Vol 71, No 2 (2016)

Problems related to the interpretation of data obtained during testing of potential geroprotectors in cytogerontological experiments are considered. It is emphasized that such compounds/physical factors should influence the processes leading to the age-related increase of death probability of multicellular organisms (primarily human, in whose aging gerontologists are mainly interested). However, in the authors’ opinion, compounds that can be used to treat age-related diseases can hardly be classified as geroprotectors. It is noted that, in the model systems using cultured cells, researchers usually evaluate their viability, the criteria of which strongly depend on the aging theory that is shared by the experimenters. In addition, it is very important what cells are used in the studies—normal or transformed cells of multicellular organisms, unicellular eukaryotic or prokaryotic organisms, etc. In particular, the biologically active compounds that decrease the viability of cultured cancer cells, similarly to the compounds that increase the viability of normal cultured cells, may increase the life span of experimental animals and humans. Various problems with interpretation of data obtained with the Hayflick model, the stationary phase aging model, and the cell kinetics model, as well as in experiments on evaluation of cell colony-forming efficiency, are analyzed. The approaches discussed are illustrated on the example of the results of gerontological studies of rapamycin, a well-known mTOR inhibitor. It is assumed that factors retarding the stationary phase aging (chronological aging) of cultured cells are, apparently, the most promising geroprotectors, although the specific mechanisms of their action may vary considerably.

Plum pox virus (PPV, genus Potyvirus, family Potyviridae) is the most important virus pathogen of stone fruit cultures of the genus Prunus from an economical standpoint. Winona strain (PPV-W) is the most variable of nine known virus strains and one of the most widespread in the European part of Russia. Six new PPV-W isolates were first discovered in green plantations of Moscow (Kp2U, Avang, Pulk, Pulk-1), in Taldomsky district of Moscow region (Karm), and in Kovrovsky district of Vladimir region (Vlad-4) on wild trees of plum Prunus domestica. 3’-Terminal segment of genome of new isolates was notable for high variability level. The study on the relationship with other isolates of this strain by means of phylogenetic analysis of gene sequence of the coat protein showed the lack of clusterization of Russian PPV-W isolates according to geographical principle. Inoculation of Nicotiana benthamiana plants by hop plant louse Phorodon humili from plum trees infected with Avang and Pulk isolates and by thistle aphid Brachycaudus cardui from the tree infected with Kp2U isolate led to the systemic viral infection in indicator plants, suggesting the possibility of PPV-W spread by both aphid species in nature. Transmission of PPV-W through seeds was not observed.

Endophytic fungi were found in natural populations of giant fescue (Festuca gigantea (L.) Vill.) and bearded wheatgrass (Elymus caninus (L.) L.) on the territory of S.N. Skadovsky Zvenigorod Biological Station (Moscow oblast). Endophytes were isolated from infected seeds of both grass species. All isolates were identified as Epichloë festucae Leuchtm., Schardl & M.R. Siegel.

We studied the possibility of using a broadly neutralizing anti-influenza A antibody as a module for the development of different protein constructs for diagnostics. For this purpose, we constructed two recombinant proteins—a Fab-fragment of the antibody and Fab-mCherry, which is a hybrid of the Fab-fragment and the mCherry fluorescent protein. Both proteins were expressed in Escherichia coli cells and purified in a functionally active state from culture medium. The antibody Fab-fragment was shown to bind all 11 tested strains of the influenza A H1N1 and H3N2 subtypes. A stronger binding was observed for group I hemagglutinins; this correlates with the immunochemical profile of the parental antibody. Comparison of the dissociation constants of complexes of the antibody Fab-fragment and Fab-mCherry with A(H1N1)/Solomon Islands/03/06 virus particles demonstrated that the attachment of the mCherry protein did not interfere with the antigen-binding properties of the antibody Fab-fragment.

An experimental setup for study of immobilized molecules and their complexes by fluorescence microscopy with sensitivity at a single fluorophore level was developed. The installation records fluorescence images of immobilized molecules in two spectral ranges simultaneously allowing analysis based on the Förster resonance energy transfer effect. The fluorescence excitation is caused by evanescent light wave formed by the total internal reflection effect. Registration of signal is conducted with a highly sensitive detection system that allows measurements with a temporal resolution of approximately 100 ms. Glass surface modification protocol was developed for immobilization of nucleosomes via high-affinity streptavidin-biotin interactions. To ensure immobilization, one of the ends of the fluorescently labeled nucleosomal DNA was biotinylated. The algorithm of image processing for analysis of structural rearrangements at a single nucleosome level was developed. Fluorescence microscopy of single immobilized molecules and their complexes allows the analysis of nucleosome structural dynamics during transcription, and its interactions with various nuclear proteins.

A method for fluorescently labeled DNA synthesis, which makes it possible to assemble mononucleosomes with 40 bp linkers, was developed. Cy3 and Cy5 labels were introduced into the linkers at distances of 10 bp before the first nucleotide and 15 bp after the last nucleotide of the nucleosome positioning DNA sequence, respectively. Without histone H1.5, f luorescence microscopy of single complexes revealed two equally probable states of nucleosomes. The states were different in the linker conformation: the open one with the energy transfer efficiency (E) between the labels of 0.06 and the closed one with E = 0.37, when the linkers are brought together. Binding of histone H1.5 with nucleosomes occurs in a range of nanomolar concentrations, and the complex formation rate is significantly higher as compared with its dissociation rate. The significant convergence of the DNA linkers (E = 0.73) is observed in these complexes together with the higher conformation uniformity in the region where the labels are localized. The developed nucleosomal constructs represent highly sensitive f luorescent sensors that can be used for the analysis of structural linker rearrangements. Also, in combination with microscopy of single complexes, they are suitable for studying the structure of complexes of nucleosomes with different chromatin architectural proteins.