[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.

[STEM CELLS PLAY NO CONSIDERABLE ROLE IN CARDIOMYOCYTE REPOPULATION OF ADULT HUMAN HEART].

There are two viewpoints concerning cardiac regeneration. One assumes that the myocardium of an adult human heart has a weak regenerative capacity. According to another, myocardium can renew at a high rate due to the presence of resident stem cells. This study was aimed to test the role of stem cells in myocardium repopulation in adult humans of different age by examining the distribution of cardiomyocytes as to their size and ploidy. Cytofluorimetry and interferometry were used to determine the dry weight, volume and ploidy of myocytes isolated from the left ventricle of the normal heart of 12 men aged 20-30 years (n = 7) and 40-50 years (n = 5). Dry weight of cardiomyocytes made up 6906 ± 182 pg (10(-12) g) aged 20-30 years and 9126 ± 263 pg in men aged 40-50 years. There were no cells with an intermediate volume between amplifying and mature myocytes. The number of candiomyocytes in the left ventricle made up (3.18 ± 0.05) x 10(9) cells in the age group 20-30 years and (2.06 ± 0.6) x 10(9) cells in the age group 40-50 years. Most of the myocyte population was represented by mononucleate cells with tetraploid nuclei (41.3%). Proportion of myocytes of different ploidy classes did not change in the interval from 20 to 50 years. Our results strongly suggest that stem cells of the heart are not involved in the regeneration of human myocardium during aging. The function of the aging heart is mostly compensated by the hypertrophy of the remaining myocytes.

[Rat heat structural and functional characteristics and gas exchange parameters after experimental myocardial infarction].

Rat heart structural and functional changes and gas exchange parameters were investigated in six months after experimental myocardial infarction. Left ventricular end-systolic and end-diastolic dimensions in rats with chronic heart failure were 78 and 30% higher than in control respectively. Left ventricle cavity volume in systole and diastole were 5 and 2 times increased respectively. Left ventricular cavity stretching was accompanied by thinning of the interventricular septum. Left ventricular structural changes leads to its functional deterioration. Left ventricular contraction fraction was reduced by 60%, and the ejection fraction--by 52% in comparison with control. Gas exchange investigation revealed that in six month after myocardial infarction oxygen consumption of operated rats was increased by 30% and production of carbon dioxide by more than 40%. Respiratory quotient, which reflects the nature of oxidized substrates, in rats with myocardial infarction was amounted to 0.85, indicating significant increase in the contribution of carbohydrates as an energy substrate for myocardial metabolism.

Sphingosine-1-phosphate: distribution, metabolism and role in the regulation of cellular functions.

The role of sphingosine-1-phosphate (S1P) in regulation of cellular functions and cell protection is reviewed. S1P, along with other sphingolipid metabolites, is believed to act as an intracellular second messenger and as an extracellular mediator molecule. S1P chemistry, production and metabolism are described. Cellular receptors for S1P and their tissue specificity are described. Platelets and erythrocytes have a crucial significance in blood transport of S1P. Hypoxic conditions induce an increase in S1P, which initiates a set of cytoprotective events via its cellular receptors. S1P involvement in regulation of cell migration, myogenesis, control of skeletal muscle function is described. It is shown that S1P balance disturbances may mediate pathological state. S1P system implication in regulation of the most important cellular functions allows considering it as a prospective remedial target.

[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.

Heterogeneity of left ventricular cardiomyocytes from rat heart.

Contractile cardiomyocytes in various parts of the heart differ in shape, size, ploidy, and other parameters. However, it is not known whether their population is heterogeneous within each heart chamber. In this paper, dry weight and ploidy of cardiomyocytes were estimated in different parts of rat left ventricle. It was found that the dry weight of cardiomyocytes in medial part of left ventricular anterior wall is higher than in other parts of the ventricle. Cardiomyocyte ploidy is the same in different regions of the left ventricle.

[Cellular mechanisms of regeneration of rats' liver after experimental myocardial infarction].

