Avaliação da concentração de pectinase no processo de hidrólise-sacarificação do farelo de mandioca para obtenção de etanol

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LEONEL M.

2000-01-01

Full Text Available Neste trabalho objetivou-se avaliar a concentração de pectinase (Pectinex Ultra SP-L no processo de hidrólise-sacarificação do farelo de mandioca para produção de etanol. Foram avaliadas quatro concentrações da enzima pectinase com enzima complemetar as amilases e o tratamento com apenas as amilases. Realizou-se a caracterização do hidrolisado e resíduo fibroso resultantes do processo, e os resultados obtidos demonstraram que a concentração mínima de pectinase para um bom rendimento do processo foi 8Kg enzima/t fibras, com. 89,4% do amido hidrolisado. Quanto ao resíduo fibroso, este apresenta potencialidade de aproveitamento com base para produtos dietéticos ricos em fibras.

Avaliação comparativa da eficácia e toxicidade do antimoniato de N-metil-glucamina e do Estibogluconato de Sódio BP88® no tratamento da leishmaniose cutânea localizada

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Deps Patrícia D.

2000-01-01

Full Text Available Foi realizado ensaio clínico randomizado simples cego para comparar a eficácia e a toxicidade do estibogluconato de Na+ (ES e o antimoniato de N-metil glucamina, o Glucantime®(GL. Sessenta e três pacientes foram distribuídos em um dos dois grupos: 32 pacientes foram tratados com GL e 31 pacientes com o ES. Ambos os grupos receberam 15mg Sb+5/kg/dia durante 20 dias. A toxicidade foi avaliada através do ECG, dosagens de uréia, creatinina, TGO, TGP, fosfatase alcalina, amilase, e lipase, avaliados antes do tratamento, nos dias 10 e 20 do tratamento, e 90 dias após o término do mesmo. No grupo tratado com GL 81% (26/32 dos pacientes curaram, comparados com 77% (24/31 no grupo do ES. Um paciente em cada grupo não respondeu ao tratamento. TGO, TGP, amilase, e lipase foram mais elevadas no grupo que usou o ES (p < 0,05. Concluiu-se que, a eficácia de ambos os tratamentos foram similares, apesar da maior toxicidade observada no grupo tratado com ES.

Milhos com diferentes texturas de endosperma e adição de alfa-amilase na dieta de leitões The effect of corn kernel texture and alpha-amylase addition in performance and digestibility of diets for weaned pigs

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Vanessa Piovesan

2011-11-01

Full Text Available Dois experimentos foram realizados para avaliar a influência de milhos com diferentes texturas de endosperma e adição de alfa amilase na digestibilidade de dietas e no desempenho de leitões na fase de creche. No ensaio de digestibilidade, foram usados 12 leitões castrados (7,96±0,31kg, alojados individualmente em gaiolas de metabolismo, enquanto no desempenho utilizaram-se 75 leitões (7,87±0,67kg distribuídos em 15 gaiolas de creche. Nos dois experimentos, os tratamentos consistiram de três dietas formuladas para serem isonutritivas, mas com milhos de diferentes texturas e adição ou não de alfa amilase, conforme descrito a seguir: 1. grãos de milho semi-dentado; 2. grãos de milho duro e; 3. grãos de milho duro + alfa-amilase. Os resultados de metabolismo mostraram que os valores de energia digestível (ED e metabolizável (EM das dietas foram influenciados pelos tipos de milho, sendo o semi-dentado superior (P0,05 entre os tratamentos no consumo de ração, ganho de peso e/ou conversão alimentar. Dietas com milho duro ou semi-dentado e adição de alfa-amilase não alteram o desempenho dos leitões. Por outro lado, dietas contendo milho semi-dentado apresentam maior energia digestível que dietas com milho duro e a adição de alfa-amilase aumenta a energia de dietas com milho duro.Two experiments were carried out to evaluate the effects of diets with different corn grain textures and amylase supplementation on nutrients digestibility and nursery piglets' performance. Twelve castrated piglets (7.96±0.31kg were individually housed in metabolic crates and 75 piglets (7.87±0.67kg penned in 15 nursery pens. In both experiments the treatments consisted of three isonutritive diets: 1. semi dent corn grain; 2. flint corn grain, and 3. flint corn grain + amylase. There was effect of treatments (P0.05 the performance characteristics studied. In conclusion, diets with flint or semi dent corns and amylase supplementation did not improve

Relação lipase/amilase nas pancreatites agudas de causa biliar e nas pancreatites agudas/crônicas agudizadas de causa alcoólica Lipase/amylase ratio in biliary acute pancreatitis and alcoholic acute/acutized chronic pancreatitis

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Ricardo Custódio Pacheco

2007-03-01

Full Text Available RACIONAL: Pancreatites agudas de causas alcoólica ou biliar podem necessitar de abordagens terapêuticas diferentes. OBJETIVO: Verificar a validade da relação lipase/amilase em diferenciar as causas alcoólica ou biliar na pancreatite aguda/pancreatite crônica agudizada. MÉTODOS: Foram avaliados nove pacientes com pancreatite aguda/pancreatite crônica agudizada alcoólica, todos homens, com idade média (desvio padrão de 39,8 ± 7,0 anos (grupo I e 29 com pancreatite aguda biliar, sendo 8 homens e 21 mulheres, com idade média de 43,6 ± 19,9 anos (grupo II. As amilasemias e lipasemias foram determinadas em pacientes com sintomatologia há, no máximo, 48 horas. A relação lipase/amilase foi calculada utilizando-se valores de amilasemia e lipasemia expressas como múltiplos de seus respectivos valores superiores de referência. RESULTADOS: As médias das lipasemias (4.814 ± 3.670 U/L e amilasemias (1.282 ± 777 U/L no grupo I foram semelhantes às do grupo II (2.697 ± 2.391 e 1.878 ± 1.319 U/L, respectivamente, mas a média das relações lipase/amilase foi significantemente maior no grupo I (4,4 ± 3,6 do que no grupo II (2,2 ± 2,2. Relação lipase/amilase >3 foi significantemente mais freqüente no grupo I (66,7% do que no grupo II (24,1% e diferenciou os dois grupos com sensibilidade de 67% e especificidade de 76%. CONCLUSÕES: 1 as amilasemias e lipasemias não diferenciaram os dois grupos avaliados; 2 relação lipase/amilase >3 é mais freqüente na pancreatite aguda/pancreatite crônica agudizada alcoólica do que na pancreatite aguda biliar, e pode ser útil na diferenciação destas duas causas de pancreatite.BACKGROUND: Alcoholic or biliary acute pancreatitis may need different therapeutic approaches. AIM: Assessing the validity of lipase/amylase ratio in differentiating biliary from alcoholic acute pancreatitis/acutized chronic pancreatitis. METHODS: Nine male patients (mean age and standard deviation: 39.8 ± 7.0 years

Human apolipoprotein B (apoB) mRNA: Identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine

International Nuclear Information System (INIS)

Higuchi, K.; Hospattankar, A.V.; Law, S.W.; Meglin, N.; Cortright, J.; Brewer, H.B. Jr.

1988-01-01

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6,538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2,153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides, designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine

Effect of hyperthermia on experimental acute pancreatitis Efeito da hipertermia na pancreatite aguda experimental

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José Luiz Jesus de Almeida

2006-12-01

ceruleína e submetidos a hipertermia, e grupo II (n = 10, animais com pancreatite aguda induzida por ceruleína mantidos em normotermia. Em todos os grupos foram medidos níveis séricos de amilase, histologia, permeabilidade vascular e conteúdo de água do pâncreas. A pancreatite aguda foi induzida através da administração de duas injeções de ceruleína (20 mcg/ kg. Dose única do corante azul de Evans foi administrada juntamente com a segunda injeção de ceruleína. Todos os animais também receberam 5 mL de solução salina subcutânea. Após a indução, os animais do grupo hipertérmico foram aquecidos com duas lâmpadas de 100 W em gaiola parcialmente isolada. A temperatura corporal foi aumentada para 39,5ºC e mantida neste nível por 45 minutos. Os animais controle foram mantidos em uma segunda gaiola em temperatura ambiente RESULTADOS: Os animais controle tiveram edema, danos histológicos e níveis de amilase típicos do modelo de pancreatite aguda leve com ceruleína. O tratamento com hipertermia melhorou o edema pancreático porém não teve efeito nos nível séricos de amilase e no dano histológico pancreático CONCLUSÕES: Os resultados sugerem efeito benéfico da hipertermia no edema inflamatório da pancreatite aguda leve experimental.

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

Science.gov (United States)

Basura, G J; Walker, P D

1999-08-05

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. Copyright 1999 Elsevier Science B.V.

Perfil enzimático de α-amilase, lipase e tripsina do pâncreas e crescimento do fígado, intestino e pâncreas de frangos de corte na fase de 1 a 21 dias de idade Enzymatic profile of α-amylase, lipase and trypsin in the pancreas and the growth of the liver, intestine and pancreas in broiler chicks from one to 21 days of age

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George Henrique Kling de Moraes

2009-11-01

Full Text Available Com o objetivo de avaliar o desenvolvimento de órgãos do aparelho digestório e o perfil enzimático de α-amilase, lipase e tripsina, realizou-se um experimento com duração de 21 dias utilizando-se pintos de 1 dia, machos, Avian Farm, criados em baterias aquecidas e alimentados à vontade com água e dieta purificada contendo os L-aminoácidos essenciais, ácido L-glutâmico (fonte de nitrogênio não-específico, minerais e vitaminas. O delineamento experimental foi inteiramente casualizado com quatro repetições de dez aves. No primeiro dia de vida e aos 7, 14 e 21 dias de idade, quatro animais foram sacrificados por deslocamento cervical e o fígado, pâncreas e intestino delgado foram removidos e pesados para determinação das atividades de α-amilase, lipase e tripsina. Os pesos absolutos aumentaram com a idade. O peso relativo do pâncreas e dos intestinos reduziu aos 21 dias. As atividades específicas (UI/mg de proteína das enzimas α-amilase e lipase foram maiores no 1º dia, diminuíram no 7º dia, alcançando pico máximo no 14º dia e tornando a reduzir no 21º dia de idade. A atividade específica (UI/mg de proteína de tripsina não se alterou com a idade. As atividades relativas da a-amilase, lipase e tripsina (UI/100 g de peso corporal aumentaram do 1º ao 14º dia de idade e reduziram no 21º de idade. As atividades máximas das enzimas digestivas α-amilase e lipase ocorrem no 14º dia de idade. O perfil enzimático da tripsina, de modo geral, não se altera com a idade, o que pode ser explicado pelo fato de os animais serem criados com dietas purificadas, que não estimulam a produção de enzimas proteolíticas.With the objective of assessing the development of organs of the digestive tract and the enzymatic profile α-amylase, lipase and trypsin, an experiment lasting 21 days was carried out using one-day-old male Avian Farm broiler chicks, reared in heated batteries and fed ad libitum with water and purified diets

Caracterização da agressividade e atividade enzimática de isolados de Colletotrichum spp. associados à antracnose do abacate

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Hugo José Tozze Júnior

Full Text Available RESUMO A antracnose é a principal doença pós-colheita do abacate. O presente trabalho teve como objetivos caracterizar a agressividade e a atividade enzimática de 30 isolados de Colletotrichum spp. de abacate. Abacates ‘Fuerte’ foram inoculados com disco de BDA contendo estruturas de cada isolado e após sete dias mensuraram-se os diâmetros das lesões. Dois grupos de isolados foram distinguidos quanto à agressividade pelo teste de teste de Scott-Knott (p=0,05, um com lesões entre 34,0 e 38,2 mm e outro com lesões entre 38,7 e 44,0 mm de diâmetro. Para caracterização enzimática, avaliaram-se as áreas dos halos formados em substratos específicos para detecção de amilase, celulase, lacase, lipase, pectato liase e protease e a atividade da enzima catalase. Os isolados não produziram amilase suficiente para formação de halo em meio de cultura. Todos os isolados produziram catalase, pectato liase e protease, enquanto 46% produziram lacase e 97% tiveram as atividades da celulase e lipase detectadas. Distinguiram-se dois grupos de isolados para as enzimas catalase e pectato liase, cinco grupos para a celulase, seis grupos para a lacase e sete grupos para as enzimas lipase e protease, pelo teste de teste de Scott-Knott (p=0,05. Não houve relação entre as atividades enzimáticas e a agressividade dos isolados.

Efeito da hipertermia na pancreatite aguda grave experimental

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Maurício Gustavo Ieri Yamanari

Full Text Available OBJETIVO: O objetivo deste estudo é avaliar os efeitos da hipertermia na pancreatite aguda (PA grave experimental induzida por ácido taurocólico. MÉTODO: A PA grave foi induzida pela injeção retrógrada de ácido taurocólico a 2,5% ou 5% no ducto pancreático principal. Após a indução, os animais foram colocados numa gaiola contendo duas lâmpadas de 100 W. A temperatura corporal foi aumentada para 39,5ºC e mantida neste nível por 45 minutos. Foram estudados taxa de mortalidade em 72 horas, permeabilidade vascular no pâncreas, porcentagem de água no tecido pancreático, amilase sérica, histologia (edema, necrose acinar e infiltrado inflamatório e níveis séricos de IL-6 e IL-10. RESULTADOS: Não houve alteração em nenhum dos parâmetros avaliados. CONCLUSÃO: Não há benefício da hipertermia na PA grave experimental induzida por ácido taurocólico.

Production and characterization of amylases from Zea mays malt

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Joana Paula Menezes Biazus

2009-08-01

Full Text Available In this work the α and β-amylase enzymes were obtained from maize (Zea mays malt and were biochemistry characterized. A germination study to obtain the maize malt with good amylase activity was made. The maize seeds were germinated in laboratory and the enzymatic activity was measured daily. Activity dependence to germination time were fitted to an exponential model (A=A0eµt, which showed that the behaviour of enzymatic activity in the germination process was similar to the growth of the microorganism. Its model could be applied to describe the mechanism of α-amylase production for each maize varieties and others cereals. Maize malt characterization showed that α and β-amylase had optimal pH between 4-6.5, optimal temperature 50 and 90ºC, and molecular weight of 67.4 and 47.5kDa, respectively. This work contributed with the advances in biotechnology generating of conditions for application of a new and of low price amylases source.Neste trabalho as enzimas α e β-amilases foram obtidas de malte de milho e depois foram caracterizadas bioquimicamente. Um estudo da germinação foi feito para obtenção do malte com boa atividade amilásica. A germinação ocorreu em escala laboratorial e a atividade enzimática foi medida diariamente. Um modelo exponencial do tipo A=A0eµt foi ajustado a dependência do tempo de germinação com a atividade, mostrando que o comportamento da atividade enzimática no processo de germinação é semelhante ao crescimento de microorganismos. Este modelo pode ser aplicado para descrever o mecanismo de produção da α-amilase para cada variedade de milho e de outros cereais. A caracterização do malte de milho mostrou que as α e β-amilase têm pH ótimo entre 4,0-6,5, temperatura ótima de 50 e 90ºC, e massa molar de 67,4 e 47,5 kDa, respectivamente. Este trabalho contribuiu com os avanços da biotecnologia gerando condições de emprego de uma nova e barata fonte de amilases.

Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

Science.gov (United States)

Stefanis, N C; Bresnick, J N; Kerwin, R W; Schofield, W N; McAllister, G

1998-01-01

The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

Rendimento e processo germinativo do grão na espiga de genótipos de trigo

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Felicio João Carlos

2002-01-01

Full Text Available O objetivo deste trabalho foi estudar o processo germinativo dos grãos na espiga, o rendimento de grãos, a adaptabilidade e a estabilidade em genótipos de trigo (Triticum aestivum L. sensíveis às variações ambientais. Os experimentos foram instalados no Núcleo Experimental do Instituto Agronômico, em Campinas, SP, no período de 1996 a 1998. A germinação na espiga foi avaliada pelo método do "Falling Number", que consiste em determinar o nível de atividade da alfa-amilase nos grãos. A segunda e a terceira colheitas foram realizadas ao sete e quatorze dias após a primeira colheita. O atraso da colheita determinou redução no rendimento, notadamente na terceira colheita; os teores de alfa-amilase foram maiores também na terceira colheita, em virtude da degradação dos grãos expostos ao tempo. O genótipo IAC 289 apresentou adaptabilidade a ambientes favoráveis, mas apresentou suscetibilidade à ocorrência de germinação dos grãos na espiga. Os genótipos IAC 24, Mochis, IAC 370 e IAC 351 foram os mais resistentes à germinação dos grãos na espiga, apresentando baixa atividade de alfa-amilase.

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Chalbos, D; Westley, B; Alibert, C; Rochefort, H

1986-01-24

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant cone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that the authors previously described as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.

Caracterização da atividade amilásica do malte de milho (Zea mays L. = Characterization of amylase activity from maize (Zea mays L. malt

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Joana Paula Menezes Biazus

2006-01-01

Full Text Available Este trabalho objetivou estudar o processo de germinação e a caracterização da atividade bioquímica das amilases do malte de milho (Zea mays L. para gerar uma fonte de amilase de baixo custo e boa atividade enzimática. A atividade enzimática foi monitorada todos os dias durante a germinação das sementes para se obter a melhor condição de produção do malte. Os resultados mostraram que a atividade enzimática nas sementes foi maior no 4º. dia de germinação. A caracterização bioquímica mostrou que as amilases do malte apresentam faixa ótima de pH entre 4,3 e 6, com temperaturas ótimas a 50°C e 80ºC e os valores de Km e Vmax para hidrólise do amido foram de 7,69.10-2 g/L e 7,69.102g/L.min, respectivamente.This work aimed to study the germination process and characterization of the amylolytic activity of the maize (Zea mays L. malt aiming to obtain source amylases at lower cost. Enzymatic activity was monitored all days during the seed germination, for obtaining thebest condition of malt production. Results showed that the enzymatic activity from maize seeds was larger in 4° germination day. Enzymes characterization showed that the maize malt amylases have optimal zone of pH between 4.3 and 6, with optimal temperatures of 50°C and 80ºC. The Km and Vmax values for starch hydrolysis were 7.69.10-2 g/L and 7.69.102g/L.min, respectively.

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

Science.gov (United States)

el Meanawy, M A; Aji, T; Phillips, N F; Davis, R E; Salata, R A; Malhotra, I; McClain, D; Aikawa, M; Davis, A H

1990-07-01

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue.

Science.gov (United States)

Wang, S Y; Gudas, L J

1990-09-15

We have previously isolated several cDNA clones specific for mRNA species that increase in abundance during the retinoic acid-associated differentiation of F9 teratocarcinoma stem cells. One of these mRNAs, J6, encodes a approximately 40 kDa protein as assayed by hybrid selection and in vitro translation (Wang, S.-Y., LaRosa, G., and Gudas, L. J. (1985) Dev. Biol. 107, 75-86). The time course of J6 mRNA expression is similar to those of both laminin B1 and collagen IV (alpha 1) messages following retinoic acid addition. To address the functional role of this protein, we have isolated a full-length cDNA clone complementary to this approximately 40-kDa protein mRNA. Sequence analysis reveals an open reading frame of 406 amino acids (Mr 45,652). The carboxyl-terminal portion of this predicted protein contains a region that is homologous to the reactive sites found among members of the serpin (serine protease inhibitor) family. The predicted reactive site (P1-P1') of this J6 protein is Arg-Ser, which is the same as that of antithrombin III. Like ovalbumin and human monocyte-derived plasminogen activator inhibitor (mPAI-2), which are members of the serpin gene family, the J6 protein appears to have no typical amino-terminal signal sequence.

Mudanças relacionadas ao amaciamento da graviola durante a maturação pós-colheita Changes related to softening of soursop during postharvest maturation

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Maria Auxiliadora Coêlho de Lima

2006-12-01

Full Text Available Este trabalho teve por objetivo avaliar mudanças físicas, químicas e bioquímicas relacionadas ao amaciamento de graviola (Annona muricata L. Crioula durante a maturação. Os frutos foram colhidos no estádio de maturidade fisiológica, em plantas cultivadas na Estação Experimental da Embrapa Agroindústria Tropical, em Pacajus, Ceará, armazenados a 26,3±0,6 ºC e 88±12% de umidade relativa, durante 1, 2, 3, 4 e 5 dias. Utilizou-se o delineamento experimental inteiramente casualizado, com quatro repetições. As variáveis analisadas foram: perda de massa, amido, pectina total e solúvel, e atividades das enzimas amilase, pectinametilesterase, poligalacturonase, alfa-galactosidases e beta-galactosidases. Não houve variação significativa no conteúdo de pectina solúvel. Após cinco dias, a perda de massa atingiu 5%, mas não promoveu murcha dos frutos. As reduções nos conteúdos de amido e de pectina total coincidiram com período de expressiva atividade, respectivamente, das enzimas amilase e da poligalacturonase e beta-galactosidase de parede celular. As modificações mais acentuadas no conteúdo de amido, de pectina total, na solubilização de pectinas e na atividade das enzimas amilase, pectinametilesterase, poligalacturonase e beta-galactosidase de parede celular ocorreram no período do segundo ao quarto dia após a colheita.This work aimed to evaluate the physical, chemical and biochemical changes related to softening during maturation of Crioula soursop fruit (Annona muricata L.. Fruits were harvested at the physiological maturity stage from plants grown at Embrapa Agroindústria Tropical Experimental Station, in Pacajus, CE, Brazil, and were stored at 26.3±0.6 ºC and 88±12% relative humidity, during 1, 2, 3, 4 and 5 days. A completely randomized design was used with four replicates. The following variables were evaluated: weight loss; starch content; total and soluble pectin content and enzymatic activity of amylase

Purificação de amilases de malte de Zea mays Purification of amylases from Zea mays malt

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Joana Paula Menezes Biazus

2010-03-01

Full Text Available Neste trabalho realizou-se a purificação das enzimas α e β-amilases de malte de milho combinando os processos de adsorção em leito expandido com o fracionamento por etanol. Alimentaram-se 5 mL de uma solução a 2% de malte de milho em tampão fosfato 0,05 M a pH 5 em uma coluna contendo as resinas de troca iônica Amberlite IRA 410, em leito fixo de 4 cm fluidizado para 8 cm. A eluição foi promovida com NaCl 0,25M com 14 mL/minuto de vazão descendente e o material eluído foi fracionado com álcool etílico nas concentrações de 40-70%, 50-80% e 60-90% (v/v a 20 e 30 ºC. A recuperação da atividade e o fator de purificação foram de 95% e 23 vezes, respectivamente, na primeira etapa. A melhor condição de fracionamento das enzimas foi apresentada a 50-80% de álcool e 20 ºC, e o fator de purificação ficou acima de 100 vezes.In the present work the purification of α and β-amylase enzymes of maize malt was carried out by expanded bed adsorption and ethanol fractionation downstream processes. 5 mL of 2% maize malt solution in 0.05 M phosphate buffer at pH was injected into a chromatographic column containing Amberlite 410 ion-exchange resin, on a 4 cm fixed bed fluidized to 8 cm. 0.25 M NaCl solution at 14 mL/ minuto of descending flux was used for promotion of the elution, the eluted matter was fractioned by 40-70, 50-80 and 60-90% ethyl alcohol concentration at 20 and 30 ºC. Activity recovery and purification factor were of 95% and 23 times, respectively, in the first step. 50-80% alcohol concentration at 20ºC was the best condition, in which a purification factor of more than 100 times was achieved.

