OXIDATIVE STRESS BASED STRATEGIES FOR ENHANCING THE EFFICACY OF HISTONE DEACETYLASE INHIBITORS (HDACi)

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that primarily act upon acetylation of histones, however they also increase levels of intracellular reactive oxygen species (ROS). We hypothesized that agents that cause oxidative stress might enhance the efficacy of HDACi. To test this hypothesis, we treated acute lymphocytic leukemia cells (ALL) with HDACi and adaphostin (ROS generating agent). The combination of two different HDACi (vorinostat or entinostat) with adaphostin synergistically induced apoptosis in ALL. This synergistic effect was blocked when cells were pre-treated with the caspase-9 inhibitor, LEHD. In addition, we showed that loss of the mitochondrial membrane potential is the earliest event observed starting at 12 h. Following this event, we observed increased levels of superoxide at 16 h, and ultimately caspase-3 activation. Pre-treatment with the antioxidant N-acetylcysteine (NAC) blocked ROS generation and reversed the loss of mitochondrial membrane potential for both combinations. Interestingly, DNA fragmentation and caspase-3 activity was only blocked by NAC in cells treated with vorinostat-adaphostin; but not with entinostat-adaphostin. These results suggest that different redox mechanisms are involved in the induction of ROS-mediated apoptosis. To further understand these events, we studied the role of the antioxidants glutathione (GSH) and thioredoxin (Trx). We found that the combination of entinostat-adaphostin induced acetylation of the antioxidant thioredoxin (Trx) and decreased intracellular levels of GSH. However, no effect on Trx activity was observed in either combination. In addition, pre-treatment with GSH ethyl ester, a soluble form of GSH, did not block DNA fragmentation. Together these results suggested that GSH and Trx are not major players in the induction of oxidative stress. Array data examining the expression of genes involved in oxidative stress demonstrated a differential regulation between cells treated with vorinostat-adaphostin and entinostat-adaphostin. Some of the genes differentially expressed between the combinations include aldehyde oxidase 1, glutathione peroxidase-5, -6, peroxiredoxin 6 and myeloperoxidase. Taken together, these experimental results indicate that the synergistic activity of two different HDACi with adaphostin is mediated by distinct redox mechanisms in ALL cells. Understanding the mechanism involved in these combinations will advance scientific knowledge of how the action of HDACi could be augmented in leukemia models. Moreover, this information could be used for the development of effective clinical trials combining HDACi with other anticancer agents.

SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase

Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by Salmonella dublin, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro. SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca2+-dependent chloride secretion. In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate to yield inositol 1,4,5,6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the inhibition of chloride secretion by phosphatidylinositol 3,4,5-trisphosphate [Eckmann, L., Rudolf, M. T., Ptasznik, A., Schultz, C., Jiang, T., Wolfson, N., Tsien, R., Fierer, J., Shears, S. B., Kagnoff, M. F., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14456–14460]. Mutation of a conserved cysteine that abolishes phosphatase activity of SopB results in a mutant strain, S. dublin SB c/s, with decreased ability to induce fluid secretion in infected calf intestine loops. Moreover, HeLa cells infected with S. dublin SB c/s do not accumulate high levels of inositol 1,4,5,6-tetrakisphosphate that are characteristic of wild-type S. dublin-infected cells. Therefore, SopB mediates virulence by interdicting inositol phosphate signaling pathways.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

Histoire des juifs et des peuples voisins : depuis la décadence des Royaumes d'Israël et de Juda jusqu'à la mort de Jesus-Christ /

Mode of access: Internet.

The Artizan.

Title from caption.

Operator, organizational, direct support, and general support maintenance manual : tank and pump unit, liquid dispensing, for truck mounting (Advanced Industries model 1800) FSN 4930-070-1181.

"August 1972."

Reports and analysis handbook series.

Title varies: no. 5, Analysis handbook series.

A major quantitative trait locus for CD4-CD8 ratio is located on chromosome 11

CD4-CD8 ratio is an important diagnostic measure of immune system functioning. In particular, CD4-CD8 ratio predicts the time taken for progression of HIV infection to acquired immune deficiency syndrome (AIDS) and the long-term survival of AIDS patients. To map genes that regulate differences between healthy individuals in CD4-CD8 ratio, we typed 757 highly polymorphic microsatellite markers at an average spacing of similar to5 cM across the genome in 405 pairs of dizygotic twins at ages 12, 14 and 16. We used multipoint variance components linkage analysis to test for linkage between marker loci and CD4-CD8 ratio at each age. We found suggestive evidence of linkage on chromosome 11p in 12-year-old twins (LOD=2.55, P=0.00031) and even stronger evidence of linkage in the same region at age 14 (LOD 3.51, P=0.00003). Possible candidate genes include CD5 and CD6, which encode cell membrane proteins involved in the positive selection of thymocytes. We also found suggestive evidence of linkage at other areas of the genome including regions on chromosomes 1, 3, 4, 5, 6, 12, 13, 15, 17 and 22.

