expression are insensitive to repression of CE. c-Myc expression is also dependent on CE. Therefore, inhibiting CE provides an attractive route for selective therapeutic targeting of cancer cells which have acquired deregulated c-Myc. PMID:27756891

  19. c-Myc deregulation induces mRNA capping enzyme dependency.

    Lombardi, Olivia; Varshney, Dhaval; Phillips, Nicola M; Cowling, Victoria H


    c-Myc is a potent driver of many human cancers. Since strategies for directly targeting c-Myc protein have had limited success, upstream regulators and downstream effectors of c-Myc are being investigated as alternatives for therapeutic intervention. c-Myc regulates transcription and formation of the mRNA cap, which is important for transcript maturation and translation. However, the direct mechanism by which c-Myc upregulates mRNA capping is unclear. mRNA cap formation initiates with the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide, catalysed by mRNA capping enzyme (CE/RNGTT). Here we report that c-Myc increases the recruitment of catalytically active CE to RNA polymerase II and to its target genes. c-Myc-induced target gene expression, cell proliferation and cell transformation is highly dependent on CE, but only when c-Myc is deregulated. Cells retaining normal control of c-Myc expression are insensitive to repression of CE. c-Myc expression is also dependent on CE. Therefore, inhibiting CE provides an attractive route for selective therapeutic targeting of cancer cells which have acquired deregulated c-Myc.

  20. Ammonium Chloride Ingestion Attenuates Exercise-Induced mRNA Levels in Human Muscle.

    Johann Edge

    Full Text Available Minimizing the decrease in intracellular pH during high-intensity exercise training promotes greater improvements in mitochondrial respiration. This raises the intriguing hypothesis that pH may affect the exercise-induced transcription of genes that regulate mitochondrial biogenesis. Eight males performed 10x2-min cycle intervals at 80% VO2speak intensity on two occasions separated by ~2 weeks. Participants ingested either ammonium chloride (ACID or calcium carbonate (PLA the day before and on the day of the exercise trial in a randomized, counterbalanced order, using a crossover design. Biopsies were taken from the vastus lateralis muscle before and after exercise. The mRNA level of peroxisome proliferator-activated receptor co-activator 1α (PGC-1α, citrate synthase, cytochome c and FOXO1 was elevated at rest following ACID (P0.05; the difference in PGC-1α mRNA content 2 h post-exercise between ACID and PLA was not significant (P = 0.08. Thus, metabolic acidosis abolished the early post-exercise increase of PGC-1α mRNA and the mRNA of downstream mitochondrial and glucose-regulating proteins. These findings indicate that metabolic acidosis may affect mitochondrial biogenesis, with divergent responses in resting and post-exercise skeletal muscle.

  1. Cellular restriction of retrovirus particle-mediated mRNA transfer.

    Galla, Melanie; Schambach, Axel; Towers, Greg J; Baum, Christopher


    Analyzing cellular restriction mechanisms provides insight into viral replication strategies, identifies targets for antiviral drug design, and is crucial for the development of novel tools for experimental or therapeutic delivery of genetic information. We have previously shown that retroviral vector mutants that are unable to initiate reverse transcription mediate a transient expression of any sequence which replaces the gag-pol transcription unit, a process we call retrovirus particle-mediated mRNA transfer (RMT). Here, we further examined the mechanism of RMT by testing its sensitivity to cellular restriction factors and short hairpin RNAs (shRNAs). We found that both human TRIM5alpha and, to a lesser extent, Fv1 effectively restrict RMT if the RNA is delivered by a restriction-sensitive capsid. While TRIM5alpha restriction of RMT led to reduced levels of retroviral mRNA in target cells, restriction by Fv1 did not. Treatment with the proteasome inhibitor MG132 partially relieved TRIM5alpha-mediated restriction of RMT. Finally, cells expressing shRNAs specifically targeting the retroviral mRNA inhibited RMT particles, but not reverse-transcribing particles. Retroviral mRNA may thus serve as a translation template if not used as a template for reverse transcription. Our data imply that retroviral nucleic acids become accessible to host factors, including ribosomes, as a result of particle remodeling during cytoplasmic trafficking.

  2. Gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mRNA degradation.

    Emma Abernathy


    Full Text Available Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression.

  3. mRNA profiling for the identification of blood-Results of a collaborative EDNAP exercise

    Haas, Cordula; Hanson, E; Bär, W


    A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, usi...

  4. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    Fitzgerald, J.; Hughes-Fulford, M.


    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P gene osteocalcin was significantly decreased to 44% of control level (P basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  5. TDP-43 suppresses tau expression via promoting its mRNA instability.

    Gu, Jianlan; Wu, Feng; Xu, Wen; Shi, Jianhua; Hu, Wen; Jin, Nana; Qian, Wei; Wang, Xinglong; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei


    In the brains of individuals with Alzheimer's disease (AD) and chronic traumatic encephalopathy, tau pathology is accompanied usually by intracellular aggregation of transactive response DNA-binding protein 43 (TDP-43). However, the role of TDP-43 in tau pathogenesis is not understood. Here, we investigated the role of TDP-43 in tau expression in vitro and in vivo. We found that TDP-43 suppressed tau expression by promoting its mRNA instability through the UG repeats of its 3΄-untranslated region (3΄-UTR). The C-terminal region of TDP-43 was required for this function. Neurodegenerative diseases-causing TDP-43 mutations affected tau mRNA instability differentially, in that some promoted and others did not significantly affect tau mRNA instability. The expression levels of tau and TDP-43 were inverse in the frontal cortex and the cerebellum. Accompanied with cytoplasmic accumulation of TDP-43, tau expression was elevated in TDP-43M337V transgenic mouse brains. The level of TDP-43, which is decreased in AD brains, was found to correlate negatively with the tau level in human brain. Our findings indicate that TDP-43 suppresses tau expression by promoting the instability of its mRNA. Down-regulation of TDP-43 may be involved in the tau pathology in AD and related neurodegenerative disorders. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Endothelin converting-enzyme-1 mRNA expression in human cardiovascular disease