Efeitos de níveis de ácido L-glutâmico e de vitamina K da dieta sobre a atividade de alfa-amilase em frangos de corte Effects of dietary levels of L-glutamic acid and vitamin K in the activity of alpha-amylase of chicks

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Flavia Escapini Fanchiotti

2005-04-01

Full Text Available Foram investigados os efeitos nutricionais de dois níveis de ácido L-glutâmico (L-Glu combinados com quatro níveis de vitamina K (Vit K sobre a atividade de alfa-amilase no quimo e pâncreas de aves de corte. Frangos de corte machos de um dia foram criados em baterias aquecidas e alimentados, à vontade, com dietas contendo todos L-aminoácidos essenciais, minerais e vitaminas (exceto Vit K até os 14 dias de idade. O experimento foi realizado em esquema fatorial, em delineamento inteiramente casualizado 2x4, com quatro repetições de oito aves cada. A dieta básica foi suplementada com 6,25 e 12,5% de L-Glu combinados com 0,02; 0,2; 20,0 e 200,0 mg de Vit K/kg de ração. Efeitos significativos de L-Glu e Vit K foram observados no quimo. A atividade específica máxima (1.005,78±245,25 UI/mg de proteína foi observada nas aves alimentadas com 6,25% de L-Glu e 20,0 mg de Vit K. Houve redução da atividade com a suplementação de 12,5% de L-Glu. No pâncreas, não houve interação entre L-Glu e Vit K, todavia, foi observado efeito de L-Glu sobre as atividades relativas, expressas em UI/g de tecido e UI/100 g de peso corporal. Os resultados sugerem que a associação entre L-Glu e Vit K interfere na atividade enzimática de alfa-amilase em aves de corte submetidas a dietas purificadas. Os resultados indicam que o nível de 12,5% de L-Glu associado aos diferentes níveis de vitamina K reduziu a atividade enzimática.The effects of two levels of L-glutamic acid (L-Glu combined with four levels of vitamin K (Vit K were studied with the objective of evaluating the activities of alpha-amylase in the chyme and pancreas of chicks. Day-old male broilers were reared in eletrically heated batteries, fed with purified amino acids diets, minerals and vitamins (except Vit K at the requirement levels, for 14 days. Feed and water were provided ad libitum. The experimental design was a factorial 2x4 with four replicates with eight chicks each The

Achados bioquímicos, eletrolíticos e hormonais de cabras acometidas com toxemia da prenhez

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Rodolfo J.C. Souto

2013-10-01

Full Text Available A abrangência dos estudos sobre a toxemia da prenhez (TP tem sido constante e inovadora em alguns conceitos, porém o estudo dos casos clínicos naturais em cabras é inexistente no Brasil. Diante disso, realizou-se análise das alterações clínicas, do perfil bioquímico, eletrolítico e hormonal com o objetivo de compreender a fisiopatogenia da enfermidade. Foram avaliadas 22 cabras atendidas na Clínica de Bovinos, Campus Garanhuns/UFRPE, diagnosticadas com TP, no período de 2007 a 2012. Realizou-se exame clínico e coleta de sangue e urina, para análise do perfil bioquímico e hormonal. Acentuadas alterações clínicas foram observadas, como decúbito, apatia, dispneia, aumento da temperatura corpórea, mucosas congestas, vasos episclerais injetados, desidratação, anorexia, hipomotilidade ou atonia ruminal, edema nos membros. Os resultados laboratoriais revelaram valores elevados para ureia, creatinina, ácidos graxos não esterificados (AGNE, β-hidroxibutirato, amilase e cortisol, enquanto as variáveis: proteína total, albumina, cálcio total, cálcio ionizado, fósforo, a relação cálcio e fósforo, sódio e insulina encontraram-se diminuídos. Outros parâmetros bioquímicos e eletrolíticos, como frutosamina, globulina, cloro, potássio e magnésio mantiveram-se dentro da faixa de normalidade. Quanto a glicemia, foram constatados três condições nas cabras com TP, normoglicêmicas (50%, hiperglicêmicas (27,28% e hipoglicêmicas (22,72%. Dos 17 (77,27% animais que obtiveram alta 10 (58,82% eram normoglicêmicos. Vieram a óbito 22,8 % (5/22, no qual 60% (3/5 delas estavam hiperglicêmicas. A média de nascimento por cabra foi superior a duas crias. Verificou-se existir associação fortemente positiva dos AGNE's com a albumina (r=0,60, fraca com o β-hidroxibutirato (r=0,10 e glicose (r=0,03. Porém, existiu relação fortemente negativa dos AGNE's com a insulina (r= -0,70, moderada com o cortisol (r= -0,52 e amilase (r

EFEITO DA ADIÇÃO DE NUTRIENTES NA FERMENTAÇÃO ALCOÓLICA DE MEIO DE CULTIVO ELABORADO COM FÉCULA DE MANDIOCA

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EDWIL APARECIDA LUCCA GATTAS

2007-01-01

Full Text Available O objetivo do trabalho foi verificar o efeito da adição de sulfato de amônio ou uréia em meios contendo KH2PO4, extrato de levedura e fécula de mandioca dextrinizada na velocidade de produção de etanol em processo de sacarificação – fermentação simultâneas. Ficou demonstrada a necessidade de suplementação do meio de cultivo com nitrogênio, podendo ser utilizados com este fim tanto o sulfato de amônio como a uréia. Nos meios enriquecidos com nutrientes houve aumento na velocidade de fermentação de 41%. Na etapa da sacarificação levada a efeito na temperatura ótima da enzima, observou-se que é indiferente empregar-se as amiloglicosidases comercializadas pela Novo Industri S.A. (Spiritamylase e SAN 200 OL ou pela Pfizer, sempre precedidas pela liquefação com alfa-amilase da Okochi Ltda.

Atividade da amilase no intestino delgado de coelhos alimentados com dietas contendo diferentes níveis de amido antes e após desmame = Amylase activity in the small intestine of rabbits fed with diets containing starch levels before and after weaning

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Haroldo Garcia de Faria

2006-07-01

Full Text Available Para avaliar a atividade amilásica em coelhos após a desmama, foram utilizados 200 animais da Raça Nova Zelândia Branco, submetidos a diferentes padrões de alimentação desde a fase de lactação. Quarenta deles foram abatidos aos 35 dias de idade e 80, aos 50 e aos 70 dias de idade. Para modular o padrão de consumo de leite e ração antes da desmama, as ninhadas equalizadas em 8 láparos do nascimento até ao 16º dia de idade, a partir desta idade, até a desmama, foram submetidos à alta e baixa disponibilidade de leite, mantendo ninhadas com 4 ou 8 láparos recebendo dietas com nível normal (15% e alto (24% de amido. A partir da desmama, os animais de cada um dos 4 padrões de alimentação até a desmama passaram a receber as mesmasdietas com níveis normal e alto de amido, em um esquema fatorial 2x2x2 (ninhadas submetidas à baixa e alta disponibilidade de leite versus dois níveis de amido na dieta até a desmama versus doisníveis de amido na dieta após a desmama. Os resultados mostraram que a manutenção de ninhadas com 4 ou 8 animais entre o 16º e 35º dia de lactação não modificou (P>0,05 a atividade da amilase em qualquer das idades avaliadas após o desmame. O uso de dietas com alto teor de amido durante a lactação aumentou (POne experiment was carried out to study the amylase activity in the small intestine (jejunum and ileum of rabbits from litters maintained on different feeding patterns. Two hundred White New Zealand rabbits were analyzed, out of which 40 35- days-old rabbits, 80 50-days-old rabbits and 80 70-days-old rabbits were slaughtered. To modulate the pattern of feed and milk intake before weaning, the litters were modulated for eight young rabbits until 16 days old. From 16 days until weaning, they were submitted to low or high milkavailability, litters maintained with four or eight young rabbits receiving diets containing normal or high starch level. The animals were distributed in a factorial design 2

A Comparative study for striatal-direct and -indirect pathway neurons to DA depletion-induced lesion in a PD rat model.

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Zheng, Xuefeng; Wu, Jiajia; Zhu, Yaofeng; Chen, Si; Chen, Zhi; Chen, Tao; Huang, Ziyun; Wei, Jiayou; Li, Yanmei; Lei, Wanlong

2018-04-16

Striatal-direct and -indirect Pathway Neurons showed different vulnerability in basal ganglia disorders. Therefore, present study aimed to examine and compare characteristic changes of densities, protein and mRNA levels of soma, dendrites, and spines between striatal-direct and -indirect pathway neurons after DA depletion by using immunohistochemistry, Western blotting, real-time PCR and immunoelectron microscopy techniques. Experimental results showed that: 1) 6OHDA-induced DA depletion decreased the soma density of striatal-direct pathway neurons (SP+), but no significant changes for striatal-indirect pathway neurons (ENK+). 2) DA depletion resulted in a decline of dendrite density for both striatal-direct (D1+) and -indirect (D2+) pathway neurons, and D2+ dendritic density declined more obviously. At the ultrastructure level, the densities of D1+ and D2+ dendritic spines reduced in the 6OHDA groups compared with their control groups, but the density of D2+ dendritic spines reduced more significant than that of D1. 3) Striatal DA depletion down-regulated protein and mRNA expression levels of SP and D1, on the contrary, ENK and D2 protein and mRNA levels of indirect pathway neurons were up-regulated significantly. Present results suggested that indirect pathway neurons be more sensitive to 6OHDA-induced DA depletion. Copyright © 2018 Elsevier Ltd. All rights reserved.

Clinical significance of LUNX mRNA, CK19 mRNA, CEA mRNA expression in detecting micrometastasis from lung cancer

International Nuclear Information System (INIS)

Zhu Guangying; Liu Delin; Chen Jie

2003-01-01

Objective: To evaluate the sensitivity, specificity and clinical significance of CK19 mRNA, CEA mRNA and LUNX mRNA for detecting micrometastasis by sampling the peripheral blood and regional lymph nodes of lung cancer patients. Methods: Reverse transcriptase chain reaction (RT-PCR) was used to detect LUNX mRNA, CK19 mRNA, CEA mRNA for micrometastasis by sampling the peripheral blood of 48 lung cancer patients and 44 regional lymph nodes of such patients treated by curative resection. Peripheral blood of 30 patients with pulmonary benign lesions and 10 normal healthy volunteers and lymph nodes of 6 patients with benign pulmonary diseases served as control. Results: 1) LUNX mRNA, CK19 mRNA, CEA mRNA were expressed in all (35/35) lung cancer tissues. 2) In the peripheral blood from 48 lung cancer patients, 30 (62.5%) were positive for LUNX mRNA, 24 (50.0%) positive for CK19 mRNA and 32(66.7%) positive for CEA mRNA. The positive detection rates of micrometastasis in 44 lymph nodes from lung cancer patients were 36.4% (16 out of 44) for LUNX mRNA, 27.3% (12 out of 44) for CK19 mRNA and 40.9% (18 out of 44) for CEA mRNA. 3) In the 30 blood samples from patients with pulmonary benign diseases, 2 (6.7%) expressed CK19 mRNA, but none expressed LUNX mRNA or CEA mRNA. All the 3 molecular markers were negative in the 10 blood samples from healthy volunteers. In 11 lymph nodes from patients with pulmonary benign lesions, none was positive for any of the three markers. 4) In 44 regional lymph nodes from lung cancer patients, 6 (13.6%) were positive for metastasis by histopathological examination, with a positive rate significantly lower than that of the RT-PCR (P<0.05). 5) The micrometastatic positive rate in the peripheral blood of 40 non-small cell lung cancer (NSCLC) patients was significantly related to TNM stage (P=0.01). Conclusions: LUNX mRNA, CK19 MRNA, CEA mRNA are all appropriate target genes for the detection of micrometastasis from lung cancer. LUNX mRNA and CEA mRNA

Perfil bioquímico do soro de frangos de corte alimentados com dieta suplementada com alfa-amilase de Cryptococcus flavus e Aspergillus niger HM2003 Biochemichal serum profile of broilers fed diets suplemented with alfa-amylase from Cryptococcus flavus and Aspergillus niger HM2003

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Cibele Silva Minafra

2010-12-01

Full Text Available Avaliou-se o perfil bioquímico do soro de frangos de corte alimentados com a enzima α-amilase produzida por dois microrganismos. Produziram-se dois extratos, um com a-amilase obtida a partir de Cryptococcus flavus em meio de levedura comercial e outro com Aspergillus niger HM2003 em meio de proteína de soja e amido comercial, com atividade de 9,58 U/mL e 10,0 U/mL, respectivamente. Utilizaram-se 360 pintos de corte Cobb 500 de 1 dia de idade e com 49,72 ± 0,68 g de peso vivo inicial. As aves foram alojadas em baterias e foram criadas até os 21 dias de idade. Foram utilizados três dietas, cada uma com cinco repetições de 12 aves, em delineamento inteiramente casualizado. A primeira dieta (basal foi formulada sem adição de enzima e as outras duas receberam a suplementação de a-amilase produzida por cultivo de Cryptococcus flavus e Aspergillus niger HM2003. Dietas à base de milho e soja foram formuladas em duas fases: pré-inicial (1-7 dias e inicial (8-21 dias. Na fase pré-inicial, foram observados os seguintes valores médios para cálcio (6,90 e 5,99 mg/dL, proteína plasmática (2,0 e 2,50 g/dL e fosfatase alcalina (979,98 e 974,66 UI/L, respectivamente para Cryptococcus flavus e Aspergillus niger HM2003. A dieta acrescida de a-amilase obtida a partir de Aspergillus niger HM2003 determinou maior concentração sérica de fósforo. Na fase inicial, os resultados significativos relacionaram-se a potássio quando avaliadas dietas com adição de a-amilase pelas duas fontes. A incorporação das enzimas testadas não proporciona alterações metabólicas ou toxicidade nos animais.It was evaluated the biochemical serum profile of broilers fed rations supplemented with α-amylase produced by two microorganisms. Two extracts were produced, one was produced with a-amylase obtained from Cryptococcus flavus in a commercial yeast-based medium and the other with Aspergillus niger HM2003 produced in soybean protein and commercial starch medium

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

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Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

2018-03-29

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

Inhibition of cyclooxygenase-2 in experimental severe acute pancreatitis Inibição da Ciclo-Oxigenase-2 na pancreatite aguda grave experimental

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José Luiz Jesus de Almeida

2006-08-01

amilase, IL-6 e IL-10 e taxa de mortalidade. RESULTADOS: Os níveis séricos de IL-6 e IL-10 foram menores do que no grupo controle. Os níveis séricos de amilase e a taxa de mortalidade permaneceram inalteradas. A análise histológica também não mostraram alterações, exceto pela necrose gordurosa, que foi maior nos animais controle. CONCLUSÃO: A inibição da COX-2 pode reduzir a liberação sistêmica de pelo menos duas citocinas, mas tem pouco efeito na lesão pancreática direta causada pelo taurocolato.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

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Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

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

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Maria Letizia Di Martino

2016-11-01

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.

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

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

1999-02-01

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.)

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

International Nuclear Information System (INIS)

Nakanaga, Kazue; Maeda Shinji; Matsuoka, Masanori; Kashiwabara, Yoshiko

1999-01-01

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, 33 P of 35 P, of which degradation energy is less that 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.)

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

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Daniela Toro-Ascuy

2016-11-01

Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

Analysis of the 3’ untranslated regions of α-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas

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Kelly, Shannan; Yamamoto, Hideki

2008-01-01

Purpose We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)–dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in α-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. Methods 3’-RACE and sequencing were used to isolate and analyze the 3’-UTRs of α-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. Results The following CPE-like sequence was detected in the 3’-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3’-UTRs of α-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. Conclusions The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3’-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of α-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas. PMID:18682811

Efeitos da ligadura do ducto pancreático e da secção ductal com livre drenagem de secreções para o peritônio sobre as funções endócrina e exócrina do pâncreas: estudo clínico e laboratorial em coelhos Endocrine and exocrine consequences of the open and ligated pancreatic duct: clinical and laboratorial findings in rabbits

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Lauro Bogodar Kuczynski

2004-06-01

Full Text Available As ressecções pancreáticas seguidas de anastomoses acompanham-se de altas taxas de morbidade, que incluem: surtos de pancreatite e, principalmente, fístulas digestivas. Nos transplantes pancreáticos a drenagem da parte exócrina do enxerto para o intestino ou para a bexiga, além das peculiaridades técnicas, também não é isenta de diversas complicações. Visando evitar ou atenuar tais conseqüências e simplificar a técnica cirúrgica, têm sido usadas outras abordagens para o tratamento ductal do coto/enxerto pancreático, tais como: drenagem livre de secreções para o peritônio, com o ducto pancreático aberto, ligadura ductal e oclusão do ducto com polímeros sintéticos. OBJETIVO: O presente estudo visa avaliar clínica e laboratorialmente as funções endócrina e exócrina do pâncreas de coelhos com o ducto aberto e ligado. MÉTODOS: Foram realizadas 150 operações, divididas em 3 grupos: N - manipulação/controle (n=50, A - grupo aberto (n=50, e L - ligado (n=50. Os momentos de observação foram pré-operatório, dia 0 (dia da operação e pós-operatório (observação e sacrifício: 7 dias, 14 dias, 28 dias, 90 dias e 180 dias. Os parâmetros analisados foram: estado geral, atividade, controles do peso corporal, ingestão hídrica, ingestão alimentar e dosagens da amilase sangüínea, glicemia, glicose urinária e insulina plasmática. RESULTADOS: Todos os grupos tiveram evolução clínica similar, com bom estado geral, ganho ponderal progressivo e valores normais da ingestão hídrica e ingestão alimentar. Exceto uma significativa elevação da amilase sangüínea nas primeiras 24-48h para os 2 grupos com interferência ductal, não houve qualquer alteração dos níveis basais da glicemia, glicosúria e insulinemia entre os 3 grupos experimentais, em todos os momentos de observação. CONCLUSÃO: À exceção da amilase sangüínea, cujos níveis basais foram significativamente elevados no 1º e 2º dias de p

Primary metabolite mobilization during germination in rosewood (Aniba rosaeodora Ducke seeds Mobilização de metabólitos primários durante a germinação de sementes de pau-rosa (Aniba rosaeodora Ducke

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Renata Braga Souza Lima

2008-02-01

Full Text Available This study aimed to characterize protein, oil, starch and soluble sugar mobilization as well as the activity of alpha-amylase during rosewood seed germination. Germination test was carried out at 25°C and the following parameters were analyzed: percentage of germination, initial, average, and final germination time. Seed reserve quantification was monitored in quiescent seeds and during different stages of radicle growth. Starch mobilization was studied in function of a-amylase activity. Germination reached 87.5% at the initial, average, and final time of 16, 21 and 30 days, respectively. Oil mobilization showed a negative linear behavior, decreasing 40% between the first and the last stage analyzed, whereas protein levels increased 34.7% during the initial period of germination. Starch content (46.4% was the highest among those of the metabolites analyzed and starch mobilization occurred inversely to the observed for soluble sugars; alpha-amylase activity increased until the 15th day, a period before radicle emission and corresponding to the highest starch mobilization. The high percentage of rosewood seed germination may be related to the controlled condition used in the germination chamber as well as to high seed reserve mobilization, in special oil and starch.Este estudo teve por objetivo caracterizar a mobilização de metabólitos primários (proteínas, óleos, amido e açúcares solúveis e determinar a atividade da enzima alfa-amilase em sementes de pau-rosa durante diferentes períodos da germinação. Sementes quiescentes, após a assepsia, foram postas para germinar a 25 °C, analisando-se as seguintes variáveis: porcentagem de germinação e tempos inicial, médio e final de germinação. A quantificação das reservas orgânicas foi monitorada nas sementes quiescentes e nos diferentes estágios de crescimento da radícula. A mobilização do amido foi monitorada mediante a atividade da enzima alfa-amilase. A germinação atingiu

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

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Llopis Ana

2010-03-01

Full Text Available Abstract Background Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'→3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

Sertralina e pancreatite aguda: relato de caso Sertraline and acute pancreatitis: a case-report

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André Malbergier

2004-03-01

Full Text Available A pancreatite aguda é uma patologia grave e com considerável morbidade e mortalidade. Vários fatores são apontados como possíveis causas de pancreatite aguda. Neste relato, será apresentado um caso de pancreatite aguda com possível associação causal com um inibidor seletivo de recaptura de serotonina: sertralina. Após um mês de tratamento com sertralina, uma paciente do sexo feminino, 55 anos, desenvolveu forte dor abdominal e elevação da amilase sérica. Após internação e retirada da sertralina, seus sintomas remitiram e os níveis de amilase voltaram ao normal. Pela potencial gravidade do quadro e pelo amplo uso desta medicação, tal associação deve ser lembrada em investigações de casos de pancreatite aguda.Acute pancreatitis is a severe disease with considerable morbidity and mortality. Many risk factors are causally related to acute pancreatitis. In this report, a case of acute pancreatitis with possible causal relationship with the use of a selective serotonin reuptake inhibitor, sertraline, will be discussed. After one month of treatment with sertraline, a female patient, 55 years-old, developed a severe abdominal pain and showed a serum amylase elevation. She was admitted to the hospital and the use of sertraline was interrupted. After that, the symptoms remitted and the serum amylase level returned to normal. Because of the potential severity of this disease and the widespread use of sertraline, this association should be reminded when investigating possible causes for acute pancreatitis.

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

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Cássio de Barros Pontes

2004-03-01

Full Text Available Clinical aspects and biochemical properties in the saliva of 21 patients prior to and following radiotherapy for head and neck cancer were evaluated (experimental group and compared with the same properties in a control group of 21 subjects free of cancer. Salivary flow was evaluated by measuring the time necessary, in seconds, for the output of 2 ml of stimulated saliva; and the buffering capacity changes were determined using a simple colorimetric method. Total salivary protein concentration was determined by the Bradford4 method. Amylase activity was measured by reducing sugars released from a soluble starch substrate, quantified by the dinitrosalicylic method. The electrophoretic profile was evaluated in polyacrylamid gel (12% SDS-PAGE using samples of 5 mg of salivary protein. A statistically significant reduction (p Foram avaliados alguns aspectos clínicos e algumas propriedades bioquímicas salivares de 21 pacientes, antes e após o tratamento radioterápico para câncer de cabeça e pescoço (grupo experimental e de 21 pacientes sem câncer (grupo controle. O fluxo salivar foi avaliado pelo tempo necessário (segundos para produção estimulada de 2 ml de saliva e a capacidade tamponante determinada frente à utilização de um método colorimétrico simples. A concentração de proteína total salivar foi determinada pelo método de Bradford4. A atividade da amilase foi mensurada através dos açúcares redutores liberados e quantificados pelo método do ácido dinitrossalicílico utilizando a glicose como substrato. O perfil eletroforético foi avaliado em gel de poliacrilamida (SDS-PAGE 12% para amostras salivares contendo 5 mg de proteína. Foi observada, no grupo experimental, redução estatisticamente significativa (p < 0,01 para o fluxo salivar (162,47 s ± 28,30 antes e 568,71 s ± 79,75 após e para a capacidade tamponante (pH 5,45 ± 0,14 antes e 4,40 ± 0,15 após. Não foi observada alteração estatisticamente

mRNA Cancer Vaccines-Messages that Prevail.

Science.gov (United States)

Grunwitz, Christian; Kranz, Lena M

2017-01-01

During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

Science.gov (United States)

Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

2013-01-01

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

International Nuclear Information System (INIS)

Hanova, Monika; Stetina, Rudolf; Vodickova, Ludmila; Vaclavikova, Radka; Hlavac, Pavel; Smerhovsky, Zdenek; Naccarati, Alessio; Polakova, Veronika; Soucek, Pavel; Kuricova, Miroslava; Manini, Paola; Kumar, Rajiv; Hemminki, Kari; Vodicka, Pavel

2010-01-01

Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m 3 ) and high (above 50 mg/m 3 ) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = - 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 ± 1.00 SSB/10 9 Da), followed by high exposure group (0.72 ± 0.81 SSB/10 9 Da) and controls (0.65 ± 0.82 SSB/10 9 Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

Principles of mRNA transport in yeast.

Science.gov (United States)

Heym, Roland Gerhard; Niessing, Dierk

2012-06-01

mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

MnO2 nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

Science.gov (United States)

Ou, Min; Huang, Jin; Yang, Xiaohai; Quan, Ke; Yang, Yanjing; Xie, Nuli; Wang, Kemin

2017-01-01

The donor donor-acceptor (DD-A) FRET model has proven to have a higher FRET efficiency than donor-acceptor acceptor (D-AA), donor-acceptor (D-A), and donor donor-acceptor acceptor (DD-AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the "DD-A" FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary "D-A" model. Furthermore, MnO 2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn 2+ ions by intracellular GSH. The results indicated that the MnO 2 nanosheet mediated "DD-A" FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO 2 nanosheet mediated "DD-A" FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle.

Science.gov (United States)

Shock, Jennifer L; Fischer, Kael F; DeRisi, Joseph L

2007-01-01

The rate of mRNA decay is an essential element of post-transcriptional regulation in all organisms. Previously, studies in several organisms found that the specific half-life of each mRNA is precisely related to its physiologic role, and plays an important role in determining levels of gene expression. We used a genome-wide approach to characterize mRNA decay in Plasmodium falciparum. We found that, globally, rates of mRNA decay increase dramatically during the asexual intra-erythrocytic developmental cycle. During the ring stage of the cycle, the average mRNA half-life was 9.5 min, but this was extended to an average of 65 min during the late schizont stage of development. Thus, a major determinant of mRNA decay rate appears to be linked to the stage of intra-erythrocytic development. Furthermore, we found specific variations in decay patterns superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes. Elucidation of Plasmodium mRNA decay rates provides a key element for deciphering mechanisms of genetic control in this parasite, by complementing and extending previous mRNA abundance studies. Our results indicate that progressive stage-dependent decreases in mRNA decay rate function are a major determinant of mRNA accumulation during the schizont stage of intra-erythrocytic development. This type of genome-wide change in mRNA decay rate has not been observed in any other organism to date, and indicates that post-transcriptional regulation may be the dominant mechanism of gene regulation in P. falciparum.

Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

International Nuclear Information System (INIS)

Kwon, Y.K.; Hecht, N.B.

1991-01-01

The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA

Partial characterization of amylases of two indigenous Central Amazonian rhizobia strains

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Arlem Nascimento de Oliveira

2010-02-01

Full Text Available Amylase production and partial characterization of crude enzyme preparations from two rhizobia strains (R-926 and R-991 were evaluated. For both the strains, maximal amylase activities were achieved during the early-to-mid- exponential growth phase; both were active over a pH range from 4.5 to 8.5 and temperature from 30 to 50 ºC. None of the ions studied (K+, Na+, Ca2+, Hg2+, Mg2+, Mn2+, Cu2+ and Zn2+ was required for the catalytic activity of strain R-926; amylase activity of strain R-991 was stimulated in the presence of K+, Hg2+ and Zn2+. The surfactants SDS, Triton X-100 and Tween-80 did not have a pronounced inhibitory effect on enzyme activities; SDS and Tween-80 caused the highest stimulatory effects. Amylase activities from the rhizobia strains were reduced by up to 30% in the presence of EDTA; amylase activity of R-926 was also inhibited by HgCl2, suggesting that Ca2+and cysteine residues could be important for activity of this strain.A produção e parcial caracterização de extratos brutos de amilase de duas estirpes de rizóbio (R-926 e R-991 foram avaliadas. Para ambas as estirpes, as máximas atividades amilolíticas foram obtidas no início/meio da fase exponencial de crescimento. As amilases rizobiais foram ativas numa variação de pH de 4,5 a 8,5 e temperatura de 30 a 50 ºC. Nenhum dos íons testados (K+, Na+, Ca2+, Hg2+, Mg2+, Mn2+, Cu2+ e Zn2+ foi exigido para a atividade catalítica da estirpe R-926. A amilase produzida pelo R991 foi estimulada na presença de K+, Hg2+ e Zn2+. Os surfactantes SDS, Triton X-100 e Tween-80 não exerceram um pronunciado efeito inibitório sobre as atividades enzimáticas, e SDS e Tween-80 causaram os maiores efeitos estimulatórios. A atividade amilolítica rizobial foi reduzida em até 30% na presença de EDTA; a amilase produzida pela estirpe R-926 também foi inibida pelo HgCl2, sugerindo, ao menos em parte, a importância de Ca2+ e resíduos de cisteína na atividade amilolítica dessa

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

Science.gov (United States)

Di Martino, Maria Letizia; Romilly, Cédric; Wagner, E Gerhart H; Colonna, Bianca; Prosseda, Gianni

2016-11-08

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, VirF 30 (30 kDa), and the shorter VirF 21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF 30 and VirF 21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF 21 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 VirF 21 The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF 30 is responsible for activation of the virulence system, VirF 21 negatively autoregulates virF expression itself. Since VirF 21 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. Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF 30 and VirF 21 , that are functionally distinct. The major form, VirF 30 , activates the genes

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

DEFF Research Database (Denmark)

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

2008-01-01

volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... 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...

