Spatial Structure of Glycogen Molecules in Cells.

Glycogen is a strongly branched polymer of α-D-glucose, with glucose residues in the linear chains linked by 1→4-bonds (~93% of the total number of bonds) and with branching after every 4-8 residues formed by 1→6-glycosidic bonds (~7% of the total number of bonds). It is thought currently that a fully formed glycogen molecule (ß-particle) with the self-glycosylating protein glycogenin in the center has a spherical shape with diameter of ~42 nm and contains ~ 55,000 glucose residues. The glycogen molecule also includes numerous proteins involved in its synthesis and degradation, as well as proteins performing a carcass function. However, the type and force of bonds connecting these proteins to the polysaccharide moiety of glycogen are significantly different. This review presents the available data on the spatial structure of the glycogen molecule and its changes under various physiological and pathological conditions.

[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.

[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.

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.

Metabolic heterogeneity of glycogen in hepatocytes of patients with liver cirrhosis: the glycogen of the liver lobule zones in cirrhosis.

The concentrations of total glycogen (TG) and its labile (LF) and stable (SF) fractions were determined in hepatocytes of portal and central zones of the normal human liver and in the liver of patients with cirrhosis of viral and alcohol aetiologies. Using PAS reaction, TG, LF and SF were revealed in histological sections of the material obtained by the liver punch biopsies. The concentrations of TG and its fractions were measured by televisional cytophotometry. In liver cirrhosis, the concentrations of TG, LF and SF in both zones of the hepatic lobule have been found to be much higher than in the normal liver. It has been shown that the ratio of the hepatocyte TG concentrations in the portal zone to the central zone both in the normal liver and in viral cirrhosis exceeds 1.0, amounting to 1.264 +/- 0.021 and 1.030 +/- 0.009, respectively. The glycogen fraction composition in the cells of both the liver lobule zones in viral cirrhosis does not differ significantly from the norm. On the contrary, in the liver of patients with alcoholic cirrhosis, the ratio of the TG concentrations in the portal zone to the central zone is reduced to 0.815 +/- 0.016 and is accompanied by qualitative changes of the glycogen composition.

The metabolic zonation of glycogen synthesis in rat liver after fasting and refeeding.

The quantitative analysis of total glycogen and two fractions of the glycogen content was made by means of cytophotometry in hepatocytes with respect to the portal and central zones of the liver lobule after 48 hr starvation and 15, 30, 60, 120 min after refeeding using the Magiscan image analyzer. It was shown that glycogen content was minimal after 48 hr starvation, although a few cells of the central zone contained a noticeable glycogen quantity. Glycogen synthesis initiation was observed after 15 min refeeding. Glycogen synthesis has been characterized by an increasing glycogen content in the portal zone of the liver lobule compared to the pericentral zone, and this difference increased with time. The distinctive morphological changes were observed in the total glycogen content as well as fractions with different optical density in the process of glycogen synthesis after starvation of rats.

Glycogen-forming function of hepatocytes in the rat regenerating cirrhotic liver after a partial hepatectomy.

Rat liver punctate biopsies were used for cytofluorimetric determinations of the content of glycogen and its fractions in hepatocytes, and also for microchemical measurements of the activity of glucose-6-phosphatase, glycogen phosphorylase, and glycogen synthase, in liver tissue with cirrhosis produced by carbon tetrachloride (CCl4) poisoning, during regeneration of the liver after the cessation of poisoning and after a partial resection of the cirrhosed liver. The liver cirrhosis was shown to be characterized by an accumulation of glycogen (predominantly of its metabolically less active fraction) in hepatocytes and by a decrease in the activities of the glycogenolytic enzymes in the liver parenchyma. On the cessation of poisoning, there was a partial or complete return to normal levels of the glycogen metabolism parameters. Some of them returned to normal more quickly if a partial hepatectomy was performed after the cessation of poisoning.

Quantitative analysis of glycogen content in hepatocytes of portal and central lobule zones of normal human liver and in patients with chronic hepatitis of different etiology.

