Interaction of multicomponent anionic liposomes with cationic pyridylphenylene dendrimer: Does the complex behavior depend on the liposome composition?

Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D350+) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.

Structural Properties of Potexvirus Coat Proteins Detected by Optical Methods.

It has been shown by X-ray analysis that cores of coat proteins (CPs) from three potexviruses, flexible helical RNA-containing plant viruses, have similar α-helical structure. However, this similarity cannot explain structural lability of potexvirus virions, which is believed to determine their biological activity. Here, we used circular dichroism (CD) spectroscopy in the far UV region to compare optical properties of CPs from three potexviruses with the same morphology and similar structure. CPs from Alternanthera mosaic virus (AltMV), potato aucuba mosaic virus (PAMV), and potato virus X (PVX) have been studied in a free state and in virions. The CD spectrum of AltMV virions was similar to the previously obtained CD spectrum of papaya mosaic virus (PapMV) virions, but differed significantly from the CD spectrum of PAMV virions. The CD spectrum of PAMV virions resembled in its basic characteristics the CD spectrum of PVX virions characterized by molar ellipticity that is abnormally low for α-helical proteins. Homology modeling of the CP structures in AltMV, PAMV, and PVX virions was based on the known high-resolution structures of CPs from papaya mosaic virus and bamboo mosaic virus and confirmed that the structures of the CP cores in all three viruses were nearly identical. Comparison of amino acid sequences of different potexvirus CPs and prediction of unstructured regions in these proteins revealed a possible correlation between specific features in the virion CD spectra and the presence of disordered N-terminal segments in the CPs.

Effect of chaperonin encoded by gene 146 on thermal aggregation of lytic proteins of bacteriophage EL Pseudomonas aeruginosa.

Investigation of the chaperonin encoded by gene 146 of bacteriophage EL Pseudomonas aeruginosa that we characterized earlier has been continued. To reveal the mechanism of its functioning, new recombinant substrate proteins, fragments of gene product (gp) 183 containing the lysozyme domain were prepared. Their interaction with gp146 was studied. The influence of the phage chaperonin on the thermal aggregation of one of these gp183 fragments and endolysin (gp188) was investigated in both the presence and the absence of ATP by dynamic light scattering. In the absence of ATP, the phage chaperonin forms stable complexes with substrate proteins, thereby protecting them against thermal aggregation. Experimental data obtained for different substrate proteins are analyzed.

Complexes between anionic liposomes and spherical polycationic brushes. An assembly of assemblies.

This paper has at its objective the assembling of liposomal assemblies onto nanoparticles. In this manner, one generates nanoparticles with a high loading capacity. Thus, spherical spherical polycationic "brushes" (SPBs) were synthesized by graft polymerizing a cationic monomer, (trimethylammonium)ethylmethacrylate chloride, onto the surface of monodisperse polystyrene particles, ca. 100 nm in diameter. These particles were complexed with small unilamellar anionic liposomes, 40-60 nm in diameter, composed of egg lecithin (EL) and anionic phosphatidylserine (PS(1-)) in PS(1-)/EL ratios from 0.10 to 0.54, a key parameter designated as ν. These complexes were then characterized according to electrophoretic mobility, dynamic light scattering, conductivity, fluorescence, and cryogenic transmission electron microscopy, with the following main conclusions: (a) All added liposomes are totally associated with SPBs up to a certain saturation concentration (specific for each ν value). (b) The number of liposomes per SPB particle varies from 40 (ν = 0.1) to 14 (ν = 0.5). (c) At sufficiently high liposome concentrations, the SPBs experience an overall change from positive to negative charge. (d) SPB complexes tend to aggregate when their initial positive charge has been precisely neutralized by the anionic liposomes. Aggregation is impeded by either positive charge at lower lipid concentrations, or negative charge at higher lipid concentrations. (e) The liposomes remain intact (i.e., do not leak) when associated with SPBs, at ν ≤ 0.5. (f) Complete SPB/liposome dissociation occurs at external [NaCl] = 0.3 M for ν = 0.1 and at 0.6 M for ν = 0.5. Liposomes with ν = 0.54 do not dissociate from the SPBs even in NaCl solutions up to 1.0 M. (g) Complexation of the PS(1-)/EL liposomes to the SPBs induces flip-flop of PS(1-) from the inner leaflet to the outer leaflet. (h) The differences in the ability of PS(1-) (a cylindrical lipid) and CL(2-) (a conical lipid) to create membranes defects are attributed to geometric factors.

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

Novel inhibitors of glyceraldehyde-3-phosphate dehydrogenase: covalent modification of NAD-binding site by aromatic thiols.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) is a glycolytic enzyme catalyzing the formation of 1,3-diphosphoglycerate from glyceraldehyde-3-phosphate and inorganic phosphate. In cooperation with E3 ubiquitin-kinase Siah1, GAPDH directly participates in the apoptotic death of neurons in Parkinson's disease. Potential GAPDH inhibitors were screened in silico, and three compounds with high affinity to the NAD-binding site and theoretically capable of forming a disulfide bond with amino acid residue Cys149 were found among cysteine and glutathione derivatives. The inhibitory effect of these compounds was tested on GAPDH from rabbit muscles using isothermal calorimetry and kinetic methods. As a result of experimental screening, we selected two compounds that inhibit GAPDH by forming disulfide bonds with the Cys149 residue in the enzyme active site. Since Cys149 is the key residue not only for the catalyzed reaction, but also for interaction with Siah1, the compounds can be assumed to inhibit the formation of the proapoptotic complex GAPDH-Siah1 and therefore have potential effect against Parkinson's disease.