Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.

Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.

Orchestration of energy efficiency capabilities for a sustainable network management

The energy demand for operating Information and Communication Technology (ICT) systems has been growing, implying in high operational costs and consequent increase of carbon emissions. Both in datacenters and telecom infrastructures, the networks represent a significant amount of energy spending. Given that, there is an increased demand for energy eficiency solutions, and several capabilities to save energy have been proposed. However, it is very dificult to orchestrate such energy eficiency capabilities, i.e., coordinate or combine them in the same network, ensuring a conflict-free operation and choosing the best one for a given scenario, ensuring that a capability not suited to the current bandwidth utilization will not be applied and lead to congestion or packet loss. Also, there is no way in the literature to do this taking business directives into account. In this regard, a method able to orchestrate diferent energy eficiency capabilities is proposed considering the possible combinations and conflicts among them, as well as the best option for a given bandwidth utilization and network characteristics. In the proposed method, the business policies specified in a high-level interface are refined down to the network level in order to bring highlevel directives into the operation, and a Utility Function is used to combine energy eficiency and performance requirements. A Decision Tree able to determine what to do in each scenario is deployed in a Software Defined Network environment. The proposed method was validated with diferent experiments, testing the Utility Function, checking the extra savings when combining several capabilities, the decision tree interpolation and dynamicity aspects. The orchestration proved to be valid to solve the problem of finding the best combination for a given scenario, achieving additional savings due to the combination, besides ensuring a conflict-free operation.

O uso de medidas antidumping como mecanismo de barreira à entrada no mercado brasileiro

As medidas antidumping são uma exceção ao livre comércio e como tal deveriam ser usadas com restrição. No entanto, desde o GATT 1947 há preocupação com o uso abusivo de tais medidas, podendo ser o mecanismo utilizado não apenas para possibilitar a recuperação da indústria doméstica prejudicada pelas importações objeto de dumping, objetivo do Acordo Antidumping (AAD), mas com o fim de proteger a indústria nacional da concorrência estrangeira. Desta forma, o objetivo do presente trabalho é apresentar as principais características do AAD firmado ao final da Rodada Uruguai que culminou na constituição da Organização Mundial do Comércio (OMC), incluindo seus antecedentes históricos e a possibilidade de aplicação discricionária, demonstrando como uma norma surgida no plano internacional em decorrência de um acordo multilateral vem sendo utilizada pelo Brasil, com a finalidade de se demonstrar a possibilidade de uso das medidas antidumping como barreira à entrada no mercado brasileiro. Confirmada a possibilidade de uso abusivo desse instrumento de defesa comercial, ainda que no plano teórico, uma vez que não é possível analisar os efeitos reais das medidas aplicadas, serão apresentadas as formas de contrabalanço ao protecionismo atualmente existentes no próprio AAD e sua incorporação e utilização pelo Brasil bem como a possibilidade de questionamento de tais medidas como práticas anticompetitivas com fundamento na legislação antitruste perante as autoridades de defesa da concorrência. Serão ainda analisadas outras possibilidades de contrabalanço ao uso exacerbado das medidas antidumping em debate e viabilidade das mesmas no cenário atual em que se verifica, de um lado, o aumento de uso de tais medidas pelos países Membros da OMC, destacando-se o Brasil e, de outro lado, impasse nas negociações multilaterais, cabendo aos Membros tomarem decisões unilaterais sobre a aplicação de tais medidas, seu grau de intensidade e forma de aplicação a depender da proteção que se pretende garantir à indústria nacional.

Prisão preventiva: direito à razoável duração e necessidade de prazo legal máximo

O direito à razoável duração do processo, inserido expressamente no ordenamento jurídico brasileiro a partir do advento da Emenda Constitucional 45/2004, já poderia ser inferido desde a incorporação da Convenção Americana de Direitos Humanos, bem como ser considerado um corolário da garantia do devido processo legal. Todo indivíduo tem o direito a um processo sem dilações indevidas, em especial aquele que se encontre submetido a uma prisão preventiva, medida cautelar pessoal de extrema gravosidade. Nesse contexto, exsurge o direito que o indivíduo preso preventivamente tem de que o seu processo seja julgado em um prazo razoável ou de que ele seja desencarcerado, caso preso além da necessidade fática contida no caso concreto. Entretanto, a interpretação da garantia não pode restar somente à livre vontade dos aplicadores do direito, sendo necessária uma regulamentação legal efetiva da duração da prisão preventiva, por meio de prazos concretos nos quais o sujeito deverá ser posto em liberdade, ante a desídia estatal. Incorporando experiências estrangeiras, deve o legislador pátrio adotar marcos temporais legais, em que a prisão preventiva deverá cessar, caso excessivamente prolongada. Muito embora no ano de 2011 tenha sido reformada a tutela das medidas cautelares pessoais no Código de Processo Penal, o legislador ordinário não aprovou a imposição de limites de duração da prisão preventiva, permanecendo ao livre arbítrio das autoridades judiciárias a interpretação da garantia em referência. Assim, o Projeto de Lei do Novo Código de Processo Penal, atualmente em trâmite no Congresso Nacional, ao prever limites máximos de duração da prisão preventiva, dá uma efetiva regulamentação à garantia da duração razoável do imputado preso, devendo ser, espera-se, mantido no eventual texto final aprovado.

Will, 1747, June 24

Handwritten copy of Last Will and Testament of Andrew Bordman II, with an approval of the document by Samuel Danforth, Judge of the Probate of Wills, Middlesex County. The document provides detail on the inheritance of Bordman's wife Elizabeth.