Glycogen content was determined in hepatocytes of different lobule zones of the normal human liver (23 patients without any liver pathology) and the liver of patients with chronic viral B hepatitis (30 patients) and chronic alcohol hepatitis (28 patients). All the patients were males and aged between 17-50 years. Quantitative analysis of the glycogen content in hepatocytes of portal and central lobule zones was carried out in sections of the human liver (material of functional biopsies) stained with PAS-reaction. The measurements were carried out using an image analyser 'Magiscan' which allows combined cytophotometric analysis of a substance in cells and determination of the cell localization in tissue. The results showed significant differences of the glycogen content in different lobule zones in the normal liver and in the liver in chronic viral and alcohol hepatitis. Ratios of glycogen content in hepatocytes of the portal and the central zones of liver lobule were 1.128 +/- 0.004 and 1.061 +/- 0.003 in normal human liver, and liver of patients with chronic viral hepatitis respectively, i.e. the glycogen content in hepatocytes of the portal lobule zone was much higher than in the central lobule zone in the normal liver and in the liver of patients with chronic viral B hepatitis. The ratio in patients with chronic alcohol hepatitis was less than 1.0 (0.930 +/- 0.003), i.e. a significantly higher glycogen content was found in hepatocytes of the central liver lobule zone. Possible mechanisms of this phenomenon are discussed. Thus, the pattern of the glycogen content in hepatocytes of different lobule zones can be used as an indicator of etiology of chronic hepatitis.

Glycogen-forming function of hepatocytes in cirrhotically altered rat liver after treatment with chorionic gonadotropin.

Using cytofluorimetric and biochemical studies on serial supravital liver punctate biopsies, effects of chorionic gonadotropin (CG) on recovery of hepatocyte glycogen-forming function in the cirrhotically altered rat liver were analyzed. The biopsies were taken first from rats with experimental cirrhosis produced by their 6-month-long poisoning with the hepatotoxic poison CCl4, then from the same animals in 1, 3, and 6 month after cessation of their poisoning, either on treatment with CG or with no treatment. In smears of isolated hepatocytes, the contents of the total glycogen (TG) and of its labile and stable fractions (LF and SF, respectively) were measured. In liver homogenates, activities of glucose-6-phosphatase (G6Pase), glycogen phosphorylase, and glycogen synthetase were determined. It was found that the threefold increased TG content in hepatocytes of cirrhotic liver returned to the normal level in 3 months without treatment, while as soon as in 1 month in the case of the treatment with CG. The CG treatment for 3 months resulted in normalization of the glycogen fraction composition that had been changed in cirrhotic liver, whereas without treatment, the glycogen LF/SF ratio remained changed even after 6 months after cessation of the poisoning with CCl4. Activity of G6Pase was fourfold reduced in cirrhosis; in 3 months after the end of poisoning, under effect of CG, the activity increased to the normal level, but somewhat decreased subsequently. In the animals that were not treated with CG, the decrease in the G6Pase activity after the cessation of the CCl4 poisoning was even more marked than in the CG-treated rats. Activities of two other enzymes of glycogen metabolism did not differ statistically significantly from the norm throughout the entire experiment. The data obtained indicate that the use of CG for rehabilitation of the glycogen-forming function of the cirrhotically altered liver is more efficient than other ways of treatment studied previously, such as partial hepatectomy or a high-carbohydrate diet.

Functional activity of human hepatocytes under traumatic disease.

Absorption and fluorescent cytophotometry techniques were applied to studies of RNA as well as of total glycogen and its fractions as the parameters of functional activity of the hepatocytes in patients with severe mechanical trauma, both with and without autointoxication (AI). Slides were stained with gallocyanine-chromalums to determine the RNA content and were processed by the fluorescent PAS-reaction for the glycogen content. To trace the dynamics of RNA and glycogen contents in the liver punction biopsies were done in the same patients. A quick increase in the RNA content took place in both groups of patients at the first period (within the first 3 days) of traumatic disease. At the second period of disease the hepatocyte RNA content in patients without AI was found to decrease up to the initial level whereas that in patients with AI increased on the average by 36% of the initial values. The total glycogen content in hepatocytes of all the patients changed insignificantly in the course of disease but its labile fraction in patients with AI decreased to 70% of the total. The increase of hepatocyte synthetic activity and the maintenance of the high glycogen level are indicative of the large compensatory potential of the liver that enables it to carry an intensive functional load under AI conditions.