Atividade das glicosidases na presença de chá verde e de chá preto

OpenAIRE

Pereira,L.L.S.; Souza,S.P.; Silva,M.C; Carvalho,G.A.; Santos,C.D.; Corrêa,A.D.; Abreu,C.M.P.

2010-01-01

Várias plantas têm sido consideradas produtos terapêuticos, dentre elas destacam-se os chás verde e preto, popularmente utilizados para controle da hiperglicemia e obesidade. Objetivou-se neste trabalho avaliar o potencial inibitório sobre as enzimas α-amilase, α e β-glicosidases e o teor de compostos fenólicos do chá verde e do chá preto. O teor de compostos fenólicos encontrados foram de 80,8 ± 0,43 mg g-1 no chá preto e 32,0 ± 0,12 mg g-1 no chá verde. O chá verde e o chá pr...

Self-amplifying mRNA vaccines.

Science.gov (United States)

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

2015-01-01

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

Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

OpenAIRE

Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

1982-01-01

Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity.

Directory of Open Access Journals (Sweden)

Jun Ling

Full Text Available Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T, BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl phthalate (MEHP as a major metabolite of another important phthalate di (2-ethylhexyl phthalate (DEHP inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29 growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

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Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

2013-01-01

Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT–PCR, western blotting, IHC, and copy number analysis. Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (Ptumour size (PALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. Conclusion: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour. PMID:24149177

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

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Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

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

International Nuclear Information System (INIS)

Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

2016-01-01

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.

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

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

2016-08-05

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

Carboidratos em dietas para o jundiá, Rhamdia quelen

OpenAIRE

Rosa, Maria do Carmo Gominho

2012-01-01

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Aquicultura Coeficientes de digestibilidade aparente (CDA) do amido do farelo de trigo, farelo de mandioca, milho moído e quirera de arroz foram comparados entre jundiá e tilápia-do-Nilo. Independente da fonte, tilápia apresentou maior CDA que jundiá, provavelmente devido a seu longo intestino e não à maior atividade de carboidrases, já que amilase e maltase do jundiá foram ...

A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3.

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Djaffar, I; Chen, Y P; Creminon, C; Maclouf, J; Cieutat, A M; Gayet, O; Rosa, J P

1994-05-15

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C, the first alternative transcript showing an altered extracellular domain of a beta integrin.

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

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Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

2012-04-25

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.

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

International Nuclear Information System (INIS)

Li, Long; Pintel, David J.

2012-01-01

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.

Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.

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Kröger, M; Tschesche, H

1997-09-01

The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

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

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

1981-05-01

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.

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

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Hesham F. Alharby

2016-11-01

Full Text Available 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. Keywords: Tomato, Salt stress, Nanoparticles, Gene expression, Real-time PCR, Polymorphism

mRNA localization mechanisms in Trypanosoma cruzi.

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Lysangela R Alves

Full Text Available Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

All-in-one detector of circulating mRNA based on a smartphone

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Cmiel, Vratislav; Gumulec, Jaromir; Svoboda, Ondrej; Raudenska, Martina; Hudcova, Kristyna; Sekora, Jiri; Balogh, Jaroslav; Masarik, Michal; Provaznik, Ivo

2016-03-01

Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

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

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

2017-07-01

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

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

Science.gov (United States)

Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

Pancreatite necro-hemorrágica fatal associada ao uso de valproato de sódio: relato de caso

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Munhoz Renato P.

2001-01-01

Full Text Available Das complicações idiossincrásicas do uso do VPA, a pancreatite é a mais rara, porém não menos importante pelo curso potencialmente fatal. Relatamos caso de paciente de 5 anos de idade, com diagnóstico de epilepsia desde os 3 anos de idade, em uso de CBZ e PB que, após estado de mal epiléptico três meses atrás, iniciou tratamento com dose de 40 mg/kg/dia de VPA, associado a CBZ 20 mg/kg/dia e PB 3 mg/kg/dia em retirada. Reinternando por vômitos, dor abdominal, febrícula, abdome tenso, doloroso e amilase de 288 UI, evoluiu com hemorragia digestiva alta, choque e amilase de 564 UI. Submetido a laparotomia exploradora, observaram-se ascite hemorrágica, hematoma retroperitoneal, aumento da cabeça do pâncreas com edema e hemorragia, com diagnóstico de pancreatite necro-hemorrágica. Evoluiu a óbito. Complicações pancreáticas do uso do VPA flutuam entre hiperamilasemia assintomática e pancreatite aguda fulminante. No caso relatado, encontramos dados congruentes com a literatura, existindo 7 casos semelhantes descritos, 4 com óbito.

Determinação da energia metabolizável do farelo residual do milho com e sem enzima em dietas para frangos de corte

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C.G. Valadares

2016-06-01

Full Text Available RESUMO O objetivo deste trabalho foi avaliar o valor nutricional e determinar a energia metabolizável do farelo residual de milho (FRM sem e com o uso da enzima alfa- amilase. Foi realizado um experimento de metabolismo com 180 pintos machos Cobb com 14 dias, distribuídos em um delineamento inteiramente ao acaso, com seis tratamentos, cinco repetições e seis aves por parcela. As dietas experimentais foram: T1: ração referência (RR, T2: 60% T1 + 40% de FRM, T3: RR + enzima, T4: 60% T1 + 40% de FRM com adição de enzima, T5: RR com substituição de 100% do milho pelo FRM e T6: RR com substituição de 100% do milho pelo FRM com adição de enzima. A composição química do FRM foi: 88,33% de matéria seca (MS, 10,23% de proteína bruta (PB, 15,44% de extrato etéreo (EE, 4,33% de cinzas (CZ e 4555kcal/kg de energia bruta (EB. Os valores dos coeficientes de metabolizabilidade aparente para o FRM sem e com adição de enzima foram, respectivamente, de 73,37% e 76,33% para MS (p=0,0136, 70,44% e 70,39% para PB (p=0,9595 e de 74,79% e 76,77% para EB (p=0,0128. Os valores da energia metabolizável aparente (EMA e da EMA corrigida para retenção de nitrogênio (EMAn para o FRM (na base natural foram de 3322±19 e 3241±18kcal/kg e de 3334±16 e 3261±17kcal/kg, respectivamente, sem e com adição de enzima. A adição da enzima não teve efeito estatístico significativo sobre os valores de EMA e EMAn, entretanto houve efeito positivo no coeficiente de metabolização da energia.

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

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Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2008-01-01

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A Sequences

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Alexandra E Grier

2016-01-01

Full Text Available Increasing demand for large-scale synthesis of in vitro transcribed (IVT mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A tail lengths to ≃120 base pairs (bp. Here, we have developed a novel method for generation of extended poly(A tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A tracts up to ≃500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s.

Expressão do mRNA para IGF-2 em oócitos e células do cumulus extraídos de folículos antrais e pré-antrais de ovelhas nativas do Estado de Pernambuco

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Arthur N. Melo

Full Text Available RESUMO: Objetivou-se avaliar a expressão do mRNA para o gene do fator de crescimento IGF-2 em oócitos e células do cumulus de ovelhas em diferentes estágios do desenvolvimento folicular. Os folículos classificados morfologicamente como antrais (terciários e pré-ovulatórios foram aspirados manualmente para obtenção dos oócitos e células do cumulus. Os folículos pré-antrais (secundários foram extraídos do córtex ovariano, por microdissecção, e os oócitos retirados. Nos dois grupos, os oócitos foram desnudados e agrupados em “pools” de dez células cada (Grupo A, n=10; Grupo B, n=10 e dez amostras com grupos de células do cumulus (Grupo A1, n=10, B1, n=10. O mRNA foi extraído e convertido em cDNA utilizando a técnica da RT-PCR, utilizando Oligo DT randômico para o mRNA. A análise da expressão confirmou a expressão gênica para IGF-2 nos grupos de oócitos e células do cumulus. Houve um aumento da expressão relativa do mRNA para IGF-2 nos grupos de oócitos durante a fase mais tardia do desenvolvimento folicular e as diferenças foram consideradas significantes (p<0,05. Não houve variação significante da expressão de IGF2 entre os grupos de células do cumulus. Conclui-se que o fator de crescimento IGF-2 tem níveis mais elevados de expressão em oócitos ovinos, na segunda fase do desenvolvimento folicular, mas expressão semelhante em células do cumulus durante as fases estudadas do desenvolvimento folicular.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

Science.gov (United States)

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

PrP mRNA and protein expression in brain and PrP(c) in CSF in Creutzfeldt-Jakob disease MM1 and VV2.

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Llorens, Franc; Ansoleaga, Belén; Garcia-Esparcia, Paula; Zafar, Saima; Grau-Rivera, Oriol; López-González, Irene; Blanco, Rosi; Carmona, Margarita; Yagüe, Jordi; Nos, Carlos; Del Río, José Antonio; Gelpí, Ellen; Zerr, Inga; Ferrer, Isidre

2013-01-01

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrP(c)). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrP(sc) (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrP(sc) levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrP(sc) deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrP(c), the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrP(c) levels in brain varies from one disease to another. Reduced PrP(c) levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.

Biosynthesis of 10 kDa and 7.5 kDa insulin-like growth factor II in a human rhabdomyosarcoma cell line

DEFF Research Database (Denmark)

Nielsen, F C; Haselbacher, G; Christiansen, Jan

1993-01-01

In the present study we have analysed the expression of insulin-like growth factor II (IGF-II) in the human rhabdomyosarcoma cell line IN157.IN157 cells express high levels of three IGF-II mRNAs of 6.0 kb, 4.8 kb and 4.2 kb. In contrast, normal skeletal muscle expresses a negligible amount of IGF......-II mRNA. Two forms of IGF-II with molecular masses of 7.5 kDa and 10 kDa, corresponding to the mature IGF-II and IGF-II with a C-terminal extension of 21 amino acids (IGF-IIE21), were secreted into the culture medium at amounts of 17 ng/ml (2.3 nM) and 15 ng/ml (1.5 nM), respectively. IN157 cells also......-II and IGF-IIE21 with Kd values of 0.5 nM and 2 nM, respectively, and IGF-I with about 500 times lower affinity. IGF-II and IGF-IIE21 stimulated DNA synthesis via the IGF-I receptor, whereas the IGF-II/Man 6-P receptor mediated their rapid internalization and inactivation. During culture of IN157 cells about...

A glimpse at mRNA dynamics reveals cellular domains and rapid trafficking through granules

NARCIS (Netherlands)

Gemert, Alice Myriam Christi van

2011-01-01

mRNA transport and targeting are essential to gene expression regulation. Specific mRNA sequences can bind several proteins and together form RiboNucleoProtein particles (RNP). The various proteins within the RNP determine mRNA fate: translation, transport or decay. RNP composition varies with

A CPMI da Violência Contra a Mulher e a Implementação da Lei Maria Da Penha

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Carmen Hein de Campos

2015-05-01

Full Text Available Este artigo analisa as conclusões da Comissão Parlamentar Mista de Inquérito da Violência contra a Mulher, criada pelo Congresso Nacional Brasileiro em 2012, no que tange à aplicação da Lei Maria da Penha. Após um ano e meio de trabalho, o relatório da CPMI apontou a fragilidade das políticas públicas de enfrentamento à violência contra mulheres e os obstáculos na implementação da Lei Maria da Penha, dentre os quais, destacam-se: a precariedade da rede de serviços e o reduzido número de juizados especializados em atendimento às mulheres em situação de violência doméstica e familiar; o descumprimento da decisão do Supremo Tribunal Federal que proíbe a aplicação da suspensão condicional do processo; a resistência de operadores/as do direito em entender a proposta da nova lei e romper com a lógica familista e o insuficiente orçamento para o desenvolvimento e a manutenção das políticas públicas de enfrentamento a essas situações de violência. A análise do relatório da CPMI permite concluir que os estados brasileiros investem muito pouco em políticas públicas específicas e que a implementação integral da Lei Maria da Penha depende de uma nova compreensão jurídica, da articulação entre os poderes públicos e de uma política orçamentária de gênero.

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

Science.gov (United States)

Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

Science.gov (United States)

Cusick, M E

1992-12-29

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

Science.gov (United States)

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

Science.gov (United States)

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

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.

Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

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Clément Chevalier

2010-03-01

Full Text Available Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

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Mariana Serpeloni

Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

mRNA in exosomas as a liquid biopsy in non-Hodgkin Lymphoma: a multicentric study by the Spanish Lymphoma Oncology Group.

Science.gov (United States)

Provencio, Mariano; Rodríguez, Marta; Cantos, Blanca; Sabín, Pilar; Quero, Cristina; García-Arroyo, Francisco R; Rueda, Antonio; Maximiano, Constanza; Rodríguez-Abreu, Delvys; Sánchez, Antonio; Silva, Javier; García, Vanesa

2017-08-01

To determine the feasibility of mRNAs ( C-MYC, BCL-XL, BCL-6, NF-κβ, PTEN and AKT ) in exosomes of plasma as a liquid biopsy method for monitoring and prognostic evolution in B-cell lymphomas. Exosomes were isolated from 98 patients with B-cell Lymphoma and 68 healthy controls. mRNAs were analyzed by quantitative PCR. An additional 31 post-treatment samples were also studied. In the general and follicular lymphoma series, the presence of AKT mRNA was associated with poor response to rituximab-based treatment. Patients with first relapse or disease progression showed a lower percentage of PTEN and BCL-XL mRNA. The presence of BCL-6 mRNA was associated with a high death rate. The absence of PTEN mRNA in the general series, and presence of C-MYC mRNA in follicular lymphomas, were associated with short progression-free survival. BCL-6 and C-MYC mRNA were independent prognostic variables of overall survival. C-MYC mRNA may provide prognostic information with respect to overall survival. BCL-XL mRNA and increase of BCL-6 mRNA in post-treatment samples could serve as molecular monitoring markers. This is the first large study to evaluate the prognostic and predictive values of pretreatment tumor-associated mRNA in exosomes. BCL-6 and C-MYC mRNA positivity in pretreatment samples were predictors of worse PFS compared to patients with mRNA negativity. C-MYC mRNA positivity was also a statistically significant predictor of inability to obtain complete response with first-line therapy.

Dental mineralization and salivary activity are reduced in offspring of spontaneously hypertensive rats (SHR A mineralização dental e a atividade salivar estão reduzidas em filhotes de ratas espontaneamente hipertensas (SHR

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Gracieli Prado Elias

2006-08-01

Full Text Available Several pathologies have been diagnosed in children of hypertensive mothers; however, some studies that evaluated the alterations in their oral health are not conclusive. This study analyzed the salivary gland activity and dental mineralization of offsprings of spontaneously hypertensive rats (SHR. Thirty-day-old SHR males and Wistar rats were studied. The salivary flow was evaluated by injection of pilocarpine, the protein concentration and salivary amylase activity, by the Lowry method and kinetic method at 405 nm, respectively. Enamel and dentin mineralization of the mandibular incisors was quantified with aid of the microhardness meter. The results were analyzed by the ANOVA or Student's t test (pDiversas patologias têm sido diagnosticadas em filhos de mães hipertensas, entretanto alguns estudos que avaliaram as possíveis alterações na saúde oral não são conclusivos. Neste trabalho foram analisadas a atividade das glândulas salivares e a mineralização dental de filhotes de ratas espontaneamente hipertensas (SHR. Ratos machos, com 30 dias de vida, SHR e Wistar, foram estudados. O fluxo salivar foi avaliado pela injeção de pilocarpina e a concentração de proteínas e atividade da amilase na saliva, pelo método de Lowry e método cinético a 405 nm, respectivamente. A mineralização do esmalte e dentina de incisivos inferiores foi quantificada através da medida da microdureza. Os resultados foram analisados pelo teste ANOVA ou t de Student's não pareado (p<0,05. Verificou-se que o fluxo salivar (0,026 mL/min/100 g ± 0,002 e a concentração de proteínas na saliva (2,26 mg/mL ± 0,14 de filhotes SHR estavam reduzidos em comparação com filhotes normotensos Wistar (0,036 mL/min/100 g ± 0,003 e 2,91 mg/mL ± 0,27, respectivamente, porém sem nenhuma alteração da atividade da amilase (SHR: 242,4 U/mL ± 36,9, Wistar: 163,8 U/mL ± 14,1. A microdureza mostrou-se menor tanto no esmalte (255,8 KHN ± 2,6 quanto na dentina (59

Composição química e antinutrientes presentes nas amêndoas cruas e torradas de chicha (Sterculia striata A. St. Hill & Naudin Chemical composition and antinutrients of raw and roasted chicha almonds (Sterculia striata A. St. Hill & Naudin

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Aline Gomes de Moura e Silva

2011-04-01

Full Text Available OBJETIVO: Analisar os componentes químicos e antinutrientes antes e após torrefação da amêndoa de chichá da espécie Sterculia striata A. St. Hill & Naudin, oriunda de Corrente, Piauí. MÉTODOS: Foram realizadas análises: centesimal, perfil de ácidos graxos, antinutrientes (taninos, inibidor de tripsina, inibidor de amilase, lectina, fitatos, compostos fenólicos e atividade de peroxidase e polifenoloxidase. RESULTADOS: Os resultados da análise centesimal da amêndoa crua e torrada foram: lipídeos de 25,1% e 26,2%, carboidratos de 44,4% e 45,6%, proteína de 20,8% e 22,1%, cinzas de 3,7% e 4,0%, fibra alimentar total de 12,3% e 10,4%, respectivamente. Nas amêndoas cruas e torradas não foram encontradas lectinas, taninos, inibidores de tripsina e alfa-amilase. Verificou-se a ausência de peroxidase e polifenoloxidase e conteúdo de compostos fenólicos de 107,7mg/100g e 108,9mg/100g para amêndoas cruas e torradas, respectivamente. CONCLUSÃO: A torrefação realizada a 205°C por 11 minutos diminuiu o teor de fitatos de 10,6mg/g para 5,5mg/g. Por fim, as amêndoas de chichá, cruas e torradas, apresentam alto teor de proteínas, fibras, ácidos graxos monoinsaturados e saturados.OBJECTIVE: This study analyzed the chemical components and antinutrients present in raw and roasted chichá almonds from the species Sterculia striata A. St. Hill & Naudin harvested in Corrente, Piauí. METHODS: The following were determined: percent composition, fatty acid profile, antinutrients (tannins, trypsin inhibitor, amylase inhibitor, lectin, phytates, phenolic compounds and peroxidase and polyphenol oxidase activities. RESULTS: The percentage composition of the raw and lipid almonds were respectively: 25.1% and 26.2% fats, 44.4% and 45.6% carbohydrates, 20.8% and 22.1% protein, 3.7% and 4.0% ashes, and 12.3% and 10.4% total fiber. Neither raw nor roasted almonds contained lectins, tannins, trypsin inhibitors, alpha-amylase, peroxidase and

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

Science.gov (United States)

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-04-15

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Importance of a 5′ Stem-Loop for Longevity of papA mRNA in Escherichia coli

OpenAIRE

Bricker, Angela L.; Belasco, Joel G.

1999-01-01

High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5′ untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA ...

A PROBLEMÁTICA DA BIOPIRATARIA: REFLEXÕES A PARTIR DA GEOPOLÍTICA E DOS DIREITOS DA SOCIOBIODIVERSIDADE

OpenAIRE

Nunes, Denise Silva; UFSM; Tybusch, Jerônimo Siqueira; UFSM

2013-01-01

O bioimperialismo configura-se como uma característica da geopolítica da atualidade, com alicerces nas estruturas da globalização e do capitalismo. Nesse contexto, a problemática da pesquisa consiste na violação dos direitos dos povos locais e a exploração do meio ambiente, em que o atual sistema internacional de propriedade intelectual contribui para a biopirataria dos recursos naturais, por meio da infiltração, em áreas ricas em diversidade biológica, das grandes corporações, multinacionais...

mRNA processing in yeast

International Nuclear Information System (INIS)

Stevens, A.

1982-01-01

Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

A pilot trial assessing urinary gene expression profiling with an mRNA array for diabetic nephropathy.

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Min Zheng

Full Text Available BACKGROUND: The initiation and progression of diabetic nephropathy (DN is complex. Quantification of mRNA expression in urinary sediment has emerged as a novel strategy for studying renal diseases. Considering the numerous molecules involved in DN development, a high-throughput platform with parallel detection of multiple mRNAs is needed. In this study, we constructed a self-assembling mRNA array to analyze urinary mRNAs in DN patients with aims to reveal its potential in searching novel biomarkers. METHODS: mRNA array containing 88 genes were fabricated and its performance was evaluated. A pilot study with 9 subjects including 6 DN patients and 3 normal controls were studied with the array. DN patients were assigned into two groups according to their estimate glomerular rate (eGFR: DNI group (eGFR>60 ml/min/1.73 m(2, n = 3 and DNII group (eGFR<60 ml/min/1.73 m(2, n = 3. Urinary cell pellet was collected from each study participant. Relative abundance of these target mRNAs from urinary pellet was quantified with the array. RESULTS: The array we fabricated displayed high sensitivity and specificity. Moreover, the Cts of Positive PCR Controls in our experiments were 24±0.5 which indicated high repeatability of the array. A total of 29 mRNAs were significantly increased in DN patients compared with controls (p<0.05. Among these genes, α-actinin4, CDH2, ACE, FAT1, synaptopodin, COL4α, twist, NOTCH3 mRNA expression were 15-fold higher than those in normal controls. In contrast, urinary TIMP-1 mRNA was significantly decreased in DN patients (p<0.05. It was shown that CTGF, MCP-1, PAI-1, ACE, CDH1, CDH2 mRNA varied significantly among the 3 study groups, and their mRNA levels increased with DN progression (p<0.05. CONCLUSION: Our pilot study demonstrated that mRNA array might serve as a high-throughput and sensitive tool for detecting mRNA expression in urinary sediment. Thus, this primary study indicated that mRNA array probably could be a

Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

Energy Technology Data Exchange (ETDEWEB)

Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

2015-12-15

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Rift Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.

A Lei Maria da Penha sob a perspectiva da ética da vingança / Maria da Penha law unde the ethics of revenge approach

OpenAIRE

Nunes, Thaísa Silva de Oliveira; Faculdade de Direito de Vitória

2011-01-01

RESUMO: Analisa-se a lei 11.340/2006, conhecida como Lei Maria da Penha, sob a perspectiva da ética da vingança, considerando-se a situação de seu uso de forma equivocada. Nesse sentido, argui-se que o judiciário deve tomar um cuidado maior ao julgar casos de violência doméstica. Aborda-se também a possibilidade de uma mulher abandonada ou ultrajada pelo seu companheiro reagir de forma a resgatar a confiança em si, ao invés de se utilizar da prática da vingança permitida pelos mecanismos jurí...

A utilização da teoria da aprendizagem significativa no ensino da Enfermagem

OpenAIRE

Alana Tamar Oliveira de Sousa; Nilton Soares Formiga; Simone Helena dos Santos Oliveira; Marta Miriam Lopes Costa; Maria Júlia Guimarães Oliveira Soares

2015-01-01

RESUMO Objetivo: 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...

Translation initiation in bacterial polysomes through ribosome loading on a standby site on a highly translated mRNA

Science.gov (United States)

Andreeva, Irena

2018-01-01

During translation, consecutive ribosomes load on an mRNA and form a polysome. The first ribosome binds to a single-stranded mRNA region and moves toward the start codon, unwinding potential mRNA structures on the way. In contrast, the following ribosomes can dock at the start codon only when the first ribosome has vacated the initiation site. Here we show that loading of the second ribosome on a natural 38-nt-long 5′ untranslated region of lpp mRNA, which codes for the outer membrane lipoprotein from Escherichia coli, takes place before the leading ribosome has moved away from the start codon. The rapid formation of this standby complex depends on the presence of ribosomal proteins S1/S2 in the leading ribosome. The early recruitment of the second ribosome to the standby site before translation by the leading ribosome and the tight coupling between translation elongation by the first ribosome and the accommodation of the second ribosome can contribute to high translational efficiency of the lpp mRNA. PMID:29632209

mRNA transfection of mouse and human neural stem cell cultures

OpenAIRE

McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

2013-01-01

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 ...

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

Science.gov (United States)

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

2014-09-01

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.

A Sintaxe da Janela

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Luís Antônio Jorge

1993-12-01

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

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

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Negi S

2007-01-01

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.

[Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

Science.gov (United States)

Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

2009-06-01

To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

Lower glutamic acid decarboxylase 65-kDa isoform messenger RNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia.

Science.gov (United States)

Glausier, Jill R; Kimoto, Sohei; Fish, Kenneth N; Lewis, David A

2015-01-15

Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc

A dignidade da pessoa humana na doutrina social da igreja

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Manzone, Gianni

2010-01-01

Full Text Available O texto faz uma análise panorâmica sobre a compreensão da dignidade humana nos diversos textos da Doutrina Social da Igreja, mostrando o entendimento da mesma nos diversos documentos dos Papas, notadamente Leão XII, Pio XI, Pio XII e João Paulo II, além de apontar aspectos históricos e culturais que influenciaram cada pronunciamento, o que foi determinante para a evolução do pensamento do Magistério na questão social. O ponto de partida é a visão antropológica da Doutrina Social da Igreja, resumida em termos de “homem criado por Deus a sua imagem e resgatado pelo sangue de Jesus Cristo” (Sollicitudo Rei Socialis 29. A partir daí, há a defesa do primado do ser humano sobre o social e reivindicações totalitaristas que perpassaram a história recente da humanidade, embora a pessoa seja sempre um ser-em-relação e situado em uma sociedade determinada

A utilização da teoria da aprendizagem significativa no ensino da Enfermagem

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Alana Tamar Oliveira de Sousa

2015-08-01

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.

