Staphylococcal protein A: Unfolding pathways, unfolded states, and differences between the B and E domains

We present multiple native and denaturation simulations of the B and E domains of the three-helix bundle protein A, totaling 60 ns. The C-terminal helix (H3) consistently denatures later than either of the other two helices and contains residual helical structure in the denatured state. These results are consistent with experiments suggesting that the isolated H3 fragment is more stable than H1 and H2 and that H3 forms early in folding. Interestingly, the denatured state of the B domain is much more compact than that of the E domain. This sequence-dependent effect on the dimensions of the denatured state and the lack of correlation with structure suggest that the radius of gyration can be a misleading reaction coordinate for unfolding/folding. Various unfolding and refolding events are observed in the denaturation simulations. In some cases, the transitions are facilitated through interactions with other portions of the protein—contact-assisted helix formation. In the native simulations, the E domain is very stable: after 6 ns, the Cα root-mean-square deviation from the starting structure is less than 1.4 Å. In contrast, the native state of the B domain deviates more and its inter-helical angles fluctuate. In apparent contrast, we note that the B domain is thermodynamically more stable than the E domain. The simulations suggest that the increased stability of the B domain may be due to heightened mobility, and therefore entropy, in the native state and decreased mobility/entropy in the more compact denatured state.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein α gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPα genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPα genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPα gene (xC/EBPα). The –1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPα or xC/EBPβ. Deletion analysis showed that the –321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPα promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPα promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPα expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

Purification and Molecular Genetic Characterization of ZPU1, a Pullulanase-Type Starch-Debranching Enzyme from Maize1

This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.

Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors.

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both RAC-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKalpha activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.

Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.

Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.

The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo.

The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA-binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo.

La prevalencia del estrés laboral asistencial entre los profesores universitarios

El burnout, o estrés laboral asistencial, es un síndrome psicológico caracterizado por agotamiento emocional (CE), despersonalización (DP) e insatisfacción personal con los logros (RP). Se conoce poco acerca de la presencia de este síndrome en muestras representativas de profesores universitarios. Los objetivos del estudio son (a) conocer la prevalencia del burnout en un contexto universitario, (b) examinar la presencia del burnout en relación con la edad, género y categoría académica en un estudio transversal mediante cuestionario anónimo enviado por correo, y (e) explorar la relación entre el burnout y diversas variables de calidad de vida, satisfacción laboral y salud. Respondieron el cuestionario un total de 331 profesores en el contexto de un programa de calidad de vida de la Universidad de Alicante (España), lo que supone una tasa de respuesta del 56,2%. Se remitieron por correo a todos los profesores, seleccionados al azar del conjunto de todos los centros, un ejemplar del cuestionario junto con las instrucciones y sobre de devolución. El estrés laboral asistencial se midió a través del Maslach Burnout lnventory (MBI) estableciéndose una situación definida por altas puntuaciones en CE y DE, y bajas en RP. Este instrumento presenta un total de 22 ítems con siete alternativas de respuesta, desde 0 (nunca experimento este sentimiento) hasta 6 (todos los días experimento este sentimiento). Nuestros resultados muestran que un reducido porcentaje de profesores, el 1,8%, experimentan el síndrome de burnout. Cuando se analizan por separado las tres dimensiones que componen el burnout se observa que un 17,8% de los profesores se siente emocionalmente exhausto en su trabajo (puntuación e» 25), un 4,2% ha desarrollado una actitud negativa hacia los estudiantes puntuación e» 10), y un 42% se siente un escasa autorrealización personal en el trabajo (puntuación d»32). CE presenta unas correlaciones medias moderadas con las variables de salud (r= 0.42) y con calidad de vida (r=0.33). Un patrón relacional menos consistente se da en las restantes dimensiones del burnout. La edad, el género y la categoría académica de los profesores no se relacionan con los niveles de burnout. Se concluye que el burnout es un síndrome poco común en profesores universitarios.

