[DNA CONTENT IN INDIVIDUAL CHROMOSOMES OF NORMAL CHINESE HAMSTER KARYOTYPE AND CHROMOSOMES OF CONSTANT HAMSTER CELL LINES CHL V-79 RJK AND Vebr-5].

Using cytometry and an microfluorimetry, we have determined the genome size in Chinese hamster Cricetulus griseus, as well as absolute and relative DNA content of its individual chromosomes and of chromosomes in the transformed Chinese hamster cell lines V-79 RJK and Vebr-5 after prolonged cultivation. It has been shown that the genome size in male and female Chinese hamster is 6.660 and 6.746 pg, respectively. Absolute content of chromosomal DNA of both studied cell lines differed significantly from the content of the corresponding chromosomal DNA of the Chinese hamster normal karyotype. During long-term cellular cultivation, changes in the DNA content of certain chromosomes in both cell lines (generally upward) reached 20-25 %. The level of DNA amplification in the p-arm of chromosome Z6, registered at the beginning of the experiment, in the course of further cellular cultivation (over 20 years) remained stable. The data obtained allow us to conclude that the malignant transformation of cells and subsequent adaptation to the conditions in vitro leads to a profound restructuring of its genome, which affects almost all chromosomes.

[DNA image-fluorimetry of individual human chromosomes].

Mucrofluorimetric method for the determination of DNA content in individual human chromosomes has been developed. The method is based on a preliminary identification of chromosomes with Hoechst 33258, followed by staining of the chromosomes with Feulgen reaction using Schiffs reagent type ethidium bromide-SO2, then measuring the fluorescence intensity of the chromosomes using an image analyzer. The method allows to determine the DNA content of individual chromosomes with accuracy up to 4.5 fg. DNA content of individual human chromosomes, their p-and q-arms as well as homologous chromosomes were measured using the developed method. It has been shown that the DNA content in the chromosomes of normal human karyotype is unstable. Fluctuations in the DNA content in some chromosomes can vary 35-40 fg.

[Analysis of glycogen structure in rat hepatocytes using cytochemical and FRET methods].

Using cytochemical and FRET (Forster, Resonance Energy Transfer) methods, the glycogen structure in rat hepatocytes was investigated during fasting and at different time intervals after per os glucose administration to animals. Hepatocytes on slides were stained with fluorescent PAS-reaction. Staining the slides with ethidium bromide-SO2 (EtBr-SO2) for 40 min revealed a labile glycogen fraction (LE), and the subsequent staining the same samples with auramine-SO2 (Au-SO2) for 50 min showed a stable glycogen fraction (SF) in the cells. The total glycogen content (LF and SF) in the hepatocytes at different stages of refeeding was determined by means of cytofluorimetry, and then efficiency of FRET was measured in the same cells. Registration of FRET in several areas of the cells was carried out on a laser scanning confocal microscope Leica TCS SP5 with application of FRET AB (Acceptor Photobleaching) procedure. In this procedure, auramine served as a donor (D) and ethidium bromide was an acceptor (A). It was shown that the efficiency of FRET varied from 10 to 14 % during refeeding, while the glycogen structure had a marked influence on the value of this parameter. FRET efficiency was shown to correlate with the ratio A/D in the cells of hungry rats and at the early stages after glucose administration to animals, which reflected the degree of filling of the external tiers of glycogen molecules of glucose residues. At later stages, this correlation was either less pronounced or absent. It was found that the FRET efficiency can vary by 3-4 times at the same value of A/D. Since the probability of energy transfer from D to A is proportional to 1/R6, where R is a distance between D and A, such variations of the FRET efficiency indicate that the glycogen molecules possess a labile structure in which the chain of glucose residues can deviate from its axis by a distance of about half their diameter.

[Effects of antimicrobial peptides of neutrophils on tumor and normal cells in culture].

We performed a comparative study of effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine bactenecin 5 (Bac5) on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic beta-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic properties. Bac5 belongs to the group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a non-lytic mechanism. We have shown that while PG1 exerts distinct and fast cytotoxic effects on most of used tumor cells being slightly less toxic for nontransformed host cell, the proline-rich Bac5 is much less cytotoxic for all the cells tested. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as had been previously established for human defensins by Panyutich et al., 1995). Pre-incubation of PG1 with alpha1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of alpha1-antitrypsin was reduced after interaction of the serpin with PG1 (not with Bac5). Confocal microscopy experiments allowed to monitor the internalization of fluorescent labeled (by BODIPY FL) peptides into target cells and their intracellular distribution. Bac5-BODIPY (at 5 microM) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations was also able to enter the cells without significant damage to them. The comparative study of the kinetics of the peptides uptake into the target cells and the influence of low temperature, energy-depletion and endocytosis inhibitors on the process of the internalization of the peptides into the cells was carried out using flow cytometry.

