[Problems and prospects of cell therapy for critical ischaemia of lower limbs]. / Problemy i perspektivy kletochnoi terapii kriticheskoi ishemii nizhnikh konechnostei.

BACKGROUND: Cell therapy was proposed as a procedure of indirect revascularization for patients with critical ischaemia of lower extremities for whom endovascular and surgical revascularization is impossible. We present herein a review of the state of the art of studies in the field of cell therapy of this cohort of patients. BASIC PROVISIONS: Cell therapy has proved safe, however, the results of studies of efficacy are relatively ambiguous and unconvincing. The number of patients in separately taken clinical trials is minimal. The reviewed studies differed not only by heterogeneity of the cell types used but by the routes of administration of cells (cells were delivered either intramuscularly (predominantly) or intraarterially) and the duration of follow up (time of assessment and duration of follow up varied from 1 month to 2 years). One of the problems became the lack of the routine study of the angiogenic potential of stem cells prior to their clinical application. It is known that the angiogenic activity of multipotent cells of apparently healthy patients may differ from that of patients suffering from atherosclerosis, chronic renal failure, diabetes. CONCLUSIONS: It is supposed that treatment with stem cells or precursor cells is more efficient as compared to protein or gene therapy not only owing to direct vasculogenic properties but a paracrine action through excretion of proangiogenic biologically active substances. More studies with larger cohorts are necessary to provide stronger safety and efficacy data on cell therapy. Besides, a promising trend in the field of cellular approaches is modulation of regenerative capability of stem cells, which may help overcome difficulties in understanding the place of cell therapy in therapeutic angiogenesis.

[Functional analysis of the Xist promoter region in mouse Mus musculus].

X-chromosome inactivation which takes place in early embryogenesis of all higher mammals is largely determined by the Xist gene activity. This gene encodes long untranslated RNA, which provides transcriptional silencing of the genes on chromosome. In the present study, three enhancer and three silencing transcriptional elements were identified in the Xist promoter region. In these regions, location of putative transcription factors was demonstrated, including the ER site, which was discovered in two positions. The effect of estradiol and retinoic acid on the promoter activity was investigated. The estradiol-induced increase of the promoter activity was demonstrated. A model of the estrogen effect on X chromosome inactivation was suggested.

[Expression of early developmental genes in vole Microtus rossiaemeridionalis].

The expression of genes Sox2, Klf4, Myc, Sall4, Gata6, Foxa2, Hnf4a, Cdx2, Esrrb, Hand1 in cultivated cells, embryos and organs of adult voles Microtus rossiaemeridionalis was studied. High resemblance of the expression patterns of these genes in the organs of adult voles, mice and humans was demonstrated. It was established that genes Gata6, Foxa2 and Hnf4a were specifically expressed in vole extraembryonic endoderm cells, while Cdx2 and Handl genes, in trophoblast stem cells. This shows that these genes can be used markers for corresponding vole cell lines. Indirect confirmation pointing to the fact that Oct4 gene is a marker gene for epiblast cells both in the vole and mouse was obtained.

[Investigation of the Tsix gene regulatory region in vole Microtus rossiaemeridionalis].

The Tsix regulatory region was examined in vole Microtus rossiaemeridionalis. The minimal promoter region, three potential enhancer regulatory elements and one transcription suppressor element were identified. The enhancer regions contained potential binding sites of transcription activators, while in the region of putative silencer contained potential binding site of the ARP1 (NR2F2) protein. This protein can play the role of either activator or repressor depending on the promoter context.

[Role of G(-43)A polymorphism in the promoter region of the Xist gene in non-random X-chromosome inactivation in intraspecific hybrid voles].

Interaction of transcription factor CTCF with the minimal promoter of Xist gene was investigated in intraspecific hybrids ofcommon voles. CTCF was shown to bind with the minimal promoter region in vivo. However, the experiments of the delay in gel resulted in the absence of interaction between the CTCF factor and its potential binding site. Probably, G(-43)A substitution influences binding efficacy of another transcription factor such as activator protein 2, AP2.

[Comparative analysis of the DXPas34 regulatory region in rodents].

Mouse X chromosome inactivation center contains the DXPas34 minisatellite locus which plays an important role in expression regulation of the Tsix and Xist genes, involved into female dosage compensation. Comparative analysis of the DXPas34 locus from mouse, rat, and four common vole species revealed similar organization of this region in the form of tandem repeat blocks. A search for functionally important elements in this locus showed that all the species examined carried the conservative motif monomers, which could be involved in regulation of X inactivation.

[Induced pluripotent stem cells].

Induced pluripotent stem cells (iPS) result from a reprogramming of somatic cells via transduction with viral vectors expressing the Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28 genes, which are essential for the establishment and maintenance of the pluripotent state. In properties, iPS are almost fully similar to embryonic stem cells (ESC). To date, iPS have been obtained from various differentiated cells of mice and humans. Along with ESC, iPS are highly promising for research and medicine.