    Bohnemeier, H; Pinto, YM; Horkay, F; Toth, M; Juhasz-Nagy, A; Orzechowski, HD; Bohm, M; Paul, M


    Endothelin-1 converting-enzyme (ECE-1) cleaves the precursor, big-endothelin-1, to the active peptide endothelin-1. The aim of this study was to investigate whether ECE-1 mRNA expression is modified in human cardiovascular disease. Tissue samples from the left human atrium were analyzed for ECE-1 ex

  7. Differential regulation of renal cyclooxygenase mRNA by dietary salt intake

    Jensen, B L; Kurtz, A


    Experiments were done to investigate the influence of dietary salt intake on renal cyclooxygenase (COX) I and II mRNA levels. To this end rats were fed either a low NaCl diet (LS; 0.02% NaCl wt/wt) or a high NaCl diet (HS diet; 4% NaCl wt/wt) for 5, 10 and 20 days. After 10 days Na excretion...... differed 760-fold, plasma renin activity and renin mRNA were increased eight- and threefold in LS compared to HS animals. Total renal COX I mRNA decreased 50% following the LS diet and did not change after the HS diet. Conversely, COX II mRNA declined after HS intake and transiently increased after salt...... depletion. COX I and II mRNAs were unevenly distributed along the cortical-medullary axis with ratios of the cortex:outer medulla:papilla of 1:3:23 and 1:1:2, respectively. Cortical COX mRNAs were inversely regulated by salt intake with eightfold changes in COX II. Conversely, in medullary zones, COX I m...

  8. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    Meller, Amit


    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  9. A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg


    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

  10. Dendritically targeted Bdnf mRNA is essential for energy balance and response to leptin.

    Liao, Guey-Ying; An, Juan Ji; Gharami, Kusumika; Waterhouse, Emily G; Vanevski, Filip; Jones, Kevin R; Xu, Baoji


    Mutations in the Bdnf gene, which produces transcripts with either short or long 3' untranslated regions (3' UTRs), cause human obesity; however, the precise role of brain-derived neurotrophic factor (BDNF) in the regulation of energy balance is unknown. Here we show the relationship between Bdnf mRNA with a long 3' UTR (long 3' UTR Bdnf mRNA), leptin, neuronal activation and body weight. We found that long 3' UTR Bdnf mRNA was enriched in the dendrites of hypothalamic neurons and that insulin and leptin could stimulate its translation in dendrites. Furthermore, mice harboring a truncated long Bdnf 3' UTR developed severe hyperphagic obesity, which was completely reversed by viral expression of long 3' UTR Bdnf mRNA in the hypothalamus. In these mice, the ability of leptin to activate hypothalamic neurons and inhibit food intake was compromised despite normal activation of leptin receptors. These results reveal a novel mechanism linking leptin action to BDNF expression during hypothalamic-mediated regulation of body weight, while also implicating dendritic protein synthesis in this process.

  11. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong


    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism.

  12. Dynamics of translation by single ribosomes through mRNA secondary structures.

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L; Reiche, Michael; Farrell, Ian; Cooperman, Barry S; Goldman, Yale E


    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

  13. Quantitative imaging of single mRNA splice variants in living cells

    Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph


    Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

  14. Hepatic lipase gene is transcribed in rat adrenals into a truncated mRNA

    A.J.M. Verhoeven (Adrie); D. Carling; H. Jansen (Hans)


    textabstractRat adrenals contain a lipase activity that is indistinguishable from hepatic lipase (HL) present in liver. Expression of HL mRNA in adrenals was studied using the method of reverse transcription-polymerase chain reaction (RT-PCR). A 596-bp fragment of HL cD

  15. Induction of interleukin-1β mRNA after focal cerebral ischaemia in the rat

    Buttini, M.; Sauter, A.; Boddeke, H.W.G.M.


    The expression of interleukin-1β (IL-1β) mRNA in the brain in response to cerebral ischaemia in rats was examined using in situ hybridization histochemistry. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent occlusion of the left middle cerebral artery (MCAO). Wher

  16. The human mRNA encoding the Goodpasture antigen is alternatively spliced.

    Bernal, D; Quinones, S; Saus, J


    The noncollagenous (NC1) domain of the human collagen alpha 3(IV)-chain is the primary target of autoantibodies produced in Goodpasture syndrome and, therefore, has been designated as the Goodpasture antigen. In this report, we show that Goodpasture antigen mRNA undergoes processing to at least two alternatively spliced forms in a variety of human tissues, resulting in the exclusion of sequence encoded by either one or two exons. Interestingly, no alternatively spliced forms were observed in bovine or rat tissues. The derived amino acid sequences of the two variant mRNA forms are identical and significantly shorter than that arising from the complete Goodpasture antigen mRNA. They lack the carboxyl-terminal region contributing to the formation of the Goodpasture epitope and all but one of the cysteines found in the complete form. These sequence characteristics suggest that, if translated, the variant Goodpasture antigen is likely to be defective in triple helix formation and no longer reactive with Goodpasture autoantibodies. Although each tissue expressing Goodpasture antigen displayed a specific mRNA pattern, the complete form was always the most abundant and was present at levels apparently unrelated to whether or not the organ of origin is a potential target in Goodpasture syndrome. Furthermore, the antigen sequence was identical in the kidneys of normal and Goodpasture-affected individuals, and no major differences in the expression of the complete and spliced forms were observed.

  17. Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes

    Jones Sophie


    Full Text Available Abstract Background Accurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored. Methods Three gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA and reverse-transcriptase PCR (RT-PCR. Results Successful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p  Conclusions This study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.

