[Infrared fluorescent markers for microarray DNA analysis on biological microchip].

To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

[Preparation stimulating hair growth possibly acts by inhibiting hair follicle ageing experiments on mice and transcriptome analysis].

Preparation stimulating hair growth (PSHG) was studied on mice of various strains (Balb/c, CBA, C57BI/6, and outbred). It was shown that a long-term (44 months) application of PSHG does not reliably affect the appearance of young healthy mice but does induce increase in the hair follicle size. No adverse consequences of the PSHG application were observed. Naturally occurring propagating regenerative hair waves peculiar to mice were preserved. In older mice (more than 2 years) with signs of alopecia, application of PSHG caused an overgrowing of bald patches within two months. Transcriptome analysis of the PSHG effect performed in fibroblast cell culture showed that PSHG stimulates processes of tissue development and remodeling. These observations together with previous findings showing that PSHG stimulates autophagy and induces death of cells subjected to oxidative stress may suggest that the mechanism of the PSHG effect involves stimulation of regeneration of skin and its derivatives owing to more efficient elimination of senescent and damaged follicle cells.

[Stimulation of proliferation by carnosine: cellular and transcriptome approaches].

Concentration of endogenous dipeptide carnosine in human muscle tissue reaches tens of millimoles. For more than 100 years of research, a lot of data concerning carnosine functions were accumulated, among which anti-aging effects are regarded most important. Heire, effect of carnosine in cell cultures was studied. It has been found that apart from the known action--an increase of the Hayflick limit and morphological rejuvenation--carnosine stimulates cell division in colony-forming assays and in the course of transition of cells to the quiescent state. The analysis of the transcriptome showed that carnosine-induced changes are mainly related to positive regulation of the cell cycle at all levels, from the onset of the DNA synthesis to chromosome condensation. One can suppose that the revealed stimulation of the cell cycle account for the carnosine-induced rejuvenation processes and a high concentration ofcarnosine in muscle tissue is required for the muscle recovery (regeneration) after excess loads.

Modification of human fibroblast properties in microtransplant.

We studied the properties of human skin fibroblast in filamentous polyglycolic microtransplant. Fibroblast adhesion to the microtransplant filaments is followed by the formation of a network cross-linked with fibroblasts. The cells rapidly proliferate during the first few days; after transfer of the microtransplant to the standard culture flask, the cells migrate to the plastic and continue proliferation. The cells are uniform and exhibit high colony-formation capacity. The bundles of microtransplant filaments persist in the culture for several days and through the cells completely leave them, the area around these filaments remains the most populated for 40 days. Mitotic cells are seen in the immediate proximity to the degrading filaments of the transplant. The effect of cell "rejuvenation" in the microtransplant can be explained by selection of cells by their adhesion to relatively thin (about 15 µ) filaments, which excludes large old cells.

[Biophysical methods for biochip analysis. Use of wide field digital fluorescent microscopy].

This paper discusses an issue on the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitive throughput recording analyses by biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides the higher rate of the analysis compared to laser scanning devices. With this analyzer a scope where biological microchips can be applied becomes wider, the development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for the analysis performance can be reduced.

Comparative analysis of expression of human telomerase catalytic subunit at the transcription level in cell cultures of different origin.

The expression of human telomerase catalytic subunit in HL-60 and HT-1080 malignant transformed cells and telomerized fibroblasts was studied by quantitative PCR. It was found that the number of transcripts of human telomerase catalytic subunit per cell in telomerized fibroblasts could be hundreds of times higher than in HL-60 and HT-1080 cells. Telomerized fibroblast cultures are suggested as experimental systems for selection of basal compounds for creation of anticancer drug prototypes, the molecular target of which is human telomerase catalytic subunit. The effects of human telomerase catalytic subunit expression on the fibroblast proteome are analyzed.

[Analytical characteristics micromatrix semiquantitative method used in serial tests with video digital registration].

A multiplex analytical system has been developed to carry out microformat latex agglutination tests with video digital registration. The system makes it possible to apply less than 1 microl drops of latex and samples in the matrix format to a carrier and to make the test in 30 points at once, as well as to record and to interpret the results of the test, by keeping analytical information for each sample. Comparison of the results of determination of the serum levels of C-reactive protein, rheumatoid factor, and antistreptolysine in the system developed by the authors with the results obtained in the macroagglutination test by the immunoturbidimetric technique leads to the conclusion that the methods are comparable. The proposed type of a latex agglutination test based on a matrix approach and miniaturization assures efficient production and the quality of laboratory studies.

[Enhanced control of proliferation in telomerized cells].

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

[A scanner-based system for documentation, objective assessment, and recording of the results of latex-agglutination tests].

The present paper describes the use of the scanner "Expert-Lab Agglutination" system to record the results of latex-agglutination tests in making a reaction on the lids of 96-well immunoassay plates. The described system provides a possibility of having a primary image of all tests carried out on a carrier. Software offers the prospect of contrasting the images of individual and control samples, increasing the size of images, and comparing the latter. The use of special image treating techniques may also yield objective figures characterizing the rate of a reaction. The possibility of archiving primary information and retrospectively appraising the correctness of the result of an analysis at any required moment is of fundamental importance to the latex-agglutination test.

[Role of telomerase in reactivation of macrophage nuclei in heterokaryons].

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.

[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.