Identification of a novel inositol phosphate recognition site: specific [3H]inositol hexakisphosphate binding to brain regions and cerebellar membranes

[3H]Inositol hexakisphosphate (InsP6) binds with a heterogeneous distribution to frozen sections of unfixed rat brain and is displaced by unlabelled InsP6. The pattern of binding correlates with binding to neuronal cell bodies. [3H]InsP6 binding to cerebellar membranes has been further characterised, is reversible, and saturable, and exhibits high specificity for inositol polyphosphates. The IC50 for competition by unlabelled InsP6 is approximately 100nM, whereas inositol 1,3,4,5,6 pentakisphosphate (Ins(13456)P5), inositol 1,3,4,5 tetrakisphosphate (Ins(1345)P4), and inositol 1,4,5 trisphosphate (Ins(145)P3) bind with an affinity at least one order of magnitude lower. [3H]InsP6 binding is clearly distinct from previously characterised Ins(145)P3 (ref. 1, 2) and Ins(1345)P4 (ref. 3) binding, both in terms of pharmacology and brain distribution.

The formation of phosphatidylcholine oxidation products by stimulated phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814 m/z) and bis-hydroperoxides (846 m/z) of PAPC was observed. However, the major oxidized product occurred at 828 m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E-2)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.

Sistemas de produção de milho sob adubação nitrogenada e plantas de cobertura do solo

The no-tillage system is the predominant model in the agricultural scenario of southern Brazil. Thus, the use of cover crops is significant due to the addition of biomass to protect the soil surface, and contribute to the cycling and/or fixing of nutrients, and in particular nitrogen (N) with liberation for the subsequent culture. Among the cool season species, it was found predominant use of oat to obtain straw to system. Though large quantities input of residue is not the preferred species to precede the corn, cereal with relevant importance in the Paraná Southwest region. It was aimed to evaluate the productivity capacity of corn in no-tillage, in the absence or presence of nitrogen fertilization, on waste of winter cover crops on soil and climatic conditions of the Paraná Southwest region. The installation of no-tillage was held in 2010 in the experimental area belonging to UTFPR, Campus Dois Vizinhos, on a Red Latosol. For the present study, we used data relating to three agricultural years (2012/2013, 2013/2014 and 2014/2015). The experimental design was randomized block design with split plots with three replications. The main plots consisted of systems composed by cover crops (black oat, ryegrass, rye, turnip, vetch, white lupine, aot+vetch consortium and oat+vetch+turnip), preceding corn. In the subplots were used two doses of nitrogen fertilization (0 and 180 kg ha N) coverage in maize.The biggest coverage rates occurred in the consortium with 95% at 62 days after sowing. The residual effect of 180 kg ha cool season plants following year. The residual effect of 180 kg ha systems, reduced in 21% the C/N ratio of poaceae. The common vetch accumulated 32 kg N per ton of MS added. The oat and rye keeps more than 50% waste to the land cover, after 120 days, while the ryegrass and vetch provide low soil protection. Consortium oat+vetch+turnip, vetch and white lupine, released the largest amounts of N, between 52 and 59 kg ha brassica and consortia positively influencing the diameter and length of cobs, number of kernels per row and, total number of grains per ear of corn, in the absence of mineral N. The weight of a thousand grains was increased by 12.4% by the addition of 180 kg ha increase in productivity of grain by the addition of 180 kg ha N, was 2.1 Mg ha 5.6 Mg ha 6.4 Mg ha components when cultivated on vetch. Systems containing fabaceae, brassica and consortium oat+vetch+turnip, predating the corn, in the absence of mineral N, provided similar grain yelds inrelation to the systems with the addition of 180 kg ha Keywords: Cover crops. No-tillage. Grain yield. Zea mays - 1 -1 N, increased 4.8% coverage rate in the of N in corn/cover crops -1 -1 . Fabaceae, -1 N mineral. The average N, in relation to dose 0 kg ha corn kernels on fabaceae, brassica and consortium oat+vetch+turnip, and poaceae the grains in succession. The consortium added amount between 4.0 the DM in the years of study. There was no effect of mineral N rate for corn yield components when cultivated on vetch. Systems containing fabaceae, brassica and consortium oat+vetch+turnip, predating the corn, in the absence of mineral N, provided similar grain yelds inrelation to the systems with the addition of 180 kg ha-1 N.