  18. In situ hybridization of cytokine mRNA using alkaline phosphatase-labelled oligodeoxynucleotide probes

    Clausen, Bettina Hjelm; Fenger, Christina; Finsen, B.


    In situ hybridization is a powerful tool for visualizing cellular gene expression in morphologically preserved brain tissue giving precise information on the regional expression of specific mRNA sequences in cells of diverse phenotype. Here, we describe a sensitive, simple, and robust method using...

  19. DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients.

    Sasaki, Hidefumi; Shitara, Masayuki; Yokota, Keisuke; Okuda, Katsuhiro; Hikosaka, Yu; Moriyama, Satoru; Yano, Motoki; Fujii, Yoshitaka


    Discoidin domain receptor 2, DDR2, is a tyrosine kinase receptor for fibrillar collagen that is involved in postnatal development, tissue repair and primary and metastatic cancer progression. Recently, mutations in the DDR2 kinase gene were identified in squamous cell lung cancer from large-scale Sanger sequencing. The present study investigated the DDR2 gene mutations and mRNA expression in surgically treated non-small cell lung cancer (NSCLC) of squamous histology cases. The presence or absence of DDR2 mutations at the kinase and discoidin domain was analyzed by direct sequencing. In this cohort, DDR2 mutations were not observed in the 166 patients with lung cancer, although DDR2 polymorphisms were observed (H136H, n=14) at the discoidin domain. mRNA levels of DDR2 in lung tumor samples and the adjacent normal lung samples were simultaneously analyzed. DDR2 mRNA levels were significantly decreased in tumor samples compared with normal lung samples. However, the DDR2 mRNA levels were elevated in the DDR2 polymorphism cases.

  20. Exercise increases hexokinase II mRNA, but not activity in obesity and type 2 diabetes.

    Cusi, K J; Pratipanawatr, T; Koval, J; Printz, R; Ardehali, H; Granner, D K; Defronzo, R A; Mandarino, L J


    Glucose phosphorylation, catalyzed by hexokinase, is the first committed step in glucose uptake in skeletal muscle. Hexokinase II (HKII) is the isoform that is present in muscle and is regulated by insulin and muscle contraction. Glucose phosphorylation and HKII expression are both reduced in obese and type 2 diabetic subjects. A single bout of exercise increases HKII mRNA and activity in muscle from healthy subjects. The present study was performed to determine if a moderate exercise increases HKII mRNA expression and activity in patients with type 2 diabetes. Muscle biopsies were performed before and 3 hours after a single bout of cycle ergometer exercise in obese and type 2 diabetic patients. HKII mRNA and activity and glycogen synthase activity were determined in the muscle biopsies. Exercise increased HKII mRNA in obese and diabetic subjects by 1.67 +/- 0.34 and 1.87 +/- 0.26-fold, respectively (P obese nondiabetic or diabetic subjects. Exercise increased glycogen synthase activity (GS(0.1) and GS(FV)) significantly in both obese nondiabetic and type 2 diabetic patients. The present results indicate that there is a posttranscriptional defect in the response of HKII expression to exercise in obese and type 2 diabetic subjects. This defect may contribute to reduced HKII activity and glucose uptake in these patients.

  1. Changes of mRNA expression of enkephalin and prodynorphin in hippocampus of rats with chronic immobilization stress

    Jia-Xu Chen; Wei Li; Xin Zhao; Jian-Xin Yang; Hong-Yan Xu; Zhu-Feng Wang; Guang-Xin Yue


    AIM: To observe the changes of enkephalin mRNA and prodynorphin mRNA in hippocampus of rats induced by chronic immobilization stress.METHODS: Thirty rats were randomly divided into three groups of 10 each: the normal control group (group A),the group induced by chronic immobilization stress for 7 d (group B) and the group induced by chronic immobilization stress for 21 d (group C). The changes of the enkephalin mRNA and prodynorphin mRNA in the rat hippocampus were detected by reverse transcription-polymerase chain reaction (RT-PCR).RESULTS: Expression levels of enkephalin mRNA and prodynorphin mRNA in rat hippocampus were significantly increased under chronic immobilization stress, and the expression of prodynorphin mRNA in the rat hippocampus in group C was remarkably higher than that in group B (0.624±0.026; n = 5; P<0.01).CONCLUSION: The increased enkephalin mRNA and prodynorphin mRNA gene expressions in rat hippocampus were involved in chronic stress.

  2. Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.

    Kirschman, Jonathan L; Bhosle, Sushma; Vanover, Daryll; Blanchard, Emmeline L; Loomis, Kristin H; Zurla, Chiara; Murray, Kathryn; Lam, Blaine C; Santangelo, Philip J


    The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Expression and its Clinical Signiifcance of CK19 mRNA in Peripheral Blood of Patients with Gastric Cancer

    Zhu Minghui; Zhu Li; Qin Youjuan; Chen Lin


    Objective:To investigate the expression of targeted gene CK19 mRNA in peripheral blood of the patients with gastric cancer and its correlation with biological behaviors of gastric cancer. Methods: CK19 mRNA in peripheral blood of 56 patients with gastric cancer was detected by RT-PCR method. Meanwhile, the peripheral blood of 12 healthy volunteers and 12 gastric cancer tissues were respectively selected as negative and positive controls. The correlation between CK19 mRNA expression in peripheral blood of the patients with gastric cancer and clinical pathological characteristics was analyzed. Results: The positive rate of CK19 mRNA in peripheral blood of 56 patients with gastric cancer was 58.9% (33/56). The expression rate of CK19 mRNA in 12 gastric cancer tissues came up to 100.0% (12/12), whereas CK19 mRNA in peripheral blood of 12 healthy volunteers was expressed negatively. CK19 mRNA expression was signiifcantly related to the clinical staging of gastric cancer and lymphatic metastasis (P0.05). Conclusion: The application of RT-PCR was highly sensitive and speciifc in detecting the CK19 mRNA in peripheral blood of patients with gastric cancer, in which CK19 mRNA is expected to be a tumor marker for judging the metastasis and recurrence and evaluating the efifcacyof gastric cancer.

