A mediator microbial biosensor for assaying general toxicity.

A mediator biosensor based on Paracoccus yeei bacteria for assaying the toxicity of perfumery and cosmetics samples was developed. An approach to selecting an electron-transport mediator based on the heterogeneous electron transfer constants for investigated mediators (ks) and the mediator-biomaterial interaction constants (kinteract) was proposed. Screening of nine compounds as potential mediators showed a ferrocene mediator immobilized in graphite paste to have the highest efficiency of electron transfer to the graphite-paste electrode (the heterogeneous transfer constant, 0.4 ±â€¯0.1 cm/s) and a high constant of interaction with P. yeei (0.023 ±â€¯0.001 dm3/(g·s)). A biosensor for toxicity assessment based on the ferrocene mediator and P. yeei bacteria was formed. The biosensor was tested on samples of four heavy metals (Cu2+, Zn2+, Pb2+, Cd2+) and two phenols (phenol and p-nitrophenol). Proceeding from the EC50 index, it was found that the use of the ferrocene mediator made the biosensor more sensitive to investigated toxicants than most analogues described. Toxicity determination of four perfumery and cosmetics samples by the developed biosensor showed prospects of using this system for real-time toxicity monitoring of samples.

Inhibition of Neutrophil Elastase and Cathepsin G As a New Approach to the Treatment of Psoriasis: From Fundamental Biology to Development of New Target-Specific Drugs.

Psoriasis therapy remains an extremely relevant area of modern drug design, due to necessity of adverse reaction reduction, inherent for actual methods of therapy. It was established that two serine proteases-neutrophil elastase 1 (HNE1) and cathepsin G (CatG)-are the key agents in psoriasis development. The collected molecular data for the presented targets form the basis for the molecular modeling strategy for the search for and identification of new target-specific inhibitors. The result of this work is a group of high-priority small-molecule compounds with double-targeted affinity, which are able to suppress the pro-psoriatic processes induced by the considered serine proteases at the initial stage of the disease.

An Integrated Method of Instrumental Microbiotesting of Environmental Safety of Various Products, Wastes, and Territories.

The described microbiotesting technique provides the recording of pH changes, redox potential, electrical conductivity, optical density, light scattering and luminescence intensities, and other parameters of samples with viable test microorganisms incubated in a liquid nutrient medium in the presence and absence of the tested factors. The results of using this system for the analysis of pro- and antibiotic activity of various oil products, as well as weak electromagnetic fields of the megahertz range, are presented. It has been shown that the proposed technique, compared to the standard methods, yields more detailed and objective information and is less time- and labor-consuming.

Optimization of Heterologous Expression of Insulin Receptor-Related Receptor Ectodomain.

In this paper, we present an approach to optimize the heterologous expression of the receptor tyrosine kinase IRR, which further simplifies the purification of the IRR from the medium and increases the final yield. The approach proposed by us can find application in the biotechnological production of other large-scale recombinant proteins produced for medical purposes.

Biotechnological Method of Preparation and Characterization of Recombinant Antimicrobial Peptide Avicin A from Enterococcus avium.

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.

New Method of Integrated Photofluorescence Microbiotesting.

A biotesting procedure that makes it possible to record the changes in the intensities of elastic light scattering, light absorption, and intrinsic photofluorescence of the protein component, as well as the determination of the concentration and structuring coefficients of the genomic component, of the samples with viable unicellular test organisms incubated in a liquid nutrient medium in the presence and absence of various external chemical factors, is described. The results of the analysis of the antibiotic activity of various metal cations with the use of this technique are presented. This technique is much more rapid, objective, and comprehensive for the assessment of the effect on the reproduction rate, metabolic activity, and genome structure of the test organisms from the samples of various products, wastes, etc. than standard visual microbiotesting methods.

Fluorescent DNA Probes: Study of Properties and Methods of Application.

The results of long-term author's studies of the optical and complex-forming properties of more than 30 synthetic low-molecular-weight fluorophores specific for DNA are described. These studies made it possible to significantly expand the already existing database of properties of such compounds, clarify the ideas about the patterns linking the mentioned properties of fluorophores with their structure, and formulate recommendations on designing new effective DNA-specific fluorophores. The results of these studies can be used, in particular, in the development of new rapid methods for diagnosing various diseases, biotesting of probiotic and antibiotic properties of various products and wastes, etc.

Development of target delivery system based on actinomycin class drugs and recombinant alpha-fetoprotein.

A recombinant alpha-fetoprotein (rAFP) was obtained in the yeast P. pastoris system, and its functional activity was confirmed. A method for producing polymer particles loaded with dactinomycin was developed, and a conjugate of these nanoparticles with rAFP was synthesized. The efficiency of the obtained conjugate on the HeLa, SKOV3, and MG-63 tumor cells and the absence of toxicity on the normal cells was shown. Experiments in vivo demonstrated a significant increase in the antitumor efficacy of the conjugate at a lower general toxicity as compared to the commercially available dactinomycin.