Novas estratégias para entender o sistema de influxo de antibióticos e a resistência antimicrobiana a nível molecular

As fluoroquinolonas são antibióticos que têm um largo espectro de ação contra bactérias, especialmente Gram-negativas. O seu mecanismo de ação assenta na inibição de enzimas responsáveis pela replicação do DNA. Porém, devido ao seu uso indevido, o surgimento de resistência bacteriana a estes antibióticos tem-se tornado um grave problema de saúde pública. Uma vez que os seus alvos de ação se situam no meio intracelular, a redução da permeabilidade da membrana externa de bactérias Gram-negativas constitui um dos mecanismos de resistência mais conhecidos. Esta redução é associada à baixa expressão ou mutações em porinas necessárias para permitir o seu transporte, mais concretamente, da OmpF. Estudos prévios demonstraram que a coordenação de fluoroquinolonas com iões metálicos divalentes e 1,10-fenantrolina (genericamente designados metaloantibióticos) são potenciais candidatos como alternativa às fluoroquinolonas convencionais. Estes metaloantibióticos exibem um efeito antimicrobiano comparável ou superior à fluoroquinolona na forma livre, mas parecem ter uma via de translocação diferente, independente de porinas. Estas diferenças no mecanismo de captura podem ser fundamentais para contornar a resistência bacteriana. De forma a compreender o papel dos lípidos no mecanismo de entrada dos metaloantibióticos, estudou-se a interação e localização dos metaloantibióticos da Ciprofloxacina (2ª geração), da Levofloxacina (3ª geração) e Moxifloxacina (4ª geração) com um modelo de membranas de Escherichia coli desprovido de porinas. Estes estudos foram realizados através de técnicas de espectroscopia de fluorescência, por medições em modo estacionário e resolvida no tempo. Os coeficientes de partição determinados demonstraram uma interação mais elevada dos metaloantibióticos relativamente às respetivas fluoroquinolonas na forma livre, um facto que está diretamente relacionado com as espécies existentes em solução a pH fisiológico. Os estudos de localização mostraram que estes metaloantibióticos devem estar inseridos na membrana bacteriana, confirmando a sua entrada independente de porinas. Este mecanismo de entrada, pela via hidrofóbica, é potenciado por interações eletrostáticas entre as espécies catiónicas de metaloantibiótico que existem a pH 7,4 e os grupos carregados negativamente dos fosfolípidos da membrana. Desta forma, os resultados obtidos neste estudo sugerem que a via de entrada dos metaloantibióticos e das respetivas fluoroquinolonas deve ser diferente. Os metaloantibióticos são candidatos adequados para a realização de mais testes laboratoriais e uma alternativa promissora para substituir as fluoroquinolonas convencionais, uma vez que parecem ultrapassar um dos principais mecanismos de resistência bacteriana a esta classe de antibióticos.

As TIC, as práticas de ensino supervisionadas, a investigação e a inovação : os blogues educativos & a segurança na internet

Presentemente, em Portugal, estima-se que a utilização das TIC (Tecnologias da Informação e da Comunicação), pelos designados nativos digitais, corresponde a cerca de 98,S°/o. Considerando que os nativos digitais correspondem aos cidadãos que nasceram somente há 15 anos atrás, significa que toda a atual população escolar vive e convive rodeada de dispositivos e de ferramentas digitais no âmbito das suas rotinas diárias. Contudo, em termos gerais, não se tem verificado a mesma realidade no espaço educativo onde a presença de computadores e de uma ligação à Internet continua a ser muito escassa e incipiente. As Práticas de Ensino Supervisionadas, no âmbito do Mestrado em Educação Pré-escolar e Ensino do 1° Ciclo do Ensino Básico, têm vindo a constituir uma «janela de oportunidades» para a realização de investigações sobre a implementação das TIC no processo de ensino aprendizagem. Apesar de todos os constrangimentos, tem sido possível promover-se uma investigação que se pode considerar inovadora e exploratória pela inexistência de investigações anteriores similares. Para o efeito, é objetivo apresentarem-se os resultados de uma investigação que envolveu a utilização de blogues, no sentido destas ferramentas digitais promoverem novos contextos de aprendizagem e, ainda, com o objetivo de serem uma «ponte» que permitiu a aproximação entre a escola e a família. Um outro exemplo de investigação a apresentar diz respeito à utilização segura da Internet. Dos resultados obtidos foi possível verificar que crianças de 7/8 anos já fazem uma utilização autónoma da internet mas, em vários casos, foi possível averiguar-se que os respetivos pais não têm noção relativamente às utilizações que os seus educandos fazem no chamado ciberespaço. Em termos globais, foi possível apurar-se que as crianças aderem muito facilmente à utilização das tecnologias digitais na sala de aula, os alunos mostraram-se sempre muito envolvidos e motivados. Para terminar, é importante referir que, apesar de todas as vantagens apuradas, quer os alunos quer os pais, continuam a afirmar que a presença dos professores é fundamental e imprescindível no processo de ensino e de aprendizagem.

