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Objectives. To develop an effective technology for the cultivation of Chinese hamster ovary (CHO) cells stably producing GamP2C5 antibody which is a component I of the GamCoviMab candidate drug for emergency prevention and therapy of infection caused by SARS-CoV-2 virus; to select optimal cultivation parameters and to scale this technology in production.Methods. The study was performed on CHO GamP2C5 (clone 78) cell culture, producing a single-domain antibody fused to the Fc fragment of human IgG1 GamP2C5. Different culture media and supplements were used. Cells were cultured in Erlenmeyer flasks, Biostat® RM 20 wave-mixed bioreactor, Ambr® 250 mini bioreactors, STR 200 stirred-tank bioreactor.Results. Using molecular-genetic and biotechnological methods, a stable clone producer of CHO GamP2C5 antibody, clone 78, was obtained. Then a technique was worked out for the cultivation of the obtained clone producer on different culture media. The most suitable cultivation regimes, culture media, and optimal supplements were selected. This technology was tested in laboratory conditions in a 10-L reactor, and then successfully scaled up for production at the MedGamal Branch of the Gamaleya National Research Center for Epidemiology and Microbiology.Conclusions. This study demonstrates the fundamental feasibility of developing and scaling up a culture technology, in order to produce a drug based on a modified single-domain antibody with virus neutralizing activity against different strains of SARS-CoV-2 virus.

Aim. In this study we aimed to develop the methodology to change the antigen specificity of chimeric antibodies by replacing the variable region genes in the previously designed universal plasmid constructions pLK DT-17 and pHG DT-17 encoding the DT-17 antibody against the diphtheria toxin (DT) to the genes of antibody binding to another DT epitope — DT-22. Materials and methods. The genes of the light and heavy chain variable regions of mouse anti-DT antibodies — DT-22 were amplified from the hybridoma producing monoclonal antibodies to DT by reverse transcription and PCR methods. Genetic engineering methods were used to replace the variable regions of DT-17 antibody in the recombinant plasmids pLK DT-17 and pHG DT-17 encoding the light and heavy chains of DT-17 antibody, respectively to the relevant genes of DT-22. Subsequently, a «supervector» pSV DT-22, containing the genes of both chains of the chimeric antibody, was designed. CHO cells were transfected with a «supervector» and a highly productive clone, secreting chimeric antibodies to DT was obtained. Immunochemical and cultural methods were used to evaluate antibody activity. The affinity chromatography was used to purified preparative amounts of antibodies. Results. The yield of purified secreted chimeric DT-22 antibodies was 4 mg from per liter of culture medium. The minimum concentration of chimeric antibodies at which DT was neutralized in the CHO cells was 22 μg/mL of medium. Conclusion. Thus it has been shown how to generate new vector coding synthesis of light and heavy chains of a chimeric DT-22 antibody specific to another DT epitope using previously constructed universal recombinant plasmids pLK DT-17 and pHG DT-17 encoding, light and heavy chains of antibodies against DT DT-17, respectively.

Previously obtained highly immunogenic Env-VLPs ensure overcoming the natural resistance of HIV-1 surface proteins associated with their low level of incorporation and inaccessibility of conserved epitopes to induce neutralizing antibodies. We also adopted this technology to modify Env trimers of the ZM53(T/F) strain to produce Env-VLPs by recombinant vaccinia viruses (rVVs). For VLP production, rVVs expressing Env, Gag-Pol (HIV-1/SIV), and the cowpox virus hr gene, which overcomes the restriction of vaccinia virus replication in CHO cells, were used. The CHO Lec1 engineered cell line lacking GlcNAc-TI was used for generating VLPs with Env proteins containing a cytoplasmic (CT) domain affecting the surface subunit (SU) conformation. This has created the opportunity to modulate the glycan composition, and refine the conditions for their production, and optimize approaches to overcoming HIV-1 resistance associated with abundant glycosylation.

Perfusion cultivation process for CHO cells producing recombinant B-domen deleted coagulation factor VIII was developed. Cell culture medium and feed supplement were optimized by screening of big panel of prototype media and supplements. Cultivation temperature was optimized for better expression of complex protein as well. Cultivation process was tested in a laboratory-scale perfusion bioreactor. Significant increase of productivity was shown as a result of the development.

Fully­human antibodies have a great therapeutic importance; however, the development of stable strains providing a high level of production of full­size antibodies is a challenging task, as antibody molecules contain two types of polypeptide chains. To develop the producing strain, random integration of the plasmid containing the gene encoding the target protein into the genome of the host cells is commonly used. The aim of this study was the development of an original expression system, using gene targeting to integrate the gene encoding the fully­human antibody into the transcriptionally active region of the genome of eukaryotic suspension cells CHO­S. To develop a stable strain, the cassette vector plasmid pCDNA5/FRTDHFR­CH­CL containing the site of homologous recombination and the genes encoding heavy and light chains of the fully human antibody of the IgG1/kappa class was constructed at the first step. Notably, DNA of the plasmid pCDNA5/FRT­DHFR­CH­CL was organized in such a way that the restriction sites for rapid cloning of DNA fragments encoding the variable domains of heavy and light chains were inserted upstream of the sequences encoding constant domains of the heavy and light chains of the antibody. Secondly, DNA fragments encoding the variable domains of the heavy and light chains of antibody against orthopoxvirus protein p35 were inserted into the pCDNA5/FRT­DHFRCH­CL cassette plasmid. Then, CHO­S/FRT cells, which contain the FRT­site for homologous recombination and are able to produce green fluorescence protein GFP, were transfected with the constructed plasmid. After the insertion of the target genes into the FRT­site, GFP production was supposed to stop. Using this selection system, a stable clone producing target antibody fh8E was selected with the level of production of about 100 μg/ml. The binding affinity of purified antibody fh8E with the targeted protein, measured by surface plasmon resonance, was 12 nM. In addition, antibody fh8E demonstrated anti­vaccinia virus activity in the plaque reduction neutralization test in vitro.

