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.     

Microbiological dehydrogenation of polyfunctional Δ5-3β-acetoxy steroids by Corynebacterium mediolanum strain B-964

Summary A high-yield microbiological transformation of polyfunctional ⁵-3-acetoxy steroids, containing an additional ring E, by Corynebacterium mediolanum strain B-964 was carried out, resulting in the corresponding ⁴-3-ketones. It was shown that the type of transformation and the yield of the reaction depend on the degree of saturation of the ring E and on the position of the oxygen-containing substituents in it.     

Genetic differentiation in a captive population of the endangered Siberian crane (Grus leucogeranus Pall.)

DNA fingerprinting, followed by multivariate analysis of data, was used to characterize genetic heterogeneity in captive populations of the endangered Siberian and sandhill cranes. The genetic structure revealed reflected the natural population and species distributions. The relevant groups differed not only from each other, but also from interspecies and inter-population hybrids bred in captivity. In this study we have tested an approach to the analysis of population structure based on individual genotypes. Interpretation of fingerprinting data by means of the analytical system applied here is a useful and reliable procedure for the estimation of genetic relationships between individuals.     

Trophic-based diversification in benthivorous charrs (Salvelinus) dwelling littoral zones of Northern lakes

Charrs of the genus Salvelinus form distinct trophic morphs living in sympatry in numerous postglacial lakes. Here we demonstrate that charrs can diversify into amphipod foraging specialists and sedentary macrobenthos consumers in the shallow-water ecosystems. This pattern was revealed in three out of six lakes under exploration supported by differences in stomach content, trophic-transmitted parasite, and stable isotope ratio analyzes. The body shape and growth rate comparison indicates that this kind of trophic-based diversification emerges at a juvenile stage and is maintained throughout the whole life. The restriction in gene flow found between the morphs allows to propose the possibility for the hereditable-based specialization to evolve. We found that those diversification phenomena are possible only in the lakes situated in vicinity of the ocean coastline, while no evidence of this split was found for inland mountain lakes. We suggest that the trophic-based diversification in the littoral ecosystems is accounted for the heterogeneity in the ecological conditions and food resources’ distribution linked to coastal wind action. This phenomenon was previously reported for the charr in Lake Fjellfrosvatn, Scandinavia, so it seems to be some universal yet poorly described kind of disruptive selection pressure for northern latitude fishes.

     

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type–specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.     

Prevalence of alleles associated with HIV resistance in Russia

Genetic polymorphisms of CCR5, CCR2, and SDF1 genes have been associated with resistance during human immunodeficiency virus type 1 (HIV-1) infection and disease progression. In the present report, we studied the frequency and co-occurrence of CCR5Delta32, CCR5-59029A/G, CCR2-64I, and SDF1-3'A allelic variants among HIV-1-seronegative individuals (n = 171) in Moscow. Observed allelic frequencies were 0.0906 [95% confidence interval (CI), 0.06-0.1212] for CCR5Delta32, 0.4072 (95% CI, 0.3542-0.4602) for CCR5-59029G, 0.1061 (95% CI, 0.0728-0.1394) for CCR2-64I, and 0.2218 (95% CI 0.1715-0.2721) for SDF1-3'A. A significant linkage disequilibrium (p = 0.0034) between CCR2-64I and SDF1-3'A alleles was observed.     

The effect of calcium on the conformation of cobalamin transporter BtuB

BtuB is a β‐barrel membrane protein that facilitates transport of cobalamin (vitamin B12) from the extracellular medium across the outer membrane of Escherichia coli. It is thought that binding of B12 to BtuB alters the conformation of its periplasm‐exposed N‐terminal residues (the TonB box), which enables subsequent binding of a TonB protein and leads to eventual uptake of B12 into the cytoplasm. Structural studies determined the location of the B12 binding site at the top of the BtuB's β‐barrel, surrounded by extracellular loops. However, the structure of the loops was found to depend on the method used to obtain the protein crystals, which—among other factors—differed in calcium concentration. Experimentally, calcium concentration was found to modulate the binding of the B12 substrate to BtuB. In this study, we investigate the effect of calcium ions on the conformation of the extracellular loops of BtuB and their possible role in B12 binding. Using all‐atom molecular dynamics, we simulate conformational fluctuations of several X‐ray structures of BtuB in the presence and absence of calcium ions. These simulations demonstrate that calcium ions can stabilize the conformation of loops 3–4, 5–6, and 15–16, and thereby prevent occlusion of the binding site. Furthermore, binding of calcium ions to extracellular loops of BtuB was found to enhance correlated motions in the BtuB structure, which is expected to promote signal transduction. Finally, we characterize conformation dynamics of the TonB box in different X‐ray structures and find an interesting correlation between the stability of the TonB box structure and calcium binding. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

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.