Linking tourism, retirement migration and social capital

A general trend in the study of international retirement migration has been the increased attention paid to the social contacts and network connections of the migrants in both the destination and the origin areas. These studies have examined the extent to which migrants build social relationships with their neighbours and the host society while also maintaining social links with their countries of origin, addressing the central role that leisure travel plays in sustaining increasingly dispersed social networks and maintaining the social capital of these networks and of the individuals involved in them. Using a case study approach to examine British retirement migration to Spain, we explore the relevance of transnational social networks in the context of international retirement migration, particularly the intensity of bidirectional visiting friends and relatives (VFR) tourism flows and the migrants’ social contacts with friends and/or family back in their home country. Building on the concept of social capital and Putnam's distinction between bonding and bridging social capital, we propose a framework for the analysis of the migrants’ international social networks. The results of a study conducted based on a sample of 365 British retirees living in the coast of Alicante (Spain) show both the strength of the retirees’ international bonding social capital and the role of ‘VFR's travel and communication technologies in sustaining the migrants’ transnational social practices and, ultimately, their international bonding social capital. It also provides evidence for the reinforcing links between tourism-related mobility and amenity-seeking migration in later life.

Water adsorption in hydrophilic zeolites: experiment and simulation

We have measured experimental adsorption isotherms of water in zeolite LTA4A, and studied the regeneration process by performing subsequent adsorption cycles after degassing at different temperatures. We observed incomplete desorption at low temperatures, and cation rearrangement at successive adsorption cycles. We also developed a new molecular simulation force field able to reproduce experimental adsorption isotherms in the range of temperatures between 273 K and 374 K. Small deviations observed at high pressures are attributed to the change in the water dipole moment at high loadings. The force field correctly describes the preferential adsorption sites of water at different pressures. We tested the influence of the zeolite structure, framework flexibility, and cation mobility when considering adsorption and diffusion of water. Finally, we performed checks on force field transferability between different hydrophilic zeolite types, concluding that classical, non-polarizable water force fields are not transferable.

Welfare state regimes and gender inequalities in the exposure to work-related psychosocial hazards

Background: Gender inequalities in the exposure to work-related psychosocial hazards are well established. However, little is known about how welfare state regimes influence these inequalities. Objectives: To examine the relationship between welfare state regimes and gender inequalities in the exposure to work-related psychosocial hazards in Europe, considering occupational social class. Methods: We used a sample of 27, 465 workers from 28 European countries. Dependent variables were high strain, iso-strain, and effort-reward imbalance, and the independent was gender. We calculated the prevalence and prevalence ratio separately for each welfare state regime and occupational social class, using multivariate logistic regression models. Results: More female than male managers/professionals were exposed to: high strain, iso-strain, and effort–reward imbalance in Scandinavian [adjusted prevalence ratio (aPR) = 2·26; 95% confidence interval (95% CI): 1·87–2·75; 2·12: 1·72–2·61; 1·41: 1·15–1·74; respectively] and Continental regimes (1·43: 1·23–1·54; 1·51: 1·23–1·84; 1·40: 1·17–1·67); and to high strain and iso-strain in Anglo-Saxon (1·92: 1·40–2·63; 1·85: 1·30–2·64; respectively), Southern (1·43: 1·14–1·79; 1·60: 1·18–2·18), and Eastern regimes (1·56: 1·35–1·81; 1·53: 1·28–1·83). Conclusion: Gender inequalities in the exposure to work-related psychosocial hazards were not lower in those welfare state regimes with higher levels of universal social protection policies.

Effect of economic recession on psychosocial working conditions by workers' nationality

Background: Several publications have documented the effects of economic recessions on health. However, little is known about how economic recessions influence working conditions, especially among vulnerable workers. Objective: To explore the effects of 2008 economic crisis on the prevalence of adverse psychosocial working conditions among Spanish and foreign national workers. Methods: Data come from the 2007 and 2011 Spanish Working Conditions Surveys. Survey year, sociodemographic, and occupational information were independent variables and psychosocial factors exposures were dependent variables. Analyses were stratified by nationality (Spanish versus foreign). Prevalence and adjusted prevalence ratios (aPRs) of psychological job demands, job control, job social support, physical demands and perceived job insecurity were estimated using Poisson regression. Results: The Spanish population had higher risk of psychological and physical job demand (aPR = 1.07, 95% CI = [1.04–1.10] and aPR = 1.05, 95% CI = [1.01–1.09], respectively) in 2011 compared to 2007. Among both Spanish and foreign national workers, greater aPR were found for job loss in 2011 compared to 2007 (aPR = 2.47, 95% CI = [2.34–2.60]; aPR = 2.44, 95% CI = [2.15–2.77], respectively). Conclusion: The 2008 economic crisis was associated with a significant increase in physical demands in Spanish workers and increased job insecurity for both Spanish and foreign workers.