When the nasopharynx is colonized with toxigenic strains of the diphtheria pathogen, toxin is released, which contributes to the death of epithelial cells. But in bacterial carriers, the development of the clinical picture of the disease does not occur. This is due to the peculiarities of the state of their immune system, as well as the peculiarities of the production of diphtheria exotoxin by corynebacteria in the biofilm. Goal. Determining the nature of the cytopathic effect of C. diphtheriae as part of a biofilm in CHO-K1 cell culture. The planktonic and biofilm (120- and 720-hour) cultures of the strains were studied: C. diphtheriae gravis tox + № 665, C. diphtheriae gravis tox + № 6765, C. diphtheriae mitis tox + № 269, C. diphtheriae gravis tox + isolated from a patient with a diagnosis Localized oropharyngeal diphtheria C. diphtheriae gravis with a silent tox-gene. Biofilm (120- and 720-hour) cultures of diphtheria pathogen strains were obtained according to the Watnik method. The cytopathic effect of corynebacterial strains was studied on a CHO-K1 cell culture, taking into account in an inverted microscope. When studying the cytopathic effect of planktonic cultures of toxigenic strains of corynebacteria, it was found that the number of living CHO-K1 cells after 24 hours was insignificant (25.3±1.2%) and sharply decreased (2.5±0.5%) after 72 hours of cultivation. Under the influence of biofilm and, especially, 720-hour cultures, a different cytopathic effect dynamics was found: the number of living cells after 24 hours remained significant (82.5±2.2%), while at 72-hour it decreased to 25.0±3.0%. In the study of filtrates of planktonic and biofilm cultures of C. diphtheriae strain with a «silent» tox-gene, similar patterns were revealed. However, the number of live CHO-K1 cells when exposed to the filtrate of a 720-hour biofilm culture was significantly higher (p≤0.05) than when studying toxigenic strains of corynebacteria. Considering the nature of the cytopathic action, it was found that planktonic cultures of toxigenic strains of corynebacteria are characterized by a change in the cell monolayer, manifested by their thinning and elongation. The study of 720-hour biofilm cultures at 72-hour exposure revealed the appearance of a large number of rounded cells (63-69%). The cytopathic effect, formed under the influence of filtrates of planktonic and biofilm cultures of C. diphtheriae with a «silent» tox-gene, as well as strains of non-diphtheria corynebacteria, is characterized by rounding of cells and the formation of symplasts. In the biofilm, the intensity of the cytopathic effect of toxigenic C. diphtheriae strains and C. diphtheriae strain with a silent tox-gene decreased. CPD, manifested by thinning and lengthening of CHO-K1 cells, is associated with the action of diphtheria exotoxin, and rounding is associated with corynebacterial enzymes and, apparently, fragments of surface structures - adhesins. Decreased release of toxin and enzymes beyond the C. bihfilm matrix is a significant cause of the «asymptomatic» carriage of diphtheria., Competing Interests: The authors declare no conflict of interest.

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

We have generated and characterized HER2-specific targeted toxin based on the low-immunogenic variant of Pseudomonas exotoxin A (LoPE), in which most of the human immunodominant B-cell epitopes have been inactivated. Nonimmunoglobulin DARPin-based HER2-specific protein was used as a targeting module for toxin delivery to the cellular target. Using confocal microscopy, it has been found that both domains in this hybrid toxin retained their functionality, i.e., the specific interaction with HER2 receptor, as well as the internalization and effective transport to ER typical of the wild-type Pseudomonas exotoxin A. The HER2-dependent cytotoxic effect correlated with receptor expression level at the cell surface, as shown in vitro using cell lines with different levels of HER2 expression. Due to the very high selective cytotoxicity against HER2-positive human tumor cells, as well as expected low immunogenicity, we believe that this new targeted toxin may be promising for future in vivo studies as a therapeutic agent for HER2-positive tumors.

Introns can frequently enhance transgene expression, and sometimes they are absolutely substantial. Based on an analysis of murine genes, in which mRNA does not have alternative splicing, a universal design of the efficiently spliced artificial introns of small sizes has been proposed. These introns are shown to be efficiently spliced in CHO cells from hamster ovaries. The proposed strategy can be used to include introns in cDNA, which would elevate the production of recombinant proteins in cell culture, as well as in transgenic animals.

The HER2/neu receptor is over-expressed on the surface of many types of cancer cells, and is widely used for tar- geted delivery of anticancer drugs. We have created geneti- cally engineered construct expressing the PE40 fragment of Pseudomonas toxin bound with the DARPin molecule which recognizes the HER2/neu receptor with high specificity. The construct destroyed transfected tumor cells in vitro. Intra-tumor injections of the construct complexed with polyethyleneimine led to growth retardation of D2F2/E2 tumors in mice. These results suggest a possibility of using this approach to develop new anticancer drugs.

