miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

Abstract Background Hypoxic conditions induce the expression of hypoxia-inducible factors (HIFs) that allow cells to adapt to the changing conditions and alter the expression of a number of genes including the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a low abundance mRNA in airway epithelial cells even during normoxic conditions, but during hypoxia its mRNA expression decreases even further. Methods In the current studies, we examined the kinetics of hypoxia-induced changes in CFTR mRNA and protein levels in two human airway epithelial cell lines, Calu-3 and 16HBE14o-, and in normal primary bronchial epithelial cells. Our goal was to examine the posttranscriptional modifications that affected CFTR expression during hypoxia. We utilized in silico predictive protocols to establish potential miRNAs that could potentially regulate CFTR message stability and identified miR-200b as a candidate molecule. Results Analysis of each of the epithelial cell types during prolonged hypoxia revealed that CFTR expression decreased after 12 h during a time when miR-200b was continuously upregulated. Furthermore, manipulation of the miRNA levels during normoxia and hypoxia using miR-200b mimics and antagomirs decreased and increased CFTR mRNA levels, respectively, and thus established that miR-200b downregulates CFTR message levels during hypoxic conditions. Conclusion The data suggest that miR-200b may be a suitable target for modulating CFTR levels in vivo

Utilizing Genome-Wide mRNA Profiling to Identify the Cytotoxic Chemotherapeutic Mechanism of Triazoloacridone C-1305 as Direct Microtubule Stabilization

Rational drug design and in vitro pharmacology profiling constitute the gold standard in drug development pipelines. Problems arise, however, because this process is often difficult due to limited information regarding the complete identification of a molecule’s biological activities. The increasing affordability of genome-wide next-generation technologies now provides an excellent opportunity to understand a compound’s diverse effects on gene regulation. Here, we used an unbiased approach in lung and colon cancer cell lines to identify the early transcriptomic signatures of C-1305 cytotoxicity that highlight the novel pathways responsible for its biological activity. Our results demonstrate that C-1305 promotes direct microtubule stabilization as a part of its mechanism of action that leads to apoptosis. Furthermore, we show that C-1305 promotes G2 cell cycle arrest by modulating gene expression. The results indicate that C-1305 is the first microtubule stabilizing agent that also is a topoisomerase II inhibitor. This study provides a novel approach and methodology for delineating the antitumor mechanisms of other putative anticancer drug candidates

Chloroplast PetD protein: evidence for SRP/Alb3-dependent insertion into the thylakoid membrane

Abstract Background In thylakoid membrane, each monomer of the dimeric complex of cytochrome b 6 f is comprised of eight subunits that are both nucleus- and plastid-encoded. Proper cytochrome b 6 f complex integration into the thylakoid membrane requires numerous regulatory factors for coordinated transport, insertion and assembly of the subunits. Although, the chloroplast-encoded cytochrome b 6 f subunit IV (PetD) consists of three transmembrane helices, the signal and the mechanism of protein integration into the thylakoid membrane have not been identified. Results Here, we demonstrate that the native PetD subunit cannot incorporate into the thylakoid membranes spontaneously, but that proper integration occurs through the post-translational signal recognition particle (SRP) pathway. Furthermore, we show that PetD insertion into thylakoid membrane involves the coordinated action of cpFTSY, cpSRP54 and ALB3 insertase. Conclusions PetD subunit integration into the thylakoid membrane is a post-translational and an SRP-dependent process that requires the formation of the cpSRP-cpFtsY-ALB3-PetD complex. This data provides a new insight into the molecular mechanisms by which membrane proteins integration into the thylakoid membrane is accomplished and is not limited to PetD

Comparative studies on detergent-assisted apocytochrome b6 reconstitution into liposomal bilayers monitored by Zetasizer instruments.

The present paper is a systematic, comparative study on the reconstitution of an apocytochrome b6 purified from a heterologous system using a detergent-free method and reconstitution into liposomes performed using three different detergents: SDS, Triton X-100 and DM, and two methods of detergent removal by dialysis and using Bio-Beads. The product size, its distribution and zeta potential, and other parameters were monitored throughout the process. We found that zeta potential of proteoliposomes is correlated with reconstitution efficiency and, as such, can serve as a quick and convenient quality control for reconstitution experiments. We also advocate using detergent-free protein purification methods as they allow for an unfettered choice of detergent for reconstitution, which is the most crucial factor influencing the final product parameters

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

28 septembre 19131913/09/28 (A14,N4731)

Additional file 4: Figure S5. of Chloroplast PetD protein: evidence for SRP/Alb3-dependent insertion into the thylakoid membrane

MALDI-TOF mass spectra. (PDF 112 kb

Additional file 3: Figure S4. of Chloroplast PetD protein: evidence for SRP/Alb3-dependent insertion into the thylakoid membrane

The Kyte–Doolittle hydropathy profile of the first 75 amino acids of pea PetD and ceQORH (Chloroplast Envelope Quinone Oxido-Reductase Homologue). (PDF 146 kb

Additional file 7: Figure S7. of Chloroplast PetD protein: evidence for SRP/Alb3-dependent insertion into the thylakoid membrane

Autoradiograph of isolated free and membrane bound ribosomes isolated during cell-free expression of PetD. (PDF 235 kb