Vol 71, No 1 (2016)

Alzheimer’s disease (AD) is a neurodegenerative disorder that becomes a cause of dementia during atrophic brain changes. There are two distinguished forms of AD: familial early-onset form (FAD, approximately 5% of all cases, develops before age 65, most commonly 40–50) and sporadic late-onset form (SAD, approximately 95% of all cases, develops after 65). Identification of genetic determinants of FAD development and evidence of amyloid-beta peptide’s (Aβ) neurotoxicity as a central event in the cascade of pathological processes significantly expanded the conception of molecular and genetic mechanisms of the disease. However, the question of whether or not the accumulation of Aβ is the triggering factor of more widespread SAD remains open. There are a growing number of arguments for Aβ overproduction being the secondary, concomitant event of AD pathological processes: synaptic failure, hyperphosphorylation of tau protein, neuroinflammation, neuronal loss, and cognitive decline. As one of triggering risk factors of AD development, mitochondrial dysfunction is considered, with the decrease in ATP synthesis and oxidative stress becoming the consequences. However, the specific molecular and genetic mechanisms of AD remain unclear. This is caused by the lack of relevant animal models for studying mechanisms of the disease and objective estimation of pathogenically justified methods of AD prevention and treatment.

Development and functional characterization of novel, high-affinity protein compounds able to selectively kill human cancer cells is an urgent task of modern biomedical research. In this work, we studied the cytotoxicity of a recombinant phototoxic protein DARPin-miniSOG against the HER2-positive human breast adenocarcinoma cells. It was found that targeted phototoxin DARPin-miniSOG interacts specifically with HER2 receptor and causes the light-induced death of HER2-positive cells by the mechanism of necrosis. Irradiation of the cells in the presence of ascorbic acid eliminates the light-induced cytotoxicity of DARPin-miniSOG, which proves the prooxidant mechanism of phototoxin action.

Balb/С mice were immunized with recombinant Ebola virus glycoprotein. Following the selection, screening, and cloning of murine hybridomas, we obtained five genetically stable clones of monoclonal antibodies GPE118 (IgG), GPE274 (IgM), GPE325 (IgM), GPE463 (IgM), and GPE534 (IgG). These antibodies were isolated and purified from the ascitic fluid of Balb/С mice using Protein G affinity chromatography (for IgG) and euglobulin precipitation (for IgM). To select at least three candidate antibodies for testing in biological assays as components of an antibody cocktail for the prophylaxis and treatment of hemorrhagic fever, we carried out an immunochemical analysis of the epitope specificity of the isolated antibodies. Based on the data of immunoblotting and sandwich ELISA, it became evident that the epitope recognized by GPE 534 differs from the epitopes recognized by the monoclonal antibodies GPE 118 and GPE 325. The last two antibodies also have different epitope specificity: it follows from the immunoblotting data and from the data on the binding of these antibodies with the intact and oxidized (partly deglycosylated) recombinant glycoprotein. For the biological activity studies and the development of recombinant counterparts, we selected three candidate high-affinity monoclonal antibodies GPE 534, GPE 118, and GPE 325.

We discuss the question of constructing three-dimensional models of DNA in complex with proteins using computer modeling and indirect methods of studying the conformation of macromolecules. We consider the methods of interpreting the experimental data obtained by indirect methods of studying the three-dimensional structure of biomolecules. We discuss some aspects of integrating such data into the process of constructing the molecular models of DNA–protein complexes based on the geometric characteristics of DNA. We propose an algorithm for estimating conformations of such complexes based on the information about the local flexibility of DNA and on the experimental data obtained by Forster resonance energy transfer (FRET) and hydroxyl footprinting. Finally, we use this algorithm to predict the hypothetical configuration of DNA in a nucleosome bound with histone H1.

An immunochromatography test system has been developed for molecular diagnostics of the potato virus Y and PLRV infection. To increase a low yield of PLRV and raise antibodies against the PLRV antigen, chimerical virus was constructed comprising the PLRV coat protein and recombinant RNA of a tobamovirus, in which capsid protein gene was replaced by the PLRV coat protein gene. Binary vector containing the DNA copy of the recombinant RNA was infectious, and yield of the chimerical virus increased up to 800 times in comparison with the wild type PLRV. On the basis of experience in the development of the diagnostics of viral and viroid infections, rational tactics are proposed for the mass laboratory and field diagnosis of viral infections on the molecular level.

In recent years, it has been shown that LOV (light, oxygen, voltage) and BLUF (Blue Light sensing Using FAD) photosensory proteins are functioning as photoreceptors of light-regulated processes not only in eukaryotes but also in numerous prokaryotes. In bacterial photoreceptors, LOV and BLUF domains with attached flavin chromophores are often associated with different effector domains, which possess enzymatic and other functions, constituting modular light-switchable systems. At present, some progress in uncovering the photoactivation mechanisms of such systems has been achieved. They are based on the chromophore photoreaction-induced changes in the photosensory domain structures and subsequent signal transduction to the effector domains. Understanding of the signal transduction principles in LOV and BLUF photosensors is important for designing, on their basis, photo-switchable enzymes and transcriptional systems applied in optogenetics—a new field in cell biology and biotechnology. The structural aspects of signal transduction by light-activated LOV and BLUF photoreceptors and their regulatory functions in bacteria, as well as some recent advances in using LOV and BLUF photosensors as activators in optogenetic systems for regulation of cellular processes are discussed.

It was shown that extracellular proteinases produced by the strains of micromycetes A. ochraceus L-1 and A. ustus 1 differ by the activity at various pH as well as by the intensity of the effect on fibrillar proteins. It was revealed that the proteinases of A. ochraceus L-1 demonstrated maximum activity during the growth of the producer in the nitrate-free growth medium (the pH of enzyme reaction was 8.0), whereas those of A. ustus 1 showed maximal activity during the growth of the micromycete in the medium containing sodium nitrate (the pH of enzyme reaction was 6.0). Values of specific fibrinolytic and collagenolytic activities of A. ochraceus L-1 were 2.2 and 1.6 times higher than those of A. ustus 1. At the same time, A. ustus 1 showed very low values of total proteolytic (caseinolytic) activity and had a high ratio of fibrinolytic activity to total proteolytic (caseinolytic) activity (6.92). It makes the strain a promising producer of proteinases, which hydrolyze fibrin and collagen.