Previously, we have shown that ginkgolic acid has an ability to potentiate currents, mediated by αl subunits of glycine receptor. In order to define the mechanism of subunit specific action of ginkgolic acid we have performed comparative analysis of the amino acid sequences of at and α2 subunits of glycine receptor. Amino acids that contribute to the different action of ginkgolic acid on glycine receptors formed by these subunits were determined. Using whole-cell configuration of patch-clamp recording, we have demonstrated that mutation of three residues in α2 subunit for corresponding ones from αl subunit makes α2 receptors sensitive to the potentiation by ginkgolic acid. Currents, mediated by α2 mutant receptors, increased by 89±14% after application of ginkgolic acid. Similarly to ai receptors α2 mutant receptors have shown a decrease in EC₅₀. for glycine under the action of ginkgolic acid. Thus, subunit selectivity of the ginkgolic acid is in strong connection with three amino acid residues that are different in α1 And α2 subunits of glycine receptor.

The karyotypes of CHO(dhfr-) and CHO(dhfr-)/susp Chinese hamster ovary cell lines were investigated with the use of GTG-staining. Modal chromosome set consists of 20 and 18 chromosomes respectively. The karyotypes of both cell lines were stable with constant ratio of normal chromosomes and chromosomes with structural rearrangements. Monosomy for chromosomes 1, 2, 4, 5, 8 was observed in both cell lines and for chromosome 9 in CHO(dhfr-)/susp cell line. The differences between CHO(dhfr-) cell lines studied by us consists of inclusion of part of chromosome 7 in der(6)t(1;6), rearrangement of del(5) and monosomy of chromosome 9. It was shown that in karyotypes of all CHO cell lines studied up today there are 5 common structurally chromosome rearrangements: del(2), inv(3), add(6), del(9) and mar1. In both CHO(dhfr-) cell lines investigated by us three unique chromosome rearrangements: del(1), der(6)t(1,6) and mar3 were revealed. Necessity of simultaneous GTG and FISH analysis of chromosomes rearrangements in the CHO cell lines under study is discussed.

Tight association of peripheral chromatin with nuclear lamina unavoidably creates topological constraints during replication. Additional complications are associated with high stability of lamina meshwork, which may hinder an access of replication factors to the sites of DNA synthesis in highly condensed template with limited mobility. In the current work we studied structural organization and dynamics of lamina as a function of replicative status of associated peripheral heterochromatin. The studies of molecular mobility of laminas at various stages of S-phase in vivo and using super-resolution microscopy showed no correlation between lamina dynamics and replicative status of attached heterochromatin. These data support the hypothesis that lamina-chromatin interactions during S-phase are regulated at the level of adapter proteins. Ultrastructural studies have demonstrated that temporal break of lamina-chromatin connections during replication does not cause noticeable spatial separation of replicating domains from nuclear periphery.

Recently, it was shown that pacemaker f/HCN channel might be permeable not only for Na+ and K+, but also for calcium ions. Pacemaker channel HCN2 was expressed in ovarian cell culture of Chinese hamster. In the patch clamp experiments the calcium current with low amplitude 0.06±-0.87 nA and open probability 0.48% (±3.02 at -110 mV, n = 6, Ca2+ 2 mmol) was measured. HCN2-Ca2+ current significantly activated with cyclic adenosinemonophosphate by increasing of open probability and inhibited by specific If-blocker ivabradine. The calcium ion current was also shown in f-channels of neonatal rats ventriculocytes. Thus, the calcium ions conduction through HCN2 and native f-channel at physiological concentrations of extracellular fluid and physiological membrane potentials of cardiomyocytes was demonstrated.

Currently, neutron capture therapy is a promising cancer treatment. This method is based on the reaction of the thermal neutron capture by some non-radioactive elements (e.g., Gds57), which results in subsequent emission of electrons and gamma rays. An effective instrument for delivery of gadolinium into the tumor tissue are the particles of the "rigid" nanostructures (NS) based on double-stranded DNA complexes with gadolinium (NS-Gd). The local concentration of Gd in such nanostructures may reach 40%. To optimize the process of neutron capture therapy it is very important to investigate possible penetration mechanisms of NS-Gd particles into the tumor cells. In this work, the dynamics of interaction NS-Gd with cultivated chinese hamster ovary cells (CHO) was studied by confocal and electron microscopy. It is shown that NS-Gd are able to enter CHO cells. This process begins in about 1 hour after the start ofincubation. After 6 h NS-Gd particles were detected in almost all cells. A further increase of the incubation time does not lead to significant changes in cell morphology, although the number NS-Gd inside the cells increases. The plasma membrane of the cells remains intact. The NS-Gd particles, which entered the cells, remain inside the cells for a long time. The data obtained show that NS-Gd are relatively low-toxic and suggest that the presence of NS-Gd in the tumor cells does not prevent their division. The data obtained are important for improving the efficiency of the neutron capture therapy method.

