Cell Cycle-dependent Metabolism of Pyrimidine Deoxynucleoside Triphosphates in CEM Cells

We incorporated 3H-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G1 and S phase cells were separated by centrifugal elutriation, and the size and specific activity of dNTP pools were determined to study the cell cycle-dependent regulation of specific dNTP synthesizing enzymes in their metabolic context. With [3H]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivity from [5-3H]deoxycytidine into dCTP occurred almost exclusively in G1 cells. During S phase, de novo synthesis by ribonucleotide reductase was switched on, resulting in a 70-fold dilution of [3H]dCTP, confirming that ribonucleotide reductase is an S phase-specific enzyme, whereas deoxycytidine kinase is not. [5-3H]Cytidine appeared in dCTP almost to the same extent in G1 as in S phase, despite the S phase specificity of ribonucleotide reductase. During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding increase in ribonucleotide reductase activity. During G1, the enzyme maintained activity to provide dNTPs for DNA repair and mitochondrial DNA synthesis. The poor incorporation of isotope from deoxycytidine into DNA earlier led to the suggestion that the nucleoside is used only for DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem. 270, 631-637). The poor phosphorylation of deoxycytidine in S phase provides a better explanation

Effects of mutational loss of nucleoside kinases on deoxyadenosine 5'-phosphate/deoxyadenosine substrate cycle in cultured CEM and V79 cells.

The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there

Quantitation of cellular deoxynucleoside triphosphates

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP

O HÁBITO DA LEITURA SENDO MOTIVADO E DESENVOLVIDO POR MEIO DE PROJETOS DE LEITURA

O presente Relatório de Estágio Curricular Supervisionado em Letras – Língua Portuguesa tem por objetivo conhecer e interagir com a Escola de Educação Básica. Esse processo de conhecimento e descoberta além de desafiante é de extrema necessidade para a formação docente. Até a conclusão deste Relatório foram inúmeras etapas desenvolvidas. Dentre elas estão a Observação e a Docência em Língua Portuguesa e Literatura, ambas desenvolvidas na Escola de Educação Básica Romildo Czepanhik, no município de Xanxerê-SC; entre 2015 e 2016. A primeira etapa do Estágio foi a observação escolar – docentes, discentes e gestão. Em seguida, foi preciso buscar materiais que ressaltassem a importância da leitura nos Ensinos Fundamental e Médio; para depois elaborar um planejamento  que contemplasse a leitura  e a produção textual, tema central deste relatório. Foram traçadas metas e, para que se concretizassem foi preciso muito estudo e dedicação. Com isso, neste relatório estão apresentados inúmeros fatores que além de justificar a problemática elencada remetem a possíveis soluções para que seja possível intervir positivamente na Escola de Educação Básica

p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage.

Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-(3)H]cytidine, [6-(3)H]deoxycytidine, and [C(3)H(3)]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication

Adaptación de un procedimiento psicoterapéutico para el estrés post-desastre aplicado después del terremoto y tsunami del 27 de febrero de 2010

136 p.El 27 de Febrero de 2010 un terremoto, de magnitud 8.8 en la escala de Magnitud de Momento, y un posterior tsunami sacudió y arrasó con la zona central de Chile. Frente a una catástrofe como esta, un porcentaje de la población afectada desarrollará estrés post-desastre. Con el fin de tratar éste, la presente investigación tiene como objetivo adaptar la Terapia Cognitivo Conductual para Estrés Post-Desastre, desarrollada en E.E.U.U por Hamblen, Gibson, Mueser y Norris, para intervenir a los sobrevivientes del 11-S y el Huracán Katrina. Para esto la terapia fue aplicada en una versión preliminar por siete terapeutas a 33 sujetos de la región del Maule, que tenían sintomatología de estrés post-desastre. Los primeros fueron entrevistados luego de finalizar la aplicación, y la información obtenida fue sometida a un análisis de contenido, que se tradujo en 47 aportes que fueron incorporados a cada etapa de la terapia original, destacándose por ejemplo, la factibilidad de aplicar la terapia en modalidad grupal, la integración del ejercicio de reentrenamiento de la respiración al finalizar cada sesión, la adaptación de la lista de actividades placenteras al contexto chileno, así como contar con reuniones clínicas semanales para los clínicos, como una instancia de desahogo y entrenamiento, entre otros aportes. Con esto se logró contar con una versión de la terapia adaptada en el contexto local. Se sugiere para futuras investigaciones evaluar la efectividad de dicha terapia a largo plazo, así como también considerar su adaptación para otra clase de situaciones traumáticas. Palabras claves: Terapia cognitiva conductual, estrés post-desastre, terapia cognitiva conductual para estrés post-desastre, terremoto, desastre natural

Fatal adenoviral necrotizing bronchiolitis case in a post-cardiac surgery intensive care unit

We report a case of a 67 year-old-male patient admitted to the intensive care unit in the post-coronary bypass surgery period who presented cardiogenic shock, acute renal failure and three episodes of sepsis, the latter with pulmonary distress at the 30th post-operative day. The patient expired within five days in spite of treatment with vancomycin, imipenem, colistimethate and amphotericin B. At autopsy severe adenovirus pneumonia was found. Viral pulmonary infections following cardiovascular surgery are uncommon. We highlight the importance of etiological diagnosis to a correct treatment approach