[A study of DNA depolyploidization and depolytenization of the heterochromatized gonosomal chromatin bodies in the secondary giant trophoblast cells of the field vole Microtus rossiaemeridionalis using cytophotometry].

A study was made of the distribution of the heterochromatized gonosomal chromatin bodies (GCB) material in the course of nuclear fragmentation of secondary giant trophoblast cells resulting in polykaryocyte formation at the late stage of their differentiation. A simultaneous DNA cytophotometry in GCBs and nuclear fragments showed a progressive GCB DNA content decrease proportional to that of DNA content in nuclear fragments. DNA contents in the nuclear fragments corresponded to 2c, 4c and 8c. In most cases 1-2 GCBs were found in the nuclear fragments of different ploidy levels. Both the total DNA content in GCBs and the DNA content in separate GCBs well correlated with the ploidy levels of fragments. The data obtained demonstrate a regular, whole-genome distribution of chromosomal materials into the nuclear fragments exemplified by sex chromosome distribution in compliance with the ploidy of nuclear fragments. We discuss a possible mechanism of nuclear fragmentation that may ensure substantially a balanced genome of nuclear fragments without leading to mitotic cycle renewal in the giant trophoblast cell population.

[The effect of image analyser noises in studies of cell structure]. / Vliianie shumov analizatorov izobrazhenii pri issledovanii tekstury kletok

Using image analysers, the influence of noises on the quality of images obtained from three types of digital CCD videocameras was studied. Algorithms for calculating the heterogeneity coefficient of cell structures have been proposed, which take into account the noises on the images. Application of procedures of image smoothing or averaging from a few shots, calculation of differences in heterogeneity coefficients of the object and a free field, and a combination of these methods have significantly reduced the influence of noises and increased the informativeness of texture features.

[Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine]. / Odnovremennaia fotometriia DNK i chisla zeren serebra v kletkakh pecheni, mechennykh 3H-timidinom ili 3H-leitsinom.

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.

[A morphometric study of the silver-stained nucleoli in mononuclear and binuclear rat hepatocytes]. / Morfometricheskoe issledovanie okrashennykh serebrom iadryshek v odnoiadernykh i dvuiadernykh gepatotsitakh krysy.

Investigation of silver-stained nucleoli has been made in mono- and binuclear rat hepatocytes of different ploidy. Morphometric parameters of nucleoli and cell ploidy (after silver removing and Feulgen staining) were measured by image analyzer "Videotest". It has been shown that the total area and total volume of nucleoli correlate with cell ploidy, but the found dependence deviates from the proportional one. Possible reasons of such a deviation have been discussed. Marked correlation of total area or total volume of nucleoli was detected between pairs of nuclei of binuclear hepatocytes, whereas between accidental pairs of mononuclear hepatocytes such correlation is practically lacking.

[Characteristics of trophoblast cell reproduction in the connective zone of the rat placenta. I. Determination of the degree of ploidy and the number of Barr bodies in interphase nuclei]. / Osobennosti reproduktsii kletok trofoblasta soedinitel'noi zony platsenty krysy. I. Opredelenie stepeni ploidnosti i chisla telets Barra v interfaznykh radrakh.

The degree of ploidy in the interphase nuclei was determined in the connective zone of the rat's placenta on days 13 and 14 of embryo development. On day 13, the ploidy in the majority of nuclei was 2c or 4c; on day 14, the 4c nuclei were dominating, the share of 8c nuclei increasing. The number of Barr's bodies in each nucleus of the placental connective zone tends to increase with the increase in ploidy level. This is an evidence of a "genuine polyploidy" as a mechanism of the initial polyploidization of the given cell population.

[Characteristics of trophoblast cell reproduction in the connective zone of the rat placenta. II. Determination of the degree of ploidy of mitotic shapes]. / Osobennosti reproduktsii kletok trofoblasta soedinitel'noi zony platsenty krysy. II. Opredelenie stepeni ploidnosti mitoticheskikh figur.

Morphological and cytophotometric studies have been made on polyploidization of placenta connective zone cells. Measurement of the DNA content in mitotic figures show that within a period of development ranging from day 13 to day 14 the bulk of mitoses (up to 25%) become tetraploid and octaploid. This may suggest that polyploidization of placenta connective zone cells proceeds via incomplete polyploidizing mitoses. Among tetraploid and octaploid mitotic figures, there are those corresponding to all the mitotic stages, from prophase to telophase. Consequently, mitosis in tetraploid and octaploid cells can reach telophase. In such cases polyploidization is likely to follow the acytokinetic mitotic pattern. A question of a certain maximum level of polyploidy that may be reached by cells due to the incomplete mitosis is discussed.