Aim: Study of plant extracts that have the ability to neutralize cytotoxic activity of hemolysin., Materials and Methods: Preparations of purified and recombinant V. cholerae eltor hemolysin as well as supernatants of V. cholerae strains were used. Determination ofcytotoxic activity of hemolysin and neutralizing activity of plant extracts were carried out by using cell cultures CHO-K1 and CaCo2., Results: Out of 9 water extracts only 3 - extracts of Rhei rhizome, Limonium gmelinii and Quercus robur neutralized hemolysin in cell culture CHO-K1 and CaCo2, whereas the other extracts--Humulus lupulus, Ocimum basilicum, Chelidonium majus, Juglans regia, Achillea milefolium and Hypericum perforatum did not have anti-cytotoxic effect. Neutralizing properties of extracts are exhibited during their co-incubation with hemolysin preparations and supernatants of V. cholerae strains already within 10 minutes., Conclusion: Plant extracts that have anti-cytotoxic activity against hemolysin are perspective for development oftherapeutic-prophylaxis preparations.

BMPs are osteoinductive proteins which are used in treatment of acute fractures. Large quantities of recombinant proteins are usually needed to achieve efficacy in the clinic. This translates to severe complications and high costs. Different strategies have been developed to improve the efficacy and safety of BMPs. Modification of the heparin-binding site in order to increase the local retention time of the morphogen is one of these approaches. Aiming at further improvement in properties of BMP-7, a novel form of this protein was designed and expressed successfully in Chinese Hamster Ovarian (CHO) cells. Substitution of the Bone morphogenetic protein-7 N-terminus by the heparin-binding site of Bone morphogenetic protein-2 was carried out to increase the heparin binding capacity of the novel protein. It was found that the novel variant, retained its in vitro biological activity and the heparin binding capacity of this protein was approximately 20% higher than that of the wild-type at a protein concentration of 100 ng/mL. The novel protein as the first variant of hBMP-7 with the enriched heparin-binding site may offer more advantages in clinical use as compared to the existing commercial form.

Aim: Study the activity of plant extracts against cholera toxin (CT) of Vibrio cholerae O1., Materials and Methods: Antitoxic activity of plant extracts was determined by using enzyme immunoassay and CHO-K1 cell culture., Results: 8 water extracts of plants were studied. Extracts of nut, tutsan, milfoil, basil do not have effect on CT activity in EIA or CHO-K1 cell culture. Celandine and rhubarb extracts do not reduce CT immunochemical activity but prevent elongation of CHO-K1 cells. Oak and hop extracts suppress binding in EIA of cholera toxin and GM1 receptors and insignificantly reduce its activity in cell culture., Conclusion: Antitoxic activityofplant extracts against CT is perspective for the development of preparations possessing inhibition effect.

Dependence of growth characteristics of cell population and the monolayer formation on the initial plating concentrations of cells were studied on the established CHO cell line. The cells were cultivated under standard conditions on plastic substrate. Initial plating concentration was varied as: 1000, 2000, 3000, 4000, 5000, and 6000 cell/cm2. It was shown that growth of the cell culture can be formally described by standard S-shaped dependence. However, a more detailed analysis revealed a discrepancy between the experimental and expected data. Specifically the arrest of cell growth at the monolayer formation does not coincide with the time when the theoretical curves approach plateau. It has been concluded that cell proliferation and the formation of a monolayer are independent processes (at last in CHO cells). Both processes may be considered as analogues of proliferation and morphogenesis in metazoan. In addition, it has been shown that the arrest of cell division occurs not only by contact inhibition of proliferation during monolayer formation but also by reducing the size of cells to some limiting dimension and by increasing the polarization of cells.

Non-Long Terminal Repeats (non-LTR or LINE) retrotransposons belong to the class of mobile genetic elements that are transposed into the host genome by reverse transcription of the RNA intermediate. Most of non-LTR retrotransposons contain two open reading frames (ORFs). The ORF1 codes for a gag-like protein, while the ORF2 codes for a reverse transcriptase (RT). We cloned two constructs based on Jockey-like non-LTR retrotransposon from genome Chironomus thummi (NLR1Cth). The retroposition assay performed in Chinese hamster ovary (CHO) cells demonstrated genome integrations of both constructs. The finding that the insect mobile element NLR1Cth is functional in mammalian cells demonstrates that this element possess universal enzymatic machinery allowing for active propagation in the genome of distant taxa. This suggests that the NLR1Cth transposon system may represent a useful tool for genetic analysis and manipulation in vertebrate cells.

A series of experiments to study the delayed effects of gamma-radiation exposure in different generations of the progenies of the Chinese hamster ovary CHO-K1 irradiated cells has been conducted. It has been shown that in the progenies of the cells irradiated with a dose of 1 Gy, the following effects are observed: in the 9-27 cell generations - increase in the genome damage, intracellular production of reactive oxygen species, apoptotic cells percentage and cell sensitivity to additional exposure (irradiation at a dose of 10 Gy); in the 30-42 cell generations - decrease of the studied parameters up to control values and increased resistance to additional exposure. It is assumed that the decrease of the studied parameters up to the control values in the 30-42 postirradiation generations of cells is caused by elimination of damaged cells or transition of genomic instability into a hidden (latent) condition.

