Recombination Phenotypes of Escherichia coli greA Mutants

<p>Abstract</p> <p>Background</p> <p>The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination.</p> <p>Results</p> <p><it>Escherichia coli </it>mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A <it>greA </it>mutant and a <it>greA </it>deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination.</p> <p>Conclusion</p> <p>These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.</p

Paroxetine suppresses recombinant human P2X7 responses

P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC50 of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC50 of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14+ monocytes was suppressed with trifluoperazine and paroxetine

Identification of aptamers against the DNA template for in vitro transcription of the HIV-1 TAR element

We have extracted from a random population of about 10⁹ oligodeoxynucleotides a series of 21-mers that are able to bind to a folded DNA 76-mer used as a template for in vitro transcription of the TAR element of the retrovirus HIV-1, by the T7 RNA polymerase. Five aptastrucs, that is, aptamers able to bind to the structure, out of 15 analyzed sequences, share the consensus motif 5'-PyGGG(TG)PyC, complementary in part to a weak double-stranded region of the target. (The parentheses indicate that either T or G is missing in one of these aptastrucs.) A dissociation constant of about 3 microM was evaluated by electrophoretic mobility shift assay for the winner sequence. Interactions between the aptastruc and the target sequences involve more than Watson-Crick base pairing of the consensus octamer. The binding is chemistry dependent. Phosphorothioate oligodeoxyribonucleotides and 2'-O-methyl oligoribonucleotides derived from the selected aptastrucs exhibit a weak if any affinity for the target