Antisense RNA-mediated suppression of Bmi-1 gene expression inhibits the proliferation of lung cancer cell line A549

The oncogene Bmi-1 regulates cell proliferation and senescence. It is reported that it controlled the self-renewal of leukemic and breast cancer stem cell and was overexpressed in some solid tumors and hematologic malignancies. In this study, the effects of inactivation of Bmi-1 mediated by a plasmid-expressing antisense Bmi-1 RNA on the proliferation of lung cancer cell line A549 were investigated. As a result, when the plasmid was stably introduced into the cell line, the Bmi-1 protein level was specifically downregulated, and the cell proliferation was significantly inhibited as shown by the cell growth curve and colony forming assay. The cells were found mostly in the phase of G(0)/G(1) and cells in S phase were significantly decreased. Our results suggest that targeting Bmi-1 might be a therapeutic potential for the treatment of non-small-cell lung cancer.     

Study of the inhibition of cyclin-dependent kinases with roscovitine and indirubin-3′-oxime from molecular dynamics simulations

Molecular dynamics simulations were performed to elucidate the interactions of CDK2 and CDK5 complexes with three inhibitors: R-roscovitine, S-roscovitine, and indirubin-3′-oxime. The preference of the two complexes for R-roscovitine over the S enantiomer, as reported by the experiment, was also found by the simulations. More importantly, the simulations showed that the cause of the stronger affinity for the R enantiomer is the presence of an important hydrogen bond between R-roscovitine and the kinases not found with S-roscovitine. The simulations also showed two amino acid mutations in the active site of CDK5/R-roscovitine that favor binding-enhanced electrostatic contributions, making the inhibitor more effective for CDK5 than for CDK2. This suggests that the effectiveness of roscovitine-like inhibitors can be improved by enhancing their electrostatic interaction with the kinases. Finally, molecular mechanics–Possion–Boltzmann/surface area calculations of the CDK5/indirubin-3′-oxime system in both water-excluded and water-included environments gave significantly different electrostatic contributions to the binding. The simulations detected the displacement of a water molecule in the active site of the water-included CDK/indirubin-3′-oxime system. This resulted in a more conserved binding pattern than the water-excluded structure. Hence, in the design of new indirubin-like inhibitors, it is important to include the water molecule in the analysis. Figure Hydrogen bonding networks at the active sites of both CDK5/R-roscotivine (light grey) and CDK2/R-roscovitine (black).     

Verprolin function in endocytosis and actin organization. Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain

Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity.     

The Raf-like kinase Raf36 negatively regulates plant resistance against the oomycete pathogen Phytophthora parasitica by targeting MKK2

Oomycetes represent a unique group of plant pathogens that are phylogenetically distant from true fungi and cause significant crop losses and environmental damage. Understanding of the genetic basis of host plant susceptibility facilitates the development of novel disease resistance strategies. In this study, we report the identification of an Arabidopsis thaliana T-DNA mutant with enhanced resistance to Phytophthora parasitica with an insertion in the Raf-like mitogen-activated protein kinase kinase kinase gene Raf36. We generated additional raf36 mutants by CRISPR/Cas9 technology as well as Raf36 complementation and overexpression transformants, with consistent results of infection assays showing that Raf36 mediates Arabidopsis susceptibility to P. parasitica. Using a virus-induced gene silencing assay, we silenced Raf36 homologous genes in Nicotiana benthamiana and demonstrated by infection assays the conserved immune function of Raf36. Mutagenesis analyses indicated that the kinase activity of Raf36 is important for its immune function and interaction with MKK2, a MAPK kinase. By generating and analysing mkk2 mutants and MKK2 complementation and overexpression transformants, we found that MKK2 is a positive immune regulator in the response to P. parasitica infection. Furthermore, infection assay on mkk2 raf36 double mutant plants indicated that MKK2 is required for the raf36-conferred resistance to P. parasitica. Taken together, we identified a Raf-like kinase Raf36 as a novel plant susceptibility factor that functions upstream of MKK2 and directly targets it to negatively regulate plant resistance to P. parasitica.     

Associations between daytime and nighttime plasma orexin A levels and cognitive function in patients with obstructive sleep apnea

Sleep and Biological Rhythms - The relationship between plasma orexin A (OXA) levels and cognitive function in patients with obstructive sleep apnea (OSA) remains unclear. This study aimed to...