The advances of the method of atomic force microscopy for investigating the animal cells and an analysis of its development have been reviewed, with much attention being given to studies of living cells. The features and problems of the method have been considered, and a number of special methods based on the use of atomic force microscopy have been analyzed. The problems of choosing the geometry of probes for studies of animal cells, determination of cell adhesion on substrate, mapping of the cell surface using chemically modified cantilevers, and the distribution of molecular components inside the cell with the use of micro- and nanosurgical approaches have been discussed. The problems of combining the atomic force microscopy with optical and laser scanning confocal microscopy have been considered. Possible applications of the method in biotechnology and medicine are discussed.

Aim: Determination of non-O1/non-O139 Vibrio cholerae toxin (CT) gene expression by using EIA, and biological effect of non-O1/non-O139 V. cholerae supernatant on cell cultures evaluation., Materials and Methods: 39 V. cholerae strains from various serological groups were studied. Hemolytic activity of strains was determined by using Greig test, and cholera toxin production--in GM1-EIA and in continuous cell lines by registering cytotonic, cytotoxic and proteolitic effect., Results: GM1-EIA method does not detect CT production in 29 museum strains of non-O1/non-O139 V. cholerae in vitro. CT was detected only in 1 non-O1/non-O139 V. cholerae strain supernatant with OD = 0.577 that is substantially lower than in O1 V. cholerae strains (OD = 2.176). In cell cultures non-O1/non-O139 V. cholerae supernatants diluted to 1:100 caused elongation only in single cells., Conclusion: Cytological model is a more sensitive technique to evaluate toxin producing abilities of non-O1/non-O139 V. cholerae strains and is appropriate for use.

Changes in the cell area during cultivation of the CHO line cells were studied using time-lapse technique (start of registration in one day after cell plating). It was established that the size of the daughter cells after mitosis remains lees than the size of the mother cell for a long time (up to 6 h). Nevertheless, the average cell area of the whole population is constant throughout the observation period (up to 18 h). We assume that this phenomenon could be a result of interaction among dividing and not-dividing cells. The experimental data confirming this conclusion are presented.

Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.

The aim of the study carried out on cells of lines L-929 (NCTC a clone 929) and CHO was to examine whether the position of cleavage furrow is random or not. CHO cells were seeded in the traditional way (evenly across the surface of a Petri dish), and L-cell were put as colonies to monitor the migration of cells emanating from them. The time-lapse filming (imaging) was used for registration of behavior of cells. Analyzing the obtained images of cells, the angle between the axis of polarization of a cell and its cleavage furrow was measured. Besides, the angle between an axis of cell polarization or its cleavage furrow and the horizontal axis of image field was measured. It was shown that position of CHO cells in a dish plane was random as well as the value of the angle between the axis of polarization of these cells and their cleavage furrows. The L-929 cells migrating from colony were orientated so, that their polarization axis was directed to the colony center, and the cleavage furrow was perpendicular with this axis. Assumptions about nonrandom position of both cultivated cells during mitosis and their cleavage furrows were made.

We have recently shown that epithelial sodium channels (ENaC) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. However, it has been also demonstrated that GTPase, dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. F. i., treatment of mpkCCD(c14) cells monolayers with dynasore (in concentrations of 10 and 100 microM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, the same concentrations of dynasore caused reduced currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel's activity and is not suitable for studies of a role of dynamin in the clathrin-mediated endocytosis of ENaC. This effect is most likely caused either by dynasore's toxic effect upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments designed to study the role of dynamin different plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin K44A transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC subunits only (control); additional transfection of cortactin in this system resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease if ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but apart from that dynamin and related processes also might be involved into ENaC regulation by cortactin.

Data obtained by other authors for mammalian cells treated by sequential action of ionizing radiation and hyperthermia were used to estimate the dependence of synergistic enhancement ratio on the ratio of damages induced by these agents. Experimental results were described and interpreted by means of the mathematical model of synergism in accordance with which the synergism is expected to result from the additional lethal damage arising from the interaction of sublesions induced by both agents.

Haponin (HLDF-alike protein) was previously identified from the human promyelocytic leukemia HL-60 cell line. For the functional study of this protein, we obtained recombinant haponin with an N-terminal hexahistidine tag using a baculovirus expression system. Antibodies against 6xHis-haponin were produced, and the expression of endogenous haponin was demonstrated in mammalian cell lines of different origin. Using affinity chromatography and immunoprecipitation methods, we have shown that in CHO-K1 cells haponin interacts with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is one of the vital glycolytic enzymes with a diverse set of noncanonical functions.

The DNA comet assay (neutral version) showed that irradiation of CHO cells in a dose of 1 Gy (gamma-radiation, (60)Co) is followed by an increase in the degree of DNA fragmentation. These changes were observed immediately after irradiation and on days 7-21. On days 2-4 and 23-28 after irradiation, the degree of DNA fragmentation in the descendants of irradiated cell did not differ from that in control samples. The increase in the degree of DNA fragmentation on days 7-21 probably results from induction of apoptosis. This assumption is confirmed by the study of cell death. The sensitivity of cells to repeated irradiation in a dose of 10 Gy significantly increased on days 9, 11, 16, and 18 after irradiation. However, these cells were resistant to repeated irradiation on days 21-28. Our results confirm the hypothesis that genomic instability is a selective mechanism, which mediates the formation of radioresistant cell clones.