Consequences of metaphase II oocyte cryopreservation on mRNA content.

Science.gov (United States)

Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

A inclusão da subjetividade no ensino da Psicopatologia

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Octavio Domont de Serpa Junior

2007-08-01

Full Text Available O estudo da Psicopatologia tem sido freqüentemente apresentado em sua dimensão descritiva. Apesar da relevância para o ensino - auxiliar o aluno a reconhecer a dimensão sintomatológica dos quadros psicopatológicos -, exclui a dimensão subjetiva da experiência do adoecimento e seus aspectos relacionais. Partindo da tradição antropológico-existencial, apresentamos uma experiência de ensino de Psicopatologia que considera tais dimensões relevantes para a compreensão do sofrimento mental. Trabalhando com estudo de caso descritivo, são apresentadas duas novas modalidades de práticas na disciplina Psicopatologia Especial I, oferecida para alunos do curso de Psicologia da UFRJ. A proposta é ampliar o ensino prático, tradicionalmente centrado no modelo da extensa entrevista clínica, realizada diante de um grande grupo de alunos que a tudo assistem passivamente. Pretendemos aproximar o ensino e a prática da Psicopatologia dos ideais da Reforma Psiquiátrica Brasileira, que concebe o adoecimento mental como fenômeno complexo, envolvendo a relação do sujeito com o mundo.

Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite

International Nuclear Information System (INIS)

Keyse, S.M.; Tyrrell, R.M.

1989-01-01

We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage

A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

Science.gov (United States)

Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

2015-01-29

Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

Pressure overload stimulated cardiac hypertrophy leads to a rapid decrease in the mRNA for creatine kinase

International Nuclear Information System (INIS)

Boheler, K.; Popovich, B.; Dillmann, W.H.

1987-01-01

Cardiac hypertrophy (CH) leads to a decrease in creatine kinase (CK) enzymatic activity. To determine if the mRNA for CK also decreases with CH, they performed the following studies. Cardiac RNA was isolated from rats subjected to either abdominal aortic stenosis (AS) or sham surgery. Through Northern blot analysis, total cardiac RNA was quantitated with a CK specific 32 P-labelled cDNA clone. At 3 and 8 days post-constriction, the mRNA for CK decreases by 54.6 +/- 7% and 65.3 +/- 18% respectively, whereas the heart weight increases by 19% and 37% relative to controls. Further studies indicate that CK mRNA also decreases by 41.8% in hypothyroid rats (Tx) but decreases by a total of 68.1% in Tx rats subjected to 8 days of AS. Pressure overload stimulated CH leads to a rapid decrease in CK mRNA in normal and Tx rats. This CK mRNA decrease may account for the decreased efficiency of contraction seen in CH

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

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Beatriz M. A. Fontoura

2013-07-01

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

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

Science.gov (United States)

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

2013-01-01

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

Neurotrophin-3 mRNA a putative target of miR21 following status epilepticus.

Science.gov (United States)

Risbud, Rashmi M; Lee, Carolyn; Porter, Brenda E

2011-11-18

Status epilepticus induces a cascade of protein expression changes contributing to the subsequent development of epilepsy. By identifying the cascade of molecular changes that contribute to the development of epilepsy we hope to be able to design therapeutics for preventing epilepsy. MicroRNAs influence gene expression by altering mRNA stability and/or translation and have been implicated in the pathology of multiple diseases. MiR21 and its co-transcript miR21, microRNAs produced from either the 5' or 3' ends of the same precursor RNA strand, are increased in the hippocampus following status epilepticus. We have identified a miR21 binding site, in the 3' UTR of neurotrophin-3 that inhibits translation. Neurotrophin-3 mRNA levels decrease in the hippocampus following SE concurrent with the increase in miR21. MiR21 levels in cultured hippocampal neurons inversely correlate with neurotrophin-3 mRNA levels. Treatment of hippocampal neuronal cultures with excess K(+)Cl(-), a depolarizing agent mimicking the episode of status epilepticus, also results in an increase in miR21 and a decrease in neurotrophin-3 mRNA. MiR21 is a candidate for regulating neurotrophin-3 signaling in the hippocampus following status epilepticus. Copyright © 2011 Elsevier B.V. All rights reserved.

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

DEFF Research Database (Denmark)

Nielsen, M. E.; Rasmussen, I. A.; Kristensen, S. G.

2011-01-01

significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. 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 follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

A aplicação dos recursos da seguridade social, a partir da perspectiva da teoria dos sistemas, da teoria comunicacional e do caos

OpenAIRE

Júnior, Dávio Antonio Prado Zarzana

2011-01-01

Este trabalho, focalizando o custeio da Seguridade Social, pretende realizar três verificações: a primeira, a existência ou não de desvios na aplicação dos recursos arrecadados para este financiamento, inclusive a partir da formulação de um conceito de desvio. A segunda, examinar os desequilíbrios que esses possíveis desvios produziriam, sob a ótica da Teoria dos Sistemas, utilizando-se, em adição, terminologia própria da Teoria Comunicacional. E a terceira, perscrutar a pos...

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

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Susanne Huch

2016-10-01

Full Text Available The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization.

Apoptose e expressão de VP2 e GAPDH na infecção precoce pelo vírus da doença infecciosa da bursa de Fabricius em pintos SPF Apoptosis and expression of VP2 and GADPH in an experimental infectious bursal disease in SPF chicks

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J.J. Batista

2007-04-01

Full Text Available Vinte e nove pintos SPF de um dia foram inoculados com o vírus da doença infecciosa da bursa de Fabricius (VDIB para avaliar a ocorrência precoce de apoptose e a expressão da proteína viral 2 (VP2 e da enzima gliceraldeído fosfato dehidrogenase (GAPDH. Os animais foram distribuídos em cinco grupos: 1-controle; e 2 a 5- com 24, 48, 72 e 96 horas pós-inoculação, respectivamente. Fragmentos da bursa de Fabricius foram colhidos para processamento histológico e extração de RNA. Lâminas coradas em HE e TUNEL (marcação in situ da fragmentação do genoma com transferase terminal de deoxinucleotídeo foram utilizadas na morfometria do índice apoptótico. Amostras de mRNA foram testadas para a expressão dos genes VP2 e GAPDH utilizando-se transcrição reversa e RT-PCR. Utilizou-se um kit SYBR GREEN PCR, e a reação foi desenvolvida em ABI Prism 7000 SDS. Os índices apoptóticos cresceram progressivamente indicando uma relação na atrofia bursal causada pelo VDIB. Paralelamente, os resultados da PCR em tempo real demonstraram queda da carga viral nas células linfóides da bursa nos diferentes intervalos de tempo do experimento. Esses resultados sugerem um papel protetor da apoptose na diminuição da replicação viral.Twenty-nine SPF 1-day-old chicks were inoculated with infectious bursal disease virus (IBDV to evaluate early apoptosis and the expression of viral protein 2 (VP2 and glyceraldehyde-3-phosphate dehydrogenease (GAPDH. Five groups were formed: G1-control -and G2 to G5, - 24, 48, 72 and 96 hours post inoculation, respectively. Half of each BF was fixed and processed by routine techniques. To quantify apoptosis, 5µm-thick sections were stained with HE and submitted to TUNEL (terminal transferase UDP nick end labeling technique. mRNA was extracted from pooled samples of 3 animals/group and used for the expression of VP2 and GADPH genes using the reverse transcription and real-time polymerase chain reaction (RT-PCR. A SYBR

Accumulation of defence-related transcripts and cloning of a chitinase mRNA from pea leaves (Pisum sativum L.) inoculated with Ascochyta pisi Lib

DEFF Research Database (Denmark)

Vad, Knud; de Neergaard, Eigil; Madriz-Ordeñana, Kenneth

1993-01-01

The race specific resistance of pea to Ascochyta pisi Lib. was shown to be exhibited as a hypersensitive response associated with the production of polyphenolic substances in epidermal and mesophyll cells. The levels of transcripts representing a pathogenesis-related (PR) protein (chitinase......) and an enzyme of phytoalexin biosynthesis (chalcone synthase) were shown to accumulate more rapidly during the hypersensitive response than during lesion development in the compatible interaction. A full-length (1143 bp) cDNA sequence of a pea chitinase (EC 3.2.1.14) (coding for an approx. 34 500 Da protein......) was deduced by combining the overlapping sequences of three clones obtained following PCR amplification of cDNA prepared from mRNA isolated 24 h after inoculation of pea leaves with Ascochyta pisi. The combined sequences were identified as a class I chitinase corresponding to the basic A1-chitinase enzyme...

Inibição da guanilato ciclase pelo azul de metileno no choque circulatório causado por pancreatite aguda necrosante: uma palavra de cuidado embasada em modelo suíno

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Caroline Floreoto Baldo

Full Text Available OBJETIVO: estudar o uso terapêutico do bloqueio da guanilato ciclase pelo azul de metileno em um modelo experimental de pancreatite aguda grave em suínos. MÉTODOS: a pancreatite aguda necrotizante foi induzida em porcos anestesiados por infusão ductal pancreática retrógrada de 1ml/kg de taurocolato de sódio a 5% e 8U/kg de enteroquinase. Três grupos foram estudados (n=5: controle (C, pancreatite (PA, "bolus" de azul seguido por pancreatite (AM+PA. Os dados incluíram enzimas séricas e do líquido abdominal, variáveis hemodinâmicas, hemogasometria arterial, volume de líquido abdominal, marcadores inflamatórios plasmáticos, nitrito/nitrato e mieloperoxidase e malondialdeído plasmático. Aplicou-se a análise de variância seguida do pós-teste de Bonferroni (p<0,05. RESULTADOS: os valores de amilase e lipase foram três e dez vezes mais elevados no grupo PA. A atividade da mieloperoxidase foi 50% superior no grupo PA. Os dados hemodinâmicos indicaram choque hipovolêmico precoce seguido de choque cardiogênico. Observou-se grave translocação de líquidos para a cavidade peritoneal. A nitrito/nitrato plasmática permaneceu inalterada. O grupo AM+PA teve aumento de cinco vezes do mieloperoxidase em comparação com o grupo C. CONCLUSÕES: a utilização de azul de metileno em suínos com pancreatite não demonstrou efeitos significativos sobre variáveis hemodinâmicas e inflamatórias. Seu uso terapêutico na pancreatite necro-hemorrágica pode ser inadequado e extremo cuidado deve ser tomado dado o aumento da peroxidação lipídica evidenciado pelo aumento dos valores do malondialdeído.

Assessing mRNA nuclear export in mammalian cells by microinjection.

Science.gov (United States)

Lee, Eliza S; Palazzo, Alexander F

2017-08-15

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

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

Science.gov (United States)

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-12-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

Thermal characterization of partially hydrolyzed cassava (Manihot esculenta starch granules

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Luiz Gustavo Lacerda

2008-12-01

Full Text Available Cassava starch, partially hydrolyzed by fungal á-amylase, was characterized using thermal analysis, light microscopy and X-ray diffraction. Thermal degradation was initiated at lower degradation temperatures after enzymatic treatment and the DSC (Differential scanning calorimetry analysis showed almost similar range of gelatinization temperature, but the enthalpies of gelatinization were quite increased for the partially hydrolyzed starch granules. The results suggested that the partial degradation of the starch granules was concentrated in the amorphous regions.Amilases fúngicas são comumente empregadas a amidos com o intuito de otimizar o rendimento de leveduras, modificar a textura de produtos panificados e prolongar a vida de prateleira do produto final. A hidrólise parcial enzimática pode auxiliar no entendimento da estrutura do amido ganular. Amido de mandioca parcialmente hidrolisado por á-amilase fúngica foi investigado utilizando-se técnicas termoanalíticas, microscopia ótica e difratometria por raios X. A degradação térmica iniciou-se a temperaturas menores após o tratamento enzimático e a análise por DSC mostrou uma próxima faixa de temperatura de gelatinização, porém, a entalpia necessária para o evento foi maior para os grânulos parcialmente hidrolisados. Os resultados sugerem que a degradação parcial do amido granular foi concentrada em regiões amorfas.

mRNA: From a chemical blueprint for protein production to an off-the-shelf therapeutic.

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Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

2013-02-01

Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and "naked mRNA" for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.

Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

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Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

2017-08-01

Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

Nonsense mutations in the human β-globin gene affect mRNA metabolism

International Nuclear Information System (INIS)

Baserga, S.J.; Benz, E.J. Jr.

1988-01-01

A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

ErbB3 mRNA leukocyte levels as a biomarker for major depressive disorder

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Milanesi Elena

2012-09-01

Full Text Available Abstract Background In recent years, the identification of peripheral biomarkers that are associated with psychiatric diseases, such as Major Depressive Disorder (MDD, has become relevant because these biomarkers may improve the efficiency of the differential diagnosis process and indicate targets for new antidepressant drugs. Two recent candidate genes, ErbB3 and Fgfr1, are growth factors whose mRNA levels have been found to be altered in the leukocytes of patients that are affected by bipolar disorder in a depressive state. On this basis, the aim of the study was to determine if ErbB3 and Fgfr1 mRNA levels could be a biomarkers of MDD. Methods We measured by Real Time PCR ErbB3 and Fgfr1 mRNA expression levels in leukocytes of MDD patients compared with controls. Successively, to assess whether ErbB3 mRNA levels were influenced by previous antidepressant treatment we stratified our patients sample in two cohorts, comparing drug-naive versus drug-free patients. Moreover, we evaluated the levels of the transcript in MDD patients after 12 weeks of antidepressant treatment, and in prefrontal cortex of rats stressed and treated with an antidepressant drug of the same class. Results These results showed that ErbB3 but not Fgfr1 mRNA levels were reduced in leukocytes of MDD patients compared to healthy subjects. Furthermore, ErbB3 levels were not affected by antidepressant treatment in either human or animal models Conclusions Our data suggest that ErbB3 might be considered as a biomarker for MDD and that its deficit may underlie the pathopsysiology of the disease and is not a consequence of treatment. Moreover the study supports the usefulness of leukocytes as a peripheral system for identifying biomarkers in psychiatric diseases.

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

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Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Single step production of Cas9 mRNA for zygote injection.

Science.gov (United States)

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Arquitetura da paisagem da cidade e a importância da sistematização da análise do problema projetual

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Rodrigo Gonçalves dos Santos

2007-12-01

Full Text Available Com este artigo pretende-se levantar os conceitos próprios da atividade da Arquitetura Paisagística encarando-a como disciplina projetual e associando-a ao Design Ambiental, necessitando, assim, de linhas metodológicas específicas para apresentação de soluções coerentes aos problemas paisagísticos. Sob esta ótica, reflexões sobre o uso da vegetação no projeto dos espaços exteriores são apresentadas apontando-se uma etapa de sistematização da análise do problema de projeto, dentro da abordagem da concepção de uma metodologia projetual em arquitetura paisagística. Também foram analisadas oito vias de circulação da área central de Florianópolis, Santa Catarina, Brasil, exemplificando uma etapa de sistematização da análise do problema de projeto.

Purification, characterization and allergenicity assessment of 26kDa protein, a major allergen from Cicer arietinum.

Science.gov (United States)

Verma, Alok Kumar; Sharma, Akanksha; Kumar, Sandeep; Gupta, Rinkesh Kumar; Kumar, Dinesh; Gupta, Kriti; Giridhar, B H; Das, Mukul; Dwivedi, Premendra D

2016-06-01

Chickpea (CP), a legume of the family Fabaceae, is an important nutrient-rich food providing protein, essential amino acids, vitamins, dietary fibre, and minerals. Unfortunately, several IgE-binding proteins in CP have been detected that are responsible for allergic manifestations in sensitized population. Therefore, the prevalence of CP induced allergy prompted us towards purification, characterization and allergenicity assessment of a major ∼26kDa protein from chickpea crude protein extract (CP-CPE). Purification of CP 26kDa protein was done using a combination of fractionation and anion exchange chromatography. This protein was further characterized as "Chain A, crystal structure of a plant albumin" from Cicer arietinum with Mol wt 25.8kDa by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Further, allergenic potential of purified 25.8kDa protein was assessed using in vivo and in vitro model. Purified protein showed IgE-binding capacity with sensitized BALB/c mice and CP allergic patient's sera. Enhanced levels of specific and total IgE, MCP-1, MCPT-1, myeloperoxidase, histamine, prostaglandin D2, and cysteinyl leukotriene were found in sera of mice treated with CP ∼26kDa protein. Further, expressions of Th2 cytokines (i.e. IL-4, IL-5, IL-13), transcription factors (i.e. GATA-3, STAT-6, SOCS-3) and mast cell signaling proteins (Lyn, cFgr, Syk, PLC-γ2, PI-3K, PKC) were also found increased at mRNA and protein levels in the intestines of mice treated with CP ∼26kDa protein. In addition, enhanced release of β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 were observed in RBL2H3 cell line when treated (125μg) with CP 26kDa protein. Conclusively, in vivo and in vitro studies revealed the allergenic potential of purified CP 26kDa protein. Being a potential allergen, plant albumin may play a pivotal role in CP induced allergenicity. Current study will be helpful for better development of therapeutic approaches to

The development of clinical activity in relapsing-remitting MS is associated with a decrease of FasL mRNA and an increase of Fas mRNA in peripheral blood

NARCIS (Netherlands)

Lopatinskaya, L.; Boxel van-Dezaire, A.H.H.; Barkhof, F.; Polman, C.H.; Lucas, C.J.; Nagelkerken, L.

2003-01-01

In this longitudinal study, we examined the expression of Fas, FasL, CCR3, CCR5 and CXCR3 mRNA in peripheral blood mononuclear cells (PBMCs) of secondary progressive (SP) and relapsing-remitting (RR) multiple sclerosis (MS) patients. In RR patients, FasL, CCR3 and CCR5 mRNA levels were increased

Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo

DEFF Research Database (Denmark)

Krogh, Nicolai; Pietschmann, Max; Schmid, Manfred

2017-01-01

The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene...... in the budding yeast Saccharomyces cerevisiae presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat......-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the m(7)G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m(7)G-capped counterpart, consistent with a key role for the m(7)G cap in mRNA turnover. Our...

Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

Science.gov (United States)

Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

2014-05-01

In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Production and action of an Aspergillus phoenicis enzymatic pool using different carbon sources Produção e ação de um pool enzimático de Aspergillus phoenicis com fontes de carbono diferentes

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Vivian Machado Benassi

2012-09-01

triturada, resíduos agroindustriais, amido e glicose ou maltose. Diversos sistemas enzimáticos foram produzidos a partir dessas fontes de carbono, mas a amilase foi a mais evidente, seguida de pectinase e xilanase. Traços de atividades de CMcases, avicelase, lipase, β-xilosidase, β-glicosidase e α-glicosidase também foram detectados. Amilases foram produzidas em flocos de centeio, amido, flocos de aveia, flocos de milho, farinha de mandioca e fibra de trigo. Níveis amilolíticos significantes foram produzidos em meio de cultura contendo glicose ou quando esse açúcar não estava mais presente, sugerindo uma forma constitutiva dessa enzima. Farinha de mandioca, flocos de centeio, aveia, cevada e milho também foram utilizados como substratos nas reações de hidrólise, com o objetivo de verificar a potencial liberação de açúcares redutores. Flocos de milho induziram maior formação de açúcares redutores, quando comparados com os outros substratos. A análise por cromatografia em camada delgada dos produtos finais da reação revelou que a hidrólise da farinha de mandioca liberou malto-oligossacarídeos, mas farinha de mandioca e flocos de milho, centeio, aveia e cevada foram hidrolisados até glicose. Esses resultados sugeriram a presença de glucoamilase e α-amilase como parte da mistura enzimática de A. phoenicis.

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

Science.gov (United States)

Burns, A T; Deeley, R G; Gordon, J I; Udell, D S; Mullinix, K P; Goldberger, R F

1978-01-01

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen. PMID:273910

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

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Moisés P. Silva

2011-02-01

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

A TRANSFORMAÇÃO DA MEMÓRIA EM MERCADORIA: FIM DA PRODUÇÃO FABRIL E A CONTINUIDADE DA “MARCA”

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Sílvia Aparecida Guarnieri Ortigoza

2008-04-01

Full Text Available O resgate da história da Fábrica de Chapéus Sarkis foi retratado neste estudo. Buscou-se analisar as influências da referida fábrica no município de Itapira, principalmente na organização e produção do centro urbano e no cotidiano dos citadinos. Seu fechamento foi entendido a partir das mudanças da moda mundial e de seus reflexos no lugar Itapira – SP, Brasil. Este estudo contempla uma análise regressiva do período (décadas de 1920 a 1960 em que o chapéu era um acessório indispensável na vida cotidiana das pessoas. Reencontrando o presente, procurou-se entender o conteúdo da forma espacial que hoje se encontra em vestígios. O estudo é singular por identificar no espaço geográfico itapirense atual a peculiaridade da transformação da tradição da fábrica em mercadoria.

Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study.

Science.gov (United States)

Gredell, Joseph A; Berger, Angela K; Walton, S Patrick

2008-07-01

The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5'- and 3'-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5'-end or 3'-end were silenced, on average, approximately 10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate si

Protein Structure and the Sequential Structure of mRNA

DEFF Research Database (Denmark)

Brunak, Søren; Engelbrecht, Jacob

1996-01-01

entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......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...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

Energy Technology Data Exchange (ETDEWEB)

Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

2015-04-03

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

International Nuclear Information System (INIS)

Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping; Ye, Lihong; Zhang, Xiaodong

2015-01-01

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro

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

Science.gov (United States)

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

2015-01-01

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.

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

Science.gov (United States)

Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

2011-03-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

MiR-200a is involved in rat epididymal development by targeting β-catenin mRNA

Institute of Scientific and Technical Information of China (English)

Xiaojiang Wu; Botao Zhao; Wei Li; Yue Chen; Ruqiang Liang; Lin Li; Youxin Jin; Kangcheng Ruan

2012-01-01

The expression of 350 microRNAs (miRNAs) in epididymis of rat from postnatal development to adult (from postnatal days 7-70) was profiled with home-made miRNA microarray.Among them,48 miRNAs changed significantly, in which the expression of miR-200a increased obviously with time,in a good agreement with that obtained from northern blot analysis.The real-time quantitative-polymerase chain reaction result indicated that temporal expression of rat β-catenin was exactly inversed to that of miR-200a during rat epididymal development,implying that miR-200a might also target β-catenin mRNA in rat epididymis as reported by Saydam et al.in humans.The bioinformatic analysis indicated that 3' untranslated region of rat β-catenin mRNA did contain a putative binding site for miR-200a.Meanwhile,it was found that the sequence of this binding site was different from that of human β-catenin mRNA with a deletion of two adjacent nucleotides (U and C).But the results of luciferase targeting assay in HEK 293T cells and the overexpression of miR-200a in rat NRK cells demonstrated that miR-200a did target rat β-catenin mRNA and cause the suppression of its expression.All these results show that miR-200a should be involved in rat epididymal development by targeting β-catenin mRNA of rat and suppressing its expression.

Involvement of the 5'-leader sequence in coupling the stability of a human H3 histone mRNA with DNA replication

International Nuclear Information System (INIS)

Morris, T.; Marashi, F.; Weber, L.; Hickey, E.; Greenspan, D.; Bonner, J.; Stein, J.; Stein, G.

1986-01-01

Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human β-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication

m6A-Driver: Identifying Context-Specific mRNA m6A Methylation-Driven Gene Interaction Networks

OpenAIRE

Zhang, Song-Yao; Zhang, Shao-Wu; Liu, Lian; Meng, Jia; Huang, Yufei

2016-01-01

As the most prevalent mammalian mRNA epigenetic modification, N6-methyladenosine (m6A) has been shown to possess important post-transcriptional regulatory functions. However, the regulatory mechanisms and functional circuits of m6A are still largely elusive. To help unveil the regulatory circuitry mediated by mRNA m6A methylation, we develop here m6A-Driver, an algorithm for predicting m6A-driven genes and associated networks, whose functional interactions are likely to be actively modulated ...

A mRNA and cognate microRNAs localize in the nucleolus.

Science.gov (United States)

Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

2014-01-01

We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

Max Weber e Martin Heidegger: a centralidade da razão e a perda da liberdade

Directory of Open Access Journals (Sweden)

Edilene Maria de Carvalho Leal

2015-10-01

Full Text Available A intenção desse artigo é analisar algumas ambivalências da modernidade, com destaque para a que consideramos a principal delas: a centralidade da razão como categoria analítica e como modelo das práticas sociais. Essa proposta é, evidentemente, uma proposta de análise teórica que se dará no âmbito específico da teoria social e da filosofia, principalmente desde interpretações produzidas por Max Weber e Martin Heidegger. Trata-se, em uma primeira instância, da tentativa de discutir criticamente as “autodescrições” que esses pensadores fizeram da modernidade. De um lado, a defesa weberiana da centralidade da razão; de outro, a crítica pós-metafísica heideggeriana dessa centralidade. Desse flagrante desencontro, podemos destacar um possível encontro analítico entre eles, ou seja, a crítica à perda da liberdade no contexto moderno.

Max Weber e Martin Heidegger: a centralidade da razão e a perda da liberdade

Directory of Open Access Journals (Sweden)

Edilene Maria de Carvalho Leal

2015-09-01

Full Text Available A intenção desse artigo é analisar algumas ambivalências da modernidade, com destaque para a que consideramos a principal delas: a centralidade da razão como categoria analítica e como modelo das práticas sociais. Essa proposta é, evidentemente, uma proposta de análise teórica que se dará no âmbito específico da teoria social e da filosofia, principalmente desde interpretações produzidas por Max Weber e Martin Heidegger. Trata-se, em uma primeira instância, da tentativa de discutir criticamente as “autodescrições” que esses pensadores fizeram da modernidade. De um lado, a defesa weberiana da centralidade da razão; de outro, a crítica pós-metafísica heideggeriana dessa centralidade. Desse flagrante desencontro, podemos destacar um possível encontro analítico entre eles, ou seja, a crítica à perda da liberdade no contexto moderno.

