We developed an original reproducible 3D-technology for preparation of single dormant microspheres consisting of 2000 somatic cells. The dynamics of microsphere assembly from mesenchymal and epithelial cells of retinal pigment epithelium was traced using time-lapse microscopy: formation of a loose aggregate over 24 h followed by its gradual consolidation and formation of a compact viable microsphere with a diameter of 100-150 μ by day 7. The cell number in the formed microspheres remains unchanged. Reactivation observed upon fusion of epithelial and/or mesenchymal microspheres results in the formation of a united compact microtissue. The fusion dynamics reproduces spherogenesis irrespective of the initial amount of co-cultured microspheres. Reactivation via two-step induced angiogenesis opens new prospects for production of vascularized microspheres and microtissues.

This work is devoted to application of laser Raman spectroscopy to improve the fidelity of molecular computing by DNA strands. The developed method provides determination of concentrations of specific nitrogenous bases not lower than 0.03 g l −1 and the accuracy of determining the total concentration of DNA in solutions 0.02-0.04 g l −1 by the Raman spectra of DNA solutions. The method allows us to control the temperature of DNA solutions in the process of molecular computing with an accuracy better than 0.2 °C. The obtained results provide required control of the parameters of DNA solutions to increase the speed and accuracy of solving the problems in the results of molecular computing.

Fraction analysis of homocysteine in biological fluids is important for the diagnosis and studies of cardiovascular, nervous, urological, and other diseases. Measurements of total, free, and reduced homocysteine by mass spectrometry with HPLC by means of its modification with N-ethylmaleimide are proposed. The linearity and detection threshold were 0.025-10 and 0.001 μM, respectively, for reduced homocysteine fraction and 0.2-100 and 0.1 μM, respectively, for the rest fractions. The accuracy and reproducibility of the method were within 12%.

Reversible cerebral ischemia of medium severity was reproduced in male Wistar rats by bilateral occlusion of the common carotid arteries. Solid-phase structures (anisomorphons) were obtained by marginal dehydration of the serum. Small focal isotropic defects in the serum anisomorphon texture were found in 100% cases during occlusion of the carotid arteries. Similar signs were detected in all patients with chronic cerebral ischemia, which proved specificity of this morphological marker of the disease.

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We studied the effects of dihydroquercetin (3.3.4.5.7-pentahydroxyflavanone, a new Russian patented preparation) on functional activity of polymorphonuclear neutrophils from patients with non-insulin-dependent diabetes mellitus. Flavonoids (quercetin and its derivative dihydroquercetin) dose-dependently suppressed generation of anion radicals and hypochlorous acid and production of malonic dialdehyde during oxidation of neutrophil membranes. Dihydroquercetin decreased activities of protein kinase C and myeloperoxidase in activated polymorphonuclear neutrophils and could bind transition metals (Fe2+). These properties determine the ability of dihydroquercetin to decrease in vitro functional activity of polymorphonuclear neutrophils from patients with non-insulin-dependent diabetes mellitus.

The effects of diquertin on the content of cyclic nucleotides in human platelets was studied. Diquertin in a concentration of 5 mM increased the content of cAMP and cGMP in native and thrombin-activated platelets probably due to inhibition of phosphodiesterase. Increasing the concentration of diquertin above 5 mM did not potentiate this effect. The antiaggregation effect of diquertin was probably associated with the increase in platelet levels of cyclic nucleotides. Key Words: flavonoids; diquertin; platelet; cyclic nucleotidesFlavonoids are natural plant compounds [8]. Diquertin or dihydroquercetin (DQC,3,3',4',5,) is a new preparation (Russia) obtained from shredded larch wood ofLarix dahurica T. and Larix sibirica L. [1]. Flavonoids inhibit enzymes involved in cell activation (protein kinase C, tyrosine kinase, and phospholipase A2). Some flavonoids are strong inhibitors of cyclic nucleotide phosphodiesterase [2,7]. The most potent phosphodiesterase inhibitors display an antiaggregation effect on human platelets in vitro.Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers suppressing platelet activation. Cyclic nucleotides inhibit phospholipase C [5], reduce platelet content of cytoplasmic Ca 2+ [4], and activate Ca 2+-ATPase [6]. cAMP reduces platelet content of cytoplasmic Ca 2+ enhancing its intracellular deposition and effiux from cells [4,6]. cGMP inhibits agonist-induced Ca 2+ mobilization from intracellular stores and Ca 2 § entry, attenuates phospholipase C activation, and modulates protein phosphorylation [3]. Department of General Pathology and Pathophysiology, Russian Academy of Postgraduate Education, MoscowTaking into account the role of cyclic nucleotides in platelet activation, we studied the effect of DQC on the content of cAMP and cGMP in human platelets. MATERIALS AND METHODSVenous blood from 20 fasting donors (12 women and 8 men) was collected into tubes with 3.8% sodium citrate (9:1 ratio). Platelets were isolated by centrifugation at 190g. DQC dissolved in DMSO to final concentrations of l, 5, and 10 mM was added to the platelet suspension and incubated at 37~ and constant agitation for 2 min. Absolute ethanol and HCI were added to the suspension, mixed, and stored at -20~ To analyse the effect of DQC on cAMP and cGMP contents in activated platelets, the suspension of native platelets was incubated with various concentrations of DQC at 37~ and constant agitation for 2 min, thrombin (0.5 U/ml) was added, and platelet aggregation was recorded for 5 min. The reaction was stopped with absolute ethanol and HC1, The samples were mixed and stored at -20~ Platelet contents of cAMP and cGMP were estimated in supematants after ethanol extraction using enzyme immunoassay test systems for quantitative analysis of cAMP and cGMP (Bioimmunogen, Russia).

Methylation of CpG-islands in promoter regions as well as interaction of miRNAs with messenger RNAs of target genes are related to multilayer mechanisms regulating gene expression. The goal of this study was to assess a possibility for miRNA gene methylation to influence indirectly activation of their target genes in lung tumors. By using a unified collection of samples of non-small cell lung cancer, it was demonstrated that elevated levels of mRNA for RHOA and NKIRAS1 genes were significantly (Spearman rank correlation, P < 10(-11)) associated both with loss of methylation in their CpG-islands and methylation in a number of miRNA genes, which, according to the miRWalk database, were predicted to possess regulatory functions. Novel potential regulatory miRNAs for RHOA (miR-9-1/-3, -34b/c, -129-2, -125b-1, -375, -1258) and NKIRAS1 (miR-34b/c, -129-2, -125b-1, -193a, -124a-1/-2/-3, -212, -132) genes in lung cancer were identified.