B cell-activating factor of the TNF family (BAFF) is critical for B cell maturation and survival. Here, we constructed a stable CHO cell line, in which the expression level of soluble form of BAFF (sBAFF) was raised from 0.13 microg/ml to 0.55 microg/ml. Purified recombinant sBAFF from these CHO cells not only bound to its receptors but also co-stimulated the proliferation of human peripheral blood B lymphocyte in vitro. These results provided us with a useful basis for further studies about sBAFF-related research.

It has been found that multiply repeated tripeptide fragment GER (Gly-Glu-Arg) from different collagen types stimulates nonspecific adhesion of CHO-K1 cells. Activation of cell adhesion is accompanied by modifications in fatty acid composition of cell membrane phospholipids. Cell incubation with the synthetic peptide increases the unsaturation indexes of phosphatidylcholin (PC), phosphatidylethanolamine (PEA) and phosphatidylinositol (PI). Arachidonic (C20:4omega6) acid is mainly contributed to the increased unsaturation index of PI. In the case of PC and PEA not only arachidonic acid but also other unsaturated fatty acids: docosatetraenoic (C22:4omega6), docosapentaenoic (C22:5omega3) and docosahexaenoic (C22:6omega3) acids are implicated in the index increasing. Besides, the elevation of relative content of molecules with polyenoic fatty acids in the group of PI molecules is accompanied by decrease in monoenoic fatty acids caused mainly by decrease in the oleic (C18:1) acid level. The role of the investigated peptide: 1) in the activation of cell adhesion as a regulator of active or non active state of integrin receptors: 2) in the alterations of fatty acid composition in main classes of phospholipids as modulator of fluidity level in annular lipid zones around these adhesive molecules is discussed.

In this study, we have examined the organization of the mitotic spindle poles in CHO-K1 cells dividing after treatment with the etoposide (1 h, 25 microM). We studied at various periods after the treatment: 1) the distribution of gamma-tubulin in mitotic cells by immunofluorescent staining; 2) the level of posttranslational modification of a-tubulin in the spindle microtubules by immunoelectron microscopy; 3) the ultrastructure of the mitotic apparatus poles by standard electron microscopy. In 48 h after the addition of the agent we identified considerable changes in the ultrastructure of poles in etoposide-treated CHO-K1 cells with bipolar and multipolar spindles. The number of centrioles increased. The centrioles were unevenly distributed among the poles, and some centrioles were not explicitly involved in the organization of mitotic spindle, furthermore they can differ in the number of outgrowing microtubules. Most centrioles were without fibrillar halo. In 48 h after the addition of etoposide, electron microscopy of cells after immunoperoxidase staining with antibodies to acetylated and tyrosinated alpha-tubulin has shown that different poles of a multipolar spindle within the same cell are stained differently for tyr-tubulin but not for acet-tubulin. Immunofluorescence staining for gamma-tubulin also points to different organization of poles in the same spindle. Our findings provide the first evidence that the pattern of immunostaning and the ultrastructure of mitotic apparatus poles differ in the cells dividing at various periods after etoposide treatment.

Normal course of processes of regeneration and epitheliazation of damage tissues has been shown to be based on the capability of cells participating in these processes for selective adhesion. In the case of the complete or partial absence of this capability in the cells-participants of the wound healing process, the so-called non-healing wounds appear. In this connection, it remains actual to search for natural agents promoting healing of chronic non-healing wounds. In the present work, we studied effects of synthetic fragments of leukocytic antimicrobial peptides defensines--GER, FGER, and GERA--on aggregation and adhesion of epitheliolike cells of the CHO-K1 line. These peptides have been established to have aggregate-stimulating properties; besides, they enhance adhesion of the cells to the untreated plastic and inhibit fibronectinmediated cell adhesion. Possible pathways of regulation by peptides of processes of intercellular and cell-matrix interaction are discussed as well as ways of release of these compounds in an organism and their functional role in an organism.

Equine diphtheria antitoxins from different manufacturers were studied. Their immunochemical interaction with diphtheria toxin, toxoid, and antigens of Corynebacterium diphtheriae in ELISA and immunoblotting assays as well as biological activity in CHO cell assay were compared. The discovered differences between antitoxin samples with stated equal activity in IU/ml point to heterogeneity of antigen composition in preparations used for immunization. Mentioned methods allow to standardize antitoxins basing on their biological activity and immunochemical characteristics.

Glycine receptors (GlyRs) provide the main inhibitory neurotransmission in spinal cord and brainstem synapses of vertebrates. Fucile et al. (2000) discovered that elevation of intracellular Ca2+ caused rapid potentiation of GlyRs. This modulation develops in less than 100 ms. It is characterized by an increase in GlyR apparent affinity for glycine. It has been suggested that the phenomenon of Ca-induced potentiation involves an unknown Ca2+-binding protein (CaBP). Using the yeast two-hybrid system, screening of human brain cDNA library against the cytoplasmic loop of human alpha 1 subunit (GlyRhl) allowed us to identify five new interactors. One of them belongs to a family of Ca-binding proteins. We analyzed effect of "short" forms of this protein (CaBP-S) on functional properties of GlyRhl expressed in HEK-293 and CHO cells. Using whole-cell recordings and rapid agonist application we constructed concentration dependencies of glycine-induced currents. This analysis revealed statistical differences in EC50s between control cells (expressing only GlyRhl) and those expressing CaBP-S. In HEK-293 cells recorded under conditions of low intracellular Ca concentration (BAPTA 20 mM in the recording pipette), EC50 for glycine in control cells and expressing GlyRhl + CaBP-S were, correspondently, 68+/-49 microM (n = 29) and 409 +/-421 microM (n = 60). In CHO cells EC50 were 54+/-43 microM (n = 25) and 123 +/-104 microM (n = 28). These differences were statistically not significant at recording with intracellular solution containing high Ca concentration (50 microM). In this case EC50 were correspondently 35+/-28 microM (n = 7) and 64 +/-38 microM (n = 7). These results suggest that CaBP-S causes decrease of GlyR sensitivity to agonist through interaction with cytoplasmic domain of GlyR.

