Application of imaging technique for characterization of ionotropic glutamate receptor ligands in cultured neurons

This article presents a non-invasive microphotometrical method for identification and the quantitative analysis of the compounds interacting with glutamate NMDA, AMPA, and KA-receptors. The method is based on the registration of ligand-gated and voltage-gated calcium channels activity using ratiometric fluorescent probe Fura-2. We have found conditions when repeated short-term (up to 30 s) applications of NMDA-, AMPA- and kainate-receptor agonists induce in most cells a calcium response almost identical to the first one. In the experiment we used the cell reaction to the first ligand application as a control, which significantly simplified the process of ligand titration. The optimal culture age was determined to enhance sensitivity of the method for each ligand. Certain rules of signal normalization were established to compare the results of different experiments and to obtain more precise values of activation and inhibition constants. It has been shown that the variability of calcium responses of individual neurons to the glutamate receptor agonists significantly depends on the degree and kinetics of their desensitization. The AMPA and KA-receptor ligands were characterized in the experiments performed in the presence of desensitization inhibitors cyclothiazide and concanavalin A, respectively. The inhibitors increased the amplitude of the response and converted the transient responses into the step ones. The method permits the detection of receptor ligands under study in an unknown sample, as well as determination of the activation and inhibition constants and the type of the inhibition. The method combines, high sensitivity, specificity and performance due to the possibility of analyzing individual activities of hundreds of cells simultaneously. The method can be used to obtain both the averaged response for all of the studied neurons and the responses of the selected cell populations or single neurons.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

New gall midge taxa of the subfamilies porricondylinae and lasiopterinae (Diptera,Cecidomyiidae) from the Rovno amber

Five new gall midge taxa of the subfamilies Porricondylinae and Lasiopterinae are described from the Late Eocene Rovno amber: Adsumyia integra gen. et sp. nov. (Dicerurini), Gratomyia inexigentis gen. et sp. nov. (Holoneurini), Winnertzia recusata sp. nov. (Winnertziini), Kovaleviola injusta gen. et sp. nov., and Spungisiola insuperabilis gen. et sp. nov. (Brachineurini).     

Genetic control of chromosomal synapsis in Secale cereale L. rye meiosis: sy19 gene, causing heterologous synapsis

Analysis of manifestation and inheritance of a new mutation inducing irregular synapsis in rye showed that abnormal phenotype is determined by a recessive allele of the sy19 gene. In the homozygotes for this mutation, even at the light microscopic level, abnormal formation of bivalents is already observed at pachytene-diakinesis. At metaphase I, the univalent frequency varies from 0 to 14; in a few cells, multivalent associations of chromosomes, which are not clearly oriented in the spindle, are detected. Electron microscopy of synaptonemal complexes revealed both homologous and heterologous synapsis in homozygotes for sy19, namely partial loss of the ability to stringent homology search. Analysis of joint inheritance of sy19 and asynaptic sy1 mutations showed that they are nonallelic, inherited independently, and interact by recessive epistasis. The phenotype of double sy1sy19 mutants indicates that the sy19 gene conditioning heterologous synapsis operates at meiosis later than the synaptic gene sy1. The epistatic group of mutations, sy9 > sy1 > sy19 and sy3, was determined.     

The use of oxidant-antioxidant system indices in forming groups at high genetic risk

While studying the pathogenetic mechanisms of Down syndrome, the imbalance of oxidant-antioxidant system in donors of extrachromosome 21 was detected. This fact was a basis for screening of new prenatal biochemical markers of Down syndrome. The ratio of free radical processes intensity and SOD activity was found as most informative index reflecting the state of oxidant-antioxidant system. It was proposed as additional criterion for the formation of high risk groups to increase the efficiency of prenatal diagnostics of Down syndrome.     