[Activity of c-myc protein is necessary in the first 6 hours of the prereplicative period for 3T3 Swiss cells]. / Aktivnost' belka c-myc neobkhodima v pervye 6 ch prokhozhdeniia prereplikativnogo perioda kletkami 3T3 Swiss.

It was shown what microinjection of polyclonal antibodies to the myc protein specifically inhibits DNA synthesis in serum-stimulated 3T3 Swiss cells during the first 6 h of the prereplicative period. The effect depends on the concentration of antibodies. Microinjections of polyclonal antibodies against the whole protein were more effective when microinjections of antibodies against parts of the protein. Microinjections of five kinds of monoclonal antibodies and their mixture were in effective. It was also shown what induction of expression of the antisense myc sequence in 3T3 Swiss cells leads to potent inhibition of DNA synthesis during the first 6 h of the prereplicative period. Thus it is clear what the myc protein participates in the early stages of preparation to replication, i.e., transition of cells from G0 to G1.

[Effect of azidothymidine on reactivation of DNA synthesis in macrophage nuclei contained in heterokaryons]. / Vliianie azidotimidina na protsess reaktivatsii sinteza DNK v iadrakh makrofagov v sostave geterokarionov.

EIn heterokaryons, DNA synthesis is reactivated in macrophage nuclei only in the case of fusion with immortal cells. Assuming that telomerase is responsible for reactivation, the effect of its inhibitor azidothymidine (AZT) was studied in heterokaryons of mouse resident peritoneal macrophages and immortal 3T3 Swiss cells. AZT suppressed reactivation of DNA synthesis in macrophage nuclei and had no effect on DNA synthesis in 3T3 Swiss cell nuclei, suggesting an altered telomere structure in normal mouse macrophages.

[Positive and negative regulation of replication in hybrid cells]. / Pozitivnaia i negativnaia reguliatsiia replikatsii v gibridnykh kletkakh.

DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non-malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed.

[Proliferative senescence of embryo fibroblasts of Japanese senescence accelerated mice (SAM) is accompanied by parallel decreasing of response to various growth factors]. / Proliferativnoe starenie émbrional'nykh fibroblastov iaponskikh uskorenno stareiushchikh myshei (SAM) soprovozhdaetsia parallel'nym oslableniem otveta na razlichnye rostovye faktory.

The Japanese senescence accelerated mice (SAM) are a group of the low-longevity mouse lines and represent a new convenient model for studying the senescence process. We studied the proliferation of embryo fibroblasts of SAMP1 and SAMR1 mouse lines. It was shown that fibroblasts of the shortest longevity line SAMP1 have a markedly decreased proliferative potential of the mean 8.7 population doublings, whereas fibroblasts of a relatively high-longevity line SAMR1 have an average proliferative potential of 12.3 doublings. The fibroblast senescence in both lines is accompanied by a simultaneous lowering of the cell proliferative response to the blood serum, epidermal, fibroblast, and platelet-derived growth factors. At initial stages of the cell culture growth, lines SAMP1 and SAMR1 exhibit the same reactions to growth factors, but already beginning from the fifth doubling, the SAMP1 cell response is sharply decreased as compared with SAMR1. Lowering the proliferative reaction is accompanied by a decreased phosphorylation of tyrosine in the cell proteins responsible for mitogenic reaction. Thus, the parallel decrease of proliferative response to different growth factors during fibroblast senescence is most likely due the emergence of a regulatory block at common stages of the mitogenic signal transduction.

[Azidothymidine, blocking telomerase functioning, shortens telomeric repeats in transformed human cells]. / Azidotimidin, blokiruia funktsiiu telomerazy, umen'shaet dlinu telomernykh povtorov v transformirovannykh kletkakh cheloveka.

The long-term action of azidothymidine, reverse transcriptase inhibitor, on cultivated U-937 (human promyelocyte leukemia) and MeWo (human melanoma) cells led to the concentration-dependent decrease in the length of telomeric chromosomal repeats. Telomere shortening was accompanied by temporary retardation of cell proliferation. Combined with the data obtained previously, these results suggest that azidothymidine inhibits telomerase functioning in cultivated cells.

[The nature of a proliferation block in differentiated cells with heterokaryons as a model: various types of absence of proliferation in cells in terminal differentiation]. / Issledovanie prirody bloka proliferatsii v differentsirovannykh kletkakh na modeli geterokarionov: razlichnye tipy otsutstviia proliferatsii v kletkakh pri terminal'noi differentsirovke.

Heterokaryons obtained by fusion of proliferating and terminally differentiated cells were studied. The data obtained suggest that mechanisms of proliferation arrest are different in macrophages on one hand and nucleate erythrocytes and polymorph leukocytes on the other. Macrophages appeared to be devoid of factors preventing replication in nontransformed and spontaneously immortalized cells. Inhibition of proliferation was probably due to certain modifications of macrophage genome which arise during differentiation and can be compensated by the effect of "immortalizing" oncogenes. On the contrary, nucleate erythrocytes and polymorphs evidently contain some factors mediating negative control of proliferation. For reactivation of DNA synthesis in these cell types after fusion with other cells the latter did not have to be immortalized. After cell fusion macrophages specifically inhibit DNA synthesis in cells containing active oncogenes.

[Telomerization as a method of obtaining immortal human cells preserving normal properties]. / Telomerizatsiia--sposob polycheniia immortal'nykh kletok cheloveka, sokhraniaiushchikh normal'nye svoistva.

Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.