[Mosaic expression of the lacZ reporter-gene under control of 5'-regulatory sequences of the alpha-S1-casein gene in transgenic mice].

Phenomenon of mosaic expression at cellular level is widely presented in tissues and organs of transgenic animals. The communication is concerned a study of the mosaics in transgenic mice carrying the lacZ reporter-gene under control of the bovine and goat alpha-S1-casein genes with 5'-flanked sequences of various ex-tent: pCLZ1--721bp, pCLZ2-- 2001 bp and pCLZ3 3409 bp constructs. Five transgenic founders were generated by injection of the recombinant DNA into zygotes: pCLZ 1 - N 16, pCLZ2 - N 37 and pCLZ3 N 7, N 36, and N 48. Positive for J3-galactosidase activity cells were detected in lactating mammary glands of all transgenic females, however, distribution of the positive cells was variable. We observed two types of mosaics: clonal or "lobule" type with positive cells filling the whole of the globule or stochastic type with single positive cells scattered over one or different lobules. Two types of mosaics were characteristic of all the transgenic animals, although, females carrying the pCLZ2 transgene showed "lobule" type more often than transgenic animals with the transgenes pCLZ and pCLZ3. It is suggested that the stochastic type of mosaics occurs in the cells at terminal stage of differentiation, whereas the <> type arises from positive for P-galactosidase proliferating precursors. Analysis of the inheritance of the transgenes in different lines demonstrated that the pCLZl transgene was inserted in the X-chromosome of the founder whereas the other two localized in autosomes. Localization of the pCLZl transgene in the X-chromosome did not influence the mosaicism; it was similar to that of transgenic animals carrying the transgenes in autosomes. Ectopic expression of the reporter-gene was detected in mandibular glands from the offsprings of the founders N 16 and N 37 only, as well as in atrezed follicles in N 37. The weak ectopic expression saggests that the 5 S-flanked regulatory sequences used in the constructs are able to provide perfect tissue-specific expression of the reporter-gene.

[The effect of regulatory sequences of alphaS1-casein gene on the expression of the lacZ-gene in loach Misgurnus fossilis L. transgenic embryos].

Transient expression of recombinant plasmids carrying the lacZ gene under the control of either bovine alphaS1-casein gene tissue-specific promoter-enhancer region or highly homologous goat alphaS1-casein gene promoter-enhancer region with supplementary regulatory sequences of the goat gene were studied in Misgurnus fossilis L. loach embryos. It has been shown previously that the expression of the constructs carrying these regulatory elements in transgenic mice occurred primarily in the mammary glands. At early developmental stages, loach embryos and early prelarvae showed nonspecific and mosaic transient expression of lacZ carrying casein regulatory sequences. Transgenic activity was the highest in 1-3-day embryos. At the same time, the efficiency of expression of lacZ gene carrying regulatory sequences of the alphaS1-casein gene of goat was higher than with the promoter-enhancer region of the bovine alphaS1-casein gene. Thus, regulatory sequences of the bovine or goat alphaS1-casein gene appeared capable of providing similar transient expression of reporter gene in the loach embryos. This model can be used for rapid testing of promoter-enhancer activity of transgenes.

[The technologies of genetic engineering in treatment of chronic lower limb ischemia].

The article contains experimental data on angiogenesis stimulated by plasmid containing the angiogenin gene. After the introduction of the gene construction, the number of capillars in the chorion-allantois membrane increases 2 to 3 times; in an ischemized limb of a rat it increases by 20 to 30%. Intramuscular administration of genetic engineering construction to patients with chronic lower limb ischemia improved the patients' condition, consisting in an increase in painless walking distance and ankle-brachial index, as well as in trophic defect healing and the betterment of muscular perfusion. Positive effects were noted after 2 to 4 weeks of treatment and remained during 6 to 24 months. There were no side-effects, except low grade fever during 1 to 2 days.

Enhanced expression of trim14 gene suppressed Sindbis virus reproduction and modulated the transcription of a large number of genes of innate immunity.