A method for investigating hepatocyte polyploidization kinetics during postnatal development in mammals.

A method for investigating weakly-proliferating cell populations of liver parenchyma on the basis of a quantitative analysis of hepatocyte polyploidization during postnatal development is described. The method uses a mathematical model which characterizes the hepatocyte polyploidization process, and incorporates data concerning the time course for relative frequencies of hepatocytes in different ploidy classes. As a result of these measurements and calculations for rat liver, transition rates of hepatocytes (the relative number of cells during a given time unit) from one ploidy class to another, and a coefficient for the reduction of hepatocyte mitotic activity with an increase in its ploidy class were obtained. Calculated curves show a good correspondence with the real process of hepatocyte frequency changes as they relate to changes in the age of the animals. To check this method, experiments investigating time changes of autoradiographic label content in the different ploidy classes of hepatocytes were carried out. By mathematically modeling the label diluting process resulting from cell proliferation and polyploidization, transition rates of hepatocytes were calculated, and they reflect values calculated from the model according to changes in occurrence frequencies.

Hepatocyte polyploidy and metabolism/life-history traits: hypotheses testing.

Two alternate hypotheses explaining the causes of mammalian liver polyploidy have been tested by means of statistical analysis of more than 30 placental species. According to the first (general) hypothesis, the omission of mitosis is beneficial in rapidly growing and differentiating tissues that should early perform their specialized functions (economy on mitosis). In this case it is to be expected that the level of polyploidy is positively correlated to the rate of development. The second hypothesis suggests that hepatocyte polyploidy provides additional gene copies necessary under heavy chemical damage to metabolically active hepatocytes, which have to detoxicate noxious substances. In this case it is reasonable to expect that the level of liver ploidy is positively correlated with the rate of metabolism while inversely with the species lifespan (since it is usually assumed that DNA repair systems are more effective in more long-lived species). It is found that, if taken separately, the developmental rate, the rate of basal metabolism, the body weight (inversely), and the maximum lifespan (inversely) are all correlated with the level of hepatocyte polyploidy. However, all these parameters are intercorrelated. When the other parameters are fixed (in partial correlation analysis), only the developmental rate and the rate of basal metabolism remain significant predictors of the hepatocyte ploidy level, with only the former being a predictor of the nuclear ploidy level, while only the latter being positively correlated with the frequency of binucleated cells. These results suggest that relative importance of the two above explanations can differ for the different modes of liver cell polyploidy.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of the glycogen-forming function of hepatocytes in rats with liver cirrhosis is facilitated by a high-carbohydrate diet.

Using cytofluorimetric and biochemical methods, the content of glycogen and its labile and stable fractions, as well as activities of glucose-6-phosphatase (EC 3.1.3.9), glycogen phosphorylase (EC 2.4.1.1) and glycogen synthase (EC 2.4.1.11) were determined in the rat liver for 6 months after chronic poisoning of the animals with CCl4 and then at 1, 3, and 6 months after the end of the poisoning. One group of rats was given a standard diet, the other, a high-carbohydrate diet. The 6-month long chronic intoxication with CCl4 was shown to produce development of typical liver cirrhosis characterized by a 2.8-fold increase in the total glycogen content in hepatocytes as compared with normal cells, by a fall in the glycogen labile fraction (from 85 to 53% of the total glycogen) as well as by decreases in the activities of glycogen phosphorylase and glucose-6-phosphatase by 25 and 82% respectively. The structural rehabilitation occurred faster and more completely at the cellular level than at the tissue level. Functional variables of the cirrhotic liver tissue also recovered, after cessation of poisoning, faster and more completely than the liver structure at the tissue level: glycogen levels in hepatocytes fell dramatically, the labile: stable glycogen fraction ratio recovered completely, and the activity of glycogen phosphorylase rose to the level characteristic of the normal liver. Use of the high-carbohydrate diet promoted a somewhat faster and more complete recovery of hepatic structure and function.

