[Regulated expression systems for gene therapy].

Gene therapy is a perspective and rapidly developing field of modern medicine, which is expected to improve state of or even cure patients that are not curable by classical methods of therapy. The logics of development of gene therapy in the near future will require the use of systems in which expression of therapeutic gene can be regulated. This review critically evaluates current regulated systems of gene therapy that can be divided into two major classes. The first class is formed by systems in which induction of therapeutic gene expression is effected by an external inducer introduced into an organism. The second class includes systems based on autoregulation principle that function without an external inducer. The review evaluates the most important systems of gene expression regulation belonging to the first class, namely systems based on tetracycline regulation, dimerization induced by rapamycin derivatives, regulation by steroid hormones, regulatory RNAs and physical principles. The most important systems of the second class that are regulated by oxygen or glucose levels are also discussed.

[Mesenchymal stem cells as an antitumor therapy tool].

Development of antitumor preparations with low toxicity and high selectivity of action is one of the top priorities of cancer gene therapy. Mesenchymal stem cells possess natural tropism towards tumors, a property that makes possible their use as a vehicle for targeted delivery of therapeutic genes into tumors of various etiologies. At present, genes encoding enzymes (cytosine deaminase, thymidine kinase, carboxyl esterase), cytokines (IL-2, IL-4, IL-12, IFN-beta) and apoptosis inducing factors (TRAIL) are used as therapeutic genes. Mesenchymal stem cells, as demonstrated using experimental models of tumors of various etiologies as well as animals with metastases in brain and lungs, are able to successfully deliver therapeutic genes into tumors and produce significant antitumor effect. However, to effectively use this therapeutic strategy in clinic, one still has to solve a number of technical problems.

[Cytotoxicity of lysomustine and its isomers, and their potential use for selection of cells].

N epsilon-Nitroso-N epsilon- [N'-(2-chloroethyl)carbamoyl]-L-lysine (I) and N epsilon- [N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-lysine (II), the isomers being the constituents of antitumor agent Lysomustine, were obtained by RFHPLC. The study of cytotoxicity of the above compounds against K562 cells showed that the lesions induced by isomer (II) produce a significant cytotoxic effect but can be efficiently repaired by the action of MGMT (O6-methylaguanine DNA methyltransferase). Under similar conditions, the lesions induced by isomer (I) produce substantially smaller effect but are weakly if at all repairable by MGMT. The effects of a clinically approved agent Lysomustine, which is the mixture of isomers (I) and (II), are similar to those of isomer (II). The results obtained point to a different chemical nature of DNA lesions induced by two Lysomustine isomers. Our data indicate that Lysomustine and its isomer (II) can be used for in vitro selection of cells expressing MGMT.

[Involvement of guanine nucleotide exchange factor xLARG in epiboly of cells of the animal pole of Xenopus laevis embryos].

The development of multicellular organisms is a complicated coordinated process of the movement of groups of embryonic cells, which is controlled by many regulatory systems. At present little is known about the regulation of the earliest manifestations of the movement in the embryogenesis: epiboly and radial intercalation. The coordinators of these processes may be small GTPases of the Rho family and their activators, the factors of exchange of guanylic nucleotides. It has been shown in this work that the overexpression of the factor of exchange of guanylic nucleotides xLARG in Xenopus laevis embryos leads to an increase in the amount of the active form of xLARG. In addition, an increase in the expression of xLARG disturbs the process of radial intercalation. The data obtained suggest that xLARG is involved in maintaining the xLARG activation level necessary for the occurrence of epiboly.

[Molecular mechanisms of germ cell line determination in animals].