In the present research, we have studied an influence of enhanced expression TRIM14 on alphavirus Sindbis (SINV, Togaviridae family) infection. In the HEK293 cells transfected with human trim14 gene (HEK-trim14), SINV yield after infection was decreased 1000-10,000 times (3-4 lg of TCD50/ml) at 24 h p.i. and considerably less (1-2 lg of TCD50/ml) at 48 h p.i. Analysis of the expression of 43 genes directly or indirectly involved in innate immune machine in HEK-trim14 non-infected cells comparing with the control (non-transfected) HEK293 cells revealed that stable trim14 transfection in HEK293 cells caused increased transcription of 18 genes (ifna, il6 (ifnß2), isg15, raf-1, NF-kB (nf-kb1, rela, nf-kb2, relb), grb2, grb3-3, traf3ip2, junB, c-myb, pu.1, akt1, tyk2, erk2, mek2) and lowered transcription of 3 genes (ifnγ, gata1, il-17a). The similar patterns of genes expression observe in SINV-infected non-transfected HEK293 cells. However, SINV infection of HEK-trim14 cells caused inhibition of the most interferon cascade genes as well as subunits of transcription factor NF-κB. Thus, stable enhanced expression of trim14 gene in cells activates the transcription of many immunity genes and suppresses the SINV reproduction, but SINV infection of HEK-trim14 cells promotes inhibition of some genes involved in innate immune system.

[The induction of neovascularization in chorioallantoic membrane of chicken embryos transfected by a recombinant plasmid containing human angiogenin gene]. / Induktsiia neovaskuliarizatsii v khorion-allantoisnoi membrane kurinykh émbrionov, transfetsirovannoi rekombinantnoi plazmidoi, soderzhashchei gen angiogenina cheloveka.

A method was elaborated to evaluate the biological activity of expression products of gene in the plasmid vectors, which are crucial for the synthesis of growth factor of blood vessels. It was proven as possible that the chrioallantonic membrane (CAM) of chicken's embryos could be transfected by recombinant plasmids containing both the reporter and target genes. The efficiency of CAM transfection was assessed by a plasmid carrying the reporter gene of green fluorescent protein (GFP). Finally, it was demonstrated that, at an infiltration of the recombinant plasmid containing the human angiogenine gene, its expression products induce the neovascularization in the CAM cells of chicken's embryos and stimulate an accretion in vessels of the 1st, 2nd and 3d orders.

[The CELO-ANG recombinant avian adenovirus with human angiogenine gene inducing neovascularization in the anterior tibial muscle of rat]. / Recombinantnyi adenovirus ptits CELO-ANG, nesushchii gen angiogenina cheloveka, indutsiruet neovaskuliarizatsiiu v perednei bol'shebertsovoi myshtse krysy.

The CELO recombinant avian adenovirus carrying the gene coding the human angiogenine (ANG) synthesis was obtained. Expression of the angiogenine gene was shown in the LMH cell culture after infection with the CELO-ANG virus. The ability of CELO recombinant adenoviruses to carry out the delivery and expression of alien genes in muscle cells was demonstrated in experiments with laboratory animals (Wistar line rats). The induced neovascularization in rat muscles after the animals were administered the CELO-ANG viruses was shown.

[A protein of the annexin group in early fish development. I. Identification and biosynthesis of the protein in embryogenesis]. / Izuchenie belka gruppy anneksinov v rannem razvitii ryb. I. Identifikatsiia belka i ego biosintez v émbriogeneze.

Ca-binding proteins were isolated from the loach (Misgurnus fossilis) eggs and embryos, using their capacity for binding to acidic phospholipids in the presence of Ca ions. It was shown that the major protein present in a mixture of these proteins was synthesized at the early developmental stages. Polyclonal rabbit antibodies were raised against this protein. These antibodies proved to be monospecific both to the protein of the loach eggs and embryos and that of the Brachydanio rerio embryos. The antibodies were used for screening cDNA library from the B. rerio embryos at 6 to 9 h of development. As a result of screening, a cDNA clone was obtained, which, when converted into a peptide sequence, shows a high degree of homology with proteins of the annexin group.

[A study of annexin family protein in early development of fish. II. Analysis of complete amino acid sequence]. / Izuchenie belka gruppy anneksinov v rannem razvitii ryb. II. Opredelenie polnoi aminokislotnoi posledovatel'nosti.

