[The role of glutamate in the pathogenesis of multiple sclerosis]. / Rol' glutamata v patogeneze rasseiannogo skleroza.

The results of recent studies indicate that the hyperactivation of the glutamatergic system plays an important role in the pathophysiology of multiple sclerosis (MS). In addition to the well-known direct toxic effect of the excessive extracellular level of the glutamate neurotransmitter on neurons, additional mechanisms of glutamate-induced cell damage have been described in the literature, including effects on oligodendrocytes, astrocytes, endothelial cells and immune cells. The study of these toxic effects will reveal the possible link between various pathological hallmarks of MS, such as axonal damage, oligodendrocyte death, demyelination, neurodegeneration, autoimmune reactions and dysfunction of blood-brain barrier. Understanding the mechanisms underlying the glutamate toxicity will contribute to the development of new therapeutic approaches for the diagnosis and treatment of patients with MS. This review focuses on the mechanisms that lead to an increase in the concentration of neurotransmitter glutamate and excitotoxicity in the context of the pathogenesis of this disease. Also the authors present the data on existing and currently developed medicines and therapeutic approaches to regulate the activity of the glutamatergic system.

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 5­10 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.

[Search of (-)-Cytisine Derivatives as Potential Inhibitors of NF-κB and STAT1].

Design and synthesis ofnew derivatives of (-)-cytisine with a wide spectrum of pharmacological activity, represents the 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 N F-κB and STATI luciferase reporter constructs to screen the (-)-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. The library of compounds included the derivatives of (-)-cytisine with amino-, amide-, thio- and carboxamide groups at 3, 5 and 12 position of the starting molecule, as well as some bimolecular derivatives. Our experimental data revealed compounds with moderate activating as well as inhibitory effects for basal NF-κB and STATI activity (IC50 or EC50 values are mainly in the micromolar range). The structure-activity relationship analysis demonstrated that the character of activity (activation or inhibition of NFκ-B and STAT1) is determined by the topology of the substituents at the (-)-cytisine molecule, whereas the nature of the substituents mainly contributes to severity of the effect (introduction of aromatic and adamantyl substituents, as well as thionyl or keto groups are of the principal importance). When evaluating the effect of (-)-cytisine derivatives on activity of NF-κB and STATI, induced by specific agents (TNFα and IFNγ, respectively) we observed that some compounds inhibited basal and stimulated activity of NF-κB and STAT1, another compounds showed the dual effect (an increase of basal- and a decrease of stimulated NF-κB activity) and several compounds increase both basal and induced activity of NF-κB and STAT1. Thus, obtained results suggest that one of the possible mechanisms of biological action of (-)-cytisine derivatives is their ability to influence the components of NF-κB and STAT1-dependent signaling pathways.

[DNA amplification using PCR with abutting primers].

DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.

[Cellular test systems for the search for transcription factor activity modulators].

Test systems for monitoring activities and the search for substances activating or inhibiting transcription factors as biotargets have been designed on the basis of luciferase constructs containing binding sites for transcription factors CREB, NFAT, NF-kB, p53, STAT1, GAS, VDR, HSF1, and HIF1alpha. 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-kB, HIF1alpha, GAS, VDR, and HSF binding activity. The findings revealed for NF-kB, NFAT, and STAT1 confirm the published data concerning the mechanisms of aspirin action. The detected effects of this drug on the HIF1alpha, GAS, VDR, and CREB activity have been demonstrated for the first time.

[Morphological features of transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the Dahlia mosaic virus promoter].

Transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the 35S promoter and the promoter of dahlia mosaic virus were obtained. The transgenic plants were characterized by increase in the length of the leaves, flower sizes, stem height, and weight of seeds; at the same time, the degree of increase was greater in the case of use of the dahlia mosaic virus promoter as a regulator of transcription. Ectopic expression of the AINTEGUMENTA gene promoted prolongation of leaf growth, while sizes of epidermal cells of the leaves remained unchanged.

NMDA receptors as a possible component of store-operated Ca²âº entry in human T-lymphocytes.

Elevation of intracellular Ca²âº 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 Ca²âº concentration in T-lymphocytes follows both the Ins(1,4,5)P(3)-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 Ca²âº influx in T cells induced by thapsigargin. Combination of thapsigargin and (+)-MK801 with following incubation does not affect Ca²âº mobilization from intracellular stores, while decreased Ca²âº entry was observed. Overall data indicate that the ion channel blocker (+)-MK801 is able to inhibit the Ca²âº influx and confirm our suggestion about involvement of NMDA receptor in the store-operated Ca²âº 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), Ca²âº-dependent (pCaMKII), PI3/Akt-dependent (pGSK-3ß), 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 Ca²âº-dependent pCaMKII. These results show that NMDA receptors are mainly involved in regulation of Ras/Rac-dependent signaling in T-lymphocytes.