Cardiomyocyte ploidy levels in birds with different growth rates.

Cytofluorimetric study of ploidy levels in ventricular cardiomyocytes was carried out on 36 adult bird species belonging to 10 orders as well as on the quail Coturnix coturnix, of different ages. It was shown that polyploidization of quail cardiomyocytes occurs during the first 40 days after hatching and ends by the time growth is completed. In adult birds, the cardiomyocyte ploidy hardly changed at all. Interspecies comparison revealed that in the adult bird myocardium 2cx2 myocytes are predominant, accounting for at least 50% of the cell population. Multinuclear cells with three to eight diploid nuclei were widespread. The percentage of such cells was five to six times higher in precocial species than in altricial birds of the same weight. Myocytes with polyploid nuclei were rare. A significant interspecies variability of cardiomyocyte ploidy levels was observed. The most prominent differences were found between the precocial and the altricial birds. The mean number of genomes in cells correlated both with the body mass and with the growth rate of the birds. The differences between the precocial and altricial birds disappeared when a statistical method was used to eliminate the effect of the growth rate, but did not when the effect of body mass was eliminated. Among the altricial birds, which are generally immobile during growth, the cardiomyocyte ploidy levels also correlated more closely with growth rate than with body mass. The opposite was observed in the precocial birds, which are highly mobile from the first minutes of life. We conclude that the interspecies variability of bird cardiomyocyte ploidy levels is a result of changes in the balance between the cardiac functional load and the growth rate; this is manifested at the cellular level as a competition between the proliferation and differentiation of cardiomyocytes. J. Exp. Zool. 289:48-58, 2001.

Relationship of hepatocyte ploidy levels with body size and growth rate in mammals.

To elucidate possible causes of the elevation of genome number in somatic cells, hepatocyte ploidy levels were measured cytofluorimetrically and related to the organismal parameters (body size, postnatal growth rate, and postnatal development type) in 53 mammalian species. Metabolic scope (ratio of maximal metabolic rate to basal metabolic rate) was also included in 23 species. Body masses ranged 10(5) times, and growth rate more than 30 times. Postnatal growth rate was found to have the strongest effect on the hepatocyte ploidy. At a fixed body mass the growth rate closely correlates (partial correlation analysis) with the cell ploidy level (r = 0.85, P < 10(-6)), whereas at a fixed growth rate body mass correlates poorly with ploidy level (r = -0.38, P < 0.01). The mature young (precocial mammals) of the species have, on average, a higher cell ploidy level than the immature-born (altricial) animals. However, the relationship between precocity of young and cell ploidy levels disappears when the influences of growth rate and body mass are removed. Interspecies variability of the hepatocyte ploidy levels may be explained by different levels of competition between the processes of proliferation and differentiation in cells. In turn, the animal differences in the levels of this competition are due to differences in growth rate. A high negative correlation between the hepatocyte ploidy level and the metabolic scope indicates a low safety margin of organs with a high number of polyploid cells. This fact allows us to challenge a common opinion that increasing ploidy enhances the functional capability of cells or is necessary for cell differentiation. Somatic polyploidy can be considered a "cheap" solution of growth problems that appear when an organ is working at the limit of its capabilities.

Determination of DNA surplus in the nuclei of some cerebellar Purkinje cells with different cytochemical methods.

Cytofluorometry and cytophotometry (Feulgen staining) as well as ultraviolet cytophotometry revealed similar histograms of DNA content in squashed nuclei of rat cerebellar Purkinje cells. More than 90% diploid (2c) cells and 1% tetraploid (4c) cells were obtained. DNA content in other Purkinje cells ranged between the 2c and 4c level.

Polyploidization and endomitosis in giant cells of rabbit trophoblast.