We studied mRNA structure of 31 kDa annexin of zebra fish Brachydanio rerio using previously obtained 3'-terminal incomplete cDNA. The size of this protein mRNA was determined by Northern hybridization. PCR screening of cDNA library of zebra fish gastrula allowed us to obtain cDNA of the 5'-terminal regions of the mRNA. The primary structure of the protein deduced from the mRNA sequence allowed us to identify it as an annexin IV with threonine in position 6--a phosphorylation target for protein kinase C.

[Introduction of the human recombinant angiogenin at the early stages of postnatal development inhibits the growth of mice]. / Vvedenie rekombinantnogo angiogenina cheloveka na rannikh stadiiakh postnatal'nogo ontogeneza tormozit rost myshei.

We studied the influence of recombinant DNA containing the cloned angiogenin gene, plasmid DNA without angiogenin gene, and purified recombinant angiogenin injected to Tg8 mice at the age of two days on the body mass of 28- and 40-day old mice. The body mass of mice that were injected with the cloned angiogenin gene or purified angiogenin was less than in the control mice. The body mice of Tg8 mice injected with recombinant DNA containing the cloned angiogenin gene did not increase from day 2 to day 40, while in the mice with purified recombinant angiogenin and control mice it increased by 24 and 57%, respectively. These data suggest that the elevated level of angiogenin at the early developmental stages inhibits the increase of body mass. The effect we described as related, in al likelihood, to the known inhibitory effect of angiogenin on protein synthesis.

[Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells]. / Vliianie reguliatornykh genov virusa immunodefitsita cheloveka tipa 1 na proliferatsiiu i differentsirovku émbrional'nykh stvolovykh kletok myshi.

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.

[Production of proteinases in the process of developing cultures of Streptomyces thermovulgaris T-54]. / Produktsiia proteinaz v protsesse razvitiia kul'turury Streptomyces thermovulgaris T-54.

The secretion of some proteolytic enzymes by Streptomyces thermovulgaris T 54 was studied using artificial chromogenic substrates of proteinases. Maximum accumulation of the enzymes hydrolysing Z-Glu-pNA and Z-Ala-Ala-Leu-pNA occurred during autolysis of the culture. Two peaks of activity were observed, when DNP-Gly-Gly-Ile-Arg was used as a substrate, one of them being correspondent to prevalence of dense colonies in the culture and the other to the prevalence of friable networks of hyphae.

[Genetic engineering potential and perspectives in the management of critical ischemia]. / Vozmozhnosti i perspektivy lecheniia kriticheskoi ishemii s ispol'zovaniem genno-inzhenernykh tekhnologii.

The paper presents a new approach to management of lower limb critical ischemia which implements recent advances in molecular biology and genetic engineering technologies. A new original compound incorporating angiogenin gene was developed to activate neoangiogenesis processes after injection into living tissues. Experimental data evidence a potential efficacy of new method for complex management of critical ischemia.

Influence of pub Gene Expression on Differentiation of Mouse Embryonic Stem Cells into Derivatives of Ecto-, Meso-, and Endoderm in vitro.

The influence of low and high pub gene expression on the initial stages of the differentiation of mouse embryonic stem cells into derivatives of ecto-, meso-, and endoderm in vitro was investigated. As follows from the results of a RT -PCR analysis, the expression of the vimentin, somatostatin, GATA 4, and GATA 6 genes, being the markers of endodermal differentiation, does not vary in both the cells with high pub gene expression and the cells with low pub gene expression, as well as in the corresponding control lines. The cells with high pub gene expression are characterized by an increase in the expression of mesodermal differentiation gene-markers (trI card, trI skel, c-kit, and IL-7), whereas the cells with low pub gene expression are specified by a decrease in their expression. According to the analyses carried out, the reverse is characteristic of the expression of ectodermal differentiation gene-markers (nestin, ≤-III tubulin, gfap, and th). Expression of these genes decreases in cell lines with high pub gene expression, whereas their expression increases with the decrease in pub gene expression. Hence, it is suggested that the variations in the pub gene expression in the embryonic stem cells influence significantly the mesodermal and ectodermal differentiation of these cells.