In the internal cytoplasm of interphase cells the density of microtubules is the highest in the centrosome area and decreases to the cell periphery. As a rule, the quantity of fluorescent microtubules cannot be counted up in the internal cytoplasm, but it is possible to estimate microtubules quantity using measuring of their optical density. In living 3T3 and CHO cells the microtubules optical density decreased according to different mathematical dependences that apparently reflected the differences of their microtubule system organization. To determine appropriateness that circumscribe the reduction of microtubules optical density from the centrosome region to the direction of cell margin, we modeled cell contours with the certain ratio and interposition of centrosome-attached and free microtubules in vector schedules CorelDraw program. The decrease of optical density was analyzed in MetaMorph program as it was described earlier (Smurova et al., 2002). It was shown that fluorescent microtubules optical density decreased exponentially (y = ae(-bx)) if the system joined only microtubules growing from the centrosome up to the cell margin. The curve became smoother in the case of not all radial centrosome-attached microtubules reached the margin, and adding of free microtubules into the system led to the sharp fall in optical density in the centrosome area and to its gradual decrease at the cell periphery. The increase in free microtubules quantity changed the character of the curve describing the reduction of optical density microtubule system which included free and centrosome-attached microtubules in proportions of 5 : 1 was described by the equation of linear regression (f= k . x + b). Thus, the mathematical dependence describing the microtubules distribution from the centrosome to the cell periphery, depends on the ratio of microtubules and their relative positioning in the cell volume. The data obtained using model systems have coincided with the results of experiments. The graphs which described the increase in microtubules optical density during microtubule repolymerization after nocodazole treatment, corresponded to the graphs for model cells. Thus, the method we used allows to analyze the microtubule system in the cases when the direct observation of individual microtubules is difficult.

The results of the evaluation of the toxicity of bacterial antigens obtained from the causative agents of plaque, glanders, melioidosis, cholera on infusoria of the species P. caudatum, as well as on cell lines L-929, CHO K-1 and peritoneal macrophages of BALB/c mice, are presented. As revealed in this study, the method of toxicity determination on infusoria is similar in its sensitivity to the methods of testing on. CHO K-1 and L-929 cells, but the former is simpler, more available and permits the determination of toxic doses producing disturbances in the vital activity of the infusoria, but not leading to their death.

S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells.

The movement of cultured cells along the substratum is a convenient model for studying cell movement in the organism, occurring during embryogenesis, angiogenesis, metastasis, wound closure, etc. The moving cells must control their pseudopodial activity along the perimeter, regulate the attachment and reattachment to the substratum, and pull their body following pseudopodium during their movement along the substratum. As proven by numerous investigations, these processes directly depend on the actomyosin system of cells. The role of microtubules as components of cytoskeleton in cell locomotion still remains obscure. The role of microtubules in cell movement is commonly investigated using microtubule-destructive drugs. Therefore in the final results the accessory drug effect on, for example, signal cascades cannot be excluded. Another mode of action on the microtubule dynamics is centrosome removal from the cells, which is easily realized by their removal together with the nucleus. It has been shown that in cytoplasts of centrosome containing fibroblasts, dynamic instability of microtubules remains. Unlike, in non-centriolar cytoplasts tread milling is observed. Some literature evidence suggests that cytoplasts of cultured cells move along the substratum not worse that intact cells do. In this study cytoplasts with and without centrosome were obtained and identified, and parameters of movement along the substratum (speed, direction) were registered for both these two populations of cytoplasts, and for control intact cells and cells involved in the experiment. The model of experimental wound of monolayer was used, because it guaranteed cell synchronization in respect to movement direction and speed. Centrosome-containing CV-1 cytoplasts displayed radial microtubule array, and centrosome-lacking cytoplasts exhibited chaotic distribution of microtubules, which is characteristic of microtubule tread milling. In addition, both kinds of cytoplasts appeared to move along the substratum much slower than the whole cells. No difference was found is speed and keeping direction between centriolar and non-centriolar cytoplasts.

The humoral response of mice and rabbits to the injection of whole-cell pertussis vaccine (PV) and acellular pertussis vaccine (APV), developed at the Mechnikov Research Institute for Vaccines and Sera (Russian Acad. Med. Sci.) in Moscow, was studied. In the sera of immunized animals antibodies to the antigenic complex were determined in the direct hemagglutination (DHA) test, specific antibodies to filamentous hemagglutinin (FHA) and pertussis toxin (PT)--in the enzyme immunoassay (EIA) and antibodies neutralizing PT in a cytopathogenic dose (CPD)--in neutralization test on Chinese hamster ovary (CHO) cells. In mice and rabbits immunized with APV the antibody titers determined in the DHA test were higher than those in the animals immunized with PV. Specific antibodies titers to FHA and PT in the sera of rabbits immunized with APV were also higher than those in the sera of rabbits immunized with PV. High dilutions of sera taken from the animals immunized with APC neutralized 4-16 doses of PT in the neutralization test on CHO cells. The most important result of this study was the detection of a more pronounced immune response in the animals immunized with APV in comparison with that induced by PV according to the results obtained in EIA and in the test of PT CPD neutralization on CHO cells.

