[Response of HeLa cells to mitomycine C. III. The analysis of nucleoli of mother and daughter cells].

The comparative analysis of the number of nucleoli in cells of the established HeLa-M line was carried out before and after exposure to mitomycin C in a concentration of 10 µg/ml for 2 h. Using time-lapse microscopy, nucleoli in mother and their respective daughter cells were computed. It has been shown that the average number of nucleoli per cell is generally higher in daughter cells than in mother cells, and a standard deviation, on the contrary, decreases. An average number of nucleoli in daughter cells, whose mother cells had been treated with mitomycin C, was higher than in corresponding cells of control group. The separate analysis has been performed for the cells having from 1 to 4 nucleoli. Nonrandom complete coincidence of the number of nucleoli in mather and daughter cells has been typicaly shown for about 1/7 of the total cell population. Mitomycin C reduces this value of about 1.5 times.

[Colocalization of nucleoli in cell nuclei of HeLa line].

The pattern of localization of nucleoli relative to each other and to cell nucleus was studied in M-HeLa cell line. For this puspose, the following morphometric parameters were introduced. For the two-nucleolar cells: 1) the ratio of the nucleus long axis to the length of a segment between the centers of the nucleoli, and 2) the angle between the segment connecting the centers of the nucleoli and a longitudinal axis of cell nucleus. For the three-nucleolar cells: the ratio perimeter of the nucleus to perimeter of a triangle with vertexes in the centre of nucleoli. We have shown that the values of these parameters are individual for each cell but their values remain constant for the cell in spite of the changes in cell shape. These results allow us to conclude that, on the one hand, the nucleoli colocalization is individual for each cell, and, on the other hand, location of nucleoli in relation to nucleus is not changed during interphase. Thereby, the distance between nucleoli increases proportionally with nucleus growth.

[Morphology of NCTC cells upon a contact with type I collagen added to culture medium].

The morphometric characteristics of NCTC cells upon their contact with type I collagen added to culture medium were studied. The cells were plated on plastic in the colony form. In a day after seeding, the culture medium was changed for the same medium complemented with 0.1% type I collagen. And the cells were incubated in this medium for 30 min more. Then the cells were washed out of collagen. Using time-lapse microscopy, the cell state at a colony edge was registered during 7 h. The area, spreading, and polarization of the cells were evaluated. It is shown that the contact with collagen did not affect the cell area, decreased cell spreading and sharply reduced a portion of polarized cells. These results probably demonstrate sensitivity of NCTC cells to the presence of type I collagen in culture medium and suggest that the cell response involves inhibition of long filopodia formation.

[Response of HeLa cells to mitomycine C. I. Cell division].

Using light microscopy, time-lapse imaging, and digital image analysis, the effect of mitomycine C (10 µg/ml) on HeLa-M cells has been studied. It has been shown that, after a 2 h contact with mitomycine, the cells could be separated into 2 groups: M-1--the functional cells surviving after division but non-entering mitosis any more; M-II--the cells entering mitosis but incapable to finish it; they are lost. Mitomycine C is known to specifically block DNA replication being located in the DNA minor groove. It should inhibit PHK synthesis if one follows the standard hypothesis of a transcription bubble formation. However, increasing the cell and nucleolus area during the M-I cell growth suggests that RNA and protein synthesis is not blocked. The author concludes that the presented data confirm his hypothesis about RNA synthesis in the major DNA groove (Petrov, 2006).

[Vital measurement of optical density of HeLa cell line].

Light absorption by the live intact HeLa cells during light microscopy was studied. The light absorption may be considered as a parameter analogous to optical density used in spectrophotometry. This parameter can be used as a quantitative characteristic of life cell as well as intracellular structures. It is shown that cells from one population but belonging to two different clones were differed by their optical density. Optical density correlation between the shadow peripheral regions of the cells and a actin localization in these regions was established.

[Response of HeLa cells to mitomycine C. II. Morphometry of the cells].

