Probes bound to myosin Cys-707 rotate during length transients in contraction

It is widely conjectured that muscle shortens because portions of myosin molecules (the “cross-bridges”) impel the actin filament to which they transiently attach and that the impulses result from rotation of the cross-bridges. Crystallography indicates that a cross-bridge is articulated–consisting of a globular catalytic/actin-binding domain and a long lever arm that may rotate. Conveniently, a rhodamine probe with detectable attitude can be attached between the globular domain and the lever arm, enabling the observer to tell whether the anchoring region rotates. Well-established signature effects observed in shortening are tension changes resulting from the sudden release or quick stretch of active muscle fibers. In this investigation we found that closely correlated with such tension changes are changes in the attitude of the rhodamine probes. This correlation strongly supports the conjecture about how shortening is achieved.

Decreased blood pressure response in mice deficient of the α1b-adrenergic receptor

To investigate the functional role of different α1-adrenergic receptor (α1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the α1b-AR (α1b−/−). Reverse transcription–PCR and ligand binding studies were combined to elucidate the expression of the α1-AR subtypes in various tissues of α1b +/+ and −/− mice. Total α1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the α1b −/− as compared with +/+ mice. Because of the large decrease of α1-AR in the heart and the loss of the α1b-AR mRNA in the aorta of the α1b−/− mice, the in vivo blood pressure and in vitro aorta contractile responses to α1-agonists were investigated in α1b +/+ and −/− mice. Our findings provide strong evidence that the α1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by α1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in α1b −/− as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in α1b−/− mice. The α1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different α1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and βs transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb βs yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein–protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.

Peroxynitrite does not decompose to singlet oxygen (1ΔgO2) and nitroxyl (NO−)

According to Khan et al. [Khan, A. U., Kovacic, D., Kolbanovskiy, A., Desai, M., Frenkel, K. & Geacintov, N. E. (2000) Proc. Natl. Acad. Sci. USA 97, 2984–2989], peroxynitrite (ONOO−) decomposes after protonation to singlet oxygen (1ΔgO2) and singlet oxonitrate (nitroxyl, 1NO−) in high yield. They claimed to have observed nitrosyl hemoglobin from the reaction of NO− with methemoglobin; however, contamination with hydrogen peroxide gave rise to ferryl hemoglobin, the spectrum of which was mistakenly assigned to nitrosyl hemoglobin. We have carried out UV–visible and EPR experiments with methemoglobin and hydrogen peroxide-free peroxynitrite and find that no NO− is formed. With this peroxynitrite preparation, no light emission from singlet oxygen at 1270 nm is observed, nor is singlet oxygen chemically trapped; however, singlet oxygen was trapped when hydrogen peroxide was also present, as previously described [Di Mascio, P., Bechara, E. J. H., Medeiros, M. H. G., Briviba, K. & Sies, H. (1994) FEBS Lett. 355, 287–289]. Quantum mechanical and thermodynamic calculations show that formation of the postulated intermediate, a cyclic form of peroxynitrous acid (trioxazetidine), and the products 1NO− and 1ΔgO2 requires Gibbs energies of ca. +415 kJ⋅mol−1 and ca. +180 kJ⋅mol−1, respectively. Our results show that the results of Khan et al. are best explained by interference from contaminating hydrogen peroxide left from the synthesis of peroxynitrite.

Open randomised study of use of levonorgestrel releasing intrauterine system as alternative to hysterectomy

Objectives: To assess whether the levonorgestrel intrauterine system could provide a conservative alternative to hysterectomy in the treatment of excessive uterine bleeding.

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

To bind at an enzyme’s active site, a ligand must diffuse or be transported to the enzyme’s surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and β-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as “ionic tethering.” We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme’s surroundings even when the substrate is nonpolar.

Deed of feoffment for property in Cambridge, Massachusetts: from Thomas Sweetman to Michael Spencer, 1677 December 10

In this deed of feoffment, written on Dec. 10, 1677, Thomas Sweetman agreed to sell his dwelling house, barn, and orchard to his son-in-law, Michael Spencer, for the cost of eighty pounds sterling. The property was located in Cambridge, Massachusetts, on what was then the northwest corner of the grounds of Harvard College, and was sold "together with the wood lot upon the rocks and cow commons belonging to it." The deed specifies that both Sweetman and his wife Isabel were to be allowed to occupy the property until their deaths, and further explains that Spencer and his family were already living in the dwelling house, occupying three rooms. The document was signed, sealed, and delivered in the presence of Daniel Gookin, Jr. and John Bridgham. It was also signed by Thomas Sweetman.

Satisfação familiar com os cuidados prestados numa unidade de cuidados intensivos : uma análise quantitativa e qualitativa

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Carbon leakage: an overview. CEPS Special Report No. 79, 6 December 2013

Carbon leakage is central to the discussion on climate policy, given the confluence of issues that are currently being debated, including the 2030 Energy and Climate Framework and the review of the EU carbon leakage list by 2014. Carbon leakage is the result of asymmetrical carbon policies, especially carbon pricing, and the resulting carbon cost, which affects the international competitive position of some EU industry and could displace production and/or investment, and the emissions of the activities displaced. This paper identifies the difference between carbon price and carbon cost to leakage exposed industry as one of two fundamental issues to be understood and addressed; lack of visibility on future climate policies and anti-leakage provisions is the other key issue. While this is a global issue, most of the experience has been accumulated in the EU. Carbon leakage is only one of the factors that could affect the competitive position of sectors, but it is difficult to attribute the impact of carbon costs versus other variables such as energy costs, labour, etc. Studies have predicted the risk of a significant amount of production leakage in a number of energy-intensive industries. To address the danger, they were included in the EU ETS carbon leakage list, which gave them access to free allowances. However, a limited number of studies undertaken after the end of the second trading period (2012) show little evidence of production leakage and asks the question whether the issue has not been blown out of proportion. The paper argues that the past may not be a good representation of the future, as it was heavily influenced by a high level of free allocation, the exceptional economic downturn, CO2 prices significantly below what was anticipated, as well as the potential for changes in some fundamental variables such as the shrinking pool of allowances available for free allocation. It emphasises the need for a well-informed debate in the EU on measures to address carbon leakage post-2020, underpinned by a number of options, and objective criteria to evaluate those options. It emphasises that the debate should cover both investment and production leakage, caused by both direct and indirect carbon costs.

