[Participation of beta2-integrins CD11b/CD18 and CD11c/CD18 in adhesion and migration of cells on fibrinogen].

The role of beta2-integrins CD11b/CD18 and CD 11c/CD 18 in adhesion and migration of leukocytes on fibrinogen was studied. The monoclonal antibodies against CD11b inhibited the spontaneous adhesion of monocytic THP-1 cells on fibrinogen, whereas antibodies to CD11c more effectively inhibited the adhesion stimulated by chemokine MCP-1. By the RNA-interference method the clones of THP-1 with reduced expression of CD11b and general beta2-subunit CD18 were obtained. MCP-I stimulated the adhesion to fibrinogen of THP-1 cells of wild-type and mutant cells with reduced expression of CD11b (THP-1-CD11b-low), but not of cells with low expression of CD18 (THP-1-CD18-low). THP-1-CD18-low cells were also characterized by the impaired chemotaxis in presence of MCP-1. The data obtained suggest that spontaneous cell adhesion to fibrinogen is mediated to a greater extent by CD11b/CD18 integrins, while chemokine-stimulated adhesion and migration is mostly dependent on CD11c/CD18 molecules.

[Cultivated human hair follicle cells are capable of integrating to skin structure in vivo].

In the present study, human keratinocytes and dermal papilla cells were labeled to investigate their behaviour after intradermal transplantation. Cells were transduced by lentiviral vectors that bore marker gene encoding green fluorescent protein (copGFP) or red fluorescent protein (DsRed). A portion of transgene expressing cells was evaluated by flow cytometry. Genetic constructions that we used provided high level (> 95 %) of transduction of hair follicle cells. In vitro transduced cells were injected under the epidermis of human skin fragments, and these fragments were then transplanted under the skin of immunodeficient mice. Injected epidermal keratinocytes were found, mainly, in hair follicles and partially in a zone of interfollicular epidermis, while dermal papilla cells were found in papilla derma. The results of the present research show that the chosen genetic constructions obtained on a basis of human immunodeficiency lentivirs are capable of effective and stable transduction of human skin cells. Injected cells survived and were found in the corresponding structures of the skin.

[Caldesmon changes the structure of actin at the leading edge and suppresses cell migration].

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibrils and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced a more than 1.5-fold accumulation of stress fibrils only in control cells but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type but not in cells with a low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.

[Expression patterns of the human and mouse IFGP family genes].

The IFGP family is a recently identified group of human and mouse genes structurally related to the genes of leukocyte Fc-receptors. In this study we compared expression patterns of six human and four mouse IFGP genes. With the exception of mouse IFGP2, the genes of the family were found to be predominantly expressed in haemopoietic cells. Expression of human IFGP1-IFGP5 and mouse IFGP3 was B cell-specific. Mouse IFGP1 transcripts were mainly found in B cells, but this gene may be either expressed by nonlymphoid cells. Expression of the human IFGP6 was detected in CD8+ T cells and NK cells. We further demonstrated that alternative splicing of human IFGP1 and IFGP6 mRNA may generate transcripts coding for the previously unknown isoforms. The novel IFGP1 isoform lacks transmembrane domain, whereas the IFGP6 isoform has altered cytoplasmic tail. The data obtained indicate that the receptors of family may contribute to the regulation of development and/or functions of three effector types of lymphocytes, namely B cells, CD8 T cells and NK cells.

[The effect of 15-ketosterol analogues with a 5,6-dimethylhept-3-en-2-yl chain at C17 on cholesterol metabolism in Hep G2 hepatoma cells]. / Vliianie analogov 15-ketosterina, soderzhashchikh 5,6-dimetilgept-3-en-2-il'nuiu tsep' pri C17, na metabolizm kholesterina v kletkakh gepatomy HepG2.

The effect on cholesterol metabolism in Hep G2 hepatoma cells was studied for new analogues of 15-ketosterol [3beta-hydroxy-5alpha-cholest-8(14)-en-15-one] (I): (24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-diene-15-one (II), (24S)-3alpha-hydroxy-24-methyl-5-alpha-cholesta-8(14),22-diene-15-one (III), and (24S)-24-methyl-5alpha-cholesta-8(14),22-diene-3,15-dione (IV). Analogues (I) and (II) were found to be equally effective inhibitors of cholesterol biosynthesis after a 3-h incubation with Hep G2 cells; however, (II) produced a stronger inhibitory effect after a 24-h incubation or after an incubation of cells preliminarily treated with the inhibitor in a medium containing no ketosterol. The ability of ketosterols to inhibit cholesterol biosynthesis decreased in the order (II) > (IV) > (III). Ketosterol (II) inhibited, whereas ketosterol (III) stimulated the biosynthesis of cholesteryl esters. (IV) stimulated the biosynthesis of cholesteryl esters at a concentration of 1-10 microM and exerted no marked effect at a concentration of 30 microM. These results indicate that delta8(14)-15-ketosterols containing a modified side chain are of interest as regulators of cholesterol metabolism in liver cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[Interaction of acyl derivatives of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one and 3alpha-hydroxy-5alpha-cholest-8(14)-en-15-one with hepatoma hep G2 cells]. / Vzaimodeistvie atsil'nykh proizvodnykh 3beta-gidroksi-5alpha-kholest-8(14)-en-15-ona i 3alpha-gidroksi-5-alpha-kholest-8(14)-en-15-ona s kletkami gepatomy Hep G2.