In the present work, principles of formation of germ line cells are reviewed. Germ line cells separate themselves from the rest of the embryo at the early stages of embryogenesis. In certain animal groups, formation of precursors of germ cells occurs by induction by surrounding cells. However, for most animal taxons, formation of primordial germ cells (PGCs) is determined by inheritance of certain maternal determinants--the so-called germ plasm. It is formed by mitochondria, electron-dense granules with the complex structure, and maternal RNAs and proteins necessary for formation of germ line. In Xenopus, the source of material for germ plasm is a mitochondrial cloud, which also specifically binds and transports to the vegetal pole maternal RNAs important for PGC formation. Cis elements determining the transport of these RNAs are usually located in the 3' untranslated region of RNA, and their function is mediated by binding of trans acting protein factors. In addition to a specific localization of certain macromolecules in germ plasm, special status of germ line cells is provided by degradation of RNA and protein components of germ plasm in somatic cells, silencing of transcription in PGCs until advanced stages of embryogenesis, and specific regulation of RNA translation in somatic and germ cells. In this review, we also briefly discuss results obtained by authors regarding the properties of a novel component of Xenopus germ plasm, namely maternal RNA germes, and encoded protein.

[Immunocytochemical study of the hcs2 gene products distribution in the command neurons of grape snail]. / Immunotsitokhimicheskoe izuchenie lokalizatsii produktov gena hcs2 v komandnykh neironakh vinogradnoi ulitki.

In the present study the cellular and subcellular distribution of putative protein products of hcs2 gene in the giant command neurons of parietal ganglia of the terrestrial snail Helix lucorum L. were investigated using light- and electron-microscopic immunocytochemistry. The product of hcs2 gene is a hybrid precursor protein belongs to the EF-hand family of the Ca(2+)-binding proteins, whose processed products are neuropeptides. By use of polyclonal antibodys against a synthetic CNP3, CNP4 and C-terminus peptide immunoreactivity was observed in the cytoplasmic secretory granules. The colloidal gold density in the granules for CNP3-4 neuropeptides was twice one for the Ca(2+)-binding protein. These immunocytochemical results point to a specific connection between putative protein products of hcs2 gene and the cell secretory apparatus, that correspond to our early expressed hypothesis that products of hcs2 gene act as neuromodulators or neurotransmitters.

[Biologic doses of sun ultraviolet in various geographic regions of Ukraine]. / Biodozy solnechnogo ul'trafioletovogo oblucheniia v razlichnykh geograficheskikh zonakh Ukrainy.

The values of minimal erytemal doses (MED) were determinated for solar ultraviolet (UV) for two geographic zones of Ukraine taking in account the seasons of a year. It was calculated a daily energetic loud produced by solar UV in summer for persons in outdoor. The idea concerning seasonal alternations of human individual sensitivity to solar UV was established.

[Stem cells: properties and perspectives of therapeutic use]. / Stvolovye kletki: svoistva i perspektivy ispol'zovaniia v meditsine.

In the present work, we review the properties of some stem cell types, namely embryonic, hematopoietic and mesenchymal stem cells, which present the most significant interest for use in medicine. Stem cells are undifferentiated cells capable of both self-maintenance and differentiation into mature specialized cells. According to their origin, stem cells can be classified as embryonic and somatic ones. The first ones can be indefinitely maintained in culture, and possess the ability to differentiate into all cells of the adult organism. The second ones possess the limited capacity to differentiate and, probably, a limited proliferative potential. For therapeutic use, important but hotly debated is the plasticity of somatic stem cells, i.e. context-dependent differentiation into "non-related" cell types. It is assumed that the differentiation of the majority of stem cell types proceeds according to the principle of stepwise hierarchical maturation through the stage of intermediate rapidly proliferating progenitor cells. The use of stem cells in medicine is mostly at the preclinical stage now. Despite the fact that embryonic stem cells are highly promising as therapeutic agents, a number of circumstances substantially limits their therapeutic use in the near future. At the same time, approaches involving autotransplantation of hematopoietic or mesenchymal stem cells are beginning to be applied successfully in the clinical trials for treatment of limb ischaemia and myocardial infarction. It is clear that despite a large number of problems and unsolved questions, the use of stem cells in medicine promises a dramatic progress in the treatment of many severe diseases.