[OCT4 and NANOG are the key genes in the system of pluripotency maintenance in mammalian cells].

Embryonic stem cells are able to give rise after differentiation to derivatives of three germinal layers (ectoderm, endoderm, and mesoderm) and to functional gametes. This property of cells is referred to as pluripotency. The pluripotent status of preimplantation embryo cells and embryonic stem cells is maintained by a complicated system of molecular signaling pathways and transcription factors. The key regulators in this system are the transcription factors OCT4 and NANOG. The role and place of these factors in the pluripotency-sustaining system and their interaction with other factors are considered in the review. Data are presented on the structure, chromosomal location, expression, and regulation of the Oct4 and Nanog genes in mammals.

[Extraembryonic endoderm stem cell lines from common voles of the genus Microtus].

Twenty-eight independent extraembryonic endoderm (XEN) stem cell lines have been obtained from morula and blastocyst cells of common voles. Most cell lines form very few cell-cell contacts when growing and morphologically correspond to the XEN that were earlier described in mice. In addition, XEN cell lines with atypical morphology forming colonies have been obtained for the first time. Both types of XEN lines rapidly proliferate, retain their morphology and karyotype during more than 25 passages in cell culture, and express genes characteristic of XEN. One of two X chromosomes in XEN lines with karyotype XX has been shown to be inactive and associated with the Xist gene transcript. It has been demonstrated that the paternal X chromosome is inactive.

[The structure and evolution of the MaSMC4 gene of common vole Microtus arvalis (Arvicolidae, Rodentia)].

In eukaryotes, the SMC (Structural Maintenance of Chromosomes) gene family is represented by at least six genes. Some of these genes have tissue-specific homologs. Eukaryotic SMC structural proteins are the members of biochemical complexes responsible for cohesion of sister chromatids, recombination, repair, regulation of gene expression, and formation of mitotic chromosomes. In the present study, the structure of the SMC4 sub-family gene was examined in common vole Microtus arvalis. Comparative analysis of rodent (M. arvalis, Mus musculus. and Rattus norvegicus), human, and Xenopus SMC4 orthologous genes was carried out, and the main patterns of their organization and regulation were established. The SMC4 genes contain 24 exons; open reading frame starts at exon 2. The SMC4 5' regions contain the CpG islands, extending in the region of exon-intron I and exon 2. The SMC4 genes are characterized by the presence of multiple transcription startpoints. The region of the major transcription startpoint contains the INR CCA,1TTTT element. The SMC4 5' region is characterized by the presence of putative binding site for basal transcription factor Sp and factor E2F, typical of the genes induced in the G I/S phase of the cell cycle. The divergence level of the SMC4 coding region was examined. The mean Ka/Ks ratio for the SMC4 genes examined was 0. 123. The region of exon 2 was found to be the most variable (Ka/Ks = 0.715), while the most conservative was the region coding for the C-globular domain, which contained the DA box (Ka/Ks = 0.024).

[Chromatin modifications during X-chromosome inactivation in female mammals].

In female mammals, the process of dosage compensation occurs during early embryonic development. As a result of this, one of X-chromosomes becomes transcriptionally inactive. This process is accompanied by chromatin remodeling on inactivated X-chromosome, providing transcriptional silencing of the genes and maintenance of their inactive state. In the present review, the dynamics of modifications occurring during embryonic inactivation, their distribution over the inactive X-chromosome, interaction, and the role in establishing and maintening the inactive state are discussed. As an illustration, modifications on the inactive X-chromosome of the Microtus common vole are presented.

[Evolution of mammalian sex chromosomes: cooperation of genetic and epigenetic factors]. / Evoliutsiia polovykh khromosom mlekopitaiushchikh: vzaimodeistvie geneticheskikh i épigeneticheskikh faktorov.

The X and Y chromosomes of mammals, which significantly differ in structure and genetic composition, are thought to originate from a pair of autosomes. During evolution of sex chromosomes in placental mammals, the degradation of the Y chromosome and inactivation spreading along the X chromosome occurred gradually and in concert. Thus, at the molecular level, the genetic and epigenetic factors interacted toward greater differentiation of the X/Y pair. In this review, in context of a comparison permitting to trace this evolutionary pathway, we consider the structural features of mammalian sex chromosomes focusing on the X-chromosomal genes and the unique epigenetic mechanism of their regulation. Possible causes and consequences of the genes skipping inactivation and aspects of molecular mechanism of X-chromosome inactivation are discussed. A number of hypotheses are considered on evolutionary relationships of X-chromosome inactivation and other molecular processes in mammals.

