Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

A PARAMETRIC METHOD FOR ASSESSING DIVERSIFICATION‐RATE VARIATION IN PHYLOGENETIC TREES

Phylogenetic hypotheses are frequently used to examine variation in rates of diversification across the history of a group. Patterns of diversification‐rate variation can be used to infer underlying ecological and evolutionary processes responsible for patterns of cladogenesis. Most existing methods examine rate variation through time. Methods for examining differences in diversification among groups are more limited. Here, we present a new method, parametric rate comparison (PRC), that explicitly compares diversification rates among lineages in a tree using a variety of standard statistical distributions. PRC can identify subclades of the tree where diversification rates are at variance with the remainder of the tree. A randomization test can be used to evaluate how often such variance would appear by chance alone. The method also allows for comparison of diversification rate among a priori defined groups. Further, the application of the PRC method is not restricted to monophyletic groups. We examined the performance of PRC using simulated data, which showed that PRC has acceptable false‐positive rates and statistical power to detect rate variation. We apply the PRC method to the well‐studied radiation of North American Plethodon salamanders, and support the inference that the large‐bodied Plethodon glutinosus clade has a higher historical rate of diversification compared to other Plethodon salamanders.     

NMR and temperature-jump measurements of de novo designed proteins demonstrate rapid folding in the absence of explicit selection for kinetics

We address the importance of natural selection in the origin and maintenance of rapid protein folding by experimentally characterizing the folding kinetics of two de novo designed proteins, NC3-NCAP and ENH-FSM1. These 51 residue proteins, which adopt the helix-turn-helix homeodomain fold, share as few as 12 residues in common with their most closely related natural analog. Despite the replacement of up to 3/4 of their residues by a computer algorithm optimizing only thermodynamic properties, the designed proteins fold as fast or faster than the 35,000 s(-1) observed for the closest natural analog. Thus these de novo designed proteins, which were produced in the complete absence of selective pressures or design constraints explicitly aimed at ensuring rapid folding, are among the most rapidly folding proteins reported to date.     

Optimization of the Water-Insoluble Procedures for <Emphasis Type="Italic">USP</Emphasis> General Chapter <Emphasis Type="Italic">Residual Solvents</Emphasis> <467>

The water-insoluble procedures in US Pharmacopeia (USP) General Chapter Residual Solvents <467>, which are based on European Pharmacopoeia procedures, were optimized and modified before their inclusion in the chapter to improve their scope, performance, and ruggedness. The optimized procedures use a static headspace introduction system with a gas chromatograph equipped with a flame ionization detector. This article describes some of the key changes made to the USP published procedures, including use of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) as the solvent, addition of 5 mL of water and 1 mL of sample (dissolved in DMSO or DMF) to the headspace vial, use of a 3:1 GC split ratio, and use of new matrix-matched system suitability solutions. These procedures were verified with two different active pharmaceutical ingredients—hydroxyzine pamoate and prednisone. In the investigation, the more polar material (hydroxyzine pamoate) showed greater recoveries for the optimized procedures when prepared in DMSO. The less polar material (prednisone) typically had greater recoveries in DMF for the optimized procedures. During experimentation, insights into sample preparation, additional types of headspace instrumentation, solvent purity, and other parameters were also gained.     

Transmission characteristics of the 2009 H1N1 influenza pandemic: comparison of 8 Southern hemisphere countries

While in Northern hemisphere countries, the pandemic H1N1 virus (H1N1pdm) was introduced outside of the typical influenza season, Southern hemisphere countries experienced a single wave of transmission during their 2009 winter season. This provides a unique opportunity to compare the spread of a single virus in different countries and study the factors influencing its transmission. Here, we estimate and compare transmission characteristics of H1N1pdm for eight Southern hemisphere countries/states: Argentina, Australia, Bolivia, Brazil, Chile, New Zealand, South Africa and Victoria (Australia). Weekly incidence of cases and age-distribution of cumulative cases were extracted from public reports of countries'' surveillance systems. Estimates of the reproduction numbers, R ₀, empirically derived from the country-epidemics'' early exponential phase, were positively associated with the proportion of children in the populations (p = 0.004). To explore the role of demography in explaining differences in transmission intensity, we then fitted a dynamic age-structured model of influenza transmission to available incidence data for each country independently, and for all the countries simultaneously. Posterior median estimates of R ₀ ranged 1.2–1.8 for the country-specific fits, and 1.29–1.47 for the global fits. Corresponding estimates for overall attack-rate were in the range 20–50%. All model fits indicated a significant decrease in susceptibility to infection with age. These results confirm the transmissibility of the 2009 H1N1 pandemic virus was relatively low compared with past pandemics. The pattern of age-dependent susceptibility found confirms that older populations had substantial – though partial - pre-existing immunity, presumably due to exposure to heterologous influenza strains. Our analysis indicates that between-country-differences in transmission were at least partly due to differences in population demography.     

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.     

FCP: functional coverage of the proteome by structures

MOTIVATION: Tools and resources for translating the remarkable growth witnessed in recent years in the number of protein structures determined experimentally into actual gain in the functional coverage of the proteome are becoming increasingly necessary. We introduce FCP, a publicly accessible web tool dedicated to analyzing the current state and trends of the population of structures within protein families. FCP offers both graphical and quantitative data on the degree of functional coverage of enzymes and nuclear receptors by existing structures, as well as on the bias observed in the distribution of structures along their respective functional classification schemes. AVAILABILITY: http://cgl.imim.es/fcp CONTACT: jmestres@imim.es.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.