R. S. Yamidanov

NMDA receptors as a possible component of store-operated Ca2+ entry in human T-lymphocytes

Elevation of intracellular Ca2+ in T-lymphocytes as a consequence of T cell antigen receptor activation triggers transcriptional programs resulting in effector cytokine secretion and immune response coordination. Increase of Ca2+ concentration in T-lymphocytes follows both the Ins(1,4,5)P3-dependent release from an intracellular store and subsequent influx from extracellular milieu. Flow cytometry and the fluorescent dye Fluo-4AM have been used to demonstrate that noncompetitive NMDA receptor antagonist (+)-MK801 inhibits Ca2+ influx in T cells induced by thapsigargin. Combination of thapsigargin and (+)-MK801 with following incubation does not affect Ca2+ mobilization from intracellular stores, while decreased Ca2+ entry was observed. Overall data indicate that the ion channel blocker (+)-MK801 is able to inhibit the Ca2+ influx and confirm our suggestion about involvement of NMDA receptor in the store-operated Ca2+ entry mechanisms in human T-lymphocytes. To identify the signal transduction pathways associated with NMDA receptors in mitogen-stimulated T-lymphocytes, the cells were incubated with (+)-MK801, then activity of key phosphorylated protein kinases of MAP-activated (pERK1/2, pSAPK/JNK, p-p38), Ca2+-dependent (pCaMKII), PI3/Akt-dependent (pGSK-3β), and PKC-activated (pPKCθ) pathways were detected. The data we obtained demonstrate that (+)-MK801 treatment leads to more prominent decrease in Ras-activated protein kinases pERK1/2 and Rac-activated proteins p-p38 and pSAPK/JNK, as compared to DAG-dependent pPKCθ and Ca2+-dependent pCaMKII. These results show that NMDA receptors are mainly involved in regulation of Ras/Rac-dependent signaling in T-lymphocytes.

cDNA macroarray analysis of gene expression changes in rat brain after a single administration of a 2-aminoadamantane derivative

The Atlas™ Rat cDNA Expression Array (BD Biosciences, United States) has been used to analyze changes in the expression of 588 genes in rat brain cells in response to a single administration of Ladasten, a 2-aminoadamantane derivative that has psychostimulating and anxiolytic effects. The analysis of hybridization on macroarrays, confirmed by the results of real-time quantitative RT-PCR, has demonstrated that Ladasten alters the expression of 12 genes in the rat brain. The GAT3 and CARBH genes are presumed to be pharmacologically important targets of Ladasten. The changes in their activity explain the mechanisms of the anxiolytic and mood-stabilizing effects of the drug. Ladasten has been shown to induce the genes whose products are involved in various signal pathways (APC, Rb, PKCIP, and PMCA), as well as the genes of cytoskeletal proteins (Tubα1 and actin), synaptic proteins (SynIA&IB and PLP), and enzymes (Gapdh and NSE). The proteins encoded by these genes are presumably involved in compensatory and/or neuroplastic adaptation to the effects of Ladasten.

Cytosine demethylation in the tyrosine hydroxylase gene promoter in hypothalamus cells of rat brain under the action of 2-aminoadamantane compound Ladasten

In our previous study of mechanisms of pharmacological activity of a derivative of 2-aminoadamantane, Ladasten, we have shown more than a twofold increase in the mRNA level of the tyrosine hydroxylase (TH) gene in the rat brain hypothalamus 1.5–2.0 h after a single administration of this substance. In the present study, we employed the bisulfate sequencing technique to detect changes of GpG islands methylation status in the TH gene 5′-flanking region (−650)−(+84) in hypothalamus cells of control and experimental rats after a single exposure to Ladasten. Ladasten was shown to increase frequency of cytosine demethylation in binding sites of some transcription factors or in neighboring motifs. It is suggested that the Ladasten-induced increase of TH gene transcriptional activity in rat hypothalamus is associated with cytosine demethylation in CpG islands in some regulatory gene elements.