[Metabolic changes in wheat (Triticum aestivum L.) plants under action of bioregulator stifun].

Under action of growth-stimulating concentrations of bioregulator stifun on wheat plants, an increase of functional activity of nucleoli of meristematic cells; contents of lectin (wheat germ agglutinin); and activity of proteinases, tripsin inhibitors, and ATPase activity was established. The pool of free amino acids was increased under bioregulator use. Levels of methionine, phenylalanine, cysteine, lysine, and tyrosine were increased. It is likely that stifun could activate protein biosynthesis in wheat plants.

[New approaches to the real-time detection of nucleotide mismatches by means of chimeric hybridization probes].

New approaches to the detection of impaired nucleotides based on the allele specific ligation of a "C probe" followed by rolling circle amplification have been developed. The detection of amplification products was realized by using enzymatic and deoxyribozyme digestion of fluorescently-labeled DNA-RNA-DNA chimeric oligonucleotide structures in cycling probe technology (CPT) in real-time mode.

[Immunogenicity of N-protein of Puumala hantavirus for noninbred mice in the intramuscular administration of its gene DNA].

The outbreaks of hantavirus infections in some regions of the Russian Federation in some years involve considerable material and social losses. In this connection, the designing of the most effective types of vaccines is an urgent task. The authors have created plasmid constructions containing the gene of hemorrhagic fever with renal syndrome virus, with whose Intramuscular injection there is a specific immune response and plasmid DNA is detectable in the adjacent tissues within a month after injection.

[The symbiosis between oriental goat's rue and the root nodule bacteria Rhizobium galegae: specificity and competitiveness].

Competitiveness and genetic variation of the Rhizobium galegae strains from the collection of the All-Russia Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, causing nodulation of oriental goat's rue under conditions of Bashkortostan soils (lacking this rhizobial species) were studied. It was demonstrated that of all the tested strains, the strains CIAM 0702 and CIAM 0704, each carrying two megaplasmids of 1500 and 2000 MDa, were the most competitive. RAPD (random amplified polymorphic DNA) analysis showed that R. galegae strains were able to intensively exchange the genetic material in the host plant rhizosphere. We did not succeed in detecting the local root nodule bacteria that were either initially able to infect oriental goat's rue or had adapted to infecting this species due to various genetic rearrangements.

[Correlation between the effect of chemotherapy on complement conformation and on CA-125 antigen in the blood plasma of patients with ovarian carcinoma].

Correlation was investigated between blood-plasma levels of C3(H2)O (conformation pattern of C3 component of the complement) and tumor-associated marker CA-125 in patients with ovarian cancer before and after chemotherapy. Since a drop in CA-125 level after chemotherapy was associated with similar changes in C3(H2)O fraction, it seems reasonable to suggest that change in conformation of C3 is a response of the immune system to cancer.

[DNA macroarray analysis of gene expression changes in rat brain after single administration of 2-aminoadamantane compound].

We have used the Rat Atlas cDNA Array ("BD Bioscience") to assess changes in mRNA expression of 588 genes in rat brain after acute treatment of 2-aminoadamantane compound--Ladasten. Drug exhibits the psychostimulating and anxyolitic actions. The analysis of results of hybridization on macrochips and their corroboration by quantitative real-time RT-PCR has allowed to reveal 12 genes, expression of which changes in response to ladasten in rat brain cells. The GAT3 and CARBH genes should be considered as primary pharmacologically significant targets and the changes of their functional conditions allows to explain the distinct mechanisms of anxyolitic properties of the drug. It was shown that Ladasten induced genes are involved in the different signalling pathways (APC, Rb, PKCIP, PMCA), genes encoding the cytosceletal proteins (Tubal, actin), synaptic proteins (Syn IA&IB, PLP) and metabolism enzymes (Gapdh, NSE). It is possible to assume, that proteins, encoded by the given genes participate in the compensatory and/or neuroplastic adaptation to biochemical effects of Ladasten.

[Contribution of lectin sugar-binding peptides structure determines specificity of rhizobium-legume symbiosis in Galega orientalis and G. officinalis].

A significant heterogeneity between bacteria Rhizobium galegae bv. officinalis and R. galegae bv. orientalis forming the nitrogen-fixing symbiosis with Galega officinalis and G. orientalis, respectively, and not forming any single cross-inoculation group, was found by means of RAPD and RFEL methods. The high level of sequence similiraty between lectins of these plants indicates at their close relationship. However the sequences of lectin sugar binding peptides (SBP) of G. orientalis (TYCNPGWDPRDR) and G. officinalis (TFYNEEWDLVIKDEH) were highly diverged. Amino acids of SBP which are involved in linkage of Ca2+ and Mn2+ ions responsible for stabilization of spatial structure of carbohydrate-binding "pocket", keep their position in peptide. It suggests that lectins participate in Rhizobium-legume symbiosis and that carbohydrate-binding site plays a key role in this process.