Morphological changes and regeneration activity of the rats' liver after an experimental myocardial infarction (MI), caused by a permanent left coronary artery occlusion, were investigated. It has been shown that in 6 months after MI there were considerable changes of the rats' liver circulatory system: the quantity of vessels per unit of area increased by 118%, thickness of their walls by 19%, and the average square of vessels lumens by 159%. The percentage of connective tissue in 6 months after MI increased more than in one and a half time in comparison with control. Inflammatory and necrotic changes in rats' liver remained for 6 months after MI. The liver injury caused by MI led to activation of regeneration processes in its parenchyma. In 6 months after MI, the number of 4c- hepatocytes decreased by 12% in comparison with control, and the number of 4c x 2- and 8c-hepatocytes increased by 45 and 71%, respectively. The mean level of hepatocytes ploidy increased in 6 months after MI by 11%. The dry mass of rats' hepatocytes increased in 6 months after MI by 19% in comparison with control. Thus, liver regeneration after MI is more due to hepatocytes hypertrophy than to their polyploidization.

Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture.

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).

Glucural (amigluracyl) improves survival of isolated rat hepatocytes in suspension.

Nonsteroid anti-inflammatory drug glucural (water-soluble N-methyl-D-glucosamine complex with 6-methyluracyl) improves survival of isolated rat hepatocytes stored in suspension. This effect of glucural is presumably explained by its membranotropism.

[Regeneration of the liver of Chinese hamster Cricetulus griseus].

Using cytofluorimetry and interferometry, hepatocyte DNA, dry mass and distribution of hepatocyte ploidy classes were measured in hamsters Cricetulus griseus in 1 month after partial hepatoctomy. Ploidy of normal liver hepatocyte was 2.35 +/- 0.03 (mean +/- SD) c. Modal ploidy class was presented by mononuclear hepatocytes with diploid nuclei (82.4 +/- 1.3 %). Hepatocyte dry mass was 605.2 +/- 4.8 pg. One month after partial hepatectomy the distribution of ploidy classes and dry mass of hepatocyte did not change. A similar hepatectomy in mice resulted in significant polyploidization of liver parenchyma: the middle level of hepatocyte ploidy increased by 32% and mononuclear octaploid cells, the number of which increased 5-fold, composed modal ploidy class in place of 4cx2-hepatocytes predominated in control mice. The number of 8cx2-hepatocytes in the liver of mice creased by more than 5-fold. Thus, in contrast with mice, in hamsters Cricetulus griseus an increase in the liver mass followed partial hepatectomy depended completely on hepatocyte proliferation.

[Comparative morphofunctional analysis of rat hepatocyte cultures isolated from the normal and pathologically changed liver due to experimental toxic hepatitis].

The goal of the study was to examine the state of primary hepatocytes of rats with toxic hepatitis induced by combination of CCl4 and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed actin cytoskeleton, cell-cell and cell-matrix contacts. To investigate morphology and localization of mitochondria the hepatocytes were stained with Rhodamine 123. Glycogen and DNA contents in hepatocytes were determined by fluorescent cytophotometry during the lifetime of the culture. Cells were maintained for 5 days, and there were no changes in ploidy distribution observed. The mean ploidy was not changed too. Thus hepatocytes of different ploidy demonstrated similar survival rate. The glycogen content was 50% higher in experimental group compared to the control. The glycogen content decreased in control and cyrrotic hepatocytes after collagenase isolation. It has been found that the control hepatocytes accumulated glycogen within 3 days. On the contrary, the glycogen levels remained to be low in the pathologic hepatocytes.

Superoxide anion production by the mitochondrial respiratory chain of hepatocytes of rats with experimental toxic hepatitis.