Genes targets identification of 5-amino-exo-3-azatricyclo[5.2.1.02,6]decane-4-one in an in vivo model of arrhythmia

The molecular mechanisms of action of 5-amino-exo-3-azatricyclo[5.2.1.02,6]decane-4-one (P11), a compound possessing strong antiarrhythmic, nootropic, anti-inflammatory and analgesic activity, have been studied. Cardiac rhythm disturbances were modeled by administering the arrhythmogenic compound aconitin in a dose of 50 μg/kg to the animals from the control group. P-11 in a dose of 0.3 mg/kg was injected intravenously in the experimental group of animals 2 min before aconitin administration. P-11 target genes were identified using the Atlas™ Rat cDNA Expression macroarray (#7738-1, BD Biosciences, United States). Reproducible changes in the expression levels of 16 genes in the heart of rats treated with P-11 concommitantly to arrhythmia modeling in vivo were detected. The genes regulated by the substance coded for proteins of the extracellular matrix are (glypican 1, Gpc1; tissue inhibitor of metalloproteinase 2, 3, Timp2, Timp3), intracellular signaling proteins (rho GTPase activating protein 7, Dlc1; protein tyrosine phosphatase 4a1, Ptp4a1; phosphodiesterase 4D, PDE4D; PI3-kinase regulatory subunit alpha, PIK3R1; guanine nucleotide binding protein alpha 12, Gna12), proteins involved in glycolysis (phosphofructokinase 1, Pfk1), intercellular interactions (junction plakoglobin, Jup), and hemostasis (tissue plasminogen activator, Plat), membranebound pumps and transporters (solute carrier family 16, member 1, Slc16a1; ATPase, Na+/K+ transporting, Atp1a), and others (c-fos proto-oncogene, c-fos; telomerase protein component 1, tlp; Annexin 1, anxa1). Therefore, the data concerning the selective effect of P-11 on genes coding for proteins involved in arrhythmogenesis allow for considering this compound as a promising medication for pathogenetically oriented therapy of arrhythmias.

Mechanisms of Action of Ladasten: Activation of Gene Expression for Neurotrophins and Mitogen-Activated Kinases

We studied the effects of single treatment with ladasten (50 mg/kg) on the content of effector kinases of the mitogen-activated cascade (ERK1/ERK2), pERK1/ERK2 activity (Thr202/Tyr204), and expression of genes for neurotrophic factors BDND and NGF in the striatum, hypothalamus, and hippocampus of rats.

Preparation of fluorescent labeled nodule bacteria strains of wild legumes for their detection in vivo and in vitro

A series of expression vectors containing TurboGFP and TurboRFP genes of fluorescent proteins under the control of the T5 phage constitutive promoter was created for a vital staining of nodule bacteria. These vectors were either obtained using the broad host range pBBRI replicon for labeling of strains, where a marker gene was expressed from a transformed plasmid, or they were prepared using the pRL765 gfp plasmid for labeling of strains via the introduction of genes of fluorescent proteins into the bacterial chromosome. Transformation was shown to be the most convenient method of transfer of constructions into cells of nodule bacteria, as there exists the possibility of spontaneous plasmid mobilization and, consequently, its transition from cells of labeled strains into other soil bacteria if the mob locus is present in vectors needed for conjugation. Fluorescent labeled strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., and Agrobacterium sp. were prepared using the obtained vector constructions. The suitability of the obtained strains for both in vivo and in vitro experiments was demonstrated.

Proteomic Analysis and Identification of Ladasten Target Proteins in Rat Brain

Two-dimensional electrophoresis and mass spectrometry detection were used to evaluate the range of proteins in rat brain after single treatment with ladasten (50 mg/kg). We identified 13 proteins with various levels of expression.

Time Course of Histone Deacetylase 1 and Acetylated H3 and H4 Histones in the Brain of Rats Treated with Ladasten

We studied the effects of single intragastric administration of ladasten in a dose of 50 mg/kg on the time course of histone deacetylase 1 (HDAC1) and levels of acetylated histones H3 (Lys9) and H4 (Lys8) in the striatum, hippocampus, and hypothalamus. Ladasten reduced HDAC1 level in rat striatum and hippocampus and modifi ed H3acK9 and H4acK8 levels in various structures of rat brain.

Changes in CREB (SER133) Content and DNA-Binding Activity of Transcriptional Factors in Rat Brain Cells against the Background of Ladasten Exposure

We studied the effects of single administration of ladasten (50 mg/kg) on the level of transcriptional factor CREB (cyclic AMP response binding element protein) phosphorylated by Ser133 in rat striatum, hypothalamus, and hippocampus. Transcriptional factors with affected DNA-binding activity were identified in brain cells.