The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation

Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIα (Topo IIα), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIα inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIα decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIα decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIα decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIα inhibitors

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

Refinement of a model for evaluating the population exposure in an urban area

A mathematical model is presented for the determination of human exposure to ambient air pollution in an urban area; the model is a refined version of a previously developed mathematical model EXPAND (EXposure model for Particulate matter And Nitrogen oxiDes). The model combines predicted concentrations, information on people's activities and location of the population to evaluate the spatial and temporal variation of average exposure of the urban population to ambient air pollution in different microenvironments. The revisions of the modelling system containing the EXPAND model include improvements of the associated urban emission and dispersion modelling system, an improved treatment of the time use of population, and better treatment for the infiltration coefficients from outdoor to indoor air. The revised model version can also be used for estimating intake fractions for various pollutants, source categories and population subgroups. We present numerical results on annual spatial concentration, time activity and population exposures to PM2.5 in the Helsinki Metropolitan Area and Helsinki for 2008 and 2009, respectively. Approximately 60% of the total exposure occurred at home, 17% at work, 4% in traffic and 19% in other microenvironments in the Helsinki Metropolitan Area. The population exposure originating from the long-range transported background concentrations was responsible for a major fraction, 86%, of the total exposure in Helsinki. The largest local contributors were vehicular emissions (12%) and shipping (2%)