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Ivanov Y.D.,RAS Institute of Chemistry | Pleshakova T.O.,RAS Institute of Chemistry | Malsagova K.A.,RAS Institute of Chemistry | Kaysheva A.L.,RAS Institute of Chemistry | And 7 more authors.
Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry

A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell. © 2015, Pleiades Publishing, Ltd. Source

Bukharina N.S.,RAS Institute of Chemistry | Ivanov Y.D.,RAS Institute of Chemistry | Pleshakova T.O.,RAS Institute of Chemistry | Frantsuzov P.A.,RAS Institute of Chemistry | And 4 more authors.
Biophysics (Russian Federation)

Change in temperature is one of the factors affecting the activity of enzymes. In this work, the thermal denaturation and aggregation of cytochrome P450 BM3 were studied by atomic force microscopy. The specific temperature transitions were studied by fluorescence analysis. In the low melting temperature range (10–33°C), a decrease in the fluorescence intensity of the aromatic residues was observed simultaneously with an increase in the fluorescence intensity of the flavin groups. The protein melting in this range indicated three narrow S-shaped cooperative transitions at 16, 22, and 29°C. Atomic force microscopy analysis in this temperature range showed that the BM3 molecules retained a globular shape as compact objects (heights, h < 7 nm; lateral dimensions, d < 50 nm), but the protein oligomeric state changed. The first two transitions were accompanied by a decrease in the degree of oligomerization and the third one by its increase. © 2015, Pleiades Publishing, Inc. Source

Starodubova E.S.,RAS Engelhardt Institute of Molecular Biology | Preobrazhenskaia O.V.,RAS Engelhardt Institute of Molecular Biology | Kuzmenko Y.V.,RAS Engelhardt Institute of Molecular Biology | Latanova A.A.,RAS Engelhardt Institute of Molecular Biology | And 2 more authors.
Molecular Biology

Rabies is an infectious disease among humans and animals that remains incurable, despite its longstanding research history. The only way to prevent the disease is prompt treatment, including vaccination as an obligatory component and administration of antirabies immunoglobulin as a supplement. Since the first antirabies vaccination performed in the 19th century, a large number of different rabies vaccines have been developed. Progress in molecular biology and biotechnology enabled the development of effective and safe technologies of vaccine production. Currently, new-generation vaccines are being developed based on recombinant rabies virus strains or on the production of an individual recombinant rabies antigen—glycoprotein (G protein), either as a component of nonpathogenic viruses, or in plants, or in the form of DNA vaccines. In this review, the main modern trends in the development of rabies vaccines have been discussed. © 2015, Pleiades Publishing, Inc. Source

Ivanov Y.D.,RAS Institute of Chemistry | Malsagova K.A.,RAS Institute of Chemistry | Izotov A.A.,RAS Institute of Chemistry | Pleshakova T.O.,RAS Institute of Chemistry | And 5 more authors.
Biochemistry and Biophysics Reports

Microwave radiation at 3.4-4.2GHz frequency of the cytochrome P450 CYP102 A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10-8 and 10-9m. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment. © 2016 The Authors. Source

Speranskaya A.S.,RAS Engelhardt Institute of Molecular Biology | Krinitsina A.A.,Moscow State University | Kudryavtseva A.V.,RAS Engelhardt Institute of Molecular Biology | Poltronieri P.,CNR Institute of Sciences of Food Production | And 6 more authors.

Background: The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism. Results: KPI-A genes from the genomes of Solanum tuberosum cv. Istrinskii and the wild species Solanum palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software. The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011 and found considerable inconsistence of their data concerning the number, location, and orientation of KPI genes of groups A and B. Conclusions: The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time. © 2012 Elsevier Masson SAS. All rights reserved. Source

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