Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub)

Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol), have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as 'sym/sub'. Formation of 3D(pol)- sym/sub complexes is slow owing to a slow rate of association (0.1 μM-1 s-1) of 3D(pol) and sym/sub and a slow isomerization (0.076 s-1) of the 3D(pol)-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3D(pol) occurs at a maximal rate of 0.051 s-1 at 22 °C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3D(pol) against inactivation with a K0.5 of 37 μM. Once formed, 3D(pol)-sym/sub elongation complexes are stable (t( 1/2 ) = 2 h at 22 °C) and appear to contain only a single polymerase monomer. In the presence of Mg2+, AMP, 2'-dAMP, and 3'-dAMP are incorporated into sym/sub by 3D(pol) at rates of 72, 0.6, and 1 s-1, respectively. After incorporation of AMP, 3D(pol)-sym/sub product complexes have a half-life of 8 h at 22 °C. The stability of 3D(pol)-sym/sub complexes is temperature-dependent. At 30 °C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3D(pol) dissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3D(pol) and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases

Poliovirus RNA-dependent RNA polymerase (3D(pol)) is sufficient for template switching in vitro

We have performed a systematic, quantitative analysis of the kinetics of nucleotide incorporation catalyzed by poliovirus RNA-dependent RNA polymerase, 3D(pol). Homopolymeric primer/templates of defined length were used to evaluate the contribution of primer and template length and sequence to the efficiency of nucleotide incorporation without the complication of RNA structure. Interestingly, thermodynamic stability of the duplex region of these primer/templates was more important for efficient nucleotide incorporation than either primer or template length. Surprisingly, products greater than unit length formed in all reactions regardless of length or sequence. Neither a distributive nor a processive slippage mechanism could be used to explain completely the formation of long products. Rather, the data were consistent with a template-switching mechanism. All of the nucleotide could be polymerized during the course of the reaction. However, very few primers could be extended, suggesting an inverse correlation between the efficiency of primer utilization and that of nucleotide incorporation. Therefore, the greatest fraction of incorporated nucleotide derives from a small fraction of enzyme when radioactive nucleotide and homopolymeric primer/template substrates are employed. The impact of these results on mechanistic studies of 3D(pol)-catalyzed nucleotide incorporation and RNA recombination are discussed

Testing mapping algorithms of the cancer-specific EORTC QLQ-C30 onto EQ-5D in malignant mesothelioma

Background: In order to estimate utilities for cancer studies where the EQ-5D was not used, the EORTC QLQ-C30 can be used to estimate EQ-5D using existing mapping algorithms. Several mapping algorithms exist for this transformation, however, algorithms tend to lose accuracy in patients in poor health states. The aim of this study was to test all existing mapping algorithms of QLQ-C30 onto EQ-5D, in a dataset of patients with malignant pleural mesothelioma, an invariably fatal malignancy where no previous mapping estimation has been published. Methods: Health related quality of life (HRQoL) data where both the EQ-5D and QLQ-C30 were used simultaneously was obtained from the UK-based prospective observational SWAMP (South West Area Mesothelioma and Pemetrexed) trial. In the original trial 73 patients with pleural mesothelioma were offered palliative chemotherapy and their HRQoL was assessed across five time points. This data was used to test the nine available mapping algorithms found in the literature, comparing predicted against observed EQ-5D values. The ability of algorithms to predict the mean, minimise error and detect clinically significant differences was assessed. Results: The dataset had a total of 250 observations across 5 timepoints. The linear regression mapping algorithms tested generally performed poorly, over-estimating the predicted compared to observed EQ-5D values, especially when observed EQ-5D was below 0.5. The best performing algorithm used a response mapping method and predicted the mean EQ-5D with accuracy with an average root mean squared error of 0.17 (Standard Deviation; 0.22). This algorithm reliably discriminated between clinically distinct subgroups seen in the primary dataset. Conclusions: This study tested mapping algorithms in a population with poor health states, where they have been previously shown to perform poorly. Further research into EQ-5D estimation should be directed at response mapping methods given its superior performance in this study

High-Q2Q^2 Elastic eded-scattering and QCD predictions

In the framework of pertubative QCD it is argued that in the elastic $ed$-scattering at $Q^{2}\sim$ few $(GeV/c)^{2}$ the light-cone-frame helicity-flip amplitudes could not be omitted. The obtained $\frac{B}{A}$ ratio of Rosenbluth structure functions is shown to be in a good agreement with experimental data. The high $Q^2$ behavior of tensor analysing power $T_{20}$ is discussed.Comment: 6 pages + 2 ps figures not included, LaTeX, ITP-93-33

Single-nucleotide resolution of RNA strands in the presence of their RNA complements

Double-stranded (ds)RNA is important for a variety of biological systems. The discovery of the dsRNA-binding motif (dsRBM), coupled with the occurrence of this motif in a wide variety of functionally diverse proteins, has led to increased interest and study of - dsRNA (6,14). For example, the dsRNA- activated protein kinase (PKR), an enzyme involved in the cellular antiviral response, contains two tandem copies of the dsRBM. In addition, the dsRNA adenine deaminases (dsRADs) contain three tandem copies of this motif (7). Likewise, the study of the RNA-dependent RNA polymerase (RdRP) activity associated with RNA virus transcriptases and replicases also requires the use of dsRNA. In each of these systems, the length of the typical RNA used is in the 10–80 bp range (1,9)

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection

We have analyzed the divalent cation specificity of poliovirus RNA- dependent RNA polymerase, 3D(pol). The following preference was observed: Mn2+ > Co2+ > Ni2+ > Fe2+ > Mg2+ > Ca2+ > Cu2+, and Zn2+ was incapable of supporting 3D(pol)-catalyzed nucleotide incorporation. In the presence of Mn2+, 3D(pol) activity was increased by greater than 10-fold relative to that in the presence of Mg2+. Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn2+ was due, primarily, to a reduction in the K(M) value for 3D(pol) binding to primer/template, without any significant effect on the K(M) value for nucleotide. The ability of 3D(pol) to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn2+, and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn2+ as divalent cation permitted 3D(pol) activity to be monitored by following extension of 5'-32P-end- labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3D(pol) was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg2+. In the presence of Mn2+, 3D(pol) efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3D(pol). The ability of 3D(pol) to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg2+. Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D(pol)-catalyzed nucleotide incorporation

Échange international et distorsions internes:Comment gouverner la globalisation ?