[Analysis of the polyploidization kinetics of the parenchymal cells in the rat liver]. / Analiz kinetiki poliploidizatsii kletok parenkhimy pecheni krys.

Methodological approaches to kinetics of cell polyploidization in the rat liver parenchyma are discussed. Different ways of hepatocyte polyploidization in the course of postnatal liver growth have been assessed. The intensities of hepatocyte transitions from one ploidy class to another were determined. On the basis of literary experimental data the following is summarized: With the increase in the animal age, there is a decrease in hepatocyte transition from one ploidy class to and ther; in young animals the intensity of formation of tetraploid hepatocytes through the stage of binuclear cells (2c----2c X 2----4c) is 0.39-0.55 within two weeks, the intensity of direct transitions (2c----4c) being 0.00-0.19 within the same time. The intensity of entering to DNA synthesis is reduced with the increase in hepatocyte ploidy levels; in this case the coefficient of the reducing of mitotic activity is calculated as 0.10-0.22, and 0.01-0.05 for 4c- and 8c-hepatocytes, resp. The factors stimulating proliferation in the liver increase the intensity of the direct cell transition (2c----4c) by several times which can exceed the intensity of transition through the binuclear cell stage.

[DNA content of the nuclei of secondary giant cells of the rat trophoblast at different phases of the polytene nucleus cycle]. / Izuchenie soderzhaniia DNK v iadrakh vtorichnykh gigantskikh kletok trofoblasta krysy na raznykh fazakh tsikla politennogo iadra.

A cytophotometric study of DNA content has been made for secondary trophoblastic giant cells, which differ morphologically in relation to the stage of the cycle of the polytene nucleus. The ploidy rate varying from 16c to 512c. It is shown that the DNA content of the nuclei with polytene chromosomes in phase G is more stable, corresponding to the 2c multiple DNA content. Unlike, reticular nuclei in phase S do not present clear-cut peaks on a histogram of DNA. Ratios of nuclei with unequal ploidy differ depending on the structure of these nuclei.

[Changes in the amount of 3H-thymidine label in hepatocytes of different ploidies in rat ontogeny after a single administration of the isotope]. / Izmenenie kolichestva 3H-timidinovoi metki v gepatotsitakh raznoi ploidnosti v ontogeneze krys posle odnokratnogo vvedeniia izotopa.

Change of 3H-thymidine quantity in mono- and binuclear rat hepatocytes of different ploidy was investigated during the first 6 weeks after a single injection of isotope to newborn rats. Rates of cell transitions (arbitrary number of cells in the time unit) from one ploidy class to another, and coefficients of the reducing of hepatocyte proliferative activity with increasing the hepatocyte ploidy were calculated on the basis of ideas about the process of autoradiographic label "diluting" in the course of the postnatal development as a result of polyploidization and ordinary mitotic divisions of hepatocytes. The calculated values are close to values of parameters, which were calculated with assistance of the model, which describes the process of polyploidization in the liver, on the basis of data on the change in the arbitrary number of different ploidy hepatocytes.

[Changes in the amount and synthesis of DNA in the nuclei of large decidual cells in rats during their differentiation]. / Izmenenie kolichestva i sinteza DNK v iadrakh bol'shikh detsidual'nykhkletok krys v khode ikh differentsirovki.

Using cytophotometry of the Feulgen-stained nuclei, the quantity of DNA was measured in the nuclei of rat's large decidua cells (LDC) on tissue sections of the antimesometrial region within days 7-13 of gestation. The quantity of nuclear DNA was expressed in units of ploidy, the haploid DNA standard being the quantity of DNA in rat's spermatid nucleus. On different days of gestation, the nuclear DNA was seen to vary in cells located in different zones of decidua. The maximum DNA content was found in the LDC located on days 9-12 of gestation somewhat in the middle of the decidua thickness. On day 11, the quantity of nuclear DNA in these cells reached in average, 22c. The quantity of DNA in the nuclei of the least differentiated LDC located on the periphery of decidua never exceeded 4.9c, whereas that in the nuclei of the most differentiated LDC, located close to the embryo, varied from 2.9c to 9.3c. On days 10 and 11 of rat's false gestation, the maximum DNA contents in the nuclei were registered in the LDC located in the middle of the decidua thickness. 3H-thymidine incorporation into the nuclei of the most differentiated LDC located nearest to the embryo stopped starting from day 10 of gestation. Phenomena of lesser quantities of nuclear DNA in most differentiated LDC, compared to that in LDC in the previous steps of differentiation, are discussed.

[Change in morphometric parameters in silver-stained nucleoli from hepatocytes of rats with liver cirrhosis and during rehabilitation from it]. / Izmenenie morfometricheskikh parametrov okrashennykh serebrom iadryshekgepatotsitov krys pri tsirroze pecheni i v protsesse ee reabilitatsii.

