Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

DNA barcoding of Cyclamen species: another approach to species identification

Cyclamen is a diverse genus with some well defined and some intransigent species complexes. Here we report on an attempt to use DNA barcoding techniques to help evaluate species boundaries. DNA barcoding uses multiple samples, each from a different individual of a species, to generate a series of DNA sequences that can be compared for similarity.

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG]2 with a topoisomerase poison in the presence of Co2+Ions†,‡

The structure of the duplex d[CG(5-BrU)ACG]2 bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 Å resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co2+ ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40°, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co2+ ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA (‘hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.