[Liposomal form of lipoic acid: preparation and determination of antiplatelet and antioxidant activity]. / Liposomal'naia forma lipoevoi kisloty: poluchenie i opredelenie antiagregatsionnoi i antioksidantnoi aktivnosti.

Optimal conditions for obtaining phosphatidylholine (PC) liposomes with lipoic acid (LA) are chosen that lead to the formation of nanoparticles with a size of 175¸284 nm with efficiency (extent) of inclusion of LA in liposomes equal 85% and characterized by a slow release of substance from the nanoparticles. The effect of "empty" liposomes and liposomal form of LA on platelet aggregation induced by arachidonic acid (AA) is established. It is found that liposomes with LА inhibit platelet aggregation, caused by AА, to 80%. In addition, it is shown that "empty" liposomes slightly (to 30%) suppress platelet aggregation, caused by AА. The amount of TBA-sensitive products in samples of platelet-rich plasma (PRP) incubated with liposomal LA is determined. It is shown that LA in the composition of liposomes retains its antioxidant properties, and the amount of products of lipid peroxidation in platelet-rich plasma decreases in a dose-dependent manner when arachidonic acid is used as an inductor of platelet aggregation. It is assumed that the antiplatelet action of the liposomal form of LА is induced by inhibition of the initiation of lipid peroxidation products caused by exogenous inducer AА. It is supposed that, after additional research, the liposomal form of LA can be considered as a new drug in complex treatment of cerebral ischemia.

Design of a stable cell line producing a recombinant monoclonal anti-TNFα antibody based on a CHO cell line.

Recombinant monoclonal antibodies (mAbs) against tumor necrosis factor alpha are widely used in the biopharmaceutical therapy of autoimmune diseases. Currently, a large number of drugs based on these antibodies are available. Accordingly, the development of these products for the Russian market is an important goal. The aim of the current study is to describe the development of one such technology. CHO-DG44-derived cell lines producing mAb were developed using two strategies, one based on individual clones and the other based on cell pools. To obtain recombinant cell lines with highly amplified genes of interest, the clones underwent dihydrofolate reductase-mediated gene amplification. Using the best strategy for the selection and amplification of mAb-producing clones, we achieved the production of more than 1 g/L in small scale, non-optimized conditions.

Antibacterial Effects of Liposomes Containing Phospholipid Cardiolipin and Fluoroquinolone Levofloxacin on Mycobacterium tuberculosis with Extensive Drug Resistance.

The effects of liposomes containing phospholipid cardiolipin without antibiotic and loaded with levofloxacin on the growth of Mycobacterium tuberculosis with extensive drug resistance were studied in vitro. Liposomes consisting of cardiolipin alone in a concentration of 335 µM completely suppressed the growth of M. tuberculosis. In order to reduce the minimum inhibitory concentration of cardiolipin, complex liposome preparation consisting of phosphatidylcholin/cholesterol/cardiolipin and loaded with levofloxacin was prepared. Due to this, the cardiolipin concentration was reduced to 33.5 µM (50 µg/ml) and concentration of levofloxacin - to 2 µg/ml.

Creation and study of triterpenoid nanoparticles and radioprotective substance genistein.

This work is devoted to the study and obtaining of new radioprotective agents based on natural flavonoid genistein and spherical amorphous nanoparticles (SANPs) produced from a mixture of birch bark triterpenoids. The physicochemical characteristics of the nanoparticles were studied by electron microscopy, dynamic light scattering, and UV-VIS spectroscopy. The radioprotective efficacy of the nanodrug in vivo and the possibility of its use as a radioprotective agent was shown.

[Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].

The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.

[Activity of the inositol-containing phospholipid dimer analogues against human immunodeficiency virus].

For the purpose of finding effective inhibitors of virus adsorption the series of inositol-containing phospholipid dimer analogues were previously synthesized. In the present work, the antiretroviral activity of these compounds against HIV-1 was demonstrated on the model of cells infected with the virus. The highest effect was found in the case of dimer poliol 5, EC50 (50%-effective concentration) was 3.9 microg/ml. The development of new polyanionic compounds, which can interfere with early steps of the virus life cycle, is a promising addition to the antiretroviral therapy based on the virus enzyme inhibitors.

[Microbiological synthesis of [2H]-inosine with a high degree of isotopic enrichment by the gram-positive chemoheterotrophic bacterium Bacillus subtilis].