La concurrence des pays émergents de la taille de la Chine ou de l’Inde fait resurgir avec force le débat qui oppose partisans et adversaires du libre-échange au sein même des pays développés. Pour les premiers, la croissance du commerce international est forcément bénéficiaire pour tous. Pour les seconds, elle est la cause de tous les maux et en particulier du chômage. Les choses sont pourtant bien différentes et beaucoup moins simples. Les changements dans le commerce international, comme d’ailleurs le progrès technique, créent inévitablement des distorsions qui perturbent le fonctionnement de l’économie. Ces distorsions ne sont pas réductibles à des dysfonctionnements des marchés des biens ou du travail. Elles ne peuvent donc pas être éliminées ab initio en choisissant des institutions optimales. Pour autant, des restrictions au commerce international ne sont pas une solution car elles risquent de créer d’autres distorsions qui viennent s’ajouter aux premières. Des distorsions avant tout intérieures appellent des solutions intérieures. Pour explorer ces solutions, il faut retenir une analyse conçue pour étudier, non les propriétés des positions d’équilibre avant et après l’ouverture à l’échange, mais les caractéristiques d’un processus de transition dont le succès n’est pas assuré. Ce type d’analyse permet de mettre en évidence la nécessité d’introduire une forme d’inertie dans les mécanismes d’ajustement. Si une relative flexibilité des salaires est encore possible quand l’intensité du changement est limitée, une certaine viscosité est requise dans le cas contraire, et un accès facilité aux ressources financières externes est nécessaire. Ce résultat pourrait aider à formuler les choix institutionnels et organisationnels à mettre en œuvre dans les économies qui veulent tirer avantage du commerce international et de la globalisation.Changes in the pattern of international trade inevitably create distortions that perturb the functioning of the economy. These distortions may not be reduced to malfunctioning goods or labour markets, and hence cannot be eliminated by simply choosing the optimal institutions. Domestic distortions call for domestic solutions. To explore these solutions it is useless to analyze the properties of equilibria before and after the opening to trade. Rather, we need to build an analytical framework suited to investigate the characteristics of a transition process whose success is not guaranteed ex ante. it appears that wage rigidity and an easy access to external financial resources are necessary in presence of fast pace of change, while if change is sufficiently slow the standard recipe of wage flexibility may be appropriate. These results may help in the institutional and organizational choices to be implemented in economies willing to profit from international trade and globalization

Liquid Hydrogen Target Experience at SLAC

Liquid hydrogen targets have played a vital role in the physics program at SLAC for the past 40 years. These targets have ranged from small "beer can" targets to the 1.5 m long E158 target that was capable of absorbing up to 800 W without any significant density changes. Successful use of these targets has required the development of thin-wall designs, liquid hydrogen pumps, remote positioning and alignment systems, safety systems, control and data acquisition systems, cryogenic cooling circuits and heat exchangers. Detailed operating procedures have been created to ensure safety and operational reliability.This paper surveys the evolution of liquid hydrogen targets at SLAC and discusses advances in several of the enabling technologies that made these targets possible

The Lamé Class of Lorenz Curves.

In this paper, the class of Lamé Lorenz curves is studied. This family has the advantage of modeling inequality with a single parameter. The family has a double motivation: it can be obtain from an economic model and from simple transformations of classical Lorenz curves. The underlying cumulative distribution functions have a simple closed form, and correspond to the Singh-Maddala and Dagum distributions, which are well known in the economic literature. The Lorenz order is studied and several inequality and polarization measures are obtained, including Gini, Donaldson-Weymark-Kakwani, Pietra and Wolfson indices. Some extensions of the Lamé family are obtained. Fitting and estimation methods under two different data configuration are proposed. Empirical applications with real data are given. Finally, some relationships with other curves are included.The authors thank to Ministerio de Econom a y Competitividad, project ECO2010-15455, for partial support. The second author thanks to the Ministerio de Educaci on (FPU AP-2010-4907) for partial support. We are grateful for the constructive suggestions provided by the reviewers, which improved the paper

Hyperons analogous to the \Lambda(1405)

The low mass of the $\Lambda(1405)$ hyperon with $j^P = 1/2^-$, which is higher than the ground state $\Lambda(1116)$ mass by 290 MeV, is difficult to understand in quark models. We analyze the hyperon spectrum in the bound state approach of the Skyrme model that successfully describes both the $\Lambda(1116)$ and the $\Lambda(1405)$. This model predicts that several hyperon resonances of the same spin but with opposite parity form parity doublets that have a mass difference of around 300 MeV, which is indeed realized in the observed hyperon spectrum. Furthermore, the existence of the $\Xi(1620)$ and the $\Xi(1690)$ of $j^P=1/2^-$ is predicted by this model. Comments on the $\Omega$ baryons and heavy quark baryons are made as well.Comment: 4 pages, talk presented at the Fifth Asia-Pacific Conference on Few-Body Problems in Physics 2011 (APFB2011), Aug. 22-26, 2011, Seoul, Kore