A simple synthesis of APM ([p-(N-acrylamino)-phenyl]mercuric chloride), a useful tool for the analysis of thiolated biomolecules.

This study describes two novel synthetic procedures to prepare APM, a useful tool for the analysis and the purification of thiolated biomolecules. The methods developed are technically simple and robust and allowed the first full characterization of pure APM. Moreover, the efficacy of APM, as a biochemical tool, was demonstrated by analysis of tRNA thiolation by APM-PAGE.

RNA polymerase II CTD S2P is dispensable for embryogenesis but mediates exit from developmental diapause in C. elegans.

Serine 2 phosphorylation (S2P) within the CTD of RNA polymerase II is considered a Cdk9/Cdk12-dependent mark required for 3'-end processing. However, the relevance of CTD S2P in metazoan development is unknown. We show that cdk-12 lesions or a full-length CTD S2A substitution results in an identical phenotype in Caenorhabditis elegans Embryogenesis occurs in the complete absence of S2P, but the hatched larvae arrest development, mimicking the diapause induced when hatching occurs in the absence of food. Genome-wide analyses indicate that when CTD S2P is inhibited, only a subset of growth-related genes is not properly expressed. These genes correspond to SL2 trans-spliced mRNAs located in position 2 and over within operons. We show that CDK-12 is required for maximal occupancy of cleavage stimulatory factor necessary for SL2 trans-splicing. We propose that CTD S2P functions as a gene-specific signaling mark ensuring the nutritional control of the C. elegans developmental program.

Studies on the potential of microparticles entrapping pDNA-poly(aminoacids) complexes as vaccine delivery systems.

Poly(D,L-lactide-co-glycolide) (PLGA) microparticles containing plasmid DNA (pDNA) have potential uses as vaccine delivery systems. Nevertheless, the established double emulsion and solvent evaporation method used to produce them is characterised by a low encapsulation efficiency (about 20%) and nicks the supercoiled DNA. The aim of this work was to develop an encapsulation process to optimise the overall encapsulation efficiency and the supercoiled DNA content, to obtain a carrier suitable for mucosal delivery of DNA vaccines. Our strategy was to reduce the global negative charge of DNA which was unfavourable to its incorporation into the polymer by condensing it with cationic poly(aminoacids) which were previously reported to improve cell transfection. In this study, after characterisation of the compaction of DNA plasmid encoding for a Green Fluorescent Protein, we demonstrated that resulting complexes were successfully encapsulated into PLGA microparticles presenting a mean size around 4.5 microns. The preliminary step of complexation enhances the yield of the process by a factor 4.1 and protects the supercoiled form. In a bacteria transformation assay, we demonstrated that extracted pDNA (naked or complexed) remained in a transcriptionally active form after encapsulation. Bovine macrophages in culture phagocytosed microparticles loaded with uncomplexed/complexed with poly(L-lysine) pDNA. The production of the Green Fluorescent Protein demonstrated that these carriers could deliver intact and functional plasmid DNA probably by escaping from lysosomal degradation.

Perception of the combined effects of smoking and alcohol on cancer risks in never smokers and heavy smokers.

The issue addressed in the study is: How do people perceive the combined effect of alcohol consumption and tobacco consumption on risks of cancer? The method used was an application of Information Integration Theory. Sixty-four participants of both sexes were asked to estimate the risk of cancer associated with a number of situations described by a tobacco-consumption level associated with an alcohol-consumption level. Participants were subsequently presented with a questionnaire concerning the way alcohol and tobacco consumption can cause cancer. Results showed that French adults apparently considered that indulging in only one of these two behaviours represents a maximum health risk. The two effects were seen to combine disjunctively which runs counter to current medical data. However, there was total contradiction between the participants' answers to the questionnaire concerning their knowledge and the information integration task.

Perception of the combined effect of smoking and alcohol on health.

Because smoking is significantly associated with drinking, the consumption of alcohol can be associated with indirect consumption of tobacco (passive smoking), and alcohol and tobacco can have synergetic effects, the ways people perceive their combined effects were investigated. Information integration theory was applied, and the results showed that rather than representing the combined effects of tobacco consumption and alcohol consumption in a synergetic or summative way, the sample of 40 French adults apparently considered that indulging in only one of these two behaviors results almost unilaterally in maximal alteration of health. The two effects were seen to combine disjunctively, a way of perceiving the combined effects of the two substances that runs counter to current medical data on the subject.

Fission yeast Csk1 is a CAK-activating kinase (CAKAK).

Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).

Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast.

Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.