Anti- Japanese-Encephalitis-Viral Effects of Kaempferol and Daidzin and Their RNA-Binding Characteristics

Background: New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an a-1 translational frameshift to synthesize the NS1 ’ protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described. Methodology/Principal Findings: The 50 % cytotoxic concentration (CC50) and 50 % effective concentration (EC50) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC 50 values of Kae and Dai were 12.6 and 25.9 mM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC50 was 21.5 and 40.4 mM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent Kb value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes wit

Structural changes in Escherichia coli membranes induced by bacteriophage T4 at different temperatures.

This paper presents some further evidence for our model of DNA translocation into Escherichia coli cells by bacteriophage T4 (see Tarahovsky, Y. S., Khusainov, A. A., Deev, A. A., Kim, Y. V. 1991. FEBS Lett. 289:18-22). When lowering the temperature, we succeeded in slowing down the infection process and in observing a few separate stages by electron microscopy. Also, potassium leakage at different temperatures was measured. At 0-6 degrees C the phage was found to be irreversibly adsorbed on the cell surface, its tail to be contracted, and the outer membrane to be invaginated. Membrane fusion and formation of broad intermembrane bridges with a hole for potassium leakage were shown to start above 7 degrees C. At about 17-20 degrees C the diameter of the bridge decreased considerably, which could correspond to the sealing of the membrane

Lipid bilayer - DNA interaction mediated by divalent metal cations: SANS and SAXD study

The structure of aggregates formed due to DNA interaction with dipalmitoylphosphatidylcholine (DPPC) in presence of Ca2+ and Zn2+ is examined using small-angle synchrotron X-ray diffraction (SAXD) and small-angle neutron scattering (SANS). SAXD detected two structures: LC - condensed lamellar phase and LX - lamellar phase with DNA strands intercalated between the adjacent lipid bilayers, without regular packing at low cation concentration (~1 mM). The high concentration of Zn2+ induces a macroscopic phase separation in mixtures. The SANS curves of DPPC+ions2+ vesicles evaluated using the strip function model have shown different modes of cations binding to the DPPC bilayers.Peer reviewed: YesNRC publication: Ye

Surface Complexation of DNA with Insoluble Monolayers. Influence of Divalent Counterions

DNA interacts with insoluble monolayers made of cationic amphiphiles as well as with monolayers of zwitterionic lipids in the presence of divalent ions. Binding to dioctadecyldimethylammonium bromide (DODAB) or distearoyl-sn-glycero-3-phosphocholine (DSPC) monolayers in the presence of calcium is accompanied by monolayer expansion. For the positively charged DODAB monolayer, this causes a decrease of surface potential, while an increase is observed for the DSPC monolayers. Binding to dipalmitoyl-sn-glycero-3-phosphocholine preserves most of the liquid expanded−liquid condensed coexistence region. The liquid condensed domains adopt an elongated morphology in the presence of DNA, especially in the presence of calcium. The interaction of DNA with phospholipid monolayers is ion specific: the presence of calcium leads to a stronger interaction than magnesium and barium. These results were confirmed by bulk complexation studies