Different Effects of Regulatory Genes (tat, nef) of Human Immunodeficiency Virus Type 1 (HIV-1) on the Proliferation and Differentiation of Mouse Embryonic Stem Cells in vitro.

To examine the effects of the tat and nef regulatory genes of human immunodeficiency virus (HIV-1) on cell differentiation we used the mouse embryonic stem cells (ESC) as a model. Proliferation, embryoid bodies (EB) formation and subsequent differentiation into cardiomyocytes, glial and neuronal cells were investigated in ESC lines transfected with these genes. It has been shown that the transfection of ESC by the tat gene increased their proliferating activity, whereas the nef gene transfected ESC showed its decrease. The number of embryoid bodies formed was higher in the cultures of ESC transfected by the nef and lower in the cells transfected by the tat in comparison with controls. The percentage of embryoid bodies with contracting cardiomyocytes was higher against control in the nef transfected cells and lower in ESC transfected with the tat. There were no reliable differences in the appearance of glial cells between control and the nef and tat transfected cell lines. Spontaneous differentiation of ESC into neuronal cells was almost not observed in the nef transfected cells, in contrast to control and the tat transfected cells. However, addition of retinoic acid (RA) to the nef transfected cells caused even a slight increase in neuron formation as compared to control ESC treated with RA. Thus, for the first time we have shown that the tat and nef regulatory genes of HIV-1 had a visible effect on proliferation of ESC and some first steps of their differentiation. In general, the reverse correlation between the effects of these two viral genes on ESC proliferation and differentiation were observed.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

[Physicochemical properties of a fibrinolytic enzyme from Streptomyces thermovulgaris culture broth]. / Fiziko-khimicheskie svoistva fibrinoliticheskogo fermenta iz kul'tural'noi zhidkosti Streptomyces thermovulgaris.

Some properties of fibrinolytic enzyme from cultural fluid of Streptomyces thermovulgaris have been studied. The molecule of enzyme has been shown to consist of one polypeptide chain with molecular mass 28000 dalton, pi = 7.45-7.6. The amino acid composition of protein is determined, the protein does not contain cysteine residues. The enzyme is not thermostable, and Ca2+ ion does not exert stabilized influence. In opposition to diisopropylfluorophosphate, phenylmethylsulfonyl fluoride does not inhibit the enzyme activity.

[Isolation and properties of the fibrinolytic enzyme from the Actinomyces thermovulgaris cultural broth]. / Vydelenie i svoistva fibrinoliticheskogo fermenta iz kul'tural'noi zhidkosti Actinomyces thermovulgaris

A fibrinolytic enzyme was isolated from the cultural broth of Actinomyces thermovulgaris T-54 by precipitation with ammonium sulphate to 0.8 saturation and chromatography on GE-cellulose and CM-cellulose. The enzyme is homogeneous as was confirmed by disk electrophoresis in polyacrylamide gel. Some properties of the enzyme were studied. A modification of the method is suggested to assay the fibrinolytic activity.

Protein kinase C and casein kinase 2 phosphorylate in vitro proteins of the annexin family from eggs of loach Misgurnus fossilis.

A mixture of proteins of the annexin family was obtained from the cytoplasm of mature eggs of loach Misgurnus fossilis (by reprecipitation with acid phospholipids in the presence of Ca2+). This mixture comprised five proteins with molecular weights of 58, 38, 36, 35, and 31 kD. Polyclonal rabbit antibodies against the major 31-kD protein were obtained. Western blot analysis showed that the obtained antibodies exhibit a high specificity towards the 31-kD protein from eggs and other tissues of loach and zebrafish (Brachydanio rerio). The analysis of cDNA corresponding to the 31-kD protein by screening the zebrafish cDNA library confirmed that this protein belongs to the annexin family. Phosphorylation of the obtained annexins in vitro was studied. It is shown that the 58-kD protein is phosphorylated by casein kinase 2 (CK2), whereas the 38-, 36-, 35-, and 31-kD proteins are phosphorylated by protein kinase C (PKC).