Development of HIV infection epidemic among intravenous drug users in the Sverdlovsky region

The Sverdlovsky region takes the fourth place among the 89 subjects of the Russian Federation by the number of registered cases of HIV infection. Drug addiction has reached an excessive scale in this region: according to the data of express evaluation carried out by the Regional Narcological Hospital and the Regional AIDS Center, not less than 7-8% of persons aged 15-30 years constantly use injection drugs. The large-scale epidemic of injection drug addiction has led to the rapid spread of HIV among addicts. The first penetration of HIV into this population took place in 1996 and, starting from the year 2000, rapid, development of the epidemic began, taking the character of an avalanche. The peak of new cases of HIV infection fell on 2001 (9,230 cases were registered). The concentrated stage of the epidemic development is observed in the region at present, the prevalence of HIV infection among drug addicts being 13.8%. Children born from HIV-infected drug addicted mothers now represent a new risk group due to great difficulties in the prophylaxis of the vertical virus transmission.     

Behavioral effects of L-tryptophan and p-chlorophenylalanine after adrenalectomy or dexamethasone administration in rats

Effects of acute administration of L-tryptophan (L-TRP. 250.0 mg/kg, i.p.) on active avoidance conditioning and "open-field" behavior were studied in male rats after adrenalectomy of dexamethasone administration. L-TRP inhibited the acquisition and reproduction of active avoidance reaction in adrenalectomized and dexamethasone-treated rats. Moreover, L-TRP decreased horizontal locomotor activity and grooming behavior in the "open field" on adrenalectomized rats. On the contrary, p-CPA restored the active avoidance conditioning in adrenalectomized rats and rats with excess of glucocorticoids. Also, p-CPA increased the total locomotor activity and grooming behavior in the "open field" in adrenalectomized rats, but decreased horizontal locomotor activity and enhanced emotional reaction in dexamethasone-treated rats in the "open field".     

The Expression of Mutation sy2 Causing Nonhomologous Synapsis in Meiosis of Diploid Rye Secale cereale L.

The cytological expression of spontaneous mutation sy2 isolated from a population of weedy rye was examined. It was demonstrated that the primary defect of meiosis in the mutant plants is nonhomologous synapsis, which occurs simultaneously with the homologous one. An electron microscope study of the synaptonemal complex (SC) at prophase I showed synaptic abnormalities that were manifested as switches of synapting axial elements to the nonhomologous partner and the formation of foldbacks of lateral SC elements. The sy2 mutants are characterized by one to two such events per meiocyte. Nonhomologous synapsis leads to the appearance of univalents at metaphase I (on average 4.16 ± 0.002 per meiocyte) and multivalents (on average 0.12 ± 0.007 per meiocyte). The presence of multivalents in 12% of meiocytes at metaphase I may result from recombination in ectopic regions of homology. It is suggested that the sy2 mutation impairs a component of the system that limits synapsis in meiocytes to only homologous chromosome pairs.     

Relationships between the gall-midge genera <Emphasis Type="Italic">Trotteria</Emphasis> and <Emphasis Type="Italic">Verbasciola</Emphasis> (Diptera,Cecidomyiidae), with description of new species from the Middle Volga River Area

Gall midges of the genera Trotteria and Verbasciola of the supertribe Lasiopteridi are inquilines. The widespread genus Trotteria includes 28 species developing in galls of 30 species of gall midges; 24 species develop in galls of Asphondyliidi. Gall midges-hosts and inquilines were found on 57 plant species of 17 families. The Palaearctic genus Verbasciola comprises three species that have been recorded in galls of three gall-midge species damaging three plant species. Seven new species are described, Trotteria coronillacola, T. perkovskii, T. cytisiphila, T. kalininae, T. jigulensis, Schizomyia samaralukensis, and Verbasciola volgensis. The diagnoses of the genera were corrected. Trotteria spinosa Kovalev is restored from the synonyms of T. umbelliferarum Kieffer. Data on the distribution of all the species examined and their associations with host gall midges and taxonomic groups of plants are given.