Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the mfold program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5′ and 3′ untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, ≈70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.

An important 2′-OH group for an RNA–protein interaction

We have investigated the role of 2′-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNACys and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2′-OH by 2′-deoxy or 2′-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2′-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2′-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2′-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2′-OH group instead provides a monitor of the steric environment during the RNA–synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2′-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

The Sm domain is an ancient RNA-binding motif with oligo(U) specificity

Sm and Sm-like proteins are members of a family of small proteins that is widespread throughout eukaryotic kingdoms. These proteins form heteromers with one another and bind, as heteromeric complexes, to various RNAs, recognizing primarily short U-rich stretches. Interestingly, completion of several genome projects revealed that archaea also contain genes that may encode Sm-like proteins. Herein, we studied the properties of one Sm-like protein derived from the archaebacterium Archaeoglobus fulgidus and overexpressed in Escherichia coli. This single small protein closely reflects the properties of an Sm or Sm-like protein heteromer. It binds to RNA with a high specificity for oligo(U), and assembles onto the RNA to form a complex that exhibits, as judged by electron microscopy, a ring-like structure similar to the ones observed with the Sm core ribonucleoprotein and the like Sm (LSm) protein heteromer. Importantly, multivariate statistical analysis of negative-stain electron-microscopic images revealed a sevenfold symmetry for the observed ring structure, indicating that the proteins form a homoheptamer. These results support the structural model of the Sm proteins derived from crystallographic studies on Sm heterodimers and demonstrate that the Sm protein family evolved from a single ancestor that was present before the eukaryotic and archaeal kingdoms separated.

Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time

The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P3-1-(2-nitro)phenylethyl guanosine 5′-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP⋅Ras⋅GDP⋅AlFx transition state analog is known, which represents a “snap shot” along the reaction-pathway. As now revealed, binding of GAP to Ras⋅GTP shifts negative charge from the γ to β phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP⋅Ras⋅GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between β and γ phosphate is cleaved. In the intermediate, the released Pi is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of Pi from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein–protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

Volunteer Engagement: Does Engagement Predict the Degree of Satisfaction among New Volunteers and the Commitment of Those who have been Active Longer?

This study examines the concept of engagement in samples of volunteers from different non-profit organisations. Study 1 analyzes the psychometric properties of the abbreviated version of the Utrecht Work Engagement Scale (UWES) (Schaufeli, Bakker, & Salanova, 2006a). Two factorial structures are examined: one-dimensional and three-dimensional structures. Based on the Three-Stage Model of Volunteers’ Duration of Service (Chacón, Vecina, & Dávila, 2007), Study 2 investigates the relationship between engagement, volunteer satisfaction, and intention to remain in a sample of new volunteers and the relationship between engagement, organisational commitment, and intention to remain in a sample of veteran volunteers. Moderated mediation analysis is provided using duration of service as a moderator in order to set a splitting point between new and veteran volunteers. The results of the confirmatory factor analysis suggest that the three-factor model fits better to the data. Regarding the structural models, the first one shows that engagement is crucial to volunteer satisfaction during the first stage, while volunteer satisfaction is the key variable in explaining intention to continue. The second structural model shows that engagement reinforces the participant’s commitment to the organisation, while organizational commitment predicts intention to continue. Both models demonstrate a notable decline when samples are changed.

Ética civil y ética religiosa en España

Se muestra la existencia en España de una ética civil, coherente con el proceso de secularización y modernización experimentado por las sociedades europeas. Tras una exposición de las bases conceptuales de lo que se considera “ética civil”, y empleando los datos de la encuesta internacional Pew Global Attitudes Project, se contrastan un conjunto de hipótesis para España relativas al peso de factores estructurales (sexo, edad, educación e ideología) en la aceptación de una ética cívica o religiosa, y la influencia de esta en opiniones, actitudes y comportamientos relacionados con la religión en la vida pública. El contraste empírico de las hipótesis especificadas revela la influencia que tiene la opción ética en la opinión pública referida al Estado, la sociedad y la religión. Los resultados son acordes con los expuestos por investigaciones anteriores, validándolos, al emplear expresiones alternativas y permitiendo un análisis novedoso de la ética civil y religiosa en la sociedad española.

Las clases medias en Europa: status y poder en el siglo XXI

European societies are undergoing deep changes as a result of globalization and the international financial crisis that began in 2007. These changes affect the class structure to the extent that these societies suffer an impact on the occupational structure and consume pattern. From the data of Eurobarometer we studied the state of social mobility in the European Union. Comparing the subjective status between 2009 and 2012, we observed divergent movements in European societies. Some societies experimented upward mobility while others just the opposite. We conclude that exists an increasing gap between European societies. To consider the effects of these changes on the status structures a structural model has been tested, exploring the relationship between social class, status and power in European societies. We conclude a statistically significant effect of social class and status on the consciousness perceived to possess more or less power. Two dimensions measure power: “political alienation” and “economic alienation”.