Saving for Retirement and Investing for Growth. Report of the CEPS-RCMI Task Force on Long-term Investing and Retirement Savings. CEPS Task Force Report, September 2013

Europe is facing a double challenge: a significant need for long-term investments – crucial levers for economic growth – and a growing pension gap, both of which call for resolute action. Crucially, at a time when low interest rates and revised prudential standards strain the ability of life insurers and pension funds to offer guaranteed returns, Europe lacks a framework ensuring the quality and accessibility of long-term investment solutions for small retail investors and defined contribution pension plans. This report considers the potential to steer household financial wealth – accounting for over 60% of total financial wealth in Europe – towards long-term investing, which would achieve two goals at once: higher growth and higher pensions. It follows a holistic approach that considers both solution design – how to gear product structuring towards long-term investing – and market structure – how to engineer a competitive market setting that is able to deliver high-quality and cost-efficient solutions. The report also considers prudential rules for insurers and pension funds and the potential to build a single market for less-liquid funds, occupational and personal pensions, with improved investor protection. It urges policy-makers to act aggressively to deliver more inclusive, efficient and resilient retail investment markets that are better equipped and more committed to deliver value over the long-term for beneficiaries.

Memo to Merkel: Post-election Germany and Europe. Bruegel Policy Brief 2013/05, 24 September 2013

Recent economic data points to the seeds of an economic recovery in the European Union. However, significant risks remain and bold policies are still needed. There are three central risks. Competitiveness adjustment is incomplete, casting doubt on the sustainability of public debt. Banking remains unstable and fragmented along national lines, resulting in unfavorable financial conditions, which further erode growth, job creation and competitiveness. Rising unemployment, especially among the young, is inequitable, unjust and politically risky. Germany has a central role to play in addressing these risks. The new German government should work on three priorities: Domestic economic policy should be more supportive of growth and adjustment, with higher public investment, a greater role for high-value added services, and more supportive immigration policy. Germany should support a meaningful banking union with a centralised resolution mechanism requiring a transfer of sovereignty to Europe for all countries including Germany. The establishment of a private investment initiative combined with a European Youth Education Fund and labour market reforms should be promoted. Building on these priorities, a significant deepening of the euro area is needed, with a genuine transfer of sovereignty, stronger institutions and democratically legitimate decision-making structures in areas of common policy.

The Euro: A Crisis of the Currency or a Failure of Politics? Paper from the Colloquium: The Politics and Economics of the Euro Crisis, Institute for European Studies, University of Malta, 2 December 2011

Summary: Overall the monetary pillar of the EMU project has worked well so that it is incorrect to speak of a 'euro' crisis though it is at the epicentre of the present crisis. The origins of the problems it is facing have more to do with the economic component, particularly because of the breach of budgetary rules which points to a failure of politics. Hence the solution must be political, namely a strong commitment by governments to achieve balanced budgets and implement structural reforms so as to lay the basis for improved competitiveness, job creation and sustainable growth.

Continuous Off-the-Job Training in East Germany After Unification: Preliminary Results of an Evaluation of the Effects for Individual Workers, CES Germany & Europe Working Papers, No. 05.9, 19 July, 1995

Retraining the labor force to match the demands of a modem economy is seen as an important task during the transition process from a centrally-planned to a market economy. This need was particularly pressing in East Germany, because the transition process has proceeded much faster than in the rest of Eastern Europe. Therefore, substantial resources have been devoted to this purpose. This paper analyzes the impact of continuous off-the-job training in East Germany from the point of view of individuals who were part of the labor force before German unification in 1990. It tries to answer questions about the average gains from participating in a specific type of training. Typical outcomes considered to measure those gains are income, employment status, job security, and expected future changes in job position. The methodology used for the evaluation is the potential outcome approach to causality. This approach has received considerable attention in the statistical literature over the last fifteen years and it has recently been rediscovered by the econometric literature as well. It is adapted to allow for important permanent and transitory shocks, such as unemployment, which influence the decision to participate in the training as well as future labor market outcomes. The empirical part is based on the first four waves of the Socio-Economic Panel (GSOEP)-East (1990-1993). This panel data set has the advantage that the fourth wave contains a special survey on continuous training and that it allows keeping track of individual behavior on a monthly, respectively yearly, basis. The econometric analysis focuses on off-the-job training courses that began after unification and were completed not later than in early 1993. Although it is obviously too early to evaluate the long-term implications, the results suggest that there are no positive effects in the short run.