Cyclic AMP efflux is regulated by occupancy of the adenosine receptor in pig aortic smooth muscle cells

Cultured pig aortic smooth muscle cells respond to extracellular adenosine by activating adenylate cyclase and by initiating the efflux of cAMP. In the presence of extracellular adenosine, efflux is first order with respect to intracellular cAMP concentration up to at least 125 pmol/10(6) cells. The apparent first-order rate constant for the efflux of cAMP increases in a dose-dependent manner in response to extracellular adenosine or 5-N-ethylcarboxamide adenosine. The EC50 for adenosine for promoting cAMP efflux is 12 microM. For cells stimulated with 5-N-ethylcarboxamide adenosine, the EC50 is 5 microM. When extracellular adenosine is removed, efflux stops abruptly. Cellular cAMP content falls but is still in a range that supports cAMP efflux when agonist is present. Efflux is not affected by H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP-dependent protein kinase. These data suggest that in pig aortic smooth muscle cells, the efficiency of cAMP efflux is regulated by A2 receptor occupancy.     

Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data

The Photo-Activatable Ribonucleoside-enhanced CrossLinking and ImmunoPrecipitation (PAR-CLIP) method was recently developed for global identification of RNAs interacting with proteins. The strength of this versatile method results from induction of specific T to C transitions at sites of interaction. However, current analytical tools do not distinguish between non-experimentally and experimentally induced transitions. Furthermore, geometric properties at potential binding sites are not taken into account. To surmount these shortcomings, we developed a two-step algorithm consisting of a non-parametric two-component mixture model and a wavelet-based peak calling procedure. Our algorithm can reduce the number of false positives up to 24% thereby identifying high confidence interaction sites. We successfully employed this approach in conjunction with a modified PAR-CLIP protocol to study the functional role of nuclear Moloney leukemia virus 10, a putative RNA helicase interacting with Argonaute2 and Polycomb. Our method, available as the R package wavClusteR, is generally applicable to any substitution-based inference problem in genomics.     

Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-<Superscript>19</Superscript>F-NMR

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using ¹⁹F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.     

Thromboxane modulating agents. 1. Design of 1-[(arylsulfonyl)amino] alkylindole derivatives as dual thromboxane synthase inhibitor/thromboxane receptor antagonists.

The design of a series of dual thromboxane synthase inhibitor/thromboxane receptor antagonists based on an indole thromboxane synthase inhibitor template is described. The indole-5-propanoic acid derivatives 17, 22 and 23 were found to be potent dual agents in vitro.     

West Nile Virus and California Breeding Bird Declines

Since it was first detected in 1999, West Nile virus (WNV) quickly spread, becoming the dominant vector-borne disease in North America. Sometimes fatal to humans, WNV is even more widespread among birds, with hundreds of species known to be susceptible to WNV infection in North America alone. However, despite considerable mortality and local declines observed in American crows (Corvus brachyrhynchos), there has been little evidence of a large regional association between WNV susceptibility and population declines of any species. Here we demonstrate a correlation between susceptibility to WNV measured by large-scale testing of dead birds and two indices of overall population change among bird species following the spread of WNV throughout California. This result was due primarily to declines in four species of Corvidae, including all species in this family except common ravens (Corvus corax). Our results support the hypothesis that susceptibility to WNV may have negative population consequences to most corvids on regional levels. They also provide confirmation that dead animal surveillance programs can provide important data indicating populations most likely to suffer detrimental impacts due to WNV.     

Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking.

The upstream coagulation enzymes are homologous trypsin-like serine proteases that typically function in enzyme-cofactor complexes, exemplified by coagulation factor VIIa (VIIa), which is allosterically activated upon binding to its cell surface receptor tissue factor (TF). TF cooperates with VIIa to create a bimolecular recognition surface that serves as an exosite for factor X binding. This study analyzes to what extent scissile bond docking to the catalytic cleft contributes to macromolecular substrate affinity. Mutation of the P1 Arg residue in factor X to Gln prevented activation by the TF.VIIa complex but did not reduce macromolecular substrate affinity for TF.VIIa. Similarly, mutations of the S and S' subsites in the catalytic cleft of the enzyme VIIa failed to reduce affinity for factor X, although the affinity for small chromogenic substrates and the efficiency of factor X scissile bond cleavage were reduced. Thus, docking of the activation peptide bond to the catalytic cleft of this enzyme-cofactor complex does not significantly contribute to affinity for macromolecular substrate. Rather, it appears that the creation of an extended macromolecular substrate recognition surface involving enzyme and cofactor is utilized to generate substrate specificity between the highly homologous, regulatory proteases of the coagulation cascade.     

Thinning affects Pinus sylvestris needle decomposition rates and chemistry differently depending on site conditions

Changes in mass and chemical composition of Pinus sylvestris senescent needles were studied over a 5 year period in Mediterranean (MF) and Continental forests (CF) in the Pyrenees under varying levels of thinning (P0: reference, no thinning; P20: removal of 20% basal area, P30: removal of 30% basal area). Decomposition rates were higher in MF (k = 0.423 year^?1) than in CF (k = 0.245 year^?1). However, the maximum decomposition limit was higher in MF (87.9%) compared to CF (78.1%). The relative importance and timing of rainfall, and cellulose and lignin abundance on the decomposition process was similar among both sites. However, air temperature and degree-days only affected CF (the colder site) during the initial stages of decomposition, while litter moisture was significant only in MF (the drier site) in the latter stages of decomposition. Nutrient and carbon dynamics showed temporal patterns similar to those reported in higher latitudes (except for Ca), however, indicators of N mineralization such as C/N and lignin/N at the study sites were lower than values reported in the literature. Decreases in decomposition rates after thinning were higher in MF than in CF, indicating that this ecosystem could, in the short term, be more sensitive to human intervention. Thinning had similar temporary qualitative effects at both sites, slowing decomposition, increasing N and P immobilization and decreasing Ca immobilization. However, quantitative effects of thinning were site dependent in that the magnitude of nutrient immobilization was higher in CF. A conceptual model is presented to explain effects of thinning on litter N dynamics. These temporary changes are not trivial as nutrient immobilization and accumulated organic matter losses over a thinning cycle may affect tree growth particularly during short rotations and intensive fast-growing plantations. Under similar nutrient availability conditions, sites where nutrient release occurs faster may show higher post-thinning tree growth rates.