Will, 1783 April 15

Handwritten copy of Last Will and Testament of Sarah Phips Bordman.

Implementing the Physical Literacy Component of the 2015 Ontario Health and Physical Education Curriculum: Is the Stage Set?

Introduction: Current physical activity levels among children and youth are alarmingly low; a mere 7% of children and youth are meeting the Canadian Physical Activity Guidelines (Colley et al., 2011), which means that the vast majority of this population is at risk of developing major health problems in adulthood (Janssen & Leblanc, 2010). These high inactivity rates may be related to suboptimal experiences in sport and physical activity stemming from a lack of competence and confidence (Lubans, Morgan, Cliff, Barnett, & Okely, 2010). Developing a foundation of physical literacy can encourage and maintain lifelong physical activity, yet this does not always occur naturally as a part of human growth (Hardman, 2011). An ideal setting to foster the growth and development of physical literacy is physical education class. Physical education class can offer all children and youth an equal opportunity to learn and practice the skills needed to be active for life (Hardman, 2011). Elementary school teachers are responsible for delivering the physical education curriculum, and it is important to understand their will and capacity as the implementing agents of physical literacy development curriculum (McLaughlin, 1987). Purpose: The purpose of this study was to explore the physical literacy component of the 2015 Ontario Health and Physical Education curriculum policy through the eyes of key informants, and to explore the resources available for the implementation of this new policy. Methods: Qualitative interviews were conducted with seven key informants of the curriculum policy development, including two teachers. In tandem with the interviews, a resource inventory and curriculum review were conducted to assess the content and availability of physical literacy resources. All data were analyzed through the lens of Hogwood and Gunn’s (1984) 10 preconditions for policy implementation. Results: Participants discussed how implementation is affected by: accountability, external capacity, internal capacity, awareness and understanding of physical literacy, implementation expertise, and policy climate. Discussion: Participants voiced similar opinions on most issues, and the overall lack of attention given to physical education programs in schools will continue to be a major dilemma when trying to combat such high physical inactivity levels.

Y-box binding protein 1 (YB-1) relevance in estrogen receptor-positive (ER+) breast cancer

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016

Bosnia i Hercegowina wobec pytania o tozsamosc. Kosowo - kewstia ostatecznego statusu. Prace OSW 10/2003 = Bosnia and Herzegovina: facing the question of identity. Kosovo - the question of final status. OSW Study 10/2003

In June 2003, during a meeting held in Saloniki, the leaders of European Union member states turned to the presidents and heads of the governments of five Western Balkans nations – Croatia, Bosnia and Herzegovina, Serbia and Montenegro, Macedonia, and Albania – assuring them that Brussels sees a future for the entire region in Europe and that, without their membership in the EU, the integration of the continent would not be complete. Of these five – actually six, as Kosovo’s protectorate was represented by a separate delegation: the Prime Minister, President, the Head of the international administration, and a representative of the Serbian party – only Croatia can count on quick integration. The membership of the remaining countries is being spoken of (unofficially) in the perspective of ten to fifteen years. However, no EU diplomat is able to answer the question of how the integration of Bosnia and Herzegovina, and Kosovo, with the EU, will look in practice – these two organisms exist solely owing to the will and efforts of the international community.

Mutual recognition in goods and services: an economic perspective. Bruges European Economic Policy (BEEP) Briefing 2/2002

Mutual recognition is a remarkable innovation facilitating economic intercourse across borders. In the EU's internal goods market it has been helpful in tackling or avoiding the remaining obstacles, namely, regulatory barriers between Member States. However, there is a curious paradox. Despite the almost universal acclaim of the great merits of mutual recognition the principle has, in and by itself, contributed only modestly to the actual realisation of free movement in the single market. It is also surprising that economists have not or hardly underpinned their widespread appreciation for the principle by providing rigorous analysis which could substantiate the case for mutual recognition for policy makers. Business in Europe has shown a sense of disenc hantment with the principle because of the many costs and uncertainties in its application in actual practice. The purpose of the present paper is to provide the economic and strategic arguments for employing mutual recognition much more systematically in the single market for goods and services. The strategic and the "welfare" gains are analysed and adetailed exposition of the fairly high information , transaction and compliance costs is provided. The information costs derive from the fact that mutual recognition remains a distant abstraction for day-to-day business life. Understandably, verifying the "equivalence" of objectives of health and safety between Member States is perceived as difficult and uncertain. This sentiment is exacerbated by the complications of interpreting the equivalence of "effects". In actual practice, these abstractions are expected to override clear and specific national product or services rules, which local inspectors or traders may find problematic without guidance. The paper enumerates several other costs including, inter alia, the absence of sectoral rule books and the next-to-prohibitive costs of monitoring of the application of the principle. The basic problems in applying mutual recognition in the entire array of services are inspected, showing why the principle can only be used in a limited number of services markets and even there it may contribute only modestly to genuine free movement and competitive exposure. A special section is devoted to a range of practical illustrations of the difficulties business experiences when relying on mutual recognition. Finally, the corollary of mutual recognition - regulatory competition - is discussed in terms of a cost/benefits analysis compared to what is often said to be the alternative , that is "harmonisation" , in EU parlance the "new approach" to approximation. The conclusion is that the manifold benefits of mutual recognition for Europe are too great to allow the present ambiguities to continue. The Union needs much more pro-active approaches to reduce the costs of mutual recognition as well as permanent monitoring structures for its application to services (analogous to those already successfully functioning in goods markets). Above all, what is required is a "mutual recognition culture" so that the EU can better enjoy the fruits of its own regulatory ingenuity.