Effects of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (I), 3alpha-hydroxy-5alpha-cholest-8(14)-en-15 one (II), 3beta-hexadecanoyloxy-5alpha-cholest-8(14)-en-15-one (III), 3alpha-hehadeeanoyloxy-5alpha-cholest-8(14)-en-15-one (IV), 3beta-acetoxy-5alpha-cholest-8(14)-en-15-one (V), 3alpha-acetoxy-5alpha-cholest-8(14)-en-15-one (VI) on cholesterol metabolism in hepatoma Hep G2 cells were studied. Compound III slowly bind to Hep G2 cells followed by internalization and metabolic transformation (at a concentration of 30 microM the total binding of compound III was (3.9 +/- 0.4) nmol per 1 mg of cell protein for 24 h incubation). Compound I depressed and compound III stimulated the uptake of low density lipoproteins radiolabeled with oleyl cholesteryl ether [14C-CE]LDL (58% and 149% from control). Compounds I and II inhibited cholesterol biosynthesis from [14C]acetate (with IC50 values of 4.0 +/- 0.7 and 8.0 +/- 1.5 microM). Effects of compounds V and VI were less potent; compounds III and IV were inactive. Compound II activated cholesterol acylation, estimated by incorporation of [14C]-oleic acid into cholesteryl esters (170% from control at a concentration of 30 microM). The results indicate correlation between polarity of the compound and its ability to regulate cholesterol metabolism in Hep G2 cells.

[Triple interpolyelectrolyte complexes DNA-polycation-polyanion: a new approach to optimization of mixtures for transfection in eukaryotic cells]. / Troinye interpoliélektrolitnye kompleksy DNK-polikation-polianion: novyi podkhod k optimizatsii smesei dlia transfektsii éukarioticheskikh kletok.

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M 30-40 kDa as polycation and polyacrylic acid (PA) with M 20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA-PEG incorporation into a ternary complex (by itself or as a 1:1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA-PEG (as a 1:9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a beta-galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1:1 mixture of PA and PA-PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.

[Rare codons and gene expression in Escherichia coli]. / Redkie kodony i ékspressiia genov v Escherichia coli.

Influence of rare codons upon gene expression in E. coli was investigated. The chimeric gene was created combining CAT gene and a fragment of the gene, encoding for alpha-domain of beta-galactosidase. The synthetic oligonucleotides were inserted in different parts of the chimeric gene. The constructed synthetic oligonucleotides encoded the same amino acid sequences and contained arginine codons AGG, AGA and CGT in various combinations. It was shown that the presence of rare arginine codons AGG and AGA in the template and their mutual arrangement significantly influence the level of gene expression. At the same time the presence of leucine, isoleucine, glycine and proline rare codons does not cause such an effect. Translation of AGGAGG and AGAAGA sequences was found to lead to the formation of a considerable amount of polypeptides of incomplete length. It was shown that the presence of such a cluster of rare codons effects on the length of specific mRNA.

[Expression of human urokinase in inoculated rodent cells. Loss of introns during gene transfer using a retroviral vector]. / Ekspressiia urokinazy cheloveka v perevivaemykh kletkakh gryzunov. Utrata intronov v khode perenosa gena s pomoshch'iu retrovirusnogo vektora.

Expression of urokinase in murine and rat cells was performed by two recombinant constructs, one containing cDNA and the other--hybrid (cDNA/genome) variant of human urokinase gene conserving 7 introns of 10, in the eukaryotic retrovirus vector pPS-3-neo. DNA of both constructs was introduced into packaging cell line psi 2 by a standard Ca-phosphate transfection technique. Infection of mouse and rat fibroblasts BALB/c 3T3 and Rat I with virus particles, produced by transfected psi 2 cells, led to an integration into the host genome of one or two recombinant proviral copies. Stable expression and secretion into the culture medium of glycosylated high molecular weight human urokinase was observed for both cell types. For the hybrid gene construct, precise excision of intervening sequences was shown during transferring of genetic material from packaging to recipient cells.

[mRNA for the plasminogen activator of the urokinase type: translation in oocytes of Xenopus laevis]. / mRNK aktivatora plazminogena urokinaznogo tipa: transliatsiia v ootsitakh Xenopus laevis.

Poly(A)+-RNA from human kidney and human embryonal lung fibroblasts fractionated by sucrose gradient centrifugation was translated in Xenopus oocytes. Assay for plasminogen-dependent fibrinolytic activity detected synthesis of secreted plasminogen activator and revealed the active fraction of poly(A)+-RNA with a sedimentation coefficient of approximately 23S. Translation products of the active fraction were immunoadsorbed by antiurokinase monoclonal antibodies immobilized on sepharose. Gel electrophoretic analysis of the protein products showed that the 23S fraction of poly(A)+-RNA from human kidney contains mRNA for single-chain urokinase-type plasminogen activator with apparent molecular weight of approximately 50 kDa.

[Isolation and characteristics of urokinase-type plasminogen activator from a culture of human embryo lung fibroblasts]. / Vydelenie i kharakteristika aktivatora plazminogena urokinaznogo tipa iz kul'tury legochnykh fibroblastov émbriona cheloveka.

Plasminogen activator from conditioned medium of human embryonal lung fibroblasts was purified by phosphocellulose P11 chromatography, followed by p-aminobenzamidine-agarose chromatography. Two forms of plasminogen activators were separated by chromatography on the heparin-sepharose. The high molecular weight form (53 kDa) with specific activity 130 000 IU/mg consists of two polypeptide chains (31 kDa and 20 kDa) and exhibits strong affinity for fibrin-celite, lysine-sepharose and heparin-sepharose. The low molecular weight form (32 kDa, 190 000 IU/mg) also binds to these sorbents, but more weakly, and its properties are very similar to those of low molecular weight urokinase. Activity of both forms of plasminogen activators are inhibited by monoclonal antibodies against urokinase. A number of enzymological chromatographic and immunological properties indicates, that the plasminogen activator from lung fibroblasts is of urokinase type.