  4. Dendritic transport element of human arc mRNA confers RNA degradation activity in a translation-dependent manner.

    Ninomiya, Kensuke; Ohno, Mutsuhito; Kataoka, Naoyuki


    Localization of mRNA in neuronal cells is a critical process for spatiotemporal regulation of gene expression. Cytoplasmic localization of mRNA is often conferred by transport elements in 3' untranslated region (UTR). Activity-regulated cytoskeleton-associated protein (arc) mRNA is one of the localizing mRNAs in neuronal cells, and its localization is mediated by dendritic targeting element (DTE). As arc mRNA has introns in its 3' UTR, it was thought that arc mRNA is a natural target of nonsense-mediated mRNA decay (NMD). Here, we show that DTE in human arc 3' UTR has destabilizing activity of RNA independent of NMD pathway. DTE alone was able to cause instability of the reporter mRNA and this degradation was dependent on translation. Our results indicate that DTE has dual activity in mRNA transport and degradation, which suggests the novel spatiotemporal regulation mechanism of activity-dependent degradation of the mRNA.


    Tânia Pitombo de Oliveira


    Full Text Available Monografia de linguística que trata da linguagem de jornal dentro da área econômica. Focaliza essencialmente o léxico da linguagem técnica desta área de interesse, de um artigo de jornal. Elaborada para a disciplina de Linguística V, Departamento de Letras, Centro de Ciências Humanas, Letras e Artes da Universidade Estadual de Maringá, em 1985.

  6. Intestinal PTGS2 mRNA levels, PTGS2 gene polymorphisms, and colorectal carcinogenesis.

    Lotte K Vogel

    Full Text Available BACKGROUND & AIMS: Inflammation is a major risk factor for development of colorectal cancer (CRC. Prostaglandin synthase cyclooxygenase-2 (COX-2 encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation. METHODS: PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466, G-765C (rs20417, T8473C (rs5275 were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort. RESULTS: PTGS2 mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001 and (P<0.035 and compared to mucosa from healthy individuals (P<0.0039 and (P<0.0027, respectively. In CRC patients, PTGS2 mRNA levels were 8-9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001. PTGS2 A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR = 0.52, 95% confidence interval (95% CI: 0.28-0.99, P = 0.047. Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele (PTGS2 AGC were at increased risk of CRC as compared to homozygous carriers of the PTGS2 AGT (A-1195G, G-765C, T8473C haplotype (OR = 5.37, 95% CI: 1.40-20.5, P = 0.014. No association between the investigated polymorphisms and PTGS2 mRNA levels could be detected. CONCLUSION: High intestinal PTGS2 mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. PTGS2 polymorphisms that have been associated with altered PTGS2 mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both PTGS2 polymorphisms and PTGS2 mRNA levels may provide information


    陈颖; 赵雨杰; 郑华川; 杨雪飞; 汪桂兰; 辛彦


    Objective: To investigate the mRNA expression of PTEN and vascular endothelial growth factor (VEGF) genes in ovarian cancer. Methods:We examined mRNA expression of PTEN and VEGF165 in normal ovary (n=5), ovarian cyst (n=5), ovarian borderline tumor (n=9), epithelial ovarian cancer (n=60) and ovarian cancer cell line (CAOV-3) by RT-PCR. Their expressions were compared with clinicopathological features of ovarian cancer. The relationship between their expressions was concerned in all ovarian samples as well. Results:mRNA expression level of PTEN gene was significantly lower in ovarian borderline tumor or ovarian cancer than that in normal ovary or ovarian cyst(P<0.05). It was negatively correlated with clinicopathological staging(P<0.05),whereas positively with histological differentiation (P<0.05). mRNA expression level of PTEN gene was significantly lower in ovarian endometrioid cancer than ovarian serous or mucinous cancer(P<0.05). mRNA expression level of VEGF165 gene was significantly higher in ovarian cancer than that in normal ovary or ovarian cyst(P<0.05). It was positively correlated with clinicopathological staging(P<0.05), whereas negatively with histological differentiation (P<0.05). mRNA expression level of VEGF165 gene was significantly higher in ovarian serous cancer than in other ovarian epithelial cancers (P<0.05). mRNA expression of VEGF165 gene was inversely correlated with mRNA expression level of PTEN gene. Conclusion:Down-regulated expression of PTEN and up-regulated expression of VEGF were considered as two important events in tumorigenesis of ovarian cancer and could be used as molecular markers to indicate the pathobiological behaviors of ovarian cancer. Decreased PTEN expression and increased VEGF expression were closely associated with tumorigenesis and pathobiological behaviors of ovarian endometrioid and serous cancer respectively. Reduced expression of PTEN gene might be involved in carcinogenesis and progression of ovarian cancer by

  8. Protor-2 interacts with tristetraprolin to regulate mRNA stability during stress

    Holmes, Brent; Artinian, Nicholas; Anderson, Lauren; Martin, Jheralyn; Masri, Janine; Cloninger, Cheri; Bernath, Andrew; Bashir, Tariq; Benavides-Serrato, Angelica; Gera, Joseph