Rubella diagnostic agents for hemagglutination inhibition (HI) and enzyme immunoassay (EIA) based on rubella virus-like particles (RVLP) have been developed. Noninfectious RVLPs containing three structural E1, E2, and C proteins were expressed in transfected CHO24S cell culture. HI titer in culture medium was 1:256. Tween-80 treatment and ether increased HI titer 4-6-fold and rendered the antigen higher stability. Immunogenic properties of RVLPs were similar to the native rubella virus in HI test with international reference human rubella serum and sera from convalescents after rubella. Serum of mice immunized with RVLP reacted similarly with RVLP antigens and native virus. Antigen for EIA from RVLP was prepared by concentrating RVLP from culture fluid and partial purification by ultracentrifugation. The results of human sera testing by HI and EIA with RVLP and native virus antigens coincided. RVLP are identical to antigenic structure of the virus, are stable and easily purified, and can therefore be used for commercial production of HI and EIA antigens.

Cholera toxin, hemolysin, dermonecrotic and proteolytic factors have been detected and identified in V. cholerae O139. The production of these substances has been found to depend on the conditions of the cultivation of vibrios, and the types of proteases have been determined.

The comparative study of the preparations of V.cholerae hemolysins of different serovars with the use of continuous cell lines CHO-K1, Vero, Hela, L-929 was carried out. The preparations of hemolysins isolated from such strains as V.cholerae 569B, V.eltor 9949, V.cholerae O-mut 461/67-34 differed in their biological activity on experimental animal models and had different cytotoxic activity. The preparations exhibiting no activity when tested in vivo (V.cholerae and V.eltor hemolysins) were cetotoxins, but in lower doses (1000 and 100 times respectively) than the preparation of V.cholerae O-mut hemolysin, active in vivo. Hemolysins induced the formation of anticytotoxic antibodies in low titers.

The goal of this research is to study a dependence between dynamics of the biological parameters, characterized the state of the cell cultures, and variations of geomagnetic field (GMF) in the quiet and disturbed conditions. The investigations were carried out on geophysical recording site in high-latitude regions. Geophysical and biological observations were conducted simultaneously and at the same place. Continuous cell lines of mouse, hamster and pisces were used in experiments. Morpho-functional characteristics, including the percentage of polynuclear cells were estimated. Has been shown that dynamic of morphofunctional state of cell culture associated with GMF variations. Geomagnetic activity (GMA) abrupt change is followed by a stepwise changes the state of cell cultures. Changes is properties of cell surface are related to cell fusion, the appearance of heterokaryons, enlargement of cellular adhesion and aggregation. Enhancement of morpho-functional variety in cellular populations involving a reorganization of nuclear material demonstrate a possible regulatory and evolutionary part of GMF variations.

We investigated histone-DNA interactions in chromatin of amplified about 1000 fold dihydrofolate reductase (DHFR) domain in methotrexate resistant Chinese hamster ovary cell line CHOC 400. We explored chromatin structure of DHFR-gene and extended region lying 3'-downstream of the DHFR-gene, which includes two zones of DNA replication initiation, Ori-beta and Ori-gamma, and matrix attachment region (MAR). Using the method of hybridization with "protein images" we found that the yield of both core histones and histone H1 cross-linked with DNA in 5'-region of the DHFR-gene, in Ori-gamma and regions flanked from either side MAR is significantly decreased in comparison with 3'-transcribed region of the DHFR-gene, Ori-beta and MAR. It was shown previously that decreased yield of histones cross-linked with DNA is typical for 5'-region of actively transcribed genes. So, histone-DNA interactions in chromatin of the 5'-region of the house-keeping and moderately transcribed DHFR-gene, Ori-gamma and regions which flank MAR resemble those in 5'-regions of actively transcribed genes. Significant differences in amount of histones cross-linked with DNA in various parts of the DHFR-domain testify to high level of heterogeneity of chromatin structure in the domain.

Comparison analysis was made of the putative replication origin DARC146 and the origin determined in the DNFR domain of CHO cells. We failed to observe extensive homology between these two sequences. However, several short (8-10 bp) areas of homology were identified. Some of them are binding sites for nuclear proteins. As shown by GM and competition experiments, both origins contain high-affinity binding sites for the transcription-replication factor Oct-1 and for two unknown nuclear factors. The two unknown nuclear factors bound to TCTCTTA and CACTTAG nucleotides. The binding sites for these proteins are located at a short distance from each other. This fact suggests interaction between these polypeptides. Measurement of DNA-binding activity of Oct-1 during the cell cycle of proliferating hepatocyte demonstrated that the binding activity of Oct-1 protein increased before initiation of DNA synthesis. The results of this report suggest that Oct-1 and two other nuclear proteins participate in the regulation of mammalian DNA replication. The results published early indicated the presence of an unusual DNA structure (bent DNA) in both origins under study. We suggest that these common elements of the origins regulate their activation.