[A novel method of subtraction cDNA hybridization allowing cloning of genes showing differential expression in cell micropopulations]. / Novyi metod vychitaiushchei gibridizatsii kDNK, pozvoliaiushchii klonirovat' geny, differentsial'no ékspressiruiushchiesia v mikropopuliatsiiakh kletok.

A novel, efficient and simple technique that combines subtractive hybridization with kinetic enrichment is proposed for obtaining enriched cDNA. The method is based on the use of a set of special primers that allow for the selective amplification by PCR only of differentially distributed sequences. Using the proposed technique, cDNA of a new gene XEp-1, specifically expressed in the presumptive epidermis of Xenopus laevis, was cloned, starting with the stage of midgastrula.

[A new method of comparative analysis of gene expression and identification of differentially expressed mRNA]. / Novyi metod sravnitel'nogo analiza ékspressii genov i identifikatsii differentsial'no ékspressiruiushchikhsiia mRNK.

A novel method of comparative gene expression analysis is proposed which is based on representing a population of expressed mRNA sequences in the form of a set of discrete cDNA restriction fragments. By means of selective isolation of 3' terminal cDNA fragments, each mRNA species is represented by no more than one cDNA fragment of specific length and sequence. Populations of cDNA fragments from different cell types are separated by high-resolution gel electrophoresis, and the separation patterns are compared. Using the proposed approach, fragments of two genes differentially expressed in murine thymus and spleen were identified and cloned. One of the genes was found to encode the terminal deoxynucleotidyltransferase, the there is apparently a heretofore unknown gene.

[Optimal chips for megabase DNA sequencing]. / Optimal'nye chipy dlia megabaznogo sekvenirovaniia DNK.

The SHOM method (Sequencing by Hybridization with Oligonucleotide Matrix) developed in 1988 is a new approach to nucleic acid sequencing by hybridization to a octanucleotide matrix composed of an array of immobilized oligonucleotides. The original matrix proposed for sequencing by SHOM had to contain at least 65,536 octanucleotides. The present work describes a new family of matrices for sequencing, which allows one to reduce the number of synthesized oligonucleotides 5-15 times without essentially decreasing the resolving power of the method.

[Chromosomal proteins in chick embryo erythrocytes on transcriptionally active and inactive genes]. / Khromosomnye belki éritrotsitov émbrionov kur na transkriptsionno aktivnykh i neaktivnykh genakh.

Using chicken embryonic erythrocytes as a model, an experimental scheme for comparing the density of linker histones and high mobility group proteins on single-copy sequences of eukaryotic genome has been developed, thus permitting to probe alterations in the chromosomal protein pattern of transcribing chromatin. The report provides experimental evidence for validity of intracellular DNA-protein cross-linking, immunoaffinity chromatography and hybridization with single-stranded probes. Depletion of linker histones and enrichment of HMG 14/17 were shown to be the discriminating feature for transcriptionally active globin gene chromatin as opposed to inactive ovalbumin and lysozyme gene chromatin.

[The structure of nucleosomal core particles located on the transcribed genome regions]. / Struktura minimal'nykh nukleosom, raspolozhennykh na transkribiruemykh uchastkakh genoma.

The arrangement of histones along nucleosomal DNA within particular active and inactive genome regions was analysed by using protein-DNA crosslinking methods combined with hybridization tests. Two-dimensional gel electrophoresis was employed to compare the nucleosome conformation and nucleosomal DNA size. The arrangement of histones along DNA and general compactness of nucleosomes were shown to be very similar in transcriptionally active and inactive genomic regions. On the other hand, nucleosomes within transcriptionally active chromatin are characterized by a somewhat larger size of nucleosomal DNA produced by micrococcal nuclease digestion and some peculiarity in electrophoretic mobility. It appears that changes in transcribed chromatin structure (such as an enhanced nuclease sensitivity) occur on the supranucleosomal level rather than in the nucleosomal core structure.