Direito à palavra: interrogações acerca da proposta da justiça restaurativa

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Edson Luiz André de Sousa

Full Text Available Partindo da constatação da necessidade de alternativas ao sistema de Justiça atual, o presente artigo objetiva dar voz a uma das propostas que vem ganhando força no cenário jurídico mundial: a Justiça Restaurativa. Para tanto, este artigo explora essa proposta, promovendo uma análise crítica amparada em autores da psicanálise (freudo-lacaniana e da Filosofia (com F. Ost e J. Derrida. Através da Justiça Restaurativa, é possível questionar a potência da circulação da palavra, característica que promove o tensionamento entre psicanálise e Direito. Além disso, a questão da Lei e das leis, da performatividade dos atos da fala, da posição do Juiz, da experiência do encontro restaurativo e da função das utopias são os principais vieses que compõem esta análise. Mais voltado para a possibilidade de anunciar questionamentos sobre essa proposta tão nova no contexto brasileiro do que de encontrar respostas satisfatórias, este artigo mantém em suspenso várias conclusões, embora assuma a constatação da importante função utópica da Justiça Restaurativa.

Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

Science.gov (United States)

Zhdanov, Vladimir P.

2017-10-01

mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

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

DEFF Research Database (Denmark)

Petersen, Tonny Studsgaard; Andersen, Claus Yding

2014-01-01

Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

International Nuclear Information System (INIS)

Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

1990-01-01

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

mRNA transfection of mouse and human neural stem cell cultures.

Directory of Open Access Journals (Sweden)

Samuel McLenachan

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

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

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. PMID:24386231

mRNA transfection of mouse and human neural stem cell cultures.

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

2013-01-01

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.

A universal trend of reduced mRNA stability near the translation-initiation site in prokaryotes and eukaryotes.

Directory of Open Access Journals (Sweden)

Wanjun Gu

2010-02-01

Full Text Available Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA.

A RNA transcript (Heg) in mononuclear cells is negatively correlated with CD14 mRNA and TSH receptor autoantibodies

DEFF Research Database (Denmark)

Habekost, G.; Bratholm, P.; Christensen, Niels Juel

2008-01-01

of the poly A(-) transcript (designated Heg) in mononuclear cells was correlated with CD14 mRNA in normal subjects and with CD14 mRNA and TSH receptor autoantibodies in patients with acute and untreated Graves' disease. mRNA was expressed in amol/mu g DNA. The main study groups were: (i) normal subjects; (ii......) patients with early and untreated Graves' disease; and (iii) patients with Graves' disease studied after treatment. In 18 normal subjects and in 20 patients with treated Graves' disease CD14 mRNA was negatively correlated with Heg (P Graves' disease Heg and thyroid...

Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability

NARCIS (Netherlands)

Fernandez, J.; Yaman, I.; Mishra, R.; Merrick, W. C.; Snider, M. D.; Lamers, W. H.; Hatzoglou, M.

2001-01-01

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA

Tissue-specific mRNA expression profiling in grape berry tissues

Science.gov (United States)

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

Tissue-specific mRNA expression profiling in grape berry tissues

Directory of Open Access Journals (Sweden)

Cramer Grant R

2007-06-01

Full Text Available Abstract Background Berries of grape (Vitis vinifera contain three major tissue types (skin, pulp and seed all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin and mesocarp (pulp, not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell

mRNA profiling for the identification of blood-Results of a collaborative EDNAP exercise

DEFF Research Database (Denmark)

Haas, Cordula; Hanson, E; Bär, W

2010-01-01

of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative......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, using...

mRNA profiling for the identification of blood--Results of a collaborative EDNAP exercise

DEFF Research Database (Denmark)

Haas, C.; Hanson, E.; Bär, W.

2011-01-01

of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative......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, using...

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

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Ganesh Ambigapathy

Full Text Available Brain-derived neurotrophic factor (BDNF has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

Science.gov (United States)

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Da possibilidade de uma Teoria Pura da Ciência da Informação: reflexões iniciais entre a Ciência da Informação e a Ciência do Direito

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Max Cirino de Mattos

Full Text Available O artigo discute a fragilidade epistemológica da Ciência da Informação, a partir da adoção de um termo polissêmico, como objeto científico, e a similaridade dessa situação com o contexto da criação da Teoria Pura do Direito de Hans Kelsen. Após a apresentação de algumas definições sobre o objeto da Ciência da Informação, discutem-se alguns reflexos relacionados à identidade científica e à fundamentação teórica derivadas de um objeto polissêmico. Apresenta-se uma breve introdução à Teoria Pura do Direito e algumas reflexões sobre a possibilidade de uma Teoria Pura da Ciência da Informação.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA.

Science.gov (United States)

Ono, Ekaterina Alexandrovna Durymanova; Taniwaki, Sueli Akemi; Brandão, Paulo

The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1log decrease in virus titter and 5.16-fold reduction in P mRNA, 24h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Estado da arte da S.C.I.E. desde 1951 a 2008 e a sua aplicação como especialidade da engenharia

OpenAIRE

Borges, Carlos Avelino Ferreira

2013-01-01

No âmbito da dissertação apresentada à Universidade da Madeira para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Engenharia Civil, foi desenvolvida uma pesquisa na área da Segurança Contra Incêndio em Edifícios, abreviadamente designada por SCIE, desde 1951 a 2008 e a sua aplicação como especialidade da Engenharia, designadamente ao nível da fenomenologia da combustão, no que concerne à evolução histórica dos organismos, legislação, normas técnicas e factos his...

Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

International Nuclear Information System (INIS)

Zambetti, G.; Stein, J.; Stein, G.

1987-01-01

The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

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

International Nuclear Information System (INIS)

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

1984-01-01

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

Poly A tail length analysis of in vitro transcribed mRNA by LC-MS.

Science.gov (United States)

Beverly, Michael; Hagen, Caitlin; Slack, Olga

2018-02-01

The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.

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

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Steven J. Schnell

2014-11-01

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

Differential regulation of renal cyclooxygenase mRNA by dietary salt intake

DEFF Research Database (Denmark)

Jensen, B L; Kurtz, A

1997-01-01

RNA correlated directly with salt intake. We conclude that dietary salt intake influences renal cyclooxygenase mRNAs zone-specifically with opposite responses between cortex and medulla. Cortical COX II-mediated prostaglandin formation is probably important in low salt states whereas medullary COX I......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...

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

Science.gov (United States)

Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

2011-07-01

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

Arc mRNA induction in striatal efferent neurons associated with response learning.

Science.gov (United States)

Daberkow, D P; Riedy, M D; Kesner, R P; Keefe, K A

2007-07-01

The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

International Nuclear Information System (INIS)

Popovich, B.; Barrieux, A.; Dillmann, W.H.

1987-01-01

Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for α-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with 32 P cDNA probes for α-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D α-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized α-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and α-actin mRNAs are decreased. Insulin treatment reverses these changes

Responsabilidade Social das empresas: a contraface da sociedade civil e da cidadania

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Simionatto, Ivete

2006-01-01

Full Text Available O artigo tem como objeto o estudo das mudanças que vêm ocorrendo nas relações entre Estado e sociedade civil e sua manifestação específica nas propostas articuladas em torno da responsabilidade social das empresas. Parte-se da hipótese de que estas práticas vêm contribuindo para a formação de consensos que fortalecem a hegemonia do capital na atualidade, mediante a redução do papel do Estado e o fortalecimento da esfera privada como setor de regulação social. Este cenário indica mudanças nas relações entre Estado e sociedade civil, uma vez que a responsabilidade social das empresas manifesta-se como uma nova proposta de sociabilidade e de resposta às expressões da questão social. Por fim, identifica-se que os serviços sociais oferecidos através da responsabilidade social fortalecem o campo do voluntariado, da solidariedade e das instituições do terceiro setor, precarizam o trabalho do Assistente Social com rebatimentos na esfera da cidadania e dos demais valores e princípios que sustentam o projeto ético-político da profissão

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

Science.gov (United States)

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

A LEITURA DA GRAMÁTICA DA FORMA DO CONJUNTO HABITACIONAL CAMPINAS-F DA CDHU EM CAMPINAS

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Marcelo de Morais

2014-12-01

Full Text Available A gramática da forma constitui uma importante ferramenta que pode dar suporte para futuros estudos e melhor qualidade em conjuntos habitacionais. A utilização de sistemas generativos de projeto pode aperfeiçoar uma série de rotinas de projeto, potencializando seu uso com outras ferramentas de informática. Este trabalho apresenta os resultados de estudo exploratório com um sistema generativo de projeto em processo de simulação e imersão, por meio do qual se pode avaliar o desenvolvimento e as alterações do projeto de implantação de conjunto habitacional social, ocasionadas pela variação das regras nos algoritmos criados. Inicialmente, se realizou a leitura Analítica da Gramática da Forma de um Conjunto Habitacional específico e, posteriormente, simulou-se em ferramenta computacional a Gramática da Forma Paramétrica, para futuros estudos de melhorias em conjuntos habitacionais similares. Tal ferramenta visa desenvolver soluções inovadoras em tecnologia da informação e comunicação aplicadas à construção e arquitetura. Os resultados demonstram a potencialidade do uso da Gramática da Forma, permitindo a inserção de novos parâmetros a cada momento, criando a perspectiva de flexibilidade na concepção e gerando diversas possibilidades que podem ser analisadas e comparadas durante o processo de tomada de decisão do projeto.

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

KAUST Repository

Arae, Toshihiro

2017-04-18

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

Da onda da gestão da qualidade a uma filosofia da qualidade da gestão: Balanced Scorecard promovendo mudanças

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Andréa Cavalcanti Correia Lima

2004-06-01

Full Text Available No final da década de 80 a Gestão da Qualidade Total (GQT era apresentada como capaz de resolver todos os problemas e atender a todas as necessidades da gestão das organizações. Buscando solucionar problemas de gestão não equacionados pela GQT, muitas organizações começam a adotar o Balanced Scorecard (BSC. Uma questão ainda não discutida com profundidade no mundo acadêmico é a viabilidade de integração desses dois modelos. O presente artigo discute a possibilidade de integração dessas duas ferramentas, verificando as melhorias que podem alcançar, principalmente no gerenciamento da estratégia da organização. Assim sendo, realizou-se estudo de caso no Caesar Park Hotel Fortaleza (CPHF, empresa que vêm adotando com sucesso os dois modelos. A pesquisa revela que é possível integrar os princípios da GQT com os do BSC, à medida que os fundamentos de ambos se complementam, sendo assim a utilização conjunta das ferramentas propicia melhor gerenciamento da estratégia da organização.In recent years, many organizations have been managed according to the principles of Total Quality Management (TQM, which became known around the globe in the 1980's, when looking for explanations for the so-called "Japanese miracle". TQM was presented as being capable of solving all problems and attending to all company management needs, a stage that seems outdated now. In the search for a model that would allow them to analyze and follow up the implementation of organizational strategies, without remaining limited to Quality indicators, many companies started to adopt the Balanced Scorecard (BSC. Companies using TQM question whether it is viable together with the BSC. In order to contribute to a solution to this question, this article discusses the possibility of aligning these two tools. Therefore, a case study was carried out at the Caesar Park Hotel Fortaleza, a company that implanted TQM in 1997 and started to adopt the BSC in 2000. The

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

DEFF Research Database (Denmark)

Schlemmer, B O; Sorensen, B S; Overgaard, J

2004-01-01

of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur...... at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression...

Applying the breaks on gene expression - mRNA deadenylation by Pop2p

DEFF Research Database (Denmark)

Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich

When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ......When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems...... to be the shortening of the poly(A) tail (deadenylation), as this step is slower than the subsequent decapping and degradation of the mRNA body. The Mega-Dalton Ccr4-Not complex contains two exonucleases, Ccr4p and Pop2p, responsible for this process. It is not known at present why two conserved nucleases are needed...

The hypoxic proteome is influenced by gene-specific changes in mRNA translation

International Nuclear Information System (INIS)

Koritzinsky, Marianne; Seigneuric, Renaud; Magagnin, Michael G.; Beucken, Twan van den; Lambin, Philippe; Wouters, Bradly G.

2005-01-01

Background and purpose: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. Materials and methods: DU145 prostate carcinoma cells were exposed to 4 h of hypoxia ( 2 ). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. Results: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. Conclusions: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

Science.gov (United States)

Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

Impacto da abertura comercial sobre a produtividade da indústria brasileira

OpenAIRE

Silva, Danielle Barbosa Lopes da

2004-01-01

Dentro do atual contexto econômico brasileiro, não existem dúvidas da importância de se discutir o tema produtividade industrial. Dentre os vários benefícios que um aumento da produtividade industrial pode gerar, há de se destacar a manutenção de preços baixos, a redução das taxas de juros e aumento do investimento e consumo. Além disso, o aumento da produtividade industrial ao reduzir o custo da produção e aumentar a sua escala, torna os produtos nacionais com preços mais competitivos no com...

The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

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Tachibana Taro

2011-10-01

Full Text Available Abstract Background Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII, which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq. Results We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII. Conclusions We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

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Carlo Cattani

2011-01-01

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

Ire1 mediated mRNA splicing in a C-terminus deletion mutant of Drosophila Xbp1.

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Dina S Coelho

Full Text Available The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions (the hydrophobic region 2--HR2--and the C-terminal translational pausing site present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. Here, we generated a Drosophila Xbp1 deletion mutant (Excision101 lacking both HR2 and C-terminal region, but not the Ire1 splicing site. We show that Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. Our results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced.

A RELAÇÃO DA TELEVISÃO E DA INTERNET COM O CONSUMO: ANÁLISES DA IDEOLOGIA E DA CULTURA NAS PROPAGANDAS

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Fábio Carlos Rodrigues Alves

2016-10-01

Full Text Available Mais que um meio de comunicação, as tecnologias da comunicação, em especial a TV, transmitem valores, ideias e atitudes, ou seja, todo um conteúdo atitudinal e procedimental. O presente texto discute o papel da mídia televisiva e da internet na transmissão de valores culturais, atendo-se principalmente na questão do consumo. Analisamos propagandas oriundas do ambiente televisivo e também a veiculada pela internet a partir da qual objetivamos, à luz do conceito de Cultura e Ideologia investigar e assinalar o condicionamento dos consumidores ao ato de consumo.

Localization of insulin receptor mRNA in rat brain by in situ hybridization

International Nuclear Information System (INIS)

Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G.

1990-01-01

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35 S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus

Ação do tacrolimus na pancreatite aguda experimental induzida pela arginina

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Marlus Moreira

Full Text Available OBJETIVO: verificar se o tacrolimus administrado em ratos, em vigência de pancreatite induzida pela L-Arginina, interfere nos níveis séricos da amilase e glicose e no padrão histológico do parênquima pancreático. MÉTODOS: quarenta ratos Wistar foram distribuídos em quatro grupos com 10 ratos cada. Grupo controle (C, grupo tacrolimus (T, grupo pancreatite (P e grupo pancreatite-tacrolimus (PT. Foram avaliados os níveis séricos de amilase, glicose e tacrolimus e feitas avaliações histológicas do pâncreas, A indução de pancreatite foi feita pela inoculação de L-Arginina na dose de 500mg/100g de peso corporal por via intraperitoneal e o tratamento com tacrolimus na dose de 1ìg/kg por via subcutânea durante quatro dias. RESULTADOS: a amilasemia estava mais elevada (p=0,0000 nos grupos PT, T e P do que no grupo controle. A média do grupo PT foi maior (p=0,0009 que a do grupo T, mas não diferiu (p=0,6802 da média do grupo P. Entre os grupos P e T não houve diferença (p=0,2568. Não houve diferença nas médias de glicemia entre os grupos (p=0,4920 e os níveis séricos de tacrolimus foram similares nos grupos PT e T (p=0,7112. Não ocorreram alterações histológicas nos grupos T e C e não ocorreu hemorragia no pâncreas dos ratos dos grupos P e PT. No grupo P, em 30% não se observou edema, em 20% observou-se a forma leve e em 50%, a moderada; quanto à infiltração inflamatória, em 80% moderada e em 20% não ocorreu, e a atrofia do parênquima foi de 60% moderada e 40% acentuada. No grupo PT, houve ocorrência de edema, infiltração inflamatória e atrofia do pâncreas em todos os ratos. CONCLUSÃO: o tratamento pelo tacrolimus induziu aumento nos níveis séricos de amilase em ratos normais, não alterou a glicemia nem o padrão histológico do parênquima pancreático. Na vigência de pancreatite induzida pela L-Arginina o tacrolimus induziu edema, infiltração inflamatória e atrofia com maior gravidade no par

Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

Science.gov (United States)

Crisan, D; Anstett, M J

1995-07-01

Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

ESTIMAÇÃO DA IDADE DA REGENERAÇÃO DA VEGETAÇÃO DE CERRADO A PARTIR DE IMAGENS LANDSAT

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Philippe Maillard, Priscilla S. Costa-Pereira

2011-07-01

Full Text Available Nas regiões em que a rede de monitoramento hidrossedimentológico é precária ou inexistente, a utilização de modelos hidrológicos, de base física que proporcionem estimativas da geração de escoamento superficial e a perda de solo, se torna muito útil para fins de planejamento das bacias hidrográficas. O presente trabalho trata da aplicação do modelo hidrossedimentológico “Soil And Water Assessment Tool” nas Sub-Bacias 2 e 1 da Bacia Experimental de São João do Cariri. O modelo com a interface ArcSWAT, que se une ao SIG, necessita da entrada de “Modelo Digital de Elevação” (MDE da bacia e as simulações foram realizadas utilizando dois MDEs para fins de análise comparativa: um originado a partir da interpolação de dados de campo e outro obtido através de sensoriamento remoto. Com isto foi possível verificar a confiabilidade do uso de MDE obtido por sensoriamento remoto. Em nível das sub-bacias experimentais, foi realizada a parametrização, na Sub-Bacia 2, e a validação, na Sub-Bacia 1. O desempenho do modelo, para a geração da lâmina escoada, foi avaliado pelo coeficiente de determinação (R² e do fator da eficiência de Nash-Sutcliffe. Os resultados mostram desempenho satisfatório para ambos os MDEs, mostrando a pouca sensibilidade da topografia da geração de lâmina simulada em pequenas bacias. Por outro lado, a produção de sedimentos apresentou resultados insatisfatórios, além do que foi significativa a discrepância da distribuição da erosão do solo na superfície da bacia pelos dois MDEs utilizados.

Effect of in vitro estrogenic pesticides on human oestrogen receptor α and β mRNA levels

DEFF Research Database (Denmark)

Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

2004-01-01

of the ERα mRNA level, but only significantly for prochloraz, dieldrin, and tolchlofos-methyl. Alone no pesticides affected the ERβ mRNA level significantly, but chlorpyrifos increased the mRNA level weakly. Co-exposure with E2 elicited a significant increased ERβ mRNA level by prochloraz, fenarimol...

Influência da nutrição mineral foliar sobre doenças da parte aérea da cultura do trigo

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Rafael Gustavo Ferreira Morales

2012-02-01

Full Text Available A utilização de fungicida para o controle de doenças foliares na cultura do trigo é prática comum entre os triticultores. Contudo, devido ao impacto ambiental provocado pelo seu uso generalizado, buscam-se alternativas para controle dos fitopatógenos. Sendo assim, este trabalho teve como objetivo avaliar o efeito da nutrição mineral foliar sobre as doenças foliares do trigo, bem como seu efeito sobre o desenvolvimento da planta. O delineamento experimental foi em blocos casualizados com quatro repetições e quatro tratamentos: 1- testemunha; 2- adubação foliar aos 30 dias após a emergência (DAE e aos 75 DAE; 3- fungicida aos 30 e 75 DAE; e 4- adubação foliar aos 30 DAE e adubação foliar mais fungicida aos 75 DAE. Foram realizadas nove avaliações da incidência e da severidade do oídio, cujos dados foram utilizados para o cálculo das áreas abaixo das curvas de progresso da doença; três avaliações da severidade da ferrugem e da mancha da gluma; e duas avaliações da mancha amarela. Foram determinadas a massa de matéria fresca e seca da parte aérea e do sistema radicular e a produtividade da cultura. Foi observada redução da incidência e da severidade de oídio em função da adubação foliar, apenas na primeira avaliação de incidência e nas segunda e quinta avaliações de severidade da doença. O efeito complementar dos nutrientes com o fungicida para redução das doenças foi observado apenas para a mancha amarela, com redução de aproximadamente 11,16 % da severidade. Os maiores valores de produtividade foram observados para os tratamentos apenas com fungicida e, com fungicida mais nutrientes. A adubação foliar proporcionou aumento da massa fresca de raízes e massa seca de raízes. Apesar disso, não houve aumento da massa fresca da parte aérea em função da adubação foliar.

AthMethPre: a web server for the prediction and query of mRNA m6A sites in Arabidopsis thaliana.

Science.gov (United States)

Xiang, Shunian; Yan, Zhangming; Liu, Ke; Zhang, Yaou; Sun, Zhirong

2016-10-18

N 6 -Methyladenosine (m 6 A) is the most prevalent and abundant modification in mRNA that has been linked to many key biological processes. High-throughput experiments have generated m 6 A-peaks across the transcriptome of A. thaliana, but the specific methylated sites were not assigned, which impedes the understanding of m 6 A functions in plants. Therefore, computational prediction of mRNA m 6 A sites becomes emergently important. Here, we present a method to predict the m 6 A sites for A. thaliana mRNA sequence(s). To predict the m 6 A sites of an mRNA sequence, we employed the support vector machine to build a classifier using the features of the positional flanking nucleotide sequence and position-independent k-mer nucleotide spectrum. Our method achieved good performance and was applied to a web server to provide service for the prediction of A. thaliana m 6 A sites. The server also provides a comprehensive database of predicted transcriptome-wide m 6 A sites and curated m 6 A-seq peaks from the literature for query and visualization. The AthMethPre web server is the first web server that provides a user-friendly tool for the prediction and query of A. thaliana mRNA m 6 A sites, which is freely accessible for public use at .

Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility.

Science.gov (United States)

Yamamoto, Hikaru; Yamashita, Yoshiki; Saito, Natsuho; Hayashi, Atsushi; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

2017-06-01

The aim of this study was to investigate whether FOXO1 and FOXO3 mRNA expression in granulosa cells is the cause of unexplained infertility. Thirty-one patients aged infertility and 18 with male partner infertility as a control group) whose serum anti-Müllerian hormone level was >0.5 ng/μL were enrolled in the study. All patients underwent oocyte retrieval under a short protocol from June 2012 to October 2013. Real-time PCR was carried out using mRNA extracted from granulosa cells retrieved from mature follicles. We compared FOXO1 and FOXO3 mRNA expression ratios in granulosa cells between the unexplained infertility group and the male infertility group. The relation between FOXO1 and FOXO3 mRNA expression ratios in granulosa cells and assisted reproduction technology clinical outcome was also examined. FOXO3 mRNA expression ratio was significantly lower in the unexplained infertility group than in the male infertility group. Moreover, FOXO3 mRNA expression ratio showed a positive correlation with both the number of retrieved oocytes and serum anti-Müllerian hormone level. A positive correlation was also identified between FOXO1 mRNA expression and total dose of hMG. As well, the number of retrieved oocytes in the unexplained infertility group was statistically lower than that in the male infertility group. A lower FOXO3 mRNA expression in granulosa cells leads to poor oocyte development in patients with unexplained infertility undergoing controlled ovarian stimulation for in vitro fertilization-embryo transfer. © 2017 Japan Society of Obstetrics and Gynecology.

Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

Science.gov (United States)

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

2014-08-28

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

Studies on the role of NonA in mRNA biogenesis

International Nuclear Information System (INIS)

Kozlova, Natalia; Braga, Jose; Lundgren, Josefin; Rino, Jose; Young, Patrick; Carmo-Fonseca, Maria; Visa, Neus

2006-01-01

The NonA protein of Drosophila melanogaster is an abundant nuclear protein that belongs to the DBHS (Drosophila behavior, human splicing) protein family. The DBHS proteins bind both DNA and RNA in vitro and have been involved in different aspects of gene expression, including pre-mRNA splicing, transcription regulation and nuclear retention of mRNA. We have used double-stranded RNA interference in Drosophila S2 cells to silence the expression of NonA and to investigate its role in mRNA biogenesis. We show that knockdown of NonA does not affect transcription nor splicing. We demonstrate that NonA forms a complex with the essential nuclear export factor NXF1 in an RNA-dependent manner. We have constructed stable S2 cell lines that express full-length and truncated NXF1 fused to GFP in order to perform fluorescence recovery after photobleaching experiments. We show that knockdown of NonA reduces the intranuclear mobility of NXF1-GFP associated with poly(A) + RNA in vivo, while the mobility of the truncated NXF1-GFP that does not bind RNA is not affected. Our data suggest that NonA facilitates the intranuclear mobility of mRNP particles

Natural selection and algorithmic design of mRNA.