A 2H-labeled purine ribonucleoside inosine was microbiologically synthesized (yield, 3.9 g/L of culture liquid) using a deuterium-adapted strain ofthe gram-positive chemoheterotrophic bacterium Bacillus subtilis, cultivated in a heavy water medium with a high degree of deuteration (99.8 at % 2H) containing 2% hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum as a source of 2H-labeled growth substrate produced in an M9 minimal medium with 98% 2H20 and 2% [2H]-methanol. The inosine extracted from the culture liquid of the producer strain was fractionated by adsorption (desorption) on an activated carbon surface, extraction with 0.3 M ammonium-formate buffer (pH 8.9), subsequent crystallization in 80% ethanol, and ion exchange chromatography on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium-formate buffer containing 0.045 M NH4Cl. Fast atom bombardment (FAB) mass spectrometry demonstrated incorporation of five deuterium atoms in the inosine molecule (62.5% 2H), three of which were contained in the ribose moiety and two in the hypoxanthine moiety.

[Nucleosides of 1,2,4-triazole: potentialities and restrictions of chemo-enzymatic method for synthesis].

The potentialities and restrictions of chemoenzymatic approach to the synthesis of new structural analogues of antiviral drug Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) have been determined. Syntheses of various amides of 1H-1,2,4-triazole-3-carboxylic acid and its 5-substituted analogues, prospective substrates of purine nucleoside phosphorylase (PNP), have been reported. The comparative effectiveness of the methods for obtaining amides aforementioned and also the methods for introducing functional groups to the C5 position of the heterocyclic system has been studied. New Ribavirin analogues bearing various substituents in the carboxamide group have been synthesized. The biotechnological method for the preparation of 1-beta-D-ribofuranosyl- 1,2,4-triazole-3-carbonitrile used as the intermediate in the synthesis of Viramidine, a contemporary Ribavirin analogue, has been developed.

[The functional analysis of polymorphic insertions of Alu retroelements at acute lymphoblastic leukemia].

Human genome variability observed within patient cohorts is considered as a goal of functional genomics essential for personalized medicine progress. In the current research we implement functional analysis of 31 polymorphic Alu insertions located within gene introns for individual genomes of patients with acute lymphoblastic leukemia (ALL). As a result we demonstrated a decrease of the primary transcripts content for 21 Alu-containing alleles. The most strong inhibitory effect of 10 Alu insertions was observed in both mononuclear blood cells of healthy donors and B-lymphoblasts of ALL patients. Allele frequencies of three Alu insertions that are located in MEF2C (two of them) and TAX1BP1 genes significantly differ (p-value 0.027. 0.052. 0.014 accordingly) between cohorts of healthy donors and ALL patients. Prolong influence of the Alu insertions on intracellular content of mature mRNA was studied for corresponding allele of TARBP1 gene.

[Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei].

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C12-C18 fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the zeta-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.

[Erythrocyte lipid composition at different stages of type 1 diabetes in children].

Complete profiles of phospholipid and ceramide molecular species from erythrocyte lipid extracts of children without carbohydrate metabolism disorders, and children with type 1 diabetes were compared by means of high performance liquid chromatography/mass spectrometry. For the first time a statistically significant increase (p<0.05) of lysophosphatidylcholine content in two groups of diabetic children with different duration of the disease (less than one year and more than one year) was found. Statistically significant changes in other lipid classes were not observed. The dependence of the content of phosphatidylcholine and phosphatidylethanolamine molecular species containing arachidonic acid residue (20:4) on the duration of the disease was found. The observed shift in lipid metabolism suggests of phospholipase A2 and chronic inflammatory process at different stages of diabetes mellitus, in cells (erythrocytes), which aer not involved in the immune response.

Protective effect of L-Arginine administration on proteins of unloaded m. soleus.

Cytoskeletal and contractile proteins degenerate during functional unloading of muscle. The ratio of myosin heavy chain (MHC) expression changes simultaneously. We have supposed that NO can be a signal molecule related to the regulation of protein metabolism upon muscle unloading. To test this hypothesis, Wistar rats underwent functional unloading for 14 days without and with peroral administration of L-arginine (500 mg/kg) as NO precursor. Significant decreases in m. soleus mass, NO, nNOS, dystrophin, Hsp90, p-S6K, and type I MHC mRNA contents were found in the group of animals with unloading without preparation compared to those in control and in the group with unloading and administration of L-arginine; at the same time, increased contents of atrogin-1/MAFbx and MuRF-1 (p < 0.05) were found. No difference in the IGF-1 mRNA content between all three groups was found. Atrophy was significantly less pronounced in the group with unloading and L-arginine administration compared to that without the amino acid, and no destruction of cytoskeletal proteins was observed. We conclude that administration of L-arginine upon functional unloading decreases the extent of m. soleus atrophy, prevents the decrease in it of type I MHC mRNA, and blocks destructive changes in some cytoskeletal proteins. Such effect can be due to the absence of increase in this group of the content of some ubiquitin ligases and decreased intensity of the p70S6 kinase synthesis marker.