V. I. Popenko

Structural effects induced by gold nanoparticles in particles of cholesteric liquid-crystalline dispersion of double-stranded nucleic acids

Gold (Au) nanoparticles of diameter ~2 nm (but not 5 or 15 nm) were capable of incorporating effectively into quasi-nematic layers of particles of cholesteric liquid-crystalline dispersions (CLCD) formed by double-stranded nucleic acids of various families [B-form DNA, A-form poly(I) × poly(C)]. A study of the properties of CLCD particles treated with Au nanoparticles by various physicochemical methods demonstrated that incorporation of Au nanoparticles into quasi-nematic layers of these particles resulted in two effects. First, it caused rearrangement of the spatial cholesteric structure of LC dispersion particles that was accompanied by a decrease in the amplitude of the anomalous band in the circular dichroism spectrum of the corresponding initial CLCD. Second, it induced Au cluster formation within the LC particles that was accompanied by the appearance of a surface plasmon resonance band in the visible spectral region. It was possible that these structural effects were responsible for the genotoxicity of the Au nanoparticles.

A new bidirectional promoter from the human genome

Both human and other mammalian genomes contain a number of closely linked gene pairs transcribed in opposite directions. Bioinformatic analysis suggests that up to 10% of human genes are arranged in this way. This work reports cloning of a human genome fragment that separates two head-to-head oriented genes located at 2p13.1 and encoding hypothetical proteins with unknown functions: CCDC (Coiled Coil Domain Containing) 142 and TTC (TetraTricopeptide repeat Containing) 31. The intergenic region CCDC142-TTC31 overlaps with a CpG island and contains a number of potential binding sites for transcription factors. This fragment functions as a bidirectional promoter in the system of luciferase reporter gene expression upon transfection of human embryonic kidney (HEK293) cells. Vectors containing oppositely oriented genes of two fluorescent proteins: green (EGFP) and red (DsRed2), separated by a fragment of the CCDC142-TTC31 intergenic region, were constructed. Transfection of HEK293 cells with these vectors resulted in simultaneous expression of both fluorescent proteins. The promoter activity was also determined for truncated versions of the intergenic region. The minimal promoter fragment contained Inr, BRE, and DPE elements characteristic for TATA-less promoters. Thus, a novel bidirectional promoter was cloned from the human genome; it can be used for simultaneous constitutive expression of two different genes in human cells.

Three-Dimensional Structure of the Ciliate Didinium nasutum Nucleoli

The nucleolar organization in ciliate Didinium nasutum somatic interphase nuclei was studied using serial ultrathin sections and compared for various physiological states of the cell, namely, fed ciliates, starved ciliates, and dormant cysts. It has been shown that the interphase nucleoli are large structures with a complex architecture: the fibrillar component forms an intricate network in the macronucleus space, while the granular component is located inside this network. The structures looking as individual nucleoli in single sections are actually parts of branched nucleolar networks. The intricate nucleolar networks do not disintegrate after a 30-h starvation; however, the granular component becomes denser and develops numerous cavities filled with fine fibrils of a nonribonucleoprotein nature. In fed D. nasutum, the fibrillar structures on the periphery of nucleoli contain numerous pores (virtually absent in starved cell nucleoli), which can potentially serve for transporting newly synthesized rRNP. Branched nucleolar networks are undetectable in cysts. Their nucleoli are individual structures consisting mainly of the fibrogranular component.

Role of N-linked glycans of HCV glycoprotein E1 in folding of structural proteins and formation of viral particles

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.

Effect of deoxynojirimycin derivatives on morphogenesis of hepatitis C virus

Viral hepatitis C is a dangerous, widespread human disease. The choice of drugs for treatment of chronic hepatitis C virus (HCV) infection is limited, and prophylactic vaccines do not exist. Thus, the development of new antiviral strategies and substances is an issue of great importance. The targeting of viral morphogenesis might be used as an alternative approach to existing strategies of HCV blocking. The glycosylation of viral envelope proteins is an important step of viral particle morphogenesis, which determines the correct assembly of HCV virions. Derivatives of a glucose analog deoxynojirimycin (DNJ) act as an α-glucosidase inhibitor and can impair the assembly of structural proteins and HCV particle formation. In the present work, the effects of alkylated DNJ derivatives, N-pentyl-DNJ and N-benzyl-DNJ, on HCV morphogenesis were studied in a model system of insect cells that produce three viral structural proteins with the formation of virus-like particles. It was shown that DNJ derivatives impair the intracellular N-glycosylation of HCV envelope glycoproteins. At the concentration of 1 mM, these substances cause an increase in the levels of gpE1 and gpE2 glycoproteins and a decrease in their electrophoretic mobility, apparently due to the inhibition of α-glucosidase in the endoplasmic reticulum and the accumulation of hyperglycosylated N-glycans in HCV glycoproteins. The interaction of the latter with calnexin results in the formation of unproductive dimers and blocks the productive assembly of virus-like particles.