[SMC (structural maintenance of chromosomes) structural protein family and their role in chromatin reorganization]. / Strukturnye belki semeistva SMC (structural maintenance of chromosomes) i ikh rol' v reorganzizatsii khromatina.

Structural chromatin proteins of the SMC (Structural Maintenance of Chromosomes) family play an important role in structural DNA reorganization in pro- and eukaryotes. Eukaryotic SMC proteins are the core components of the cohesin and condensin complexes. The cohesin complex is responsible for sister chromatid and homolog cohesion in mitosis and meiosis. The condensin complex uses ATP energy to induce positive coiled-coils in DNA, which results in compaction of the latter and formation of mitotic chromosome scaffold. In addition, the SMC proteins constitute recombination and recombination repair complexes. In hermaphrodites of nematode Caenorhabditis elegans, the SMC protein-containing complex controls dosage compensation and inactivation of the X chromosome genes.

[Isolation of ES-like lines of common voles of the genus Microtus from blastocysts and germ cells and as a result of the fusion of somatic cells with mouse embryonic stem cells]. / Poluchenie ES-podobnykh linii obyknovennykh polevok roda Microtus iz blastotsist, germinal'nykh kletok i ot sliianiia somaticheskikh kletok s émbryonal'nymi stvolovymi kletkami myshi.

Three and four independent cell lines with limited pluripotency were obtained from the inner cell mass cells of blastocysts and primordial germ cells of common voles, respectively. The results of cytogenetic analysis suggest that all these lines originated from the embryos of F1 Microtus rossiaemeridionalis x M. arvalis males and had a great number of near-triploid cells already during the early passages. The cells of these lines, like those of the inner cell mass, were characterized by the alkaline phosphatase activity. Nine independent cell lines were obtained as a result of hybridization of the mouse embryonic stem cells and vole splenocytes: eight lines and one line from hybridization with the M. kirgisorum and M. rossiaemeridionalis splenocytes, respectively. The cells of these lines expressed some properties of embryonic stem lines had a chromosome complement similar to the sum of two initial diploid sets of the mouse and vole.

[Gene for structural proteins of the SMC family in the common vole Microtus arvalis]. / Geny strukturnykh belkov semeistva SMC u obyknovennoi polevki Microtus arvalis.

Genes for four subfamilies of SMC (structural maintenance of chromosomes) proteins have been isolated from the genome of a common vole Microtus arvalis. The high degree of homology between representatives of each SMC protein subfamily of different classes of organisms has been demonstrated. The full-sized copy of a mammalian gene encoding SMC4 protein has been isolated and analyzed for the first time. The SMC proteins enter into the composition of complexes responsible for cohesion of sister chromatids, formation of mitotic chromosomes, recombination, DNA repair, and regulation of gene expression. We discuss the possible participation of the SMC proteins in inactivation of the X chromosome in mammalian females. Common voles of genus Microtus group "arvalis" serve a unique model for the study of the inactivation process.

[Organization of extended repeats in heterochromatin in sex chromosomes in the common vole species Microtus group "arvalis"]. / Organizatsiia protiazhennykh povtorov v geterokhromatine polovykh khromosom u obyknovennykh polevok roda Microtus gruppy "arvalis".

Two long repeats, MS3 and MS4, are predominantly located in sex-chromosomal heterochromatin in common vole species. Their tandem arrangement was revealed by means of the PCR analysis of genomic DNAs of four Microtus species and by restriction mapping of clones selected from a M. rossiaemeridionalis genomic library. Several mobile elements proved incorporated in a monomeric unit of each repeat and amplified together with its other components. In addition, LINE inserts were found in MS4 tandem arrays. The copy number of both repeats per haploid genome was estimated at 100-300 for euchromatin and 20,000-40,000 for the M. rossiaemeridionalis genome. The repeats were assumed to be the major component of sex-chromosomal heterochromatin DNA.

[High-resolution GTG-banding and nucleolar organizer regions of chromosomes of two vole species: Microtus rossiaemeridonionalis and M. transcaspicus (Rodentia, Arvicolidae)]. / GTG-okraska vysokogo razresheniia i iadryshkoobrazuiushchie raiony khromosom dvukh vidov obyknovennykh polevok: Microtus rossiaemeridionalis i M. transcaspicus (Rodentia, Arvicolidae).

With the use of the GTG-banding of prometaphase chromosomes, 503 and 402 segments were revealed in haploid chromosome sets of voles Microtus rossiaemeridionalis and M. transcaspicus, respectively. Based on a detailed study of chromosomes at different condensation levels, idiograms of M. rossiaemeridionalis and M. transcaspicus chromosomes were constructed. Sequential Ag-staining and GTG-banding allowed nucleolar organizer regions (NORs) to be localized in 16 and 11 chromosome pairs of M. rossiaemeridionalis and M. transcaspicus, respectively.