Morphological and cytophotometric investigations have been performed on giant cells of the rabbit trophoblast to reveal a mechanism of nuclei polyploidization and define the level of polyploidy. The character of endomitotic chromosomes is found to differ and depend largely on the degree of nuclei polyploidy. Small chromosomes were found in nuclei with low levels of polyploidy. For highly polyploid nuclei, two stages are distinguished. In the first case condensed chromosomes join into bundles resembling Riesenchromosomen in plants, whereas in the second, decondensed chromosomal threads separate and disperse in the karyoplasm. The splitting does not involve nuclei-forming chromosomes in the region of the nucleolar organiser. The degree of polyploidy was determined on the 15th day of development. It was found that giant cell nuclei contain DNA in amounts corresponding to 32-512 chromosomal sets. Most of the nuclei have levels of 128c and 256c. Highly-polyploid nuclei disintegrate into small nuclei with the degree of polyploidy varying from 1c to 32c. Di- tri- and tetraploid nuclei predominate.

Human hepatocyte polyploidization kinetics in the course of life cycle.

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86-92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C x 2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.

Fluorescent PAS-reaction study of the epithelium of normal rabbit ileum and after challenge with enterotoxigenic Escherichia coli.

Fluorescent periodic acid-Schiff reaction (FPR) was used in the study of the normal rabbit ileal epithelium and its changes after injection of living cultures or enterotoxins of enterotoxigenic Escherichia coli. This reaction, with the use of auramine OO-SO2 complex as a Schiff-type reagent, demonstrates gut epithelium periodate-reactive mucosubstances more distinctly and brightly than does the common periodic acid-Schiff (PAS) reaction. It permitted the quantitative assessment of polysaccharide content in the gut sections by microfluorimetry, and examined extensively the mucosal structures, brush border, and mucous cells which participate in the interaction with enteropathogens. Fluorescent periodic acid-Schiff reaction showed that noninvasive enterotoxigenic E. coli O148:H28 B7A organisms caused restricted damage to the intestinal epithelium brush border. Invasive enterotoxigenic E. Coli O26:K60:H11 N3 organisms penetrated the epithelium and caused extensive brush border lesions and mucous cell hyperproduction. Importance of FPR in the complex morphologic analysis of enteric infections, pathogenesis of escherichoses under study, and some aspects of the intestinal epithelium histology are discussed.

Biodegradation of magnetite dextran nanoparticles in the rat. A histologic and biophysical study.

BACKGROUND: Superparamagnetic iron oxide particles represent a new class of contrast agents that increase the detectability of hepatic and splenic tumors by magnetic resonance imaging (MRI). Magnetite dextran nanoparticles, a preparation with a small mean particle diameter in solution and null zêta potential present high safety margin and efficacy. The purpose of this investigation was to define the main steps of the metabolism of the iron oxide crystals. EXPERIMENTAL DESIGN: Rats were intravenously administered a single small dose of 59Fe-labeled MD3 (3 mg Fe/kg), and the biodistribution of 59Fe was investigated in the different organs from 2 hours to 25 days postinjection. Magnetic susceptibility studies were conducted in parallel to light microscopy and immunohistochemistry from day 1 to day 14 after administration. RESULTS: Most of the dose accumulated in the carcass (45%), liver (7%), and spleen (7%) in the first 2 hours. In the spleen, a continuously iron uptake was observed up to 48 hours (44%), then decreased to 25 days (22%). The splenic magnetic susceptibility dropped sharply during the first days and then more slightly until day 14. In the liver and blood, the 59Fe-level decreased at 24 hours and then increased until day 25 (11% and 27%, respectively). Histochemistry features essentially confirmed the radiotracer data and showed that iron oxide cores were accumulated into the Kupffer cells and the macrophages of the splenic marginal zone. With time, the number of the granules was decreased whereas the fine iron granules appeared in the cytoplasm. Immunopositive staining for ferritin was markedly increased in the liver hepatocytes to 3 days after injection, and in the splenic marginal zone macrophages to 14 days after injection. CONCLUSIONS: The data point to the early biodegradation of the iron oxide crystals. MD3 thus appear as an interesting biodegradable new contrast agent first devoted to magnetic resonance imaging of liver and spleen diseases that could be further extended to heart, kidneys, and other organs.