HeLa-M cells were analyzed after the 2h incubation in the medium with mitomycin C (10 µg/ml). It has been shown that a part of the cells contacted with the cytostatic agent passes mitosis normally, but the daughter cells no longer divide. During the observation period (2 days), the area of the cells increased linearly reaching twice the size of intact cells. Thereby spreading of the cells contacted with mitomycine decreased, and their polarisation was not changed. Along with the increasing cell size, the nucleus size is also increased indicated de novo protein synthesis. Analysis of nucleoli revealed a small decrease of their area during the first hours after the contact with mitomycine followed by an increase of the area. Finally, the nucleolus area exceeded that of control cells, which can only happen if rRNA was synthesized. On the other hand, DNA cross-linking by mitomycine should inhibit transcription because the mechanism of transcription is assumed to involve local melting of DNA within its minor groove. This contradiction can be overcome by assuming that transcription occurs in a DNA major groove without separation of the chains.

[Effect of type I collagen and fibronectin on cell morphology of human MSCs in vitro].

Limited knowledge about behaviour of stem cells in culture seems to be one of the reasons for problems in their successful introduction to applied medicine. To address this issue we have studied in vitro interaction of human mesenchymal stromal cells (MSCs) with various substrates (plastic, type I collagen, fibronectin, and mixtures of these proteins at various ratios) during the 16-18 h after cell plating. Several cell morphology features such as Area, Perimeter, spreading coefficient, polarization coefficient were determined. It has been shown that MSCs respond specifically to the substrate and can be classified into several groups according to the parameters studied. Collagen preferably fibronectin have opposite effects on polarization and spreading of the cells. Collagen preferably enhances polarization of the cells, whereas fibronectin stimulates proportional spreading of cells. Effect of collagen-fibronectin mixture on the cells cannot be considered as a simple additive effect. We assume that variation in the ratio of these proteins in the extracellular matrix might be one of the possible ways to influence the morphology of stem cells when they are induced to differentiate.

[Correlation between growth of callus and a number of nodule for pea Pisum sativum].

The processes of nodulation and callusing were studied for pea Pisum sativum in a comparative aspect. Seven varieties of pea plants were used. The nodulation was characterized by the number of nodules and their mass per one plant. The frequency ratio of nodules of various weights was described by the Lorentz equation. The growth of callus was described using equation of the S-dependence. It has been shown that plants can produce maximal (optimum for nitrogen fixation) mass of nodules by an increase either in the nodules number or in the mass of each nodule. This feature is specific to the varieties of peas. It has been also found that the plants of the pea varieties, which have a large number of nodules, produce calluses of maximum size. Small calluses are characteristic of the varieties having plants with small amounts of large nodules. The data obtained has been interpreted on the assumption that the nodulation is underlined by both differentiation and proliferation, whereas the callusing is due only to the proliferation process.

[Dynamics of spreading of cells of L-929 line after the mitosis].

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".

[Two spreading states of mesenchymal cells of the human embryo in vitro].

Spreading mesenchymal cells of human embryo on plastic and type I collagens (from rat, sheep and bull) was studied. Spreading of the cells on collagens was stronger than that in the control but no differences between the different collagens were revealed. The cell perimeter, the spreading coefficient and the cell projection area on the substrate were used as morphometric parametres. The spreading of cells was monitored for 0.5-2 h after plating. During the spreading both on plastic and on collagen, the groups of small cells were revealed as separate subpopulations. As a whole, such cells comprehend 9 % of the cell population in the control and 2% in experiment. We assume that this cell type is associated with a special independent functional state of the cells that precedes cell spreading.

[Analysis of the cell cycle duration of cells of permanent line L-929].

The direct measurement of the cell cycle duration in L-929 cells was performed using time-lapse photography. The cell cycle duration was 15.77 +/- 0.08 h with a standard deviation of 1.54 +/- 0.06 h. The experimental value fit to a normal distribution with a correlation coefficient 0.999. High homogeneity of this parameter and a wide range of variability of the karyotype (58-66 chromosomes) indicate that there is no correlation between these characteristics of L-929 cells. It is also shown that the difference between cell cycle durations of daughter cells tent to zero and fits by an exponent.