    The A/U-rich RNA binding protein tristetraprolin (TTP) is an mRNA destabilizing factor which plays a role in the regulated turnover of many transcripts encoding proteins involved in immune function and cell growth control. TTP also plays a role in stress-induced destabilization of mRNAs. Here we report the interaction of TTP with a component of the mTORC2 kinase, Protor-2 (PRR5-L, protein Q6MZQ0/FLJ14213/CAE45978). Protor-2 is structurally similar to human PRR5 and has been demonstrated to bind mTORC2 via Rictor and/or Sin1 and may signal downstream events promoting apoptosis. Protor-2 dissociates from mTORC2 upon hyperactivation of the kinase and is not required for mTORC2 integrity or activity. We identified Protor-2 in a yeast two-hybrid screen as a TTP interactor using the C-terminal mRNA decay domain of TTP as bait. The interaction of Protor-2 with TTP was also confirmed in vivo in co-immunoprecipitation experiments and Protor-2 was also detected in immunoprecipitates of rictor. Protor-2 was shown to stimulate TTP-mediated mRNA turnover of several TTP-associated mRNAs (TNF-α, GM-CSF, IL-3 and COX-2) in Jurkat cells when overexpressed while the half-lives of transcripts which do not decay via a TTP-mediated mechanism were unaffected. Knockdown of Protor-2 via RNAi inhibited TTP-mediated mRNA turnover of these TTP-associated mRNAs and inhibited association of TTP with cytoplasmic stress granules (SG) or mRNA processing bodies (P-bodies) following induction of the integrated stress response. These results suggest that Protor-2 associates with TTP to accelerate TTP-mediated mRNA turnover and functionally links the control of TTP regulated mRNA stability to mTORC2 activity. PMID:21964062


    Busato, Paulo César; Universidade Federal do Paraná, Curitiba/PR


    O presente artigo trata das bases que conduzem à estruturação de uma teoria do delito a partir da filosofia da linguagem. Apresenta-se a estrutura formada pela ação significativa, a liberdade de ação e a teoria da norma, bem como uma composição breve da teoria do delito.

  10. Historicidade da filosofia em Hegel e Heidegger

    Christian Iber


    Full Text Available Gostaria de discutir, em forma de teses, a temática da historicidade da filosofia em contraste e na sua divergência em Hegel e Heidegger. Diferente do historicismo, que não soluciona o problema da historicidade da filosofia, porque ele dissolve tendencialmente a filosofia na história da filosofia e não coloca em especial a questão da verdade, Hegel e Heidegger desenvolvem uma teoria genuinamente filosófica da história da filosofia do interesse filosófico pela verdade. Esse interesse genuinamente filosófico pela verdade segue do seu conceito respectivo de filosofia, que inclui em ambos uma posição crítica à Modernidade. Ambos os pensadores não são apenas pensadores modernos, mas também pensadores críticos da Modernidade, de uma Modernidade que entrou no estado de uma autorreflexão. Em Hegel e Heidegger, a filosofia da história da filosofia é ao mesmo tempo a chave para uma filosofia crítica de Modernidade da história.

  11. Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

    Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Li Hong [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States); LeBlanc, Gerald A., E-mail: [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States)


    The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

  12. Resistance artery creatine kinase mRNA and blood pressure in humans.

    Karamat, Fares A; Oudman, Inge; Ris-Stalpers, Carrie; Afink, Gijs B; Keijser, Remco; Clark, Joseph F; van Montfrans, Gert A; Brewster, Lizzy M


    Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

  13. Sex differences in global mRNA content of human skeletal muscle.

    Amy C Maher

    Full Text Available Women oxidize more fat as compared to men during endurance exercise and several groups have shown that the mRNA content of selected genes related to fat oxidation are higher in women (e.g. hormone sensitive lipase, beta-hydroxyacyl-CoA dehydrogenase, CD36. One of the possible mechanisms is that women tend to have a higher area percentage of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that sex would influence the basal mRNA and protein content for genes involved in metabolism and the determination of muscle fiber type. Muscle biopsies from the vastus lateralis were collected from healthy men and women. We examined mRNA content globally using Affymetrix GeneChips, and selected genes were examined and/or confirmed by RT-PCR. Furthermore, we examined protein content by Western blot analysis. Stringent gene array analysis revealed 66 differentially expressed genes representing metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Stringent gene array analysis and RT-PCR confirmed that mRNA for; acyl-coenzyme A acyltransferase 2 (ACAA2, trifunctional protein beta (HADHB, catalase, lipoprotein lipase (LPL, and uncoupling protein-2 (UCP-2 were higher in women. Targeted gene analysis revealed that myosin heavy chain I (MHCI, peroxisome proliferator-activated receptor (PPARdelta were higher in women compared with men. Surprisingly, there were no significant sex based differences in protein content for HADHB, ACAA2, catalase, PPARdelta, and MHC1. In conclusion, the differences in the basal mRNA content in resting skeletal muscle suggest that men and women are transcriptionally "primed" for known physiological differences in metabolism however the mechanism behind sex differences in fiber type remains to be determined.

  14. KSRP modulation of GAP-43 mRNA stability restricts axonal outgrowth in embryonic hippocampal neurons.

    Clark W Bird

    Full Text Available The KH-type splicing regulatory protein (KSRP promotes the decay of AU-rich element (ARE-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4. Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43's ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp(+/- and Ksrp(-/- embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3' UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs.

  15. Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

    Briani, Federica; Curti, Serena; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni


    The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

  16. mRNA trans‐splicing in gene therapy for genetic diseases

    Berger, Adeline; Maire, Séverine; Gaillard, Marie‐Claude; Sahel, José‐Alain; Hantraye, Philippe


    Spliceosome‐mediated RNA trans‐splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post‐transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre‐mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans‐splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans‐splicing, review the different strategies that are under evaluation to lead to efficient trans‐splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487–498. doi: 10.1002/wrna.1347 For further resources related to this article, please visit the WIREs website. PMID:27018401

  17. Low-level lasers and mRNA levels of reference genes used in Escherichia coli

    Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.


    Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

  18. Heme Oxygenase-1 mRNA Expression in Egyptian Patients With Chronic Liver Disease

    Abeer El-Sayed Abd El-Wahab


    Full Text Available Background: Chronic liver disease (CLD is a global medical problem. This disease is associated with increased hepatic oxidative stress. One of the antioxidant enzymes that protect cells against this stress is heme oxygenase-1 (HO-1.Objectives: This study aimed to investigate the mRNA expression of HO-1 in Egyptian patients with CLD and its relation to oxidative stress biomarkers.Patients and Methods: Levels of serum ferritin, carboxyhemoglobin, malondialdehyde (MDA, and erythrocyte-reduced glutathione (GSH were measured, and HO-1 mRNA expression was detected in 45 CLD patients (15 with nonalcoholic steatohepatitis [NASH], 15 with chronic hepatitis C, and 15 with liver cirrhosis and 15 healthy controls.Results: HO-1 mRNA expression was increased in patients with NASH, chronic hepatitis C, and liver cirrhosis compared to controls. The expression in cirrhotic patients was significantly higher than that in patients with NASH and chronic hepatitis C. Compared to controls, patients with NASH, chronic hepatitis C, and liver cirrhosis had higher levels of ferritin, carboxyhemoglobin, and MDA and lower levels of GSH. HO-1 mRNA expression was positively correlated with levels of carboxyhemoglobin, serum ferritin, and serum MDA and negatively correlated with levels of erythrocyte GSH in CLD patients.Conclusions: HO-1 mRNA expression was significantly increased in CLD patients, and the increase reflected the severity of the disease. The significant relationship between the increased HO-1 expression and oxidative stress biomarkers in patients with CLD suggests that HO-1 may play an important role in protecting the liver from oxidative stress-dependent damage. Therefore, induction of HO-1 could be a novel therapeutic option for CLD.

  19. Fases da história da fotografia e a questão da aura, segundo Walter Benjamin

    Orlando José Ribeiro de Oliveira


    Full Text Available Este artigo aborda as fases da história da fotografia propostas por Walter Benjamin, no ensaio Pequena História da Fotografia, e discute as transformações que atingiram a obra de arte na modernidade, à luz do conceito de aura. A periodização estabelecida por Benjamin - apogeu, declínio, revitalização - é ilustrada com trabalhos dos fotógrafos referidos pelo autor no texto em que visa à “compreensão real da essência da arte fotográfica”.

  20. Prevenção da anemia ferropriva em lactentes da Zona da Mata Meridional de Pernambuco

    de Lourdes Perez Diaz Teixeira, Maria


    Esta tese é apresentada sob a forma de um artigo de revisão da literatura e dois artigos originais. A revisão da literatura discorre sobre a importância da deficiência de ferro e a anemia dela decorrente para os lactentes, bem como das estratégias preventivas que têm sido preconizadas. O primeiro artigo original aborda o papel da alimentação, mais especificamente do aleitamento materno exclusivo, na prevenção da anemia no lactente. Aborda ainda a relação entre a anemia materna ...

  1. Clarice Lispector - A hora da estrela: o discurso no panfleto da exposição

    Lucília Maria Sousa Romão

    Full Text Available Este trabalho intenta discutir, à luz da Análise do Discurso francesa, como o conceito de memória e heterogeneidade nos atos de linguagem pode contribuir para uma reflexão sobre os estudos da informação e da documentação. Partindo de recortes do panfleto da exposição sobre Clarice Lispector a hora da estrela, realizada no segundo semestre de 2007 pelo Museu da Língua Portuguesa (Estação da Luz, São Paulo, interpretamos as várias vozes que circulam e sustentam o sujeito e o sentido.

  2. História da fisioterapia em Portugal: da origem a 1966

    Fonseca, João Pedro da


    Mestrado em Fisioterapia. Em 1851, Gleich usa pela primeira vez o termo de Fisioterapia. Surge num momento em que a arte de curar o campo médico, procurava corresponder à necessidade que a sociedade tinha de cuidados de saúde mais eficazes. A área médica procurou a exclusividade da ginástica, da aplicação da mecanoterapia, da electroterapia, da massagem e da hidroterapia. O termo Fisioterapeuta surge em Portugal em 1918 e Serviço de Fisioterapia em 1917 num Instituto da Casa Pia de Lisboa ...

  3. Da ordem. Da cidade. Da literatura: personagens à beira do "ruim do mundo"

    Eliana Kuster


    Full Text Available As grandes cidades estão doentes. Seus sintomas: a quebra da sociabilidade e a irrupção rotineira da violência. Diante disso, ecoa na sociedade um clamor por ordem. No mesmo momento em que a polícia põe a tropa na rua, os literatos criam personagens que dão voz à sensação crescente de que a cidade está deixando de ser uma referência à constituição das identidades urbanas. É a partir da literatura, em especial da obra do escritor Rubem Fonseca, que iremos perscrutar as possibilidades apresentadas pela cidade para o convívio humano.The great cities are sick. Its indications: the sociability in addition and the routine irruption of the violence. Ahead of this, a outcry for order echoes in the society. At the same moment where the policy goes to the street, the writers create personages who give voice to the increasing sensation of that the city is leaving of being a reference to the constitution of the urban identities. It is from literature, in special of the writer Rubem Fonseca, that we will go to investigate the possibilities presented for the city for the human conviviality.

  4. O Património Cultural da Freguesia da Cumieira

    Silva, José Emílio Esteves da


    Dissertação de Mestrado em Ciências da Cultura O património cultural, material e imaterial, caracteriza e identifica uma comunidade através dos tempos. A freguesia da Cumieira, concelho de Santa Marta de Penaguião, possui, na sua área, um vasto património constituído por achados arqueológicos e edificações e por um conjunto de saberes ancestrais, ainda utilizados no seu quotidiano, que faz a história do seu povo. Exemplos dessa riqueza são os indícios de povoamento romano e mouro, a igreja...