Carbon Leakage: Options for the EU. CEPS Special Report, March 2014

This CEPS Special Report builds on the first deliverable of the project entitled “Carbon leakage: Options for the EU”. It identifies carbon costs, and the ability to pass through carbon costs, as the main risk factors that could lead from asymmetrical carbon policies to carbon leakage. It also outlines and evaluates, based on criteria discussed in the paper, options for detecting and mitigating the risk of carbon leakage in three jurisdictions, with special attention to the EU ETS (Emissions Trading Scheme). Based on the analysis of approaches currently used in a number of existing carbon pricing systems, it identifies the balance between the number of sectors identified as being at risk, and the amount of compensation provided as a risk mitigation measure, as the critical element in providing an optimum approach to address carbon leakage risks. It also identifies a risk-based approach to identifying sectors at risk as allowing for a better reflection of reality in a counterfactual argument. Finally, the paper concludes that while, with some exceptions, there has been limited carbon leakage until now, the past may not be a good reflection of the future and that measures need to be put in place for the post-2020 period. While examining a number of approaches, it identifies free allocation as the most likely way forward for mitigating the risk of carbon leakage. While other approaches may provide interesting options, they also present challenges for implementation, from a market functioning, to international trade and relations, points of view. A number of challenges will need to be addressed in the post-2020 period, with many of them part of the EU ETS structural reform package. Some of these challenges include, among others, the need to recognise, and provide for individual sectoral characteristics, as well as for changes in production patterns, due to economic cycles, and other factors. Finally, the paper emphasises the need for an open dialogue regarding the post-2020 provisions for carbon leakage as no overall Energy and Climate Package is likely to be agreed on until this matter is addressed.

The Composition and Drivers of Energy Prices and Costs in Energy-Intensive Industries: The Case of Ceramics, Glass and Chemicals. CEPS Special Report No. 85, 27 March 2014

In May 2013, the European Commission received a mandate from the European Council to “to present an analysis of the composition and drivers of energy prices and costs in Member States, with a particular focus on the impact on households, SMEs and energy intensive industries, and looking more widely at the EU's competitiveness vis-à-vis its global economic counterparts”. Following such mandate and in view of the preparation by the Commission of a Communication and a Staff Working Document, DG Enterprise and Industry commissioned CEPS to carry out a set of studies aimed at providing well-grounded evidence about the evolution and composition of energy prices and costs at plant level within individual industry sectors. A team of CEPS researchers conducted the research, led by Christian Egenhofer and Lorna Schrefler. Vasileios Rizos served as Project Coordinator. Other CEPS researchers contributing to the project included: Fabio Genoese, Andrea Renda, Andrei Marcu, Julian Wieczorkiewicz, Susanna Roth, Federico Infelise, Giacomo Luchetta, Lorenzo Colantoni, Wijnand Stoefs, Jacopo Timini and Felice Simonelli. In addition to an introductory report entitled “About the Study and Cross-Sectoral Analysis”, CEPS prepared five sectoral case studies: two on ceramics (wall and floor tiles and bricks and roof tiles), two on chemicals (ammonia and chlorine) and one on flat glass. Each of these six studies has been consolidated in this single volume for free downloading on the CEPS website. The specific objective was to complement information already available at macro level with a bottom-up perspective on the operating conditions that industry stakeholders need to deal with, in terms of energy prices and costs. The approach chosen was based on case studies for a selected set (sub-)sectors amongst energy-intensive industries. A standard questionnaire was circulated and respondents were sampled according to specified criteria. Data and information collected were finally presented in a structured format in order to guarantee comparability of results between the different (sub-)sectors analysed. The complete set of files can also be downloaded from the European Commission’s website: http://ec.europa.eu/enterprise/newsroom/cf/itemdetail.cfm?item_id=7238&lang=en&title=Study-on-composition-and-drivers-of-energy-prices-and-costs-in-energy-intnsive-industries The results of the studies were presented at a CEPS Conference held on February 26th along with additional evidence from other similar studies. The presentations can be downloaded at: http://www.ceps.eu/event/level-and-drivers-eu-energy-prices-energy-inten...

What’s the use of a transatlantic free trade area? Jean Monnet Policy Brief 05/2014

For many of those who remember the hostile EU-US trade relations of the 1980’s and the various trade disputes that have emerged between these two trade partners since then, the opening of negotiations on a joint free trade area would be good news. Strengthened trade cooperation between the partners holds the promise of expanding their mutual exchange of goods and services, not the least by solving obstacles to integration on less transparent issues such as the extent to which product characteristics should be defined by their regional characteristics (e.g. can Budweiser be produced outside the Budweis region in the Czech Republic?).

What’s the use of a transatlantic free trade area? Jean Monnet Policy Brief 05/2013

No abstract.