[The estimation of similarity in characteristics of the post-mitotic daughter L-929 cells during their migration along the substrate].

Using time-lapse microscopy, spreading of the post-mitotic daughter cells has been studied. The work was performed on non-synchronized cells of established L-929 cell line. The study was aimed to characterize the morphology of the cells as they move along the substrate and to determine whether the area of the migrating cells changes nonrandom. Two new parameters have been proposed for comparison of cell morphology: the identity indicator (II) and the synchronism indicator (SI). Time-dependent changes in the area in pairs of cells were measured to calculate these parameters. The first indicator shows the degree of coincidence between the absolute values of the area in the pair of the cells, whereas the second indicator shows synchronism of the changes in the cell areas and does not depend on their absolute values. The lower are the indicators, the higher is the similarity in the time-dependent changes in the areas of cell pairs studied. The indicators were shown to be approximately 1.5-fold lower for the pairs of the post-mitotic daughter cells than those for any other pair of the cells. The results indicate a nonrandom pattern of change in the morphology of the cells during their movement along the substrate.

[The average cell size as a factor reflecting the interaction of the CHO cells during process of proliferation].

Changes in the cell area during cultivation of the CHO line cells were studied using time-lapse technique (start of registration in one day after cell plating). It was established that the size of the daughter cells after mitosis remains lees than the size of the mother cell for a long time (up to 6 h). Nevertheless, the average cell area of the whole population is constant throughout the observation period (up to 18 h). We assume that this phenomenon could be a result of interaction among dividing and not-dividing cells. The experimental data confirming this conclusion are presented.

[Pharmacological protection of the myocardium with reamberin in coronary artery bypass grafting in patients with postinfarction angina].

AIM: To assess efficacy of reamberin in preoperative preparation and after coronary bypass (CB) in patients with macrofocal myocardial infarction (MI) complicated with postinfarction angina. MATERIAL AND METHODS: A total of 45 patients with Q-positive MI complicated with postinfarction angina pectoris entered the trial. The study group consisted of 20 (44.4%) patients given 200-400 ml injections of 1.5% reamberin solution for 3 days before coronary artery bypass grafting (CABG) and 3-5 days after it. The control group consisted of 25 (55.6%) patients given basic therapy without cardioprotection. ECTG-60, echocardiography, CM-ECG, laboratory tests were made before CABG. CABG was made in conditions of artificial blood circulation in all the patients. RESULTS: Clinical stabilization was observed after direct myocardial revascularization in hospitalized 25 (100%) patients of the study group and 22 (88%) patients of the control group. Early postoperative acute cardiac failure (ACF) developed in 3 (12%) patients from the study group and 9 (36%) from the control group (p = 0.04), arrhythmia occurred in 2(8%) and 8(32%) patients, respectively (p = 0.03). Two (8%) control patients died in early postoperative period from acute cardiac failure. Perioperative MI occurred in 2(8%) control patients. After 12 months of the follow-up, patients of the study group had no recurrent angina pectoris, while among the controls 4(16%) patients had recurrent angina of FC III. After surgical intervention at discharge and 12 months after treatment patients of both groups improved systolic and diastolic functions of the left ventricle. Normalization of the diastolic function was registered in 80% patients of the study group (p < 0.001) and in 44% from the control group (p < 0.001) after 1 year follow-up. CONCLUSION: Reamberin reduces the number of postoperative complications, ischemic damage to the myocardium, significantly improves systolic and diastolic functions of the left ventricle.

[Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes I and IV types].

The concept that stem cells form an independent subpopulations of somatic cells assumes the heterogeneity of cellular populations in adult tissues. As skin keratinocytes have natural affinity to extracellular matrix proteins, we made an attempt to reveal subpopulations of these cells depending on the time of their adhesion to substrates of collagen I and IV types and fibronectin. After selection for 10, 20 and 30 min the keratinocytes were cultivated for 24 h. The area of cell projection on a substrate and the spreading coefficient were measured (Kuzminykh, Petrov, 2004; Petrov et al., 2007). In 24 h statistically reliable morphological differences between the cells depending on the substratum were found. The size of the cells growing on collagen I type was twice as large as that of the cells cultivated on collagen IV type or in fibronectin. Irrespective of the substratum, up to 60-65% of the cells had a rounded form. The cultivation on collagens revealed the heterogeneity of keratinocytes both in the control cultures and under selection by adhesion time, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interaction with extracellular matrix proteins in the organism.

[Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and the keratin 19 to actin ratio].

Keratin 19 is reported to be a marker for skin stem cells and that assumes an independent subpopulation of these cells in keratinocyte population. In addition, keratinocytes have natural affinity to extracellular matrix proteins. The aim of this work was to reveal subpopulations ofkeratinocytes cultivated on the substrates of collagen I type, laminin-2/4 and fibronectin, and distinguished by keratin 19/actin ratio (G/R). The area of cell projection on a substrate, perimeter, the spreading coefficient and G/R were measured. Both on fibronectin and laminin-2/4, keratinocyte populations were morphologically homogeneous with large cells comprising 12 and 20 percents of the population, respectively. On fibronectin, correlation between morphological parameters of the cells and the keratin 19/actin ratio was not found. The cells growth on collagen behaved as a heterogeneous population, with the large cells compressing more than 50 percents of the population. An average, the size of the cells growing on collagen was twice as large as than of the cells cultivated on fibronectin. On the other hand, correlation between the cell size and G/R was revealed in the cells growth both on collagen and laminin-2/4. The cells with lower G/R value display a larger size and extent of spreading. We assume that this correlation may be determined by alphal and alpha2, integrins characteristic of these cells. Our results cast doubts on whether stem cells are present in the culture of human skin keratinocytes.

[Early changes in the state of the cell surface and nucleoid of irradiated Ehrlich ascites carcinoma cells]. / Rannie izmeneniia sostoianiia kletochnoi poverkhnosti i nukleoida obluchennykh kletok astsitnogo raka Erlikha.

For cells of Ehrlich ascite carcinoma, irradiated by the dose of 10 Gy, a decrease in the coefficient of cell distribution in the two-phase polymer system--dextrane-polyethylene-glycol and an increase in the nucleoid viscosity are shown. The values of both indices characterizing the state of the cell surface and nuclear DNA by the same way have changed in time after irradiation: the maximum effect was observed 1 hour after irradiation and the normalization of the both indices was marked 5-6 hours after it.

[A method of evaluation of plant culture growth]. / Sposob otsenki rosta kallusno'i kul'tury.

The callus growth in culture was evaluated using a stereomicroscope.

[Study of surface properties of chloroplasts of some beans using a binary phase polymer system]. / Izuchenie poverkhnostnykh svoistv khloroplastov nekorotykh bobovykh s pomoshch'iu dvukhfaznoi polimernoi sistemy.

The surface properties of chloroplasts in leaves of some leguminous plants--Pisum sativum, Trifolium paratense, Melilotus album, and Vicia cracca--were investigated, based on the analysis of phase formation kinetics of the aqueous polymeric two-phase system dextrane-500/polyethyleneglycol-6000. The surface properties of chloroplasts have been shown to display both species- and sort-specificity. A specific influence of chloroplasts of melilot and non-chlorophyllic pea lines on phase formation kinetics has been noticed.

[Surface properties of L cells studied by countercurrent distribution in a 2-phase polymer system before and after cyochalasin B and colcemid exposure]. / Issledovanie poverkhnostnykh svoistv L-kletok metodom protivotochnogo raspredeleniia v dvukhfaznoi polimernoi sisteme do i posle vozdeistviia na nikh tsitokhalazina B i koltsemida.

A study was made of the effect of cytochalazin B and cocemid on the line L cells of monolayer and suspension culture studied by the method of counter-current distribution in two-phase polymer system dextran-500 - polyethylenglycol-6000. It has been shown that cytochalazin B caused a decrease in the partition coefficient of both the subline cells irrespective of the growth phase of cell population. Colcemid decreases the partition coefficient in the log-phase of cell culture but increases it in the stationary phase. Effect of colcemid is not associated with the accumulation of metaphase cells in the cell population.