The progression of toxic hepatitis is accompanied by the activation of oxidative processes in the liver associated with an enhancement of the mitochondrial respiratory chain activity and superoxide anion production (O(2)(*-)). The purpose of this study was to examine our previously formulated assumption concerning the predominant contribution of the complex I to O(2)(*-) production increase by the mitochondrial respiratory chain of hepatocytes in toxic hepatitis (Shiryaeva et al. Tsitologiia, 49, 125-132 2007). Toxic hepatitis was induced by a combined application of CCl(4) and ethanol. Respiratory chain function analysis was executed with submitochondrial particles (SP) in the presence of specific inhibitors. It was shown that the rate of O(2)(*-) production by SP of animals with toxic hepatitis, when NADH was delivered, was 2.5-fold higher as compared with the control. The rates of O(2)(*-) production by SP of rats with toxic hepatitis in the presence of NADH or NADH+ rotenone were similar. The O(2)(*-) production rate by control SP in the presence of NADH + rotenone corresponded to the O(2)(*-) production rate by toxic hepatitis SP when only NADH was delivered. When NADH+ myxothiazol were delivered to the incubation system, O(2)(*-) production by toxic hepatitis SP was 72% higher than for the control. Conversely, in the presence of antimycin A, the production of O(2)(*-) by toxic hepatitis SP was lower compared to the control. Collectively, the presented data indicate that the O(2)(*-) production rate was enhanced by the complex I of the hepatocyte mitochondrial respiratory chain in experimental toxic hepatitis. Complex III contribution to the production of O(2)(*-) was insignificant. We assume that the increase in O(2)(*-) production by the respiratory chain may be considered not only as the mechanism of pathology progression, but also as a compensatory mechanism preserving the electron transport function of the mitochondrial respiratory chain when complex I functioning is blocked in part.

[Proteoglycans of L6J1 myoblast extracellular matrix. Characteristics and effect on myoblast adhesion].

Proteoglycans were isolated from extracellular matrix of L6J1 rat myoblasts and their influence on myoblast adhesion was studied. Proteoglycan digestion with chondroitinase AC and heparinase III degrading the polysaccharide moieties revealed that chondroitin sulfate proteoglycans are the main class of myoblast extracellular matrix proteoglycans. Electrophoresis of enzymatically processed proteoglycans was used to examine their core proteins. Myoblast adhesion was suppressed by proteoglycans or the mixture of proteoglycans and fibronectin/extracellular matrix. When being processed with chondroitinase AC the combined substrate of fibronectin and proteoglycans lost the capability of myoblast adhesion suppression. Thus, as a result of presented work the proteoglycans of L6J1 rat myoblast extracellular matrix were isolated and purified. The main class of proteoglycans was chondroitin sulphate proteoglycans. Isolated proteoglycans suppressed myoblast adhesion and this effect was mediated by polysaccharide moieties of proteoglycans.

Hepatocyte mitochondrion electron-transport chain alterations in CCl(4) and alcohol induced hepatitis in rats and their correction with simvastatin.

The goal of this study was to examine the state of hepatocyte mitochondrial respiratory chain of rats with toxic hepatitis induced by CCl(4) and ethanol. Oxygen consumption by hepatocytes and mitochondria was determined. Endogenous oxygen consumption by pathological hepatocytes was 1.3-fold higher compared with control. Rotenone resulted in 27% suppression of respiration by pathological hepatocytes whereas 2,4-dinitrophenol produced a 1.4-fold increase of respiration. States 3 and 4 of mitochondrial respiration with malate and glutamate were found to be higher as compared to control. State dinitrophenol and state 3 respirations were similar within every group of animals when being tested with malate and glutamate or succinate. Cytochrome c oxidase activity in hepatitis was 1.8-fold higher compared with control. Simvastatin administration resulted in a decrease in hepatocyte endogenous respiration in hepatitis. The presented data lead to the assumption that the increased oxygen consumption by the respiratory chain of pathological mitochondria to be linked mainly with the altered function of complex I.

[New protective effect of simvastatin in rats with experimental steatohepatitis].

