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The effect of inhibition of proteasome activity on direct cell-cell interactions in primary hepatocyte cultures was studied. The circahoralian rhythm of protein synthesis was a marker of cell-cell communication. The addition of the proteasome inhibitor MG132 at doses of 10 or 20 μM to the medium with hepatocyte cultures for 19 h resulted in a significant reduction in the total pool of 3H-leucine in cells. The incorporation of leucine into proteins changed slightly or negligibly, whereas the content of free labeled leucine in hepatocytes decreased. The rhythm of protein synthesis was distorted compared to the control. The rhythm was restored by external organizers, such as gangliosides and melatonin, as well as by enhancing the activity of protein kinases--the key factor in the organization of the rhythm of protein synthesis. A short-term (3-h) exposure to MG132 did not change the pool of leucine, but the rhythm of protein synthesis was also disturbed. Thus, protein catabolism affects cell-cell interactions organizing the rhythm of protein synthesis. Another factor of the downregulation of the rhythm of protein synthesis, the secretion of proteins from the hepatocytes in vivo, which was shown in vivo in many studies, was also revealed in our study when measuring the content of proteins stained with Coomassie Brilliant Blue G250 in the medium with hepatocyte cultures.

A comparative study was performed of dense 5-hour cultures of rat hepatocytes and equal-density cultures of mesenchymal stromal cells (MSC) isolated from human adipose tissue of rat bone marrow. The cells were grown on collagen-coated class slides in serum-free medium. Unlike in hepatocytes, no rhythm of protein synthesis was initially revealed in MSC, but such a rhythm manifested itself when the culture medium was supplemented with melatonin (2 nM, 5 min). The results of experiments with cytoplasmic calcium chelator BAPTA-AM and protein kinase inhibitor H7 indicate that the mechanism of protein synthesis synchronization in MSC consists in calcium-dependent phosphorylation of cell proteins.

In cultures of human keratinocytes HaCaT contained in a serum-free medium on glass, a circahoralian rhythm of protein synthesis was found similar to the one in hepatocytes in vitro. The intensity of the synthesis was determined by the inclusion of 3H-leucine corrected for the pool of free marked leucine. Rhythm was studied in washed 1- or 2-day cultures after the change of the medium. The medium conditioned with keratinocytes HaCaT synchronized the rarefied hepatocyte cultures nonsynchronous in the control. Therefore, the keratinocytes liberate synchronizing factors into the medium. A BAPTA-AM chelator of calcium ions eliminates the protein synthesis rhythm both in dense hepatocyte cultures synchronous in the control and in the HaCaT keratinocyte cultures. The effect of the H7 inhibitor of protein kinases was analogous. Thus, both in keratinocytes and hepatocytes, self-synchronization of fluctuations of the intensity of protein synthesis takes place. The mechanism of self-synchronization is the calcium-depending phosphorylation of cell proteins.

Melatonin injected intraperitoneally into rat synchronizes the ultradian rhythm of protein synthesis after 100 min in primary hepatocyte cultures isolated from this rat, which are studied after 1 or 2 days. The effective synchronization concentrations of melatonin--0.01-0.02 microg per kg of rat weight--are three orders lower than melatonin doses used in clinical practice in human treatment.

Current data confirm the fundamental statement of the cell theory concerning the cell reproduction in a series of generations (omnis cellula e cellula). Cell communities or ensembles integrated by the signaling systems established in prokaryotes and protists and functioning in multicellular organisms including mammals are considered as the structural and functional unit of a multicellular organism. The cell is an elementary unit of life and basis of organism development and functioning. At the same time, the adult organism is not just a totality of cells. Multinucleated cells in some tissues, syncytial structure, and structural-functional units of organs are adaptations for optimal functioning of the multicellular organism and manifestations of cell-cell communications in development and definitive functioning. The cell theory was supplemented and developed by studies on cell-cell communications; however, these studies do not question the main generalizations of the theory.

Comparison of current data on direct cell-cell communications in mammals, protists, and bacteria suggests that the emergence of the signaling systems of self-organization underlay the emergence of multicellular organisms. Biogenic amines, regulators of coordinated behavior and aggregation in bacteria, have been found in protists and multicellular organisms. In metazoans, biogenic amines have become specific neurotransmitters. At the same time, the studies on synchronization of protein synthesis rhythm in mammalian cell cultures demonstrated that noradrenaline and serotonin have conserved their ancient function of cell-cell cooperation in mammals, which is manifested as coordinated social behavior of cells in population in the case of bacteria and multicellular organisms.

The effect of 1 to 1000 nM melatonin was studied on daily cultures of rat hepatocytes on slides in serum-free medium. The minimum melatonin concentration (1 nM) proved to synchronize protein synthesis in asynchronous sparse cultures of hepatocytes from rats of different age, and a circahoralian rhythm of protein synthesis was revealed in them. In dense weekly synchronous hepatocytes from old rats (2.5-years-old with the weight of about 600 g), melatonin improved cell synchronization to the level of young animals. Melatonin treatment increased the mean rate of protein synthesis in rats of different age.

Considering the data on the low level of self-organization (self-synchronization) of protein synthesis rhythm in aging, we studied the possible interference of the signaling factors of self-organization, gangliosides and catecholamines, as well as catecholamine reception. Experiments were carried out on primary cultures of rat hepatocytes on slides. Inhibited ganglioside synthesis did not prevent the organization of protein synthesis rhythm by the alpha-adrenomimetic agent phenylephrine. Upon the blockade of alpha-receptors by prazosin, the protein synthesis rhythm was observed after the exposure to gangliosides. Alpha-adrenolytic agents prazosin and benoxathian abolished the synchronizing effect of the beta-adrenomimetic isoproterenol. A mixture of alpha- and beta-adrenomimetic agents inhibited the protein synthesis rhythm-organizing effect of noradrenaline. Thus, the signaling molecules of self-organization of protein synthesis function independently via specific receptors.

