V. A. Vakhitov

Functional analysis of diploid wheat rRNA promoter by transient expression.

The rRNA gene promoter regions of three diploid wheats Triticum boeoticum Boiss, Triticum urartu Tum. ex Gandil, and Triticum monococcum L. have been sequenced and analyzed. It has been found that the rRNA promoter initiation regions of diploid wheats contain the sequences, which differ from the evolutionary conserved TATAGTAGG (+1 is underlined) motif of monocots. The transient expression assay in wheat protoplasts confirmed that cloned sequences are active promoters. Deletion analysis showed that the promoter sequence localized between -113 and +15 (relative to +1) is enough to direct RNA polymerase I-dependent transcription.

Search for (-)-cytisine derivatives as potential inhibitors of NF-κB and STAT1

Design and synthesis of new derivatives of (-)-cytisine with a wide spectrum of pharmacological activity, represents a potential therapeutic interest for development of drug candidates for neurodegenerative disorders, inflammatory diseases, and treatment of nicotine addiction. We used HEK293 cell line, transiently transfected with NF-κB and STAT1 luciferase reporter constructs, to select (-)-cytisine derivatives for their potency to modulate basal and induced NF-κB and STAT1 activity. Currently, NF-κB, STAT1 and components of their signaling pathways, are considered as attractive targets for pharmacological intervention, primarily in chronic inflammation, cancer, autoimmune, neurodegenerative and infectious diseases. Library of tested compounds included derivatives of (-)-cytisine with amino, amide, thionyl and carboxamide groups at the 3rd, 5th and 12th position in the original molecule, as well as other bimolecular derivatives. Our experimental results revealed compounds with moderate inducing, as well as inhibitory, effects on basal NF-κB and STAT1 activity (IC50 or EC50 values are mainly in the micromolar range). The structure-activity relationship analysis demonstrated that mode of activity (activation or inhibition of NF-κB and STAT1) is determined by the topology of substituents in (-)-cytisine molecule, whereas the nature of substitutions determines the severity of the effect (introduction of aromatic and adamantyl substitutions, as well as thionyl or ketone groups are of principal importance). Assessments of effects of (-)-cytisine derivatives on activity of NF-κB and STAT1, induced by specific agents (TNFα and IFNγ, respectively), revealed that certain compounds inhibited both basal and stimulated activity of NF-κB and STAT1, whereas other compounds showed a dual effect (increase in basal and decrease in stimulated NF-κB activity), in turn, several compounds increased both basal and induced activity of NF-κB and STAT1. In summary, obtained results indicate that one possible mechanism of biological action of (-)-cytisine derivatives lies is their ability to influence components of NF-κB and STAT1 signaling pathways.

Cellular test systems for the search of transcription factors activity modulators

Test systems for monitoring activities and the search for substances activating or inhibiting transcription factors as biological targets have been designed on the basis of luciferase constructs containing binding sites for transcription factors CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF1α. An assessment of the functional activity of reporter constructs has been carried out using their transient transfection into HEK293 cells followed by treatment with specific inducers. The functional activity of all reporter constructs was observed based on the increased luciferase expression. In order to evaluate the efficiency of the suggested test systems, aspirin was used. Incubation of cells transfected with the above-mentioned constructs treated with aspirin was accompanied by the suppression of NF-κB, HIF1α, GAS, VDR, and HSF binding activity. The findings revealed for NF-κB, NFAT, and STAT1 confirm the published data concerning the mechanisms of aspirin action. The detected effects of this drug on the HIF1α, GAS, VDR, and CREB activity have been demonstrated for the first time.

Synthesis and Cytotoxic Activity of Conjugates of (–)-Cytisine and Thermopsin Amine Derivatives with 1,3-Dimethyl-5-Formyluracil

New conjugates of the quinolizidine alkaloids (–)-cytisine and thermopsin were synthesized by alkylating their 9-amino derivatives with 1,3-dimethyl-5-formyluracil. The cytotoxic properties of the synthesized conjugates against cell lines HEK293, HepG2, and Jurkat were studied. Screening identified the lead compound 5-{[(3-benzyl-8-oxo-1,3,4,5,6,8-hexahydro-2H-1,5-methanopyrido[1,2-a][1,5]diazocin-9-yl)amino]methyl}-1,3-dimethylpyrimidine-2,4-(1H,3H)-dione, which suppressed metabolic activity of lymphoblastic leukemia cells with inhibitory concentration (IC50) 20.6 ± 2.1 μM (IC50 of reference compound 5-fluorouracil was 18.5 ± 3.3 μM).

NMDA Receptors Regulate Genes Responsible for Major Immune Functions of Mononuclears in Human Peripheral Blood

To determine the role of NMDA receptors in the functional regulation of immunocompetent cells, comparative assay was carried out for genes expressed in the mononuclears in peripheral blood of healthy persons under normal conditions and after blockade of these receptors. The genes, whose expression changed in response to blockade of NMDA receptors in mononuclears, encode the products involved in regulation of the major functions of immune cells, such as proliferation (IL4, VCAM1, and CDKN2A), apoptosis (BAX, MYC, CDKN2A, HSPB1, and CADD45A), activation (IL4R, IL4, VCAM1, and CDKN2A), and differentiation (IL4, VCAM1, and BAX).

Inhibition of IAA oxidase activity of wheat anionic peroxidase by chitooligosaccharides

The study demonstrated that chitooligosaccharides with a molecular weight of 5–10 kDa and a degree of acetylation of 65% exhibited an auxin-like effect in wheat plants and also played an important role in regulating the activity of polysaccharide (chitin)–specific anion isoenzymes of peroxidase oxidizing indole acetic acid. Changes in the kinetic parameters of the interaction of the wheat anionic chitin-specific peroxidase with pI ∼3.5 with chitin oligomers in the presence of indoleacetic acid were pH-depended and indicated that chitooligosaccharides significantly impair the ability of the enzyme for oxidation at pH levels of 4.2 and 6.0. It can be assumed that chitooligosaccharides not only induce protective plant systems but also increase the accumulation of auxin in plant tissues, thus adversely affecting a number of components of the plant protective system against pathogens.