Silver-stained nucleoli of rat hepatocytes were studied in norm, in liver cirrhosis produced by CCl4 poisoning and after cessation of the poisoning. Morphometric parameters of nucleoli were measured using a Videotest computer image analyser. Under cirrhosis the mean number of nucleoli per nucleus was determined to exceed their normal number by 1.27 times. The total volume of nucleoli in the nucleus also exceeded the normal level (by 1.15 times). 3 months after the end of CCl4-poisoning, these parameters decreased almost to normal values. A statistically significant correlation was revealed between the number of nucleoli and their total volume (0.881). Changes of the parameters also correlated with the total protein content in the hepatocytes. Possible reasons for this correlation are discussed. The ratio of the number of chromosomal NORs to the mean number of nucleoli in the nucleus is proposed to be used as a feature for comparative analysis of functional status of nucleoli in the nuclei of different ploidy and in cells of different animal species.

[Hypertrophy of the myometrial cells in women with myoma]. / Gipertrofiia kletok miometriia zhenshchin pri miome.

A rapid growth of the uterus myoma results from the cell hypertrophy, which involves essential ultrastructural changes in the cytoplasm and the nucleus, thus suggesting the intensification cell functions. The increase in the cell volume and intensification of secretion is followed by the enlargement of the interface between the nucleus and the cytoplasm resulting in formation of numerous invaginations of the nuclear membrane. The autoradiographical data demonstrate a considerable DNA synthesis in the peripheral zone of the myoma, unlike the situation seen in the centre of the myoma and the unchanged myometrium, where DNA synthesis is not considerable. The cytospectrophotometric analysis confirms the above data showing the highest content of DNA in the peripheral zone of the myoma.

[Dynamic glycogen synthesis in the hepatocytes of different areas of the hepatic lobes in rats]. / Dinamika sinteza glikogena v gepatotsitakh razlichnykh zon dolek pecheni krys.

Using image analyser Magiscan, a quantitative analysis of the total glycogen and of its two fractions was made in hepatocytes of portal and central zones of the liver lobule of rats after a 48 hour starvation and 15, 30, 60, 120 minutes after refeeding. Glycogen content was the lowest after a 48 hour starvation and only a few cells of the central zone contained a noticeable glycogen quantity. Glycogen synthesis initiation began 15 minutes after refeeding. Glycogen synthesis is characterized by a higher glycogen content in the portal zone of liver lobule, and further this difference was even more increased. Different changes were observed in the content of glycogen fractions in the process of glycogen resynthesis after starvation of rats.

[The RNA content of hepatocytes of different ploidies]. / Soderzhanie RNK v gepatotsitakh razlichnoi ploidnosti.

The RNA contents in rat and human liver cells was measured using the scanning absorbtion photometric method after gallocyanin-chromalum staining. The RNA content was shown to increase proportionally with the increase of genome numbers in hepatocytes.

[Method of automated cytomorphological analysis for classifying leukocytes by their stage of destruction]. / Metod avtomatizirovannogo tsitomorfologicheskogo analiza dlia klassifikatsii leikotsitov po stadiiam destruktsii.

Destruction of human peripheral blood leukocytes by toxins has been studied with the aid of an automatic microimage analyzer the "Morphoquant". The most informative parameters which define destruction stages have been revealed: the area and number of fragments of nucleus images and their distance to the nucleus centre under different levels of the optical threshold. Software for morphological analysis of leukocytes has been developed in addition to their classification using four destruction stages. The results of computerized classification of blood smears will compare with those obtained by a working haemotologist (85% coincidence).

[A cytophotometric study of the RNA and glycogen content of human hepatocytes at different stages in the development of traumatic disease]. / Tsitofotometricheskoe issledovanie soderzhaniia RNK i glikogena v gepatotsitakh cheloveka na raznykh étapakh razvitiia travmaticheskoi bolezni.

Absorption and fluorescent cytophotometry techniques were applied to studies of RNA, glycogen and its fractions in hepatocytes of patients with hard mechanic trauma, both with and without endointoxication. For measuring RNA and glycogen contents, slides were stained by gallocyanin-chromalum or underwent fluorescent PAS-reaction, respectively. The repeated aspiration biopsy material was used for investigation of RNA and glycogen contents in dynamics. A quick increase in RNA content took place at the first stage (within the first 3 days) of traumatic illness of both the groups of patients. At the second stage of illness the hepatocyte RNA content in patients without endointoxication was seen to decrease to the initial level, whereas that in patients with endointoxication increased from the initial level by 36%. At different stages of illness the total glycogen content is changed insignificantly in the course of illness, but its labile fraction decreases to 70% of the total glycogen in patients with endointoxication. The increase of hepatocyte synthetic activity and the maintenance of the normal glycogen level may suggest a sufficient compensatory possibility of the liver, in spite of a high functional load under endointoxication.