  5. O papel da geografia em face da crise ambiental


    Full Text Available resumo Diante da crise ambiental atual, diversas disciplinas científicas têm se voltado para sua análise, a fim de apontar soluções para dirimir ou minimizar os danos ambientais e sociais causados pelas atividades econômicas e por outros tipos de fatores de pressão ambiental. Este artigo propõe-se inicialmente a mostrar sucintamente como mudou a visão geográfica predominante no mundo ocidental a respeito da relação entre as populações humanas e o ambiente. Uma mudança de visão que foi reflexo das mudanças que estavam em curso na geografia como um todo, resguardadas as diferenças entre os países. Discute-se ainda que a crise ambiental é uma crise do conhecimento, como afirmam cada vez mais autores. Este texto culmina com as virtudes e limitações da geografia na busca desse entendimento, no intuito de colaborar para seu avançar.

  6. A arte da fuga: Os mecanismos da liquidez

    José Maria Castro Caldas


    Full Text Available A ideia de “liquidez” enquanto característica da fase actual da modernidade, presente na obra mais recente de Zygmunt Bauman, convida a uma leitura cruzada entre este autor e Keynes. Quer em Bauman, quer em Keynes, a liquidez aplica-se a relações que podem ser facilmente revertidas, ou revertidas a baixo custo, e a sistemas que, sendo caracterizados pela precariedade dos laços que unem os seus elementos constituintes, tendem a ser, eles próprios, instáveis e precários. Em ambos os autores, a liquidez e a especulação surgem como respostas racionais à incerteza e, ao mesmo tempo, como estratégias individuais que contribuem para o aumento do risco sistémico. As duas abordagens são complementares e coerentes. A leitura cruzada sugere a existência de mecanismos da liquidez transversais a diferentes domínios institucionais cuja identificação é aqui ensaiada.

  7. Estudo fitoquímico e avaliação da atividade anticolinesterásica de extratos da casca da raiz da Mimosa tenuiflora (Willd.) Poiret

    Neves, Maiane dos Santos


    Mimosa tenuiflora (Willd.) Poiret (jurema preta) é uma espécie da família Fabaceae nativa da Caatinga, encontrada largamente em todo nordeste brasileiro. Na medicina popular, as cascas do caule e da raiz da jurema preta são utilizadas no tratamento de diversas enfermidades, como queimaduras e inflamações, além de ser empregada tradicionalmente como bebida em rituais indígenas. Com relação à casca da raiz, há poucos estudos descritos na literatura sobre seu perfil químico e propriedades biológ...

  8. Teoria da adequação econômica da conduta: significado econômico da conduta em face da tutela penal antitruste

    Luiz da Silva, Ivan


    Essa tese tem por objetivo o desenvolvimento da teoria da adequação econômica da conduta no direito penal econômico. Vale-se de uma abordagem interdisciplinar abrangendo a Economia, o direito econômico e o direito penal. Para alcançar o desiderato a investigação analisou a intervenção do direito penal em face da atividade econômica e, em especial, os fundamentos da tutela penal antitruste, para fins de estabelecer os contornos teóricos necessários à aplicação das premissas fund...

  9. Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus.

    Standaert, D G; Landwehrmeyer, G B; Kerner, J A; Penney, J B; Young, A B


    NMDA receptors are composed of proteins from two families: NMDAR1, which are required for channel activity, and NMDAR2, which modulate properties of the channels. The mRNA encoding the NMDAR2D subunit has a highly restricted pattern of expression: in the forebrain, it is found in only a small subset of cortical, neostriatal and hippocampal neurons. We have used a quantitative double-label in situ hybridization method to examine the expression of NMDAR2D mRNA in neurochemically defined populations of neurons. In the neostriatum, NMDAR2D was expressed by the interneuron populations marked by preprosomatostatin (SOM), the 67-kDa form of glutamic acid decarboxylase (GAD67), parvalbumin (PARV), and choline acetyltransferase (ChAT) mRNAs but not by the projection neurons expressing beta-preprotachykinin (SP) or preproenkephalin (ENK) mRNAs. In the neocortex, NMDAR2D expression was observed in only a small number of neurons, but these included almost all of the SOM-, GAD67-, and PARV-expressing interneurons. In the hippocampus, NMDAR2D was not present in pyramidal or granule cells, but was abundant in SOM-, GAD67-, and PARV-positive interneurons. NMDAR2D expression appears to be a property shared by interneurons in several regions of the brain. The unique electrophysiological characteristics conveyed by this subunit, which include resistance to blockade by magnesium ion and long channel offset latencies, may be important for the integrative functions of these neurons. NMDAR2D-containing receptor complexes may prove to be important therapeutic targets in human disorders of movement. In addition, the presence of NMDAR2D subunits may contribute to the differential vulnerability of interneurons to excitotoxic injury.

  10. Los daños morales

    Gallo Segoviano, Gerardo


    El daño moral es un daño cuya veracidad es muy difícil de demostrar en el mundo material; hay numerosas teoría sobre cuándo determinar la existencia del daño moral, quiénes son los sujetos susceptibles de sufrir este daño y de qué forma podemos entrar a valorar pecuniariamente este daño. Abordadas las cuestiones generales del daño, el trabajo se orienta hacia la concreción del daño moral que resulta de la lesión de los derechos al honor, a la intimidad personal y familiar y a la propia imagen...

  11. Aspectos incomuns da paracoccidioidomicose infantil

    Achiléa Lisboa Bittencourt


    Full Text Available São relatados dois casos de paracoccidioidomicose (PCM infantil. Em um deles, a principal manifestação da doença foi tumoração abdominal, simulando linfoma maligno não Hodgkin. O outro apresentou acentuado envolvimento articular, com ruptura da cápsula. Representam o segundo e terceiro casos de PCM infantil observados na Bahia, ambos procedentes de áreas urbanas.The authors present two cases of infantil para-coccidioidomycosis. In one case the main presentation was an abdominal mass that mimicked non- Hodgkin lymphoma. In the other case the child had a joint swelling with rupture of the capsule and the clinical diagnosis was piogenic arthritis. They represent the second and third cases of infantile paracoccidioidomycosis observed in the state of Bahia, all coming from urban areas.

