Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K(d) value (2.9 microM) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K(d) values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIalpha led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.     

The Impact of Viral RNA on the Association Rates of Capsid Protein Assembly: Bacteriophage MS2 as a Case Study

A large number of single-stranded RNA viruses, which form a major class of all viruses, co-assemble their protein container and their genomic material. The multiple roles of the viral genome in this process are presently only partly understood. Recent experimental results indicate that RNA, in addition to its function as a repository for genetic information, could play important functional roles during the assembly of the viral protein containers. An investigation of the impact of genomic RNA on the association of the protein subunits may therefore provide further insights into the mechanism of virus assembly. We study here the impact of viral RNA on the association rates of the capsid proteins during virus assembly. As a case study, we consider the viral capsid of bacteriophage MS2, which is formed from 60 asymmetric (AB) and 30 symmetric (CC) protein dimers. Using Brownian dynamics simulations, we investigate the effect of the binding of an RNA stem-loop (the translational repressor) on the association rates of the capsid protein dimers. Our analysis shows that translational repressor binding results in self-association of AB dimers being inhibited, whilst association of AB with CC dimers is greatly enhanced. This provides an explanation for experimental results in which an alternating assembly pattern of AB and CC dimer addition to the growing assembly intermediate has been observed to be the dominant mode of assembly. The presence of the RNA hence dramatically decreases the number of dominant assembly pathways and thereby reduces the complexity of the self-assembly process of these viruses.     

Barriers and challenges in clinical ethics consultations: the experiences of nine clinical ethics committees

Clinical ethics committees have recently been established in nearly all Norwegian hospital trusts. One important task for these committees is clinical ethics consultations. This qualitative study explores significant barriers confronting the ethics committees in providing such consultation services. The interviews with the committees indicate that there is a substantial need for clinical ethics support services and, in general, the committee members expressed a great deal of enthusiasm for the committee work. They also reported, however, that tendencies to evade moral disagreement, conflict, and 'outsiders' are common in the hospitals. Sometimes even the committees comply with some of these tendencies. The committees agree that there is a need to improve their routines and procedures, clarify the committees' profile and field of responsibility, to make the committees well-known, to secure adequate operating conditions, and to develop organizational integration and support. Various strategies to meet these challenges on a local, regional or national level are also explored in this paper.     

The Impact of Viral RNA on Assembly Pathway Selection

Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T = 3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T = 3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.     

Increased Health Risk in Subjects with High Self-Reported Seasonality

Background

Seasonal variations in mood and behaviour, termed seasonality, are commonly reported in the general population. As a part of a large cross-sectional health survey in Hordaland, Norway, we investigated the relationship between seasonality, objective health measurements and health behaviours.

Methodology/Principal Findings

A total of 11,545 subjects between 40–44 years old participated, completing the Global Seasonality Score, measuring seasonality. Waist/hip circumference, BMI and blood pressure were measured, and blood samples were analyzed for total cholesterol, HDL cholesterol, triglycerides and glucose. Subjects also completed a questionnaire on miscellaneous health behaviours (exercise, smoking, alcohol consumption). Hierarchical linear regression analyses were used to investigate associations between seasonality and objective health measurements, while binary logistic regression was used for analysing associations between seasonality and health behaviours. Analyses were adjusted for sociodemographic factors, month of questionnaire completion and sleep duration. Seasonality was positively associated with high waist-hip-ratio, BMI, triglyceride levels, and in men high total cholesterol. Seasonality was negatively associated with HDL cholesterol. In women seasonality was negatively associated with prevalence of exercise and positively associated with daily cigarette smoking.

Conclusions/Significance

High seasonality was associated with objective health risk factors and in women also with health behaviours associated with an increased risk for cardiovascular disease.     

Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions

The complex of the subunits (RIalpha, Calpha) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K(d) = 0.25 microm) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RIalpha or RIIalpha was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RIalpha or RIIalpha showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.     

Aspects of carbon metabolism in Chloroflexus

When fluoroacetate was added to aerobic, washed cells of Chloroflexus, O₂ uptake was strongly inhibited and citrate accumulated. Under anaerobic conditions in the light, fluoroacetate inhibited CO₂ uptake and caused citrate accumulation. The results are taken as evidence for the operation of a tricarboxylic acid cycle in Chloroflexus both under aerobic conditions in the dark and anaerobically in the light. 2. Organic compounds are assimilated into the storage materials polyglucose and poly--hydroxybutyric acid by washed cells of Chloroflexus. The type of storage product formed from acetate depends upon the availability of reducing power. 3. Low activities of the key enzymes of the reductive pentose phosphate cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase were detected in cell free extracts of photoheterotrophically grown Chloroflexus.Abbreviations RuBP Ribulose-1,5-bisphosphate - TCA tricarboxylic acid - PHB poly--hydroxybutyric acid     

Substrate enhances the sensitivity of type I protein kinase a to cAMP

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.     

Field Evaluation of a Semiautomated Method for Rapid and Simple Analysis of Recreational Water Microbiological Quality

An early warning system using a rapid enzymatic semiautomated method suitable for fecal coliform detection in recreational waters within 8 h was developed further and evaluated in this study. This rapid method was compared to the standard method followed in the United Kingdom. We used 1,011 samples originating from 206 different locations in Wales. When we assessed the presence or absence of fecal coliforms, targeting very low levels of contamination, we obtained 83.9% agreement between the rapid method and the lauryl sulfate broth-membrane filtration technique, whereas direct confirmation of the samples processed by the rapid method showed 89.3% agreement. Environmental enzymatic background activity was found to be the main limiting factor for this method. Owing to a specific and integrated handling of the results by the software of the instrument, the percentage of false-positive results (a consequence of enzymatic background) was successfully limited to 2.9% by the direct confirmation evaluation. However, 7.8% false-negative results due to “late-growers” had to be accepted in order to produce results within a working day. At present, the method can be used in a more conservative way to assess the environmental threshold of 100 CFU of fecal coliforms per 100 ml in recreational waters. The implications of our findings with regard to the applicability of rapid enzymatic methods are discussed.