Effect of Nucleocapsid on Multimerization of gypsy Structural Protein GAG

The structural protein (Gag) of Drosophila retrovirus gypsy contains capsid and nucleocapsid domains. Gag forms virus-like particles in a bacterial cell; furthermore, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid vary in size and are less organized than particles formed by a full-length Gag. The nucleocapsid determines the organization and structure of the particles, which is ensured by the amino acid residues at its N-terminal (a nucleocapsid proximal part). The assembly of the particle occurs in the presence of any RNAs or single-stranded DNA oligonucleotides.

Relative Position of Nucleolar Chromatin and Nucleolar Components in Ciliate Didinium nasutum Somatic Nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.

Structural protein GAG of the gypsy retrovirus forms virus-like particles in the bacterial cell

Amino acid sequence of the drosophila retrovirus MDG4 (gypsy) structural protein Gag does not contain a canonical motif known for the majority of vertebrate retroviruses. Moreover, protein translation can theoretically begin with two separated initiation codons located within its unique open reading frame. We designed constructs for expression of two theoretically possible variants of Gag polypeptide and investigated the ability of each product to form virus-like particles in the bacterial cell, i.e., in the absence of eukaryotic cell factors. The results obtained showed that both variants of the gypsy protein Gag form globular particles in the bacterial cell.

Superparamagnetic cobalt ferrite nanoparticles “blow up” the spatial ordering of double-stranded DNA molecules

A study was made of the formation of cholesteric liquid-crystalline dispersions (ChLCDs) of double-stranded DNA molecules that were treated with positively charged superparamagnetic cobalt ferrite nanoparticles and the effect the particles exert on DNA liquid-crystalline dispersions (LCDs). When magnetic nanoparticles (MNPs) were bound to linear double-stranded DNA molecules at a high ionic strength (0.3 M NaCl), subsequent phase exclusion of the complexes from a polyethylene glycol (PEG)-containing solution failed to produce a dispersion wherein particles have a spatially twisted arrangement of neighbor double-stranded DNA molecules. When MNPs were added to a DNA ChCLD (1 MNP per 1 DNA molecule), the DNA structure was distorted at MNP binding sites so that the spatial ordering of DNA LCD particles was blown up, abolishing both abnormal optical activity and the characteristic Bragg maximum on a small-angle X-ray scattering (SAXS) curve. The effect may have important biological consequences, considering that physicochemical properties of double-stranded DNA LCD particles reflect features of the DNA spatial organization in chromosomes of primitive organisms.

Immunocytochemical visualization of P185HER2 receptor using antibodies fused with dibarnase and conjugate of barstar with colloidal gold

The localization of the P185HER2 transmembrane receptor in SKOV-3 and BT-474 cancer cells was studied by fluorescence, confocal, and electron immunomicroscopy. The P185HER2 receptor is a marker of breast and ovarian tumors; it is also considered to be a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, firstly, to study the distribution of P185HER2 in cancer cells, secondarily, to remove P185HER2 from the cell surface and, thirdly, to eliminate target cells. In this study, for visualization P185HER2 we propose an immunocytotoxic system, which consists of monoclonal miniantibody 4D5 scFv to the extracellular P185HER2 domain fused with two molecules of barnase (cytotoxic RNAase from Bacillus amyloliquefaciens) and its specific inhibitor, barstar. Fluorescent microscopy showed that the module 4D5 scFc-dibarnase:barstar is efficient for identifying P185HER2 on the surfaces of cancer cells. It was found by confocal microscopy that interaction with 4D5 scFc-dibarnase results in the internalization of P185HER2. The localization of P185HER2 in human ovarian carcinoma cells (SKOV-3) and breast carcinoma cells (BT-474) was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185HER2 is distributed unequally on the cell surface with preferential localization on protrusions or close to their bases and at contacts between protrusions and the cell membrane. At 37°C, P185HER2 is internalized through coated pits and vesicles and concentrates in endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in BT-474 cells.