The intragastric introduction of carbon tetrachloride (CCl4) (0.2 ml/kg in 50% oil suspension, twice a week) and ethyl alcohol (5% solution ad libitum as the only available drink) in rats over a period of four weeks results in the development of inflammation, fibrosis, and fatty dystrophy in the liver. Such a fast formation of liver damage is obviously caused by potentiating effect of alcohol in combination with CCl4. Simultaneous injection of simvastatin (1 mg/kg, intragastrically) in rats with ethanol--CCl4 hepatitis decreased fatty dystrophy and produced certain anticytolytic and anticholestatic effects without potentiation of microsomal oxidation system damage by hepatotoxins. In addition, simvastatin shows hypolipidemic activity, which is manifested primarily by a decrease in the general holesterol level in the blood serum.

Isolation and characterization of proteoglycans synthesized by rat myoblasts L6J1 in culture.

Proteoglycans synthesized by rat myoblasts L6J1 in culture were isolated using sorbent Q-Sepharose from culture medium, extracellular matrix (ECM), and cells. Elution of the sorbed material in a NaCl gradient separated proteoglycans from the bulk of proteins eluted at low concentration of the salt. Four fractions (fractions I-IV) were obtained for each component of the cell culture, including two proteoglycan fractions for the ECM and culture medium and one fraction for the myoblasts. Proteoglycans of the culture medium were virtually completely represented by proteoglycans of fetal calf serum. With enzymes chondroitinase ABC and heparinase III chondroitin/dermatan sulfate proteoglycans were shown to prevail in all components of the myoblast culture. The core proteins of proteoglycans were characterized by electrophoresis.

[Hepatocyte mitochondrion respiratory chain in rats with experimental toxic hepatitis].

The purpose of this study was to examine hepatocyte mitochondrion respiratory chain in rats subjected to ethanol and CCl4 administration within 4 weeks to induce an experimental hepatitis. Oxygen consumption was determined as a measure of mitochondrion respiration chain function. The development of liver pathology was accompanied by fat accumulation, fibrosis, triglycerides and lipid peroxidation increase. Respiratory chain characteristics damage was found. Endogenous oxygen consumption by hepatocytes isolated from pathological liver was found 34% higher compared to control. Exogenous malate and pyruvate substrates delivery didn't stimulate cell respiration. Rotenone (the inhibitor of the I complex) decreased 27% oxygen consumption by pathological hepatocytes while dinitrophenol produced 37% cell respiration increase. States 3 (V3) and 4 (V4) mitochondrial respiration with malate + glutamate as substrates were found to be 70 and 56% higher accordingly compared to control level. V3 and Vd (dinitrophenol respiration) for mitochondria from pathological liver didn't differ from control when being tested with malate + glutamate or succinate as substrates. Cytochrome c oxidase activity increased (+ 80%) as compared to control. Administration of hypolipidemic agent simvastatin simultaneously with ethanol and CC14 resulted in decrease liver fat accumulation, fibrosis and peroxidation products. Simvastatin administration caused hepatocyte endogenous respiration decrease while malate + pyruvate, dinitrophenol or rotenone delivery produced oxygen consumption alterations similar to control. However, when isolated mitochondria from liver of simvastatin treated animals being tested the decrease of oxidative phosphorylation coupling for substrates malate + glutamate was found. While simvastatin did not cause changes in cytochrome c oxidase activity. We propose the hypothesis that the NCCR complex in rat mitochondria with experimental toxic hepatitis works extensively on superoxydanion production. Alterations of SCCR, Coenzyme Q-cytochrome c-reductase, cytochrome c oxidase and ATP-synthase activities have an adaptive nature to compensate for impaired NCCR function.

[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.

[Morphological and biochemical aspects of skeletal muscle injury and regeneration subjected to physical load and hypodynamia].

This paper reviews the literature data on morphological and biochemical aspects of skeletal muscle injury by exercises, hypodynamia and microgravity. Muscle injury depends on the duration and intensity of action. In spite of differences of muscle injury mechanisms by exercises and hypodynamia, this injury restricts muscle function and capacity to continue muscle work. Possible approaches to minimization of the muscular tissue injury and accelerating its regeneration are discussed.

[Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy].

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.