La música como lenguaje de las emociones. Un análisis empírico de su capacidad performativa

Esta investigación estudia la relación entre las emociones y la música. Se efectúa un diseño de test-retest donde los individuos expresan qué música escucharían según estados de ánimo, para posteriormente pedir a una segunda muestra, distinta a la anterior, que valore en qué grado considera que dichas canciones expresan esos mismos estados de ánimo. Se ha efectuado un análisis de los rasgos musicales de dichas canciones. Se concluye que tanto en las emociones como en los rasgos musicales existe una dimensionalidad subyacente. En el caso de las emociones corresponde con las propuestas del diferencial semántico. En los rasgos musicales, se determinan dos dimensiones aún sin etiquetar teóricamente. Finalmente, se muestra la relación empírica entre rasgos musicales y emociones mediante un modelo estructural de medición.

Crisis política y juventud en España: el declive del bipartidismo electoral

This paper shows the increasing trend of Spanish young people towards non-conventional mechanisms of political expression, in a crisis context which has reduced its reliance on traditional political actors. As an alternative to bipartisanship, political participation moves towards increased interest and attention to new players, such as emergent political parties. Using a covariance structural model, factors such as political activism, awareness and understanding of country problems, and trust in the political system, are explored in order to explain an electoral behavior that is undergoing deep changes.

Resilience by solidary ties

This article considers an empirical approach to the relationships among three well known concepts: “Benevolence” (Schwartz), Solidarity and Resilience ("Subjective wellbeing scale" - SWB). The first concept refers to cultural values, the second one to social networks and the third to the ability to recover from crisis. The measurement of solidarity has been done from the point of view of supportive ties. The baseline hypothesis considers that the presence of a high value in Benevolence contributes to the involvements in solidarity networks. Participation in supportive relationships facilitates recovery from personal crisis. Using data from the European Social Survey (ESS6), we conclude from this structural analysis that the resilience reflected in a society is partly a consequence of the supportive networks shaped by the presence of benevolence values.

Subordinate-manager gender combination and perceived leadership style influence on emotions, self-esteem and organizational commitment

A theoretical model was developed to investigate the relationships among subordinate-manager gender combinations, perceived leadership style, experienced frustration and optimism, organization-based self-esteem and organizational commitment. The model was tested within the context of a probabilistic structural model, a discrete Bayesian network, using cross-sectional data from a global pharmaceutical company. The Bayesian network allowed forward inference to assess the relative influence of gender combination and leadership style on the emotions, self-esteem and commitment consequence variables. Further, diagnostics from backward inference were used to assess the relative influence of variables antecedent to organizational commitment. The results showed that gender combination was independent of leadership style and had a direct impact on subordinates' levels of frustration and optimism. Female manager-female subordinate had the largest probability of optimism, while male manager teamed with a male subordinate had the largest probability of frustration. Furthermore, having a female manager teamed up with a male subordinate resulted in the lowest possibility of frustration. However, the findings show that the gender issue is not simply female managers versus male managers, but is concerned with the interaction of the subordinate-manager gender combination and leadership style in a nonlinear manner. (C) 2003 Elsevier Inc. All rights reserved.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Individual spirit at work and its relationship with employee work attitudes and organisational outcomes:An empirical examination in corporate Thailand

The purpose of this thesis is to conduct empirical research in corporate Thailand in order to (1) validate the Spirit at Work Scale (2) investigate the relationships between individual spirit at work and three employee work attitudinal variables (job satisfaction, organisational identification and psychological well-being) and three organisational outcomes (in-role performance, organisational citizenship behaviours (OCB), and turnover intentions) (3) further examine causal relations among these organisational behaviour variables with a longitudinal design (4) examine three employee work attitudes as mediator variables between individual spirit at work and three organisational outcomes and (5) explore the potential antecedents of organisational conditions that foster employee experienced individual spirit at work. The two pilot studies with 155 UK and 175, 715 Thai samples were conducted for validation testing of the main measure used in this study: Spirit at Work Scale (Kinjerski & Skrypnek, 2006a). The results of the two studies including discriminant validity analyses strongly provided supportive evidence that Spirit at Work Scale (SAWS) is a sound psychometric measure and also a distinct construct from the three work attitude constructs. The final model of SAWS contains a total of twelve items; a three factor structure (meaning in work, sense of community, and spiritual connection) in which the sub-factors loaded on higher order factors and also had very acceptable reliability. In line with these results it was decided to use the second-order of SAWS model for Thai samples in the main study and subsequent analysis. The 715 completed questionnaires were received from the first wave of data collection during July - August 2008 and the second wave was conducted again within the same organisations and 501 completed questionnaires were received during March - April 2009. Data were obtained through 49 organisations which were from three types of organisations within Thailand: public organisations, for-profit organisations, and notfor-profit organisations. Confirmatory factor analysis of all measures used in the study and hypothesised model were tested with structural equation modelling techniques. The results were greatly supportive for the direct structural model and partially supportive for the fully mediated model. Moreover, there were different findings across self report and supervisor rating on performance and OCB models. Additionally, the antecedent conditions that fostered employees experienced individual spirit at work and the implications of these findings for research and practice are discussed.