Science.gov (United States)

Cohen, Barry; Skiena, Steven

2003-01-01

Messenger RNA (mRNA) sequences serve as templates for proteins according to the triplet code, in which each of the 4(3) = 64 different codons (sequences of three consecutive nucleotide bases) in RNA either terminate transcription or map to one of the 20 different amino acids (or residues) which build up proteins. Because there are more codons than residues, there is inherent redundancy in the coding. Certain residues (e.g., tryptophan) have only a single corresponding codon, while other residues (e.g., arginine) have as many as six corresponding codons. This freedom implies that the number of possible RNA sequences coding for a given protein grows exponentially in the length of the protein. Thus nature has wide latitude to select among mRNA sequences which are informationally equivalent, but structurally and energetically divergent. In this paper, we explore how nature takes advantage of this freedom and how to algorithmically design structures more energetically favorable than have been built through natural selection. In particular: (1) Natural Selection--we perform the first large-scale computational experiment comparing the stability of mRNA sequences from a variety of organisms to random synonymous sequences which respect the codon preferences of the organism. This experiment was conducted on over 27,000 sequences from 34 microbial species with 36 genomic structures. We provide evidence that in all genomic structures highly stable sequences are disproportionately abundant, and in 19 of 36 cases highly unstable sequences are disproportionately abundant. This suggests that the stability of mRNA sequences is subject to natural selection. (2) Artificial Selection--motivated by these biological results, we examine the algorithmic problem of designing the most stable and unstable mRNA sequences which code for a target protein. We give a polynomial-time dynamic programming solution to the most stable sequence problem (MSSP), which is asymptotically no more complex

Optimization of drying process of Zea Mays malt to use as alternative source of amylolytics enzymes

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Joana Paula Menezes Biazus

2005-06-01

Full Text Available This work aimed to study the drying process optimization of maize (Zea Mays malt for obtaining maize malt, without affecting enzymatic activity of alpha e beta-amylases from maize malt. Results showed that dryer operation must occur in zone at 54°C and 5.18-6 h process time. The maize malt obtained had good enzymatic properties.Este trabalho objetivou a otimização da secagem do malte de milho (Zea Mays para obter um malte sem afetar a atividade das enzimas presentes neste, alfa e beta -amilases. Os resultados mostraram que a operação do secador deve ser feita a 54°C e entre 5,18-6 h de processo. O malte obtido possuiu boas propriedades enzimáticas.

A identidade à flor da pele: etnografia da prática da tatuagem na contemporaneidade

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Andrea Lissett Pérez

2006-04-01

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.

A INFLUÊNCIA DA TECNOLOGIA E DA INTERNET NO MARKETING

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Lúcia Cristina Vargas Machado

2014-09-01

Full Text Available O dinamismo do mundo globalizado vem exigindo grandes mudanças das organizações, sejam para aquelas que produzem bens tangíveis, sejam para as prestadoras de serviços. E dentro da observância das mudanças, pode-se considerar as revoluções vividas pela humanidade, considerando aquela que mais mudou o cenário mercadológico pela primeira vez que foi 1ª revolução industrial e, a outra, a revolução da informação. Neste contexto, o objetivo deste trabalho é demonstrar através da revisão de literatura, a significativa e importante transformação da informação para a área de marketing das organizações. A revolução da informação, tendo como suas precursoras e legítimas aliadas a tecnologia e a internet, respectivamente, fez com que surgisse um novo tipo de marketing: o marketing digital. Com este novo tipo de marketing, as organizações que estiverem mais presentes na internet, poderão obter uma maior vantagem competitiva e como consequência uma maior lucratividade

Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

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Barredo Julio C

2002-03-01

Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

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

DEFF Research Database (Denmark)

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

2003-01-01

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

A ruptura articulatória dos seres: a propósito da exposição da vida à dispersão da sua ontologia

OpenAIRE

Rosa, Jorge Leandro

2014-01-01

Este artigo trata da possibilidade de renovar o entendimento da eugenia no mundo contemporâneo. A biologia e a genética são fontes científicas da eugenia, mas não fazem parte de seu núcleo filosófico. Essa é a razão pela qual vemos a ontologia subjacente à eugenia como nossa principal preocupação. O estatuto da eugenia como uma prática está mudando rapidamente e a vida é tanto o objeto como a dimensão oculta dessa mudança. Entendida por meio da história dos seres, a dispersão da ontologia per...

An investigation of nutrient-dependent mRNA translation in Drosophila larvae

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Sabarish Nagarajan

2014-10-01

Full Text Available The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling.

IMPLICAÇÕES DA ANÁLISE V-A-T DA TEORIA DAS RESTRIÇÕES NA GESTÃO DA CADEIA DE SUPRIMENTOS

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Diego Augusto de Jesus Pacheco

2016-03-01

Full Text Available O objetivo dessa pesquisa é identificar o impacto da análise V-A-T da Teoria das Restrições na gestão estratégica de materiais à luz da Matriz de Posicionamento Estratégico dos Materiais (MPEM. A partir de dois estudos de caso, buscou-se verificar: i em qual classificação cada empresa se enquadrava dentro da lógica V-A-T, e ii se a gestão estratégica de materiais a partir da abordagem da MPEM pode ser considerada semelhante ou não nos casos investigados. Os resultados da pesquisa apontaram que a classificação V-A-T no caso da empresa S é do tipo A e no caso da empresa K é do tipo V. Já a análise da gestão estratégica de materiais a partir da MPEM, evidenciou que no caso S pertence ao quadrante não crítico e no caso K pertence ao quadrante estratégico. Identificou-se que a classificação V-A-T tende a impactar na gestão de materiais e no controle de estoques.

Cointegrating MiDaS Regressions and a MiDaS Test

OpenAIRE

J. Isaac Miller

2011-01-01

This paper introduces cointegrating mixed data sampling (CoMiDaS) regressions, generalizing nonlinear MiDaS regressions in the extant literature. Under a linear mixed-frequency data-generating process, MiDaS regressions provide a parsimoniously parameterized nonlinear alternative when the linear forecasting model is over-parameterized and may be infeasible. In spite of potential correlation of the error term both serially and with the regressors, I find that nonlinear least squares consistent...

A circulação da experiência da dor em narrativas no Facebook

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Marlon Santa Maria Dias

2017-07-01

Full Text Available Este trabalho reflete alguns aspectos da problemática da circulação na ambiência da midiatização. A partir de material coletado em um grupo de discussão no Facebook sobre a mobilização “Eu não mereço ser estuprada”, analisamos as estratégias discursivas que compõem os processos de enunciação da mobilização dos atores sociais. Apontamos a reconfiguração narrativa na circulação da experiência da dor e a criação de redes interativas de apoio e aconselhamento.

Nerve growth factor mRNA in brain: localization by in situ hybridization

International Nuclear Information System (INIS)

Rennert, P.D.; Heinrich, G.

1986-01-01

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons

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

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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.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Köster, Tino

2017-04-13

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.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

2017-01-01

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.

Postage for the messenger: Designating routes for Nuclear mRNA Export

Science.gov (United States)

Natalizio, Barbara J.; Wente, Susan R.

2013-01-01

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

Screening women for cervical cancer carcinoma with a HPV mRNA test: first results from the Venice pilot program.

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Maggino, Tiziano; Sciarrone, Rocco; Murer, Bruno; Dei Rossi, Maria Rosa; Fedato, Chiara; Maran, Michela; Lorio, Melania; Soldà, Marika; Zago, Fiorella; Giorgi Rossi, Paolo; Zorzi, Manuel

2016-08-23

HPV DNA-based screening is more effective than a Pap test in preventing cervical cancer, but the test is less specific. New HPV tests have been proposed for primary screening. The HPV mRNA test showed a similar or slightly lower sensitivity than the HPV DNA tests but with a higher specificity. We report the results of an organised HPV mRNA-based screening pilot program in Venice, Italy. From October 2011 to May 2014, women aged 25-64 years were invited to undergo a HPV mRNA test (Aptima). Those testing positive underwent cytological triage. Women with positive cytology were referred to colposcopy, whereas those with negative cytology were referred to repeat the HPV mRNA test 1 year later. The results of the HPV mRNA test program were compared with both the local historical cytology-based program and with four neighbouring DNA HPV-based pilot projects. Overall, 23 211 women underwent a HPV mRNA test. The age-standardised positivity rate was 7.0%, higher than in HPV DNA programs (6.8%; relative rate (RR) 1.11, 95% confidence interval (CI) 1.05-1.17). The total colposcopy referral was 5.1%, double than with cytology (2.6%; RR 2.02, 95% CI 1.82-2.25) but similar to the HPV DNA programs (4.8%; RR 1.02; 95% CI 0.96-1.08). The cervical intraepithelial neoplasia grade 2+ detection rate with HPV mRNA was greater than in the HPV DNA programs at baseline (RR 1.50; 95% CI 1.19-1.88) and not significantly lower at the 1-year repeat (RR 0.70; 95% CI 0.40-1.16). The overall RR was 1.29 (95% CI 1.05-1.59), which was much higher than with cytology (detection rate 5.5‰ vs 2.1‰; RR 2.50, 95% CI 1.76-3.62). A screening programme based on the HPV mRNA obtained results similar to those observed with the HPV DNA test. In routine screening programmes, even a limited increase in HPV prevalence may conceal the advantage represented by the higher specificity of HPV mRNA.

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease.

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Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J; Leo, Oberdan; Goriely, Stanislas

2013-08-26

Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine-rich element (ARE)-binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow-derived dendritic cells (BMDCs) from TTP(-/-) mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3' untranslated region. TTP(-/-) mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23-IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation.

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

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Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

A metodologia de projecto na aprendizagem da biodiversidade e geodiversidade na praia da concha

OpenAIRE

Rosa, Marina Maria Filipe

2012-01-01

Dissertação de mestrado em Biologia e de Geologia, apresentada ao Departamento de Ciências da Terra e ao Departamento de Ciências da Vida Este trabalho consistiu na implementação e avaliação da metodologia de projeto no estudo da Biodiversidade e Geodiversidade na Praia da Concha, Portugal Central, dando ênfase ao trabalho prático, através de uma aula de campo e de aulas laboratoriais. O trabalho foi realizado numa escola da Marinha Grande, com 11 alunos do 10º ano de escolaridade. Sendo a...

A prática do quarto passo da iniciativa hospital amigo da criança

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Eloana Ferreira D'Artibale

2014-04-01

Full Text Available O objetivo deste estudo foi analisar os fatores envolvidos na prática do quarto passo da Iniciativa Hospital Amigo da Criança, a partir da vivência das puérperas internadas em um Hospital Amigo da Criança. Métodos: Trata-se de um estudo descritivo, exploratório, com abordagem qualitativa. A coleta de dados foi realizada de novembro de 2011 a janeiro de 2012, por meio de observação sistemática não participante dos partos, e entrevista semiestruturada com 16 sujeitos. Para o tratamento dos dados, foi aplicada a análise de conteúdo modalidade temática. Resultados: Os principais obstáculos para a efetivação do quarto passo foram a prioridade dada aos cuidados de rotina após o nascimento, o parto cesáreo e o processo de trabalho díspar das determinações da Iniciativa Hospital Amigo da Criança. Conclusão: Conclui-se ser necessária a (reconstrução e a reflexão de saberes e práticas acerca deste passo.

A redescoberta da Infância e da Criança

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Gabriela Trevisan

2010-11-01

Full Text Available A(s reconstrução (ões da (s imagen(s produzida (s sobre a Infância e sobre as crianças continuam a assumir-secomo desafio actual e necessário, à consideração de uma criança-cidadã, sujeita de direitos e deveres, ecompetente para o seu exercício. O modo como estas imagens influenciam essas possibilidades é visível emdiferentes contextos de vida quotidiana da criança sendo, a escola, local por excelência de exercício democrático ede aprendizagem dos direitos das crianças. Os professores, por sua vez, constituem-se como agentes centrais napromoção, defesa, e leitura crítica dos direitos da criança, nomeadamente, o de expressar a sua voz pelos seuspróprios meios e de participar activamente na comunidade educativa.

Effect of electroacupuncture on TRPM7 mRNA expression after cerebral ischemia/reperfusion in rats via TrkA pathway.

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Zhao, Li; Shi, Jing; Sun, Ning; Tian, Shunlian; Meng, Xianfang; Liu, Xiaochun; Li, Lingli

2005-01-01

The effect of electroacupuncture (EA) on TRPM7 mRNA expression of focal cerebral ischemia in rats and further the role of EA in the relationship between TRPM7 and trkA pathway was investigated. Thirty SD rats were randomly divided into 5 groups : normal group, ischemia/reperfusion group, EA treated group (ischemic rats with EA treatment), TE infusion group (ischemic rats with EA treatment and TE buffer infusion), AS-ODN group (ischemic rats with EA treatment and antisense trkA oligonucleotide infusion). The stroke animal model was established by the modified method of middle cerebral artery occlusion. Antisense trkA oligonucleotide that blocked NGFs effects was injected into cerebroventricle before EA. The TRPM7 mRNA was detected by RT-PCR method. The results showed that there were low TRPM7 mRNA levels in cortex and hippocampus in normal group. Compared with normal group, TRPM7 mRNA expression was increased significantly in ischemia/reperfusion group (PPM7 mRNA was found in EA treated group in contrast to ischemia/reperfusion group (P<0.05). The expression of TRPM7 mRNA in AS-ODN group was remarkably increased compared with EA treated group and TE infusion group (P<0.05). The results indicated that TRPM7 channels in the ischemic cortex and hippocampus in rats might play a key role in ischemic brain injury. EA could reverse the overexpression of TRPM7 in cerebral ischemia/reperfusion rats. And the inhibitory effect of EA on TRPM7 channels might be through trkA pathway.

Regulation of mRNA translation influences hypoxia tolerance

International Nuclear Information System (INIS)

Koritzinsky, M.; Wouters, B.G.; Koumenis, C.

2003-01-01

Hypoxia is a heterogenous but common characteristic of human tumours and poor oxygenation is associated with poor prognosis. We believe that the presence of viable hypoxic tumor cells reflects in part an adaptation and tolerance of these cells to oxygen deficiency. Since oxidative phosphorylation is compromized during hypoxia, adaptation may involve both the upregulation of glycolysis as well as downregulation of energy consumption. mRNA translation is one of the most energy costly cellular processes, and we and others have shown that global mRNA translation is rapidly inhibited during hypoxia. However, some mRNAs, including those coding for HIF-1 α and VEGF, remain efficiently translated during hypoxia. Clearly, the mechanisms responsible for the overall inhibition of translation during hypoxia does not compromize the translation of certain hypoxia-induced mRNA species. We therefore hypothesize that the inhibition of mRNA translation serves to promote hypoxia tolerance in two ways: i) through conservation of energy and ii) through differential gene expression involved in hypoxia adaptation. We have recently identified two pathways that are responsible for the global inhibition of translation during hypoxia. The phosphorylation of the eukaryotic initiation factor eIF2 α by the ER resident kinase PERK results in down-regulation of protein synthesis shortly after the onset of hypoxia. In addition, the initiation complex eIF4F is disrupted during long lasting hypoxic conditions. The identification of the molecular pathways responsible for the inhibition of overall translation during hypoxia has rendered it possible to investigate their importance for hypoxia tolerance. We have found that mouse embryo fibroblasts that are knockout for PERK and therefore not able to inhibit protein synthesis efficiently during oxygen deficiency are significantly less tolerant to hypoxia than their wildtype counterparts. We are currently also investigating the functional significance

Protein functional features are reflected in the patterns of mRNA translation speed.

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López, Daniel; Pazos, Florencio

2015-07-09

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

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

Institute of Scientific and Technical Information of China (English)

匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民

2003-01-01

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

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

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Morimoto, Shimpei; Yahara, Koji

2018-03-01

Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

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Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

Protein phosphatase magnesium-dependent 1δ (PPM1D mRNA expression is a prognosis marker for hepatocellular carcinoma.

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Guang-Bing Li

Full Text Available Protein phosphatase magnesium-dependent 1δ (PPM1D is an oncogene, overexpressed in many solid tumors, including ovarian cancer and breast cancer. The current study examined the expression and the prognostic value of PPM1D mRNA in human hepatocellular carcinoma (HCC.Total RNA was extracted from 86 HCC and paired non-cancerous liver tissues. PPM1D mRNA expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR. Immunohistochemistry assay was used to verify the expression of ppm1d protein in the HCC and non-cancerous liver tissues. HCC patients were grouped according to PPM1D mRNA expression with the average PPM1D mRNA level in non-cancerous liver tissue samples as the cut-off. Correlations between clinicopathologic variables, overall survival and PPM1D mRNA expression were analyzed.PPM1D mRNA was significantly higher in HCC than in the paired non-cancerous tissue (p<0.01. This was confirmed by ppm1d staining. 56 patients were classified as high expression group and the other 30 patients were categorized as low expression group. There were significant differences between the two groups in term of alpha-fetoprotein (α-FP level (p<0.01, tumor size (p<0.01, TNM stage (p<0.01, recurrence incidence (p<0.01 and family history of liver cancer (p<0.01. The current study failed to find significant differences between the two groups in the following clinical characteristics: age, gender, portal vein invasion, lymphnode metastasis, hepatitis B virus (HBV infection and alcohol intake. Survival time of high expression group was significantly shorter than that of low expression group (median survival, 13 months and 32 months, respectively, p<0.01.Up-regulation of PPM1D mRNA was associated with progressive pathological feature and poor prognosis in HCC patients. PPM1D mRNA may serve as a prognostic marker in HCC.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

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Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

2016-05-01

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

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Li, Ping; Noegel, Angelika A

2015-11-16

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A importancia da pericia frente a iatrogenia e a responsabilidade civil no exercicio da odontologia

OpenAIRE

Sergio Duz

2002-01-01

Resumo: O presente trabalho tem por objetivo avaliar a aplicação do termo iatrogenia, face a sua relação com as questões periciais em casos que envolvam a responsabilidade civil dos profissionais da área da saúde. A importância deste trabalho baseia-se na acentuada divergência quanto a sua interpretação. Assim sendo, o escopo da presente pesquisa é avaliar o campo de abrangência de aplicação do termo iatrogenia, nos casos de litígios processuais em que pode surgir a obrigação de indenizar. Ne...

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

Energy Technology Data Exchange (ETDEWEB)

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

1994-12-31

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.

Filosofia da análise da estabilidade da liquidez

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Rodrigo Antônio Chaves da Silva

2005-07-01

Full Text Available A informação foi considerada finalidade de nosso conhecimento, até o período em os pensadores e pesquisadores da contabilidade passaram a raciocinar sobre o conteúdo e o significado dos informes. Nesta busca da razão sobre os estados patrimoniais, surgiu a análise contábil que procura por meio de relações e identidades, o significado da dinâmica expressa da estaticamente na informação. O primeiro aspecto que surgiu no objeto de análise foi o estudo da liquidez, que é um dos principais exercícios do patrimônio. A estabilidade também é outro exercício básico e imprescindível, pois este é que promove o equilíbrio do organismo administrativo. A ciência contábil após a sua dignidade científica passou a trilhar caminhos esplendorosos, amparados em doutrina que permite alcançar os píncaros filosóficos. Os estudos concernentes aos aspectos de interação da estabilidade na liquidez são, complexos e somente com os recursos filosóficos da contabilidade se pode estudá-los com o panorama holístico e sublime. A filosofia da contabilidade não é alheia às suas práticas tecnológicas, podendo buscar pontos sublimes de panoramas abrangentes, para o estudo analítico da liquidez e estabilidade, observando todas as dimensionalidades e essencialidades de acontecimentos, na comprovação e orientação dos estados de ineficácia e eficácia patrimonial.

The mRNA level of MLH1 in peripheral blood is a biomarker for the diagnosis of hereditary nonpolyposis colorectal cancer.

Science.gov (United States)

Yu, Hong; Li, Hui; Cui, Yongan; Xiao, Wei; Dai, Guihong; Huang, Junxing; Wang, Chaofu

2016-01-01

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by functional defects in mismatch repair (MMR) genes, including mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2). This study aimed to assess whether the mRNA expression of MLH1 in peripheral blood could be used as a biomarkers for the diagnosis of HNPCC. The mRNA level of MLH1 was determined in 19 HNPCC families (46 members) using real-time quantitative polymerase chain reaction (qPCR). The mRNA levels of MLH1 in HNPCC were significantly lower than controls (P MLH1 for the diagnosis of HNPCC with the area under curve of 0.858. At an optimal cut-off value (0.511), the mRNA level of MLH1 had a sensitivity of 81.3% and a specificity of 86.7% for distinguishing HNPCC from controls. In conclusion, the mRNA expression of MLH1 in peripheral blood may serve as a biomarker for the diagnosis of HNPCC.

Detection of melatonin receptor mRNA in human muscle

International Nuclear Information System (INIS)

Li Lei

2004-01-01

To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

International Nuclear Information System (INIS)

Kurkcuoglu, Ozge; Doruker, Pemra; Jernigan, Robert L; Sen, Taner Z; Kloczkowski, Andrzej

2008-01-01

The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here, the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3' end forming the Shine–Dalgarno complex at the initiation step; the 3' end may act as a 'hook' to reel in the mRNA to facilitate its exit

A tensão naturalismo/normativismo no campo da definição da doença

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Paula Gaudenzi

2014-12-01

Full Text Available Vemos uma tendência da psiquiatria contemporânea de reduzir a experiência subjetiva da doença a achados neurofisiológicos. Na contramão desse movimento estão os trabalhos de Bill Fulford e Georges Canguilhem. O primeiro aposta na precedência da illness sobre a disease; Canguilhem defende a ideia de que o pathos precede o logos e ambos afirmam a inseparabilidade entre fatos e valores. Trazem, assim, a ênfase na dimensão sócio-simbólica da experiência subjetiva, marcando a centralidade da clínica para a interrogação sobre o sofrimento humano.

Maternal plasma levels of cell-free β-HCG mRNA as a prenatal diagnostic indicator of placenta accrete.

Science.gov (United States)

Zhou, J; Li, J; Yan, P; Ye, Y H; Peng, W; Wang, S; Wang, X Tong

2014-09-01

Several biomarkers, including maternal serum creatinine kinase and α-fetoprotein, have been described as potential tools for the diagnosis of placental abnormalities. This study aimed to determine whether maternal plasma mRNA levels of the β subunit of human chorionic gonadotropin (β-HCG) could predict placenta accreta prenatally. Sixty-eight singleton pregnant women with prior cesarean deliveries (CDs) were classified into three groups: normal placentation (35 women, control group); placenta previa alone (21 women, placenta previa group); and both placenta previa and placenta accreta (12 women, placenta previa/accreta group). Maternal plasma concentrations of cell-free β-HCG mRNA were measured by real-time reverse-transcription polymerase chain reaction and were expressed as multiples of the median (MoM). Cell-free β-HCG mRNA concentrations (MoM, range) were significantly higher in women with placenta accreta (3.65, 2.78-7.19) than in women with placenta previa (0.94, 0.00-2.97) or normal placentation (1.00, 0.00-2.69) (Steel-Dwass test, P accreta group, the concentration of cell-free β-HCG mRNA was significantly higher among women who underwent CDs with hysterectomy (4.41, 3.49-7.19) than among women whose CDs did not result in hysterectomy (3.20, 2.78-3.70) (Mann-Whitney U test, P = 0.012). An increased level of cell-free β-HCG mRNA in the maternal plasma of women with placenta accreta may arise from direct uteroplacental transfer of cell-free placental mRNA molecules. The concentration of cell-free β-HCG mRNA in maternal plasma may be applicable to the prenatal diagnosis of placenta accreta, especially to identify women with placenta accreta likely to require hysterectomy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

Science.gov (United States)

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Selective translation of the measles virus nucleocapsid mRNA by La protein

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Yoshihisa eInoue

2011-08-01

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.

Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

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Ji-Hyun Yeom

Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

A batuta da morte a orquestrar a vida

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Altair Macedo Lahud Loureiro

Full Text Available Com colocações de outros estudiosos da morte, teço e apresento apontamentos rápidos sobre a morte a organizar a vida. Esposo a idéia da necessidade da morte para que a vida tenha sentido nesta busca humana da completude. Endereço o olhar para uma situação especial de relação com a morte: a reação dos que ficam ao presenciar e sentir a morte de um dos seus idosos; daquele familiar, ou não, que assume o seu cuidado e acompanha o seu fim iminente; que presencia, humanamente impotente, a hora da solitária partida. Solitária morte, pois que exclusiva, própria daquele idoso do qual cuidava, do seu fim natural, por ser humano.

O Significado da Segurança na Europa: A UE – da PESC ao Colapso da Política Externa

OpenAIRE

Charillon, Frédéric

2001-01-01

O presente artigo tem por objectivo analisar o significado da segurança na Europa. A construção do processo comunitário dada a especificidade e conteúdo ambicioso do projecto envolvente, leva o autor a passar em revista vários dossiers comunitários de onde resultam consequências sobre o conceito e práticas da segurança europeia. A reinvenção do conceito de segurança na Europa passa em muito pela inserção de novas matérias no tradicional domínio da segurança entre as quais se contam e...

Cloning of the DNA-binding subunit of human nuclear factor κB: The level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor α

International Nuclear Information System (INIS)

Meyer, R.; Hatada, E.N.; Bartsch, C.; Scheidereit, C.; Hohmann, H.P.; Haiker, M.; Roethlisberger, U.; Lahm, H.W.; Schlaeger, E.J.; van Loon, A.P.G.M.

1991-01-01

The DNA binding subunit of nuclear factor κB (NF-κB), a B-cell protein that interacts with the immunoglobulin κ light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor α (TNFα), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-κB protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-κB binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNFα or phorbol ester. Thus, both factors not only activate NF-κB protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-κB

Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

Science.gov (United States)

Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

2018-05-01

Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

Cup regulates oskar mRNA stability during oogenesis.

Science.gov (United States)

Broyer, Risa M; Monfort, Elena; Wilhelm, James E

2017-01-01

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

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Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Conjecturas da Epistemológia Jurídica e Aspectos da Teoria da Linguagem

OpenAIRE

Oliveira, Rita de Cássia Cartelli de; Cesumar; Motta, Ivan Dias; Cesumar

2008-01-01

Apresentar-se-ão reflexões em torno da epistemologia jurídica e alguns aspectos da teoria da linguagem; a necessidade de acompanhamento e aprimoramento da linguagem jurídica, para que o direito não se distancie da realidade, mantendo-se apenas como um sistema do status quo; uma breve análise de algumas teorias da ciência do direito e da linguagem; as especificidades dos termos lingüísticos para a análise da ciência do direito, pautada na contemporaneidade sob a perspectiva humanista, buscando...