  12. Prediction of microRNAs affecting mRNA expression during retinal development

    Cogliati Tiziana


    Full Text Available Abstract Background MicroRNAs (miRNAs are small RNA molecules (~22 nucleotides which have been shown to play an important role both in development and in maintenance of adult tissue. Conditional inactivation of miRNAs in the eye causes loss of visual function and progressive retinal degeneration. In addition to inhibiting translation, miRNAs can mediate degradation of targeted mRNAs. We have previously shown that candidate miRNAs affecting transcript levels in a tissue can be deduced from mRNA microarray expression profiles. The purpose of this study was to predict miRNAs which affect mRNA levels in developing and adult retinal tissue and to confirm their expression. Results Microarray expression data from ciliary epithelial retinal stem cells (CE-RSCs, developing and adult mouse retina were generated or downloaded from public repositories. Analysis of gene expression profiles detected the effects of multiple miRNAs in CE-RSCs and retina. The expression of 20 selected miRNAs was confirmed by RT-PCR and the cellular distribution of representative candidates analyzed by in situ hybridization. The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression. Highly expressed miRNAs included miR-124, miR-125a, miR-125b, miR-204 and miR-9. Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed. Conclusions This study has detected the effect of miRNAs upon mRNA expression in immature and adult retinal tissue and cells. The validity of these observations is supported by the experimental confirmation of candidate miRNA expression and the regulation of predicted target genes following miRNA over-expression. Identified miRNAs are likely to be important in retinal development and function. Misregulation of these miRNAs might contribute to retinal degeneration and disease

  13. Interaction between thymidylate synthase and its cognate mRNA in zebrafish embryos.

    Yuyan Zhang

    Full Text Available Thymidylate synthase (TS, which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 microM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 microM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

  14. Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

    Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J


    The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis

  15. Dis3- and exosome subunit-responsive 3 Prime mRNA instability elements

    Kiss, Daniel L.; Hou, Dezhi [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States); Gross, Robert H. [Dartmouth College, Department of Biological Sciences, Life Sciences Center 343, Hanover, NH 03755 (United States); Andrulis, Erik D., E-mail: [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States)


    Highlights: Black-Right-Pointing-Pointer Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. Black-Right-Pointing-Pointer Identified novel 3 Prime UTR cis-acting element that destabilizes a reporter mRNA. Black-Right-Pointing-Pointer Show exosome subunits are required for cis-acting element-mediated mRNA instability. Black-Right-Pointing-Pointer Define precise sequence requirements of novel cis-acting element. Black-Right-Pointing-Pointer Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3 Prime -5 Prime exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3 Prime untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are

  16. Tissue-specific mRNA expression profiles of human solute carrier 35 transporters.

    Nishimura, Masuhiro; Suzuki, Satoshi; Satoh, Tetsuo; Naito, Shinsaku


    Pairs of forward and reverse primers and TaqMan probes specific to each of 23 human solute carrier 35 (SLC35) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The mRNA expression of SLC35As, SLC35Bs, SLC35Cs, SLC35D1, SLC35D2, SLC35Es, and SLC35F5 was found to be ubiquitous in both adult and fetal tissues. SLC35D3 mRNA was expressed at the highest levels in the adult retina. SLC35F1 mRNA was expressed at high levels in the adult and fetal brain. SLC35F2 mRNA was expressed at the highest levels in the adult salivary gland. Both SLC35F3 and SLC35F4 mRNAs were expressed at the highest levels in the adult cerebellum. Further, individual differences in the mRNA expression levels of human SLC35 transporters in the liver were also evaluated. Our newly determined expression profiles were used to study the gene expression in 31 adult human tissues, 6 fetal human tissues, and 3 cell lines, and tissues with high transcriptional activity for human SLC35 transporters were identified. These results are expected to be valuable for research concerning the clinical diagnosis of disease.

  17. Comparative expression of the mRNA for three intestinal hydrolases during postnatal development in the rat

    Freund, J N; Torp, N; Duluc, I


    The distribution of the mRNA for intestinal aminopeptidase-N, lactase-phlorizin hydrolase and sucrase-isomaltase was compared during rat postnatal development as well as along the longitudinal axis of the intestinal tract including small-intestine and colon. We found out that each mRNA exhibited...

  18. Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia

    Chen, C; Pore, N; Behrooz, A; Ismail-Beigi, F; Maity, A


    Oncogenic transformation and hypoxia both induce glut1 mRNA. We studied the interaction between the ras oncogene and hypoxia in up-regulating glut1 mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras...

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

    Ping Xie


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

  20. Relationship between expression of somatostatin receptors subtype 2 mRNA and estrogen and progesterone receptors in breast cancer

    曾希志; 姚榛祥


    Objectives To observe the expression of somatostatin receptor subtype 2 (SSTR2) mRNA, and investigate the relationship between the expression of SSTR2 mRNA and the expressions of estrogen and progesterone receptors (ERs and PRs) in benign and malignant breast tissues.Methods Samples from a total of 23 breast carcinomas, 16 mammary hyperplasias, and 9 mammary fibroadenomas were analyzed. SSTR2 mRNA expression was examined by in situ hybridization using multiphase oligoprobes. ER and PR expressions were detected by immunohistochemical staining. A computerized image analysis system was utilized to estimate the relative content of SSTR2 mRNA.Results The rate of expression (87.0%) and relative content (0.47) of SSTR2 mRNA in breast cancer were higher than those in benign breast tissue (64%,0.26) (P<0.05). SSTR2 mRNA expression was closely correlated with ER and PR expressions in breast cancer (P<0.05). SSTR2 mRNA was also positively correlated with ER expression in benign breast tissues.Conclusions SSTR2 mRNA expression is higher or in benign breast tissues than in malignant ones. There is a significant positive correlation between SSTR2 mRNA and ER and PR expressions. Combined antiestrogen and somatostatin analogue in treatment of ER-positive breast cancers should be further investigated.