Caractérisation des mécanismes moléculaires liés à p53 régulant l’expression du gène P2RY6.

Le récepteur P2Y[indice inférieur 6] est un récepteur couplé à une protéine G responsable de l’activation de nombreuses voies de signalisation. Dans l’épithélium du côlon, il participe au maintien de l’équilibre hydrique, mais il a été montré que le récepteur P2Y[indice inférieur 6] participait à l’aggravation des symptômes inflammatoires dans la maladie de Crohn ou dans la colite ulcéreuse. Les maladies inflammatoires de l’intestin sont des facteurs pouvant mener au cancer colorectal. En effet, il existe deux types de cancers colorectaux : le cancer sporadique et le cancer associé à la colite qui se différencient notamment par la séquence d’apparition de mutations génétiques. Par exemple, le gène TP53 est muté de façon tardive dans le cancer colorectal sporadique et muté de façon précoce dans le cancer associé à l’inflammation. Puisque le récepteur P2Y[indice inférieur 6] est impliqué dans la création d’un environnement pro-inflammatoire, nous nous sommes intéressés au rôle de p53 sur l’expression du gène P2RY[indice inférieur 6] et avons formulé l’hypothèse suivante : la présence de TP53 mutant va réguler de façon différentielle l’expression du gène P2RY[indice inférieur 6] dans le cancer colorectal. L’objectif général des travaux est le suivant : caractériser les mécanismes moléculaires liés à TP53 régulant l’expression du gène P2RY[indice inférieur 6] dans le cancer colorectal. Les objectifs spécifiques pour ce projet de recherche sont donc : (1) déterminer et caractériser les régions promotrices du gène P2RY[indice inférieur 6] dans les cellules épithéliales intestinales cancéreuses et (2) étudier l’effet de la protéine p53 de type sauvage ou mutée sur l’expression du récepteur P2Y[indice inférieur 6]. Le gène P2RY[indice inférieur 6] code pour 8 variants d’ARN messagers. Les variants 1, 2, 3, 5, 6, 7 et 8 codent pour l’isoforme 1 du récepteur P2Y[indice inférieur 6], forme connue du récepteur. Le variant 9 code pour l’isoforme 2, non caractérisée. Nos travaux ont permis de mettre en évidence l’existence de quatre régions promotrices potentielles du gène P2RY[indice inférieur 6] et la présence du variant 9, codant pour l’isoforme 2 du récepteur P2Y[indice inférieur 6] dans la lignée cellulaire Caco-2. Nous avons également montré que les formes normale et mutée de p53 régulent de façon différentielle l’expression du récepteur P2Y[indice inférieur 6]. Enfin, le rôle de l’isoforme 2 reste à étudier, mais les tests effectués suggèrent qu’elle est activable par l’UDP.

Revisión de la literatura científica sobre publicidad de medicamentos de venta libre y comportamiento del consumidor

El consumo de medicamentos es un asunto que actualmente se ha convertido en una preocupación a nivel global, ya que no todos los medicamentos están sujetos a prescripción médica, y esto implica que su consumo dependa de otras fuentes de información, como la publicidad masiva o el consejo de personas legas, entre otros. Esta revisión se basó en la publicidad y el impacto que tiene esta frente al consumidor. La presente revisión se dividió en dos categorías dado la relevancia del tema, en la primera se encuentra las características de los medicamentos de venta libre donde se evidenció cómo funciona el sector de la industria farmacéutica, las características de los medicamentos en general y los riesgos del abuso de este comportamiento. Así mismo, en la segunda categoria se habló sobre la publicidad y mercadeo de ventas libres donde se evidenció el alto impacto que tiene la publicidad en el consumidor, las restricciones que hay en el contexto nacional e internacional.