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex.

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Xie, Ping

2015-10-09

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.

Associations of ACE Gene Insertion/Deletion Polymorphism, ACE Activity, and ACE mRNA Expression with Hypertension in a Chinese Population

Science.gov (United States)

He, Qingfang; Fan, Chunhong; Yu, Min; Wallar, Gina; Zhang, Zuo-Feng; Wang, Lixin; Zhang, Xinwei; Hu, Ruying

2013-01-01

Background The present study was designed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion (I/D, rs4646994) polymorphism, plasma ACE activity, and circulating ACE mRNA expression with essential hypertension (EH) in a Chinese population. In addition, a new detection method for circulating ACE mRNA expression was explored. Methods The research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation. 221 hypertensives (cases) and 221 normotensives (controls) were interviewed, subjected to a physical examination, and provided blood for biochemical and genetic tests. The ACE mRNA expression was analyzed by real time fluorescent quantitative Reverse Transcription PCR (FQ-RT-PCR). We performed logistic regression to assess associations of ACE I/D genotypes, ACE activity, and ACE mRNA expression levels with hypertension. Results The results of the multivariate logistic regression analysis showed that the additive model (ID, DD versus II) of the ACE genotype revealed an association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ACE ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, our data also indicate that plasma ACE activity (adjusted OR was 1.13(95% CI: 1.08-1.18)) was significantly related to hypertension. However, the plasma ACE mRNA expressions were not different between the cases and controls. Conclusion ACE I/D polymorphism and ACE activity revealed significant influence on hypertension, while circulating ACE mRNA expression was not important factors associated with hypertension in this Chinese population. The detection of circulating ACE mRNA expression by FQ-RT-PCR might be a useful method for early screening and monitoring of EH. PMID:24098401

Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy

DEFF Research Database (Denmark)

Fisker, Niels; Westergaard, Majken; Hansen, Henrik Frydenlund

2007-01-01

We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced ...

Produção in vitro de enzimas extracelulares por fungos e sua relação com os sintomas descritos em planta hospedeira

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Alice Elias

2015-12-01

Full Text Available RESUMOOs fungos fitopatogênicos habitantes de solo causam perdas econômicas em muitas culturas e são difíceis de serem controlados. Esses fungos podem ser agrupados pelos sintomas comuns que causam nas plantas, bem como pelas enzimas extracelulares que podem produzir. O objetivo do trabalho foi verificar a produção in vitro de enzimas extracelulares por fungos de solo e tentar relacionar essas enzimas com os sintomas que cada fungo causa em planta hospedeira. O ensaio foi delineado em esquema inteiramente casualizado, com dois fatores, sete fungos (Cylindrocladium spathiphylli, Rhizoctonia solani, Sclerotium rolfsii, Ceratocystis fimbriata, Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. dianthi e Verticillium dahliae mais testemunha e seis enzimas (amilase, carboximetilcelulase, lipase, lacase, catalase e gelatinase com 10 repetições. Catalase e gelatinase foram mensuradas por escala de notas, enquanto que as demais pelo cálculo da área da coroa circular. O ensaio foi repetido e a análise foi realizada com os dados de dois ensaios. Os fungos que causam podridão na raiz ou no colo da planta apresentaram maior produção de lacase, enquanto os que causam obstrução, fendas ou até a destruição do sistema vascular demonstraram a prevalência da lipase.

A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

Science.gov (United States)

Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

2014-09-15

mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs. Copyright © 2014 Elsevier Ltd. All rights reserved.

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

Science.gov (United States)

Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

2003-05-01

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.

Science.gov (United States)

Pardi, Norbert; Tuyishime, Steven; Muramatsu, Hiromi; Kariko, Katalin; Mui, Barbara L; Tam, Ying K; Madden, Thomas D; Hope, Michael J; Weissman, Drew

2015-11-10

In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005-0.250mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1-4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

BIOTECNOLOGIA, DESENVOLVIMENTO E INOVAÇÃO: REFLEXÕES ACERCA DA PROPRIEDADE INTELECTUAL, LEI DE CULTIVARES E DA LICENÇA COMPULSÓRIA NO ÂMBITO DA BIOTECNOLOGIA

Directory of Open Access Journals (Sweden)

Fabiane Oliveira

2014-12-01

Full Text Available Considera-se o contexto dos processos de inovação e desenvolvimento no mundo moderno, a preocupação socioambiental e a tecnologia desenvolvida em função da agroindústria. Destaca-se a crescente importância conferida à biotecnologia na sociedade contemporânea. Reflete-se criticamente o estudo da propriedade intelectual no âmbito da biotecnologia em face do Estado e da inovação e do desenvolvimento. Busca-se, primeiramente, apresentar definição, origem e aspecto relevante da biotecnologia, a fim de tornar a sapiência desta ciência mais pragmática. Posto isso, elucida-se sobre a área de abrangência da proteção da propriedade intelectual nesse ramo da ciência. Nesse viés, ocupa-se também de demonstrar a Lei de Propriedade Intelectual mediante a problemática das patentes no setor biotecnológico. Observa-se, a seguir, o estudo da Lei de Cultivares em função da utilização do mecanismo de prospecção de patentes. Demonstra-se os aspectos importantes e o impacto da Lei de Cultivares no âmbito da propriedade intelectual. Exemplifica-se as cultivares protegidas. Apresenta-se a problemática da licença compulsória no âmbito da propriedade intelectual no ramo da biotecnologia. Objetiva-se demonstrar a situação desse ramo da ciência sob o prisma da propriedade intelectual, do fortalecimento e da padronização desses direitos. Expõe-se, por último, a importância da proteção dessa ciência para o desenvolvimento e para a inovação no país. Para isso, recorta-se a analise atual da Biotecnologia e da Propriedade Intelectual no Brasil sob o enfoque da legislação especifica e da hipótese da licença compulsória. Utiliza-se de pesquisa bibliográfica na área de biotecnologia, economia e direito, sob o enfoque da propriedade intelectual, inovação e do desenvolvimento.

Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

Science.gov (United States)

Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

2013-01-01

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (Pknowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

A decomposição do Estado e o protagonismo da sociedade civil no enfrentamento da questão social no Brasil: os dilemas da cidadania e da democracia na contemporaneidade

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Freire, Silene de Moraes

2006-01-01

Full Text Available O presente artigo foi elaborado a partir de preocupações profissionais voltadas para desvelar a presença e o enfrentamento, por parte do Estado e da sociedade civil, da questão social no cenário histórico-cultural da sociedade brasileira, evidenciado a partir da década de noventa, sobretudo, quando observamos a guinada do Estado brasileiro para a “mentalidade privatizante” que as reformas estruturais de cunho neoliberal promoveram. No caso brasileiro, tal projeto reafirma uma das particularidades de nossa cultura política: o moderno se constitui por meio do “arcaico”, recriando nossa herança histórica ao atualizar aspectos persistentes e, ao mesmo tempo, transformando-os no contexto da globalização. Pretendemos, assim, discutir de que modo a permanência histórica da desigualdade social, dos limites da cidadania e da democracia serviram como elemento facilitador para a implementação da agenda neoliberal no Brasil

Expression and significance of cyclooxygenase-2 mRNA in benign and malignant ascites

Science.gov (United States)

Lu, Jing; Li, Xiao-Feng; Kong, Li-Xia; Ma, Lin; Liao, Su-Huan; Jiang, Chang-You

2013-01-01

AIM: To investigate the mRNA expression of cyclooxygensae-2 (COX-2) in benign and malignant ascites, and to explore the difference in COX-2 mRNA expression among different diseases. METHODS: A total of 36 samples were collected from the Fifth Affiliated Hospital of Sun Yat-Sen University and divided into two experimental groups: benign ascites (n = 21) and malignant ascites (n = 15). Benign ascites included cirrhotic ascites (n = 10) and tuberculous ascites (n = 5). Malignant ascites included oophoroma (n = 7), cancer of colon (n = 5), cancer of the liver (n = 6), gastric cancer (n = 2), and bladder carcinoma (n = 1). The mRNA expression of COX-2 in ascites was examined with reverse transcriptase polymerase chain reaction (RT-PCR) technology, and the positive rate of COX-2 mRNA was compared between different diseases. RESULTS: The positive rate of COX-2 mRNA in malignant ascites was 42.9% (9/21), which was significantly higher than in benign ascites, 6.7% (1/15), difference being significant between these two groups (χ2 = 4.051, P = 0.044). The proportion of the positive rate in the malignant ascites was as follows: ovarian cancers 57.1% (4/7), colon cancer 40.0% (2/5), liver cancer 33.3% (2/6), gastric cancer 50.0% (1/2), and bladder cancer 0.00% (0/1). However, there was no significant difference in COX-2 mRNA expression among various tumors with malignant ascites (χ2 = 1.614, P = 0.806). Among the benign ascites, COX-2 mRNA levels were different between the tuberculous ascites (0/5) and cirrhotic ascites (1/10), but there was no significant difference (P = 1.000). CONCLUSION: COX-2 mRNA, detected by RT-PCR, is useful in the differential diagnosis of benign and malignant ascites, which also has potential value in the clinical diagnosis of tumors. PMID:24187465

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

International Nuclear Information System (INIS)

Lazarus, Kyren A.; Zhao, Zhe; Knower, Kevin C.; To, Sarah Q.; Chand, Ashwini L.; Clyne, Colin D.

2013-01-01

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 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 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 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

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-08-30

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

Expression of galectin-9 mRNA in obese children with polymorphism of the lactase gene

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A.E. Abaturov

2018-02-01

.73 ± 52.60 RU DmRNA Gal-9/mRNA actin, which exceeded the expression level of Gal-9 mRNA in children without obesity and lactose malabsorption (313.34 ± 19.70 RU DmRNA Gal-9 / mRNA actin, p < 0.01. After the use of exogenous lactase preparations in children with genotype C/C 13910 and lactose malabsorption for a month, the level of expression of Gal-9 mRNA approximated to the level of 246.21 ± 15.70 RU DmRNA Gal-9/mRNA actin, whereas when used only the low-lactose diet the level increased insignificantly to 58.72 ± 21.10 VD DmRNA Gal-9/mRNA actin, p < 0.01. It is also interesting that in children with genotype C/C 13910 without lactose malabsorption against the background of a low-lactose diet, the level of expression of Gal-9 mRNA decreased to 388.38 ± 20.40 RU DmRNA Gal-9/mRNA actin, approaching the level of healthy children without obesity. Conclusions. In children with genotype C/C 13910 the level of expression of Gal-9 mRNA depends on the lactose malabsorption, which requires replacement therapy that combines a low-lactose diet and the use of exogenous lactase preparations. In the absence of lactose malabsorption, using only the low-lactose diet in children with genotype C/C 13910 approximates the level of expression of Gal-9 mRNA to the level of healthy children without obesity.

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance

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Shimpei Morimoto

2018-03-01

Full Text Available Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes (ADC17 and KIN1 that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after

A SEDUÇÃO DA AUSÊNCIA: O TEXTO E A EPISTEMOLOGIA DA CIÊNCIA

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Ana Fukui

2016-12-01

Full Text Available O objetivo deste estudo é mostrar como o produtor textual organiza, em artigos de divulgação científica para crianças da revista Ciência Hoje das Crianças (CHC, a passagem do senso comum para o saber científico. Para isso, estudam-se, tendo por base a Linguística Textual e a Semiolinguística, o título e os parágrafos iniciais dos artigos, observando como acontecem a instalação do tópico discursivo, a construção de objetos de discurso e a orientação argumentativa. A análise linguística proposta é iluminada pelos princípios da Epistemologia da Ciência, de Bachelard, que orientam o produtor-divulgador da ciência. Observa-se que o texto se organiza de forma a desencadear um processo de orientação argumentativa que parte de um saber comum à instalação do saber científico, base do fim discursivo de incitação ao fazer, comum aos artigos do corpus. Na perspectiva da Semiolinguística, trata-se da indicação de uma lacuna existente, seja de ação, seja de conhecimento, seguida de informações que permitem o preenchimento do espaço vazio.

Clarice Lispector and the Historical Context in "A Hora da Estrela" Clarice Lispector e a contracena da História em A hora da estrela

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Isabel Virginia de Alencar Pires

2011-07-01

Full Text Available Este artigo aborda o livro "A hora da estrela", de Clarice Lispector, à luz dos pressupostos da Teoria da Recepção, de Jauss, que leva em conta o contexto histórico nas análises literárias. Publicada em fins de 1977, essa obra – que conta a história de Macabéa, uma nordestina que, “sem saber como nem por que”, acaba indo morar no “inacreditável Rio de Janeiro” – é considerada por diversos estudiosos como a “mais explicitamente social” da autora, pois até então os livros clariceanos costumavam ser recorrentemente acusados pela crítica da época de se “alhearem” da realidade, dedicando-se predominantemente a abordagens de caráter psicológico e deixando de lado o contexto político e social. Em A hora da estrela, a temática social passa a ser mais explícita, verificando-se assim uma espécie de “ruptura” na trajetória literária clariceana, interrompida no mesmo ano de publicação da obra, com a morte da autora. Este artigo busca apreender, nas entrelinhas do texto, as marcas que o diálogo do último livro lançado em vida pela escritora, durante o período do autoritarismo brasileiro, realiza com o seu contexto de surgimento. Para tanto, o estudo faz ainda uma aproximação entre o último livro de Clarice Lispector e o cinema de Glauber Rocha. This article contemplates Clarice Lispector’s “A hora da estrela” (The Hour of the Star, based on Jauss´ Reception Theory, which takes into account the historical context in literary analyses. Published in late 1977,  this work – which tells the story of Macabéa - a natural born northeast Brazilian woman that “not knowing why or how” moves to the “unbelievable city of Rio de Janeiro” – is considered by many scholars the “most explicitly social” one  done by the author. Up to that moment, her books had frequently been accused by critics of “deviating” from reality, focusing predominantly on psychological features and leaving social

A EVOLUÇÃO DA TECNOLOGIA DA INFORMAÇÂO (TI DENTRO DA ADMINISTRAÇÃO

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Joe Yukinori Katsurayama

2017-03-01

Full Text Available Buscamos relacionar a tecnologia e os paradigmas contemporâneos que hoje são de fundamental importância na rede empresarial e como neste contexto cotidiano essa questão é primordial para o desenvolvimento. O fenômeno da globalização mostra o quanto está modernizado a crescente expansão das mídias nas empresas atuais. Diante disto, o objetivo do estudo visa realizar um levantamento bibliográfico sobre a eficácia da Tecnologia da Informação na Administração. Para isto foram levantados estudos sobre a evolução desta tecnologia na base de dados Scielo, Google Acadêmico. Observou- se com o presente estudo que aos poucos as empresas foram se sensibilizando para a importância da informação na gestão de negócios. O conceito de TI engloba software, telecomunicações, automação, recursos multimídia, recursos de organização de dados, entre outros.

Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability

DEFF Research Database (Denmark)

Vytvytska, O; Jakobsen, J S; Balcunaite, G

1998-01-01

RNA. In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has...

Hepatic chemerin mRNA in morbidly obese patients with nonalcoholic fatty liver disease.

Science.gov (United States)

Kajor, Maciej; Kukla, Michał; Waluga, Marek; Liszka, Łukasz; Dyaczyński, Michał; Kowalski, Grzegorz; Żądło, Dominika; Berdowska, Agnieszka; Chapuła, Mateusz; Kostrząb-Zdebel, Anna; Bułdak, Rafał J; Sawczyn, Tomasz; Hartleb, Marek

The aim of this study was to investigate hepatic chemerin mRNA, serum chemerin concentration, and immunohistochemical staining for chemerin and and chemokine receptor-like 1 (CMKLR1) in hepatic tissue in 56 morbidly obese women with nonalcoholic fatty liver disease (NAFLD) and to search for a relationship with metabolic and histopathological features. Chemerin mRNA was assessed by quantitative real-time PCR, chemerin, and CMKLR1 immunohistochemical expression with specific antibodies, while serum chemerin concentration was assessed with commercially available enzyme-linked immunosorbent assays. Serum chemerin concentration reached 874.1 ±234.6 ng/ml. There was no difference in serum chemerin levels between patients with BMI steatosis, and definite nonalcoholic steatohepatitis (NASH). Liver chemerin mRNA was observed in all included patients and was markedly, but insignificantly, higher in those with BMI ≥ 40 kg/m2, hepatocyte ballooning, greater extent of steatosis, and definite NASH. Hepatic chemerin mRNA might be a predictor of hepatic steatosis, hepatocyte ballooning, and NAFLD activity score (NAS) but seemed not to be a primary driver regulating liver necroinflammatory activity and fibrosis. The lack of association between serum chemerin and hepatic chemerin mRNA may suggest that adipose tissue but not the liver is the main source of chemerin in morbidly obese women.

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

Science.gov (United States)

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

2017-10-01

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.

MANEJO DA IRRIGAÇÃO DA CULTURA DA ALFACE (Lactuca sativa L. ATRAVÉS DO TANQUE CLASSE A

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ANDRADE JÚNIOR A.S. de

1997-01-01

Full Text Available Com o objetivo de avaliar o efeito de diferentes níveis de irrigação, baseados em frações da evaporação do Tanque Classe A (ECA (0,25; 0,50; 0,75 e 1,00 sobre o comportamento produtivo da cultura da alface, variedade "Mesa 659" (tipo americana, cultivada em uma estufa plástica e irrigada por gotejamento, foi conduzido um experimento durante o período de julho a setembro de 1993. O comportamento produtivo foi avaliado através da determinação da matéria fresca (MFP, produtividade (PROD e eficiência do uso de água (EUA. Os resultados de MFP e PROD mostraram resposta quadrática, indicando acréscimo em ambos à medida que aumentaram as lâminas de irrigação aplicadas até o nível 0,75 da ECA, apresentando valores máximos de 818,72 g e 90,97 t.ha-1, respectivamente. Os dados relativos à EUA revelaram resposta linear decrescente, significando que à medida que os níveis de irrigação aumentam ocorre uma diminuição na EUA.

Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA

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Raúl Ortiz

2017-07-01

Full Text Available Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs.

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

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Piazza, Carol Lyn; Smith, Dorie

2018-01-01

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

Tecnologias da não-violência e modernização da justiça no Brasil: o caso da justiça restaurativa

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Schuch, Patrice

2008-01-01

Full Text Available Este artigo investiga, sob o ponto de vista antropológico, alguns aspectos da implementação da justiça restaurativa no Brasil. Através da pesquisa etnográfica, pode-se salientar que a justiça restaurativa articula um conjunto de atores diversos e conjuga ideários de autogestão e transformação individual com modernização da justiça e promoção de uma cultura de paz. Este texto objetiva entender como se dá a emergência dessa nova tecnologia de gerenciamento da vida, assim como discutir certas tensões próprias ao processo de sua disseminação.

NOTAS SOBRE A IRRACIONALIDADE DA GUERRA

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Edilene Leal

2014-08-01

Full Text Available Nesse artigo, procedemos a uma análise sobre a irracionalidade da guerra em tempos cada vez mais caracterizados pela evolução da racionalidade técnica, ou seja, em tempos contemporâneos. Esse é o ponto a partir do qual traçamos, aqui, um percurso argumentativo dividido em duas partes. Na primeira, reconstituímos elementos da concepção de guerra de filósofos como Maquiavel, Clausewitz e Kant, destacando, nos dois primeiros pensadores, a prerrogativa de que a guerra é racional quando se configura meio para fins políticos. Na segunda parte, confrontamos essa racionalidade política da guerra com a dupla dimensão técnica que a guerra assumiu: de um lado, a guerra como um fim em si mesmo e não mais como meio da ação política; de outro, a dimensão racionalizante da guerra como resultado do progresso técnico e científico, atualizada como guerra do terror.

Interleukin-21 mRNA expression during virus infections

DEFF Research Database (Denmark)

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

2006-01-01

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

Correlation of mRNA Expression and Signal Variability in Chronic Intracortical Electrodes.

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Falcone, Jessica D; Carroll, Sheridan L; Saxena, Tarun; Mandavia, Dev; Clark, Alexus; Yarabarla, Varun; Bellamkonda, Ravi V

2018-01-01

The goal for this research was to identify molecular mechanisms that explain animal-to-animal variability in chronic intracortical recordings. Microwire electrodes were implanted into Sprague Dawley rats at an acute (1 week) and a chronic (14 weeks) time point. Weekly recordings were conducted, and action potentials were evoked in the barrel cortex by deflecting the rat's whiskers. At 1 and 14 weeks, tissue was collected, and mRNA was extracted. mRNA expression was compared between 1 and 14 weeks using a high throughput multiplexed qRT-PCR. Pearson correlation coefficients were calculated between mRNA expression and signal-to-noise ratios at 14 weeks. At 14 weeks, a positive correlation between signal-to-noise ratio (SNR) and NeuN and GFAP mRNA expression was observed, indicating a relationship between recording strength and neuronal population, as well as reactive astrocyte activity. The inflammatory state around the electrode interface was evaluated using M1-like and M2-like markers. Expression for both M1-like and M2-like mRNA markers remained steady from 1 to 14 weeks. Anti-inflammatory markers, CD206 and CD163, however, demonstrated a significant positive correlation with SNR quality at 14 weeks. VE-cadherin, a marker for adherens junctions, and PDGFR-β, a marker for pericytes, both partial representatives of blood-brain barrier health, had a positive correlation with SNR at 14 weeks. Endothelial adhesion markers revealed a significant increase in expression at 14 weeks, while CD45, a pan-leukocyte marker, significantly decreased at 14 weeks. No significant correlation was found for either the endothelial adhesion or pan-leukocyte markers. A positive correlation between anti-inflammatory and blood-brain barrier health mRNA markers with electrophysiological efficacy of implanted intracortical electrodes has been demonstrated. These data reveal potential mechanisms for further evaluation to determine potential target mechanisms to improve

Characterization of DNA polymerase. beta. mRNA: cell-cycle growth response in cultured human cells

Energy Technology Data Exchange (ETDEWEB)

Zmudzka, B Z; Fornace, A; Collins, J; Wilson, S H

1988-10-25

DNA polymerase ..beta.. (..beta..-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. The authors used a cDNA probe to study abundance of ..beta..-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the ..beta..-polymerase transcript is low abundance and is neither cell-cycles nor growth phase responsive.

Controle Externo da governança de Tecnologia da Informação

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Raimir Holanda Filho

2012-06-01

Full Text Available O crescente processo de informatização da Administração Pública brasileira contribui para uma maior agilidade e qualidade nos serviços públicos prestados para a sociedade e um consequente aumento na transparência das ações governamentais. Por outro lado, os gastos nos investimentos e na manutenção dos recursos de Tecnologia da Informação (TI vêm aumentando consideravelmente, bem como tem-se verificado uma forte dependência das instituições com relação aos sistemas informatizados e à segurança das suas bases de dados. Com o aumento da importância estratégica da área de TI, houve uma busca pela aplicação de modelos de governança, com o objetivo de tornar a área controlável, com resultados mensuráveis e orientada aos objetivos do negócio da instituição. A auditoria de TI tem como função principal avaliar o processo de gestão, no que se refere aos seus diversos aspectos, tais como a governança corporativa, gestão de riscos de TI e procedimentos de aderência às normas regulatórias, apontando eventuais desvios e vulnerabilidades, como também oferecendo alternativas de soluções para esses diversos problemas. No âmbito do controle externo, os Tribunais de Contas começam a reconhecer a necessidade de implantar áreas especializadas na realização de Auditoria de TI. Neste contexto, o presente trabalho pretende apresentar quais as abordagens utilizadas nesta área de fiscalização, destacando a importância da Governança de TI como importante instrumento na atuação do controle externo na fiscalização da gestão e do uso da Tecnologia da Informação na Administração Pública.

Regulation of mouse hepatic CYP2D9 mRNA expression by growth and adrenal hormones.

Science.gov (United States)

Jarukamjorn, Kanokwan; Sakuma, Tsutomu; Jaruchotikamol, Atika; Oguro, Miki; Nemoto, Nobuo

2006-02-01

The constitutive expression of CYP2D9 is sexually dimorphic, namely, strong in males, but diminutive in females. Repetition of mimic growth hormone (GH) secretion pattern impressively returned the mRNA expression level to that in intact mice: the GH secretion pattern's regulation of CYP2D9 mRNA expression has been predominantly disrupted by exogenous GH-administration. The extensive decline of CYP2D9 mRNA expression becoming a sexually non-specific P450 in 9-week-old male mice exposed as neonates to monosodium L-glutamate (MSG) suggested that the male GH secretion pattern is a key to the regulation of male-specific CYP2D9 mRNA expression in adult mice. Dexamethasone (Dex) showed possibility to induce CYP2D9 mRNA expression in adult MSG-neonatally treated mice of either sex. However, the antagonism was observed by co-administration of Dex and GH in the males. Dex-administration in adrenalectomized mice significantly elevated CYP2D9 mRNA expression levels. These findings suggest that an adrenal hormone participates in the regulatory mechanism of CYP2D9 mRNA expression in association with GH.

Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant

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Olsvik Pål A

2009-03-01

Full Text Available Abstract Background The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA and cytochrome P450 A1 (CYP1A expression in Atlantic salmon Salmo salar L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg of the CYP1A inducer β-naphthoflavone (BNF and intestinal tissue (mid and distal intestine; MI and DI was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by in situ hybridization (ISH and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR; two detoxifying genes (CYP1A and glutathione S-transferase; GST, a stress marker gene (heat shock protein 70; HSP70, PCNA and a gene marker of apoptosis (caspase 6A. Results PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group. Conclusion This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal

UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

International Nuclear Information System (INIS)

Dalgaard, Louise T.

2012-01-01

Highlights: ► UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. ► UCP2 mRNA up-regulation by glucose is dependent on glucokinase. ► Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. ► This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic β-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/− islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2−/− and GK+/− islets compared with GK+/− islets and UCP2 deficiency improved glucose tolerance of GK+/− mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/− mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes

OpenAIRE

Pardi, Norbert; Tuyishime, Steven; Muramatsu, Hiromi; Kariko, Katalin; Mui, Barbara L; Tam, Ying K; Madden, Thomas D; Hope, Michael J; Weissman, Drew

2015-01-01

In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA c...

A ARQUIVÍSTICA COMO DISCIPLINA APLICADA NO CAMPO DA CIÊNCIA DA INFORMAÇÃO

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Fernanda Ribeiro

2011-06-01

Full Text Available Apesar de os arquivos terem origens muito remotas, que se podem confundir com as origens da própria escrita, a Arquivística como disciplina só no século XIX e no quadro do historicismo e do positivismo, se começou a afirmar, numa perspectiva de “ciência auxiliar” da História. Esta perspectiva historicista e custodial adquiriu novos contornos por alturas da viragem do século, graças à afirmação da vertente tecnicista, que se desenvolveu ao longo de todo o século XX, por efeito das condições sociais, económicas e culturais derivadas da revolução tecnológica em curso. Neste texto põe-se em confronto o tradicional paradigma historicista, custodial e tecnicista da Arquivística com um novo paradigma emergente, apelidado de pós-custodial, informacional e científico que entende esta disciplina como um saber aplicado do campo da Ciência da Informação, sendo apresentados os fundamentos epistemológicos, teóricos e metodológicos da abordagem proposta.

Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

NARCIS (Netherlands)

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

1990-01-01

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

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

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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.

Dis3- and exosome subunit-responsive 3′ mRNA instability elements

International Nuclear Information System (INIS)

Kiss, Daniel L.; Hou, Dezhi; Gross, Robert H.; Andrulis, Erik D.

2012-01-01

Highlights: ► Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. ► Identified novel 3′ UTR cis-acting element that destabilizes a reporter mRNA. ► Show exosome subunits are required for cis-acting element-mediated mRNA instability. ► Define precise sequence requirements of novel cis-acting element. ► 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′–5′ 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′ 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 underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of mRNA

Saberes docentes da dança

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Silvia Camara Soter da Silveira

2016-12-01

Full Text Available Este artigo trata do tema saberes docentes e busca sua relação com o ensino da dança na escola. A categoria saber docente tornou-se central no campo da formação de professores a partir dos anos 1990 no Brasil. Neste trabalho, o saber docente é abordado através de dois autores que se dedicaram ao tema: Maurice Tardif (2012 e Selma Garrido Pimenta (2012, na busca de refletir sobre as especificidades do saber docente daquele que ensina dança na escola. O saber docente se constitui, de uma parte, na formação inicial e contínua em instituições de formação de professores e, de outra parte, se desenvolve ao longo da vida dos estudantes e professores, em contextos distintos destas instituições, nas vivências destes professores antes da chegada à formação inicial, nas marcas que a escolarização deixou, na articulação do que aprendem nos cursos de formação de professores com a realidade da escola e, também, pela própria prática docente no início da atividade profissional, no contexto da escola, através das trocas com seus pares. No caso da dança, no saber docente se mesclam saberes constituídos no ensino formal como no não formal, que na trajetória dos docentes de dança parece ter um papel importante.

Caracterização de isolados de Xanthomonas axonopodis pv. manihotis = Characterization of isolates of Xanthomonas axonopodis pv. manihotis

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Roberto Luis Portz

2006-07-01

Full Text Available A bacteriose (Xanthomonas axonopodis pv. manihotis e a doenca de maior importancia economica na cultura da mandioca. Para estudar a variabilidade genetica desta bacteria no Oeste do Parana, foram realizados levantamentos em Entre Rios do Oeste, Marechal Candido Rondon, Mercedes, Missal, Nova Santa Rosa e Pato Bragado. Os isolados foram caracterizados em relacao a atividade de amilase, ƒ¿ e ƒÀ-esterase e agressividade. Dos 61 materiais vegetais coletados, obtiveram-se 19 isolados da bacteria, com maior incidencia para variedades de mesa em relacao aqueles para industria. Manivas provenientes de Pato Bragado, Entre Rios do Oeste e Mercedes apresentaram incidencias de 10, 27 e 10%, respectivamente, valores inferiores aos de Marechal Candido Rondon (50% e Nova Santa Rosa (58%. Os isolados foram agrupados em cinco, seis e 12 grupos em relacao a capacidade amilolitica, agressividade e isoenzimas de esterase, respectivamente. Nao houve relacao entre atividade de amilase e agressividade. Isolados de Marechal Candido Rondon foram mais agressivos que os provenientes das outras regioes. O agrupamento com base em esterase permitiu verificar que isolados provenientes de Entre Rios do Oeste, Nova Santa Rosa e Mercedes apresentaram alto grau de similaridade. Estes resultados indicam haver diferenciacaoentre os isolados da bacteria presentes nos municipios amostrados.The bacterial blight (Xanthomonas axonopodis pv. manihotis is the most important disease of cassava. To study the genetic variability of pathogen in the West of Parana, a research was carried out at Entre Rios do Oeste, Marechal Candido Rondon, Mercedes, Missal, Nova Santa Rosa and Pato Bragado. The isolates were characterized to amylase activity, ƒ¿ and ƒÀ-esterase and aggressiveness. From 61 collected materials, were obtained 19 bacterial isolates, with larger incidence for varieties of human consume than those for industry. Stems from Pato Bragado, Entre Rios do Oeste and Mercedes

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

Science.gov (United States)

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

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Deng D

2015-04-01

Full Text Available Dawei Deng,* Yang Li,* Jianpeng Xue, Jie Wang, Guanhua Ai, Xin Li, Yueqing GuDepartment of Biomedical Engineering, China Pharmaceutical University, Nanjing, People’s Republic of China*These authors contributed equally to this workAbstract: Messenger RNA (mRNA, a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP beacon containing a bare gold nanoparticle (AuNP as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.Keywords: molecular beacon, bioinformatics, gold nanoparticle, STAT5b mRNA, visual detection

Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

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Stefanie Hoyer

2015-01-01

Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

Estudos visando a sintese total da estavamicina, um inibidor do virus da herpes

OpenAIRE

Luciana Souza Alcantara Jardim

2000-01-01

Resumo: O vírus Epstein-Barr (EBV) é um vírus causador da herpes em seres humanos, infectando linfócitos e células epiteliais. Estima-se que este vírus infecta aproximadamente 90% da população mundial. Em 1995, Estavamicina, um novo produto natural da família dos pirrolocetoindanos foi isolado da cultura líquida de Streptomyces sp. Estavamicina mostrou atividade inibidora moderada contra a ligação do fator de transcrição EBV BZLF1 com o DNA com um valor de IC50 = 50 mM. Estavamicina possui um...

Resistência genotípica e monitoramento da favorabilidade para ocorrência da ferrugem alaranjada da cana-de-açúcar

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Kelly Lana Araújo

2013-12-01

Full Text Available A ferrugem alaranjada é atualmente uma das principais doenças da cana-de-açúcar. Redução na produtividade de genótipos suscetíveis e intermediários superiores a 40% foram registradas em muitos países, inclusive no Brasil. Este trabalho foi desenvolvido com o objetivo de monitorar os principais genótipos de cana-de-açúcar plantados no Brasil, sob alta pressão de inóculo, quanto à ocorrência e ao desenvolvimento da ferrugem alaranjada. O experimento foi conduzido em área experimental da Usina Univalem, do Grupo Raízen S.A., localizada em Valparaíso (SP. O delineamento experimental utilizado foi em blocos casualizados, com 38 tratamentos (genótipos e quatro repetições. Cada parcela foi constituída por três linhas de cinco metros, espaçadas de 1,4m. Entre cada parcela foi incluída uma linha do genótipo CV14, suscetível à ferrugem alaranjada. A partir da emergência das plantas, todas as parcelas foram monitoradas mensalmente para determinar o início do aparecimento dos sintomas da doença. Depois de constatados os primeiros sintomas, a severidade da ferrugem alaranjada foi quantificada mensalmente na folha +3 em três plantas por parcela. Os meses mais favoráveis para ocorrência da doença foram Fevereiro, Março, Abril e Maio. Foram observados sintomas da ferrugem alaranjada nos genótipos CTC 9, CV 14, RB93-5641, SP842025, RB72-454, SP89-1115, SP81-3250, RB85-5156, CTC 15, RB92-579, SP83-2847 e RB92-5211. Os genótipos SP84-2025 e CV 14 foram os mais suscetíveis à doença. Este trabalho contribui para aperfeiçoar o manejo integrado da ferrugem alaranjada no Brasil, além de auxiliar os programas de melhoramento visando à obtenção de novas variedades resistentes à doença.

A regeneração hepática e os inibidores da enzima conversora da angiotensina

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Fernando Silva Ramalho

2000-01-01

Full Text Available No presente estudo, o autor aborda conceitos fundamentais acerca do fenômeno regenerativo hepático, seguido de alguns aspectos importantes relacionados ao sistema calicreína-cininas. A associação entre os inibidores da enzima conversora da angiotensina, a bradicinina e a regeneração hepática é discutida à luz de estudos recentes, que revelam um papel estimulante das cininas sobre a proliferação hepatocelular. Os inibidores da enzima conversora da angiotensina, extremamente utilizados no tratamento clínico da hipertensão arterial sistêmica, da insuficiência cardíaca congestiva e da nefropatia diabética, também são capazes de favorecer a regeneração hepática.

Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning.

Science.gov (United States)

Johnson, S A; Lampert-Etchells, M; Pasinetti, G M; Rozovsky, I; Finch, C E

1992-01-01

This study describes evidence in the adult human and rat brain for mRNAs that encode two complement (C) proteins, C1qB and C4. C proteins are important effectors of humoral immunity and inflammation in peripheral tissues but have not been considered as normally present in brain. Previous immunocytochemical studies showed that C proteins are associated with plaques, tangles, and dystrophic neurites in Alzheimer's disease (AD), but their source is unknown. Combined immunocytochemistry and in situ hybridization techniques show C4 mRNA in pyramidal neurons and C1qB mRNA in microglia. Primary rat neuron cultures also show C1qB mRNA. In the cortex from AD brains, there were two- to threefold increases of C1qB mRNA and C4 mRNA, and increased C1qB mRNA prevalence was in part associated with microglia. As a model for AD, we examined entorhinal cortex perforant path transection in the rat brain, which caused rapid increases of C1qB mRNA in the ipsilateral, but not contralateral, hippocampus and entorhinal cortex. The role of brain-derived acute and chronic C induction during AD and experimental lesions can now be considered in relation to functions of C proteins that pertain to cell degeneration and/or cell preservation and synaptic plasticity.

Characterization of the ptr5+ gene involved in nuclear mRNA export in fission yeast

International Nuclear Information System (INIS)

Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

2012-01-01

Highlights: ► We cloned the ptr5 + gene involved in nuclear mRNA export in fission yeast. ► The ptr5 + gene was found to encode nucleoporin 85 (Nup85). ► Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. ► Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. ► Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A) + RNA transport] 1 to 11, which accumulate poly(A) + RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5–1 mutant shows dots- or a ring-like accumulation of poly(A) + RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5 + gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5–1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5–1 mutation. In addition, we found that the ptr5–1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5–1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

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

DEFF Research Database (Denmark)

Krakauer, Martin; Sorensen, P; Khademi, M

2008-01-01

BACKGROUND: Interferon (IFN)-beta therapy in multiple sclerosis (MS) has been suggested to promote a deviation from T lymphocyte production of pathogenic Th1 cytokines to less detrimental Th2 cytokines, but this is still controversial. We studied patterns of in vivo blood mononuclear cell (MNC...... of any Th1 or Th2 cytokines. The largest changes in cytokine mRNA levels occurred early (~9-12 h) after an IFN-beta injection. CONCLUSION: We found no evidence of a Th1- or Th2-mRNA-promoting effect of IFN-beta therapy. The therapeutic effect of IFN-beta is more likely attributable to the induction...

Effects of intrinsic aerobic capacity and ovariectomy on voluntary wheel running and nucleus accumbens dopamine receptor gene expression.

Science.gov (United States)

Park, Young-Min; Kanaley, Jill A; Padilla, Jaume; Zidon, Terese; Welly, Rebecca J; Will, Matthew J; Britton, Steven L; Koch, Lauren G; Ruegsegger, Gregory N; Booth, Frank W; Thyfault, John P; Vieira-Potter, Victoria J

2016-10-01

Rats selectively bred for high (HCR) and low (LCR) aerobic capacity show a stark divergence in wheel running behavior, which may be associated with the dopamine (DA) system in the brain. HCR possess greater motivation for voluntary running along with greater brain DA activity compared to LCR. We recently demonstrated that HCR are not immune to ovariectomy (OVX)-associated reductions in spontaneous cage (i.e. locomotor) activity. Whether HCR and LCR rats differ in their OVX-mediated voluntary wheel running response is unknown. To determine whether HCR are protected from OVX-associated reduction in voluntary wheel running. Forty female HCR and LCR rats (age ~27weeks) had either SHM or OVX operations, and given access to a running wheel for 11weeks. Weekly wheel running distance was monitored throughout the intervention. Nucleus accumbens (NAc) was assessed for mRNA expression of DA receptors at sacrifice. Compared to LCR, HCR ran greater distance and had greater ratio of excitatory/inhibitory DA mRNA expression (both line main effects, PWheel running distance was significantly, positively correlated with the ratio of excitatory/inhibitory DA mRNA expression across animals. In both lines, OVX reduced wheel running (Pwheel running, they had greater OVX-induced reduction in wheel running than LCR such that no differences were found 11weeks after OVX between HCROVX and LCROVX (interaction, Pwheel running in HCR was associated with an OVX-mediated reduction in the ratio of excitatory/inhibitory DA mRNA expression. The DA system in the NAc region may play a significant role in motivation to run in female rats. Compared to LCR, HCR rats run significantly more, which associates with greater ratio of excitatory/inhibitory DA mRNA expression. However, despite greater inherent motivation to run and an associated brain DA mRNA expression profile, HCR rats are not protected against OVX-induced reduction in wheel running or OVX-mediated reduction in the ratio of excitatory

Os azulejos da Misericórdia: a imagem documento na história da cultura religiosa luso-brasileira

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Maria Eurydice de Barros Ribeiro

2015-06-01

Full Text Available Os azulejos portugueses em Portugal e no Brasil são considerados quase sempre como objetos da História da Arte. A proposta deste estudo consiste em abordar os azulejos não como objetos de arte, mas como documentos da história da cultura luso-brasileira. O conceito de imagem documento será desenvolvido por meio da análise de um dos ritos mais importantes da história da cultura religiosa ocidental: a procissão. A atenção do estudo recairá, em especial, sobre a Procissão dos Ossos, que se encontra representada nos painéis de azulejos (séc. XVIII da Igreja da Misericórdia em Salvador. A Procissão dos Ossos, instituída por D. Manoel em 1498, tinha como objetivo a retirada dos ossos dos justiçados, dando-lhes sepultura. Para a missão, o rei designou a Ordem da Misericórdia. O ato régio se estendeu ao Brasil, quando a primeira Casa da Misericórdia foi fundada na cidade do Salvador, em 1549. O painel, datado do século XVIII, representando a cerimônia, divide o espaço da igreja da Misericórdia com outras duas representações: a procissão do Senhor Morto, introduzida no Brasil (também pelos portugueses, e a Procissão do Fogaréu, introduzida pelos espanhóis. Entre a data da realização da primeira Procissão dos Ossos e o assentamento do painel de azulejos existe um espaço de tempo que permite avaliar a heterogeneidade dos tempos da arte, onde os ritmos diferenciados não coincidem, necessariamente, com os tempos da história.

Transcription pattern of UL131A-128 mRNA in clinical strains of ...

Indian Academy of Sciences (India)

Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by ...

O desenvolvimento da Competência Informacional nas crianças a partir da literatura infantil

OpenAIRE

Djuli Machado De Lucca; Clarice Fortkamp Caldin; João Primo Ramirez Righi

2015-01-01

Objetiva realizar uma reflexão acerca da prática de leitura de literatura infantil para o desenvolvimento da Competência Informacional nas crianças. A Competência Informacional é caracterizada por um movimento que estimula o uso crítico e reflexivo da informação. Trata da primeira fase de desenvolvimento da Competência Informacional, entendida, nesse artigo, como letramento informacional, que se constitui em um processo. É nessa fase que o indivíduo desenvolve habilidades básicas relacionadas...

Nonparametric testing for DNA copy number induced differential mRNA gene expression

NARCIS (Netherlands)

van Wieringen, W.N.; van de Wiel, M.A.

2009-01-01

The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer.

A dimensão da pobreza, da desnutrição e da fome no Brasil

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Carlos Augusto Monteiro

2003-08-01

Full Text Available Este trabalho pretende contribuir para um melhor entendimento da dimensão e possíveis soluções para a fome e a desnutrição no Brasil. Inicialmente, procuramos esclarecer a natureza distinta de cada um desses problemas e o que os distingue conceitualmente da pobreza. A seguir, examinamos as alternativas disponíveis para operacionalizar os conceitos de pobreza, desnutrição e fome em estudos empíricos que buscam aferir a freqüência desses problemas na população. Finalmente, apresentamos e comparamos resultados relativos à freqüência, distribuição geográfica e tendência secular da pobreza, da desnutrição e da fome no Brasil e destacamos suas principais implicações para o delineamento de políticas públicas que busquem o controle desses problemas em nosso meio.This paper intends to contribute to a better understanding of the dimension and possible solutions for hunger and malnutrition in Brazil. We start by clarifying conceptual distinctions between hunger and malnutrition and what distinguishes these two problems from poverty. Then we examine available alternatives to implement the concepts of poverty, malnutrition and hunger in field studies aiming to assess the magnitude of these problems in the population. Finally, we present and compare results on the frequency, distribution and secular trends of poverty, malnutrition and hunger in Brazil and we also discuss its main implications to the design of public policies aiming the control of these problems.

A Economia da Família

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Ladislau Dowbor

2015-04-01

Full Text Available A família pode ser vista como unidade de reprodução econômica: pais sustentam filhos e idosos, e serão por sua vez sustentados. Hoje, com a desarticulação da família - nos EUA apenas 26% dos domicílios tem pais e filhos -, a fragilização do Estado e a privatização dos serviços sociais, é o próprio processo de redistribuição do excedente social entre gerações que se vê prejudicado. A dinâmica econômica ajuda a entender os impactos muito mais amplos, como a tensão entre gerações, a redução da sociabilidade e o sentimento crescente de angústia que se generaliza. A fase não produtiva da infância e da juventude, bem como da terceira idade, está se prolongando de maneira muito significativa. Foi-se o tempo em que a criança de 10 anos já ia para a roça ajudar o pai, e em que os pais faleciam quando os filhos chegavam à maturidade. Os desafios são econômicos, mas também sociais e culturais, em particular nas grandes cidades. É a própria base econômica da reprodução da sociedade que está se deslocando.

Atividade das glicosidases na presença de chá verde e de chá preto Activity of glycosidases in the presence of green tea and black tea

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L.L.S. Pereira

2010-12-01

Full Text Available Várias plantas têm sido consideradas produtos terapêuticos, dentre elas destacam-se os chás verde e preto, popularmente utilizados para controle da hiperglicemia e obesidade. Objetivou-se neste trabalho avaliar o potencial inibitório sobre as enzimas α-amilase, α e β-glicosidases e o teor de compostos fenólicos do chá verde e do chá preto. O teor de compostos fenólicos encontrados foram de 80,8 ± 0,43 mg g-1 no chá preto e 32,0 ± 0,12 mg g-1 no chá verde. O chá verde e o chá preto, em condições de consumo, inibiram as enzimas em estudo, porém, após a simulação do fluido gástrico o inibidor presente no chá verde perdeu a ação. O chá preto deixou de inibir a α-amilase e apresentou inibição inalterada para α e β-glicosidases. Tais resultados sugerem que o chá preto pode ser auxiliar em dietas de restrição de carboidratos.Several plants have been considered therapeutic products, including green and black tea, popularly used to control hyperglycemia and obesity. This study aimed to evaluate the inhibitory potential of the enzymes α-amylase, α and β-glycosidases, as well as the content of phenolic compounds in green tea and black tea. The concentrations of phenolic compounds found were 80.8 ± 0.43 mg g-1 in black tea and 32.0 ± 0.12 mg g-1 in green tea. Under the tested conditions of use, green and black tea inhibited the enzyme under study. However, after simulation of the gastric fluid, the inhibitor present in green tea lost its action. Black tea no longer inhibited a-amylase and showed unaltered inhibition for α and β-glycosidases. These results suggest that black tea can be helpful in diets restricting carbohydrates.

The silence of MUC2 mRNA induced by promoter hypermethylation associated with HBV in Hepatocellular Carcinoma

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Ling Yang

2013-01-01

Full Text Available Abstract Background To evaluate the promoter methylation status of MUC2 gene and mRNA expression in patients with hepatocellular carcinoma. Methods We analyzed MUC2 methylation by MSP, and MUC2 mRNA by real-time PCR in 74 HCC. Results MUC2 mRNA were lower in HCC tissues (Mean -ΔCt = −4.70 than that in Non-HCC tissues (Mean -ΔCt = −2.98. Expression of MUC2 was elevated in only 23 (31.08% of the 74 HCC patients. MUC2 promoter was hypermethylated in 62.2% (46/74 of HCCs, and in only 18.9% (14/74 of non-tumor samples. MUC2 mRNA were lower in HCC patients with hypermethylation (Mean -ΔΔCt = −2.25 than those with demethylation (Mean -ΔΔCt = −0.22, and there is a decreased tendency for MUC2 mRNA in HCC patients with promoter hypermethylation (p = 0.011. There was a significantly correlation found between MUC2 mRNA and HBV and AFP in HCC. The loss of MUC2 mRNA and hypermethylation could be poor prognostic factors. After treated by 5-Aza-CdR and TSA, we found that MUC2 mRNA induced significantly in 7721, Huh7 and HepG2 cells. Conclusion The results suggested that MUC2 mRNA silenced by promoter hypermethylation is associated with high levels HBV in HCC.

Carcinoma diferenciado da tireóide: a validade da complementação da tireoidectomia

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José Luís Braga de Aquino

Full Text Available OBJETIVO: Analisar a invasão tumoral do lobo contralateral da glândula tireóide no carcinoma diferenciado, correlacionando o risco/benefício com as complicações decorrentes de uma segunda intervenção. MÉTODO: De outubro/93 a dezembro/96 foram operados 20 pacientes com carcinomas diferenciados da glândula tireóide. Os parâmetros analisados foram sexo, idade, tipo de operação, tipo de complicações, histopatológico da peça cirúrgica e invasão do lobo contralateral. Eram dois pacientes do sexo masculino (10% e 18 do feminino (90%; as idades variaram de 17 a 89 anos; o tipo histológico mais freqüente foi o carcinoma papilífero (13 casos, seguido do folicular (seis casos e carcinoma de células de Hürthle (um caso. Como primeiro procedimento cirúrgico houve 11 lobectomias + istmectomias, quatro lobectomias subtotais e uma istmectomia. Cinco pacientes não realizaram a totalização (um por fibrose, três por perda de seguimento e um por ser microcarcinoma. RESULTADOS: Na análise do lobo contralateral realizada em 15 pacientes, 11 resultaram negativas e outras quatro positivas (26,6%. As complicações apresentadas foram rouquidão (dois casos revertidos com tratamento fonoterápico, hipoparatireoidismo (dois casos, um transitório e um permanente. CONCLUSÃO: A totalização da tireoidectomia é um procedimento importante no tratamento do tumor maligno da tireóide pela alta porcentagem de metástase contralateral (26,6%. Além disso, é um procedimento com mortalidade nula e pequena incidência de complicações.

Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

OpenAIRE

Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

2011-01-01

An oligonucleotide representing a regulatory element of human thymidylate synthase mRNA has been crystallized as a dimer. The structure of the asymmetric dimer has been determined at 1.97 Å resolution.

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

DEFF Research Database (Denmark)

Fackenthal, James D; Yoshimatsu, Toshio; Zhang, Bifeng

2016-01-01

patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation...... to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary...... or agarose gel electrophoresis, followed by sequencing. RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. CONCLUSIONS: These naturally occurring alternate-splicing events...

Da-Bu-Yin-Wan and Qian-Zheng-San to Neuroprotect the Mouse Model of Parkinson’s Disease

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Xiao-Gang Gong

2014-01-01

Full Text Available Da-Bu-Yin-Wan (DBYW and Qian-Zheng-San (QZS, two classic traditional Chinese medicinal formulas, were clinically employed to treat Parkinson’s disease (PD. Our previous studies demonstrated neuroprotective effects of them on mitochondrial function in PD mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP. The purpose of this research was to investigate their possible mechanisms in the light of mitochondrial ATP-sensitive potassium (mitoKATP channels. The neuroprotective effect of DBYW and QZS on dopamine (DA neurons in substantia nigra (SN in the MPTP-induced PD mice was investigated by behavioral test (pole test and immunohistochemistry. Adenosine triphosphate (ATP level in the midbrain tissue was detected by firefly luciferase method. MitoKATP channel subunits SUR1 and Kir6.2 mRNA and protein expressions were tested by real-time PCR (RT-PCR and Western blot. It was observed that DBYW and/or QZS served to ameliorate MPTP-induced behavioral impairment and prevent the loss of substantia nigra dopamine neurons, as well as increase ATP level in the midbrain tissue and downregulate SUR1 expression at mRNA and protein levels with no marked influence on Kir6.2. We concluded that DBYW and QZS exhibit neuroprotective effects probably through the regulation of ATP level and mitoKATP channel subunit expressions.

In situ localization of chalcone synthase mRNA in pea root nodule development.

NARCIS (Netherlands)

Yang, W.C.; Canter Cremers, H.C.J.; Hogendijk, P.; Katinakis, P.; Wijffelman, C.A.; Franssen, H.J.; Kammen, van A.; Bisseling, T.