The aim of the review is to attract attention to the importance of a dynamic approach to symptoms of health and pathology. In this regard, long-known approximately 24-hour or circadian rhythms (CR) are very prospective. However, according to recent studies, shorter approximately hourly rhythms (AHR) are of not less importance. Unlike CR, HR are not obtruded on the organism by periodical external influence of day-night pattern, but present cell's own fundamental property, a method of their metabolism. The fractal nature of HR has been substantiated; age-dependent changes in HR have been demonstrated; the importance of AHR for diagnostics and prognosis in gastroenterology and cardiology has been determined. To perform more clinical observations is an important task.

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 microM or forskolin at 10 microM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 microM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 microM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists-->changes in the level of calcium ions in cytoplasm-->increased protein kinase activities-->protein phosphorylation-->modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.

The effect of gangliosides and phenylephrine synchronizing the protein synthesis rhythm was preserved in hepatocytes cultured in the normal serum-free medium for one-two days. Hence, the membrane signal triggers intracellular, as was shown by us earlier, calcium-dependent processes, which regulate the kinetics of protein synthesis for a certain time after the signal perception.

Cell interactions have been studied in cultures pf hepatocytes from young and old rats. The rhythm of protein synthesis is an index of cell interaction and synchronization in culture, while the amplitude of oscillations characterized cell cooperation in an aggregate rhythm. The mean rhythm amplitude in the culture of hepatocytes from old rats is twice lower than that from young rats. Gangliosides (mixture, bovine brain gangliosides) and alpha1-adrenomimetic phenylephrine enhanced synchronization of cultures of the cells from old rats and increased the amplitude of oscillations to the level of young animals. Addition of rat blood serum (10%) to the medium revealed the rhythm of protein synthesis in the culture, asynchronous in the control, i.e., led to their synchronization. In media with young and old rat blood sera, oscillations were intense, with high amplitudes, and low, respectively. Addition of bovine brain gangliosides to a medium with old rat blood serum increased the amplitudes of oscillations to a level of the rhythm stimulated by the young rat serum. Thus, the cells of old animals can fully perceive synchronizing factors and, in the case of their increased concentration, the rhythm of protein synthesis in old animals did not differ from that in young rats. Current data on biochemical mechanisms underlying intercellular cooperation in the formation of population rhythm of protein synthesis have been discussed.

The medium conditioned by dense, self-synchronized hepatocyte cultures was centrifuged at 150,000 g to obtain two fractions. The light fraction (supernatant fluid) contained ganglioside monomers and micelles, and the heavy fraction (pellet) contained gangliosides in the vesicles shed from the cell membrane. In the test populations of hepatocytes, the rhythm of protein synthesis was used as an indicator of cell synchronization resulting from their cooperative activity. Low-density hepatocyte cultures with asynchronous fluctuations of protein synthesis proved to be synchronized by both the initial conditioned medium and its vesicular fraction. Our previous studies have shown that this occurs under the effect of GM1 monosialoganglioside, which is released from cultured cells and accumulates in the conditioned medium. Liposomes consisting of GM1 and phosphatidylcholine from egg yolk (1:19 mol%), compared to free exogenous GM1, synchronized the rhythm of protein synthesis more effectively: synchronization was observed at a GM1 concentration in liposome suspension of only 0.0003 microM, compared to 0.06 microM and higher in the case of free GM1. Thus, GM1 as a component of membranes and monolayer lipid structures proved to be much more effective than free GM1 in promoting hepatocyte cooperation with respect to the rhythm of protein synthesis.

Previously we demonstrated synchronized oscillations of protein synthesis rate in the hepatocyte cultures upon accumulation of monosialognaglioside GM1 in the medium or after introduction of exogenous GM1 to the medium. The synchronized oscillations of the protein synthesis rate in dense hepatocyte cultures were blocked 30 min after their treatment with 10-20 microM BAPTA-AM--a chelating agent of cytoplasmic calcium. Enzyme immunoassay for GM1 demonstrated similar amounts of GM1 in the medium conditioned for 3 h by dense hepatocyte cultures pretreated with 20 microns]M BAPTA-AM for 1 h and in the medium of normal dense cultures--0.0060 +/- 0.0005 and 0.0055 +/- 0.0005 pmol/1000 cells, respectively. The content of GM1 was also similar in the normal and BAPTA-AM-pretreated hepatocytes--0.158 +/- 0.013 and 0.183 +/- 0.014 pmol/1000 cells, respectively. The synchronized rhythm of protein synthesis has been confirmed in the diluted cultures in the medium conditioned by the normal dense cultures. However, the medium conditioned by the dense cultures pretreated with BAPTA-AM induces no synchronization of the diluted cultures. Since GM1 concentration was normal in this medium, we propose the effect of physicochemical form of the gangliosides accumulated in the medium on their ability to synchronize the rhythm of protein synthesis.

We studied the effects of the chelating agents of extra- and intracellular calcium ions, EGTA and BAPTA-AM, and of the inhibitor of Ca2+ release from the reticulum, TMB-8, in the kinetics of protein synthesis in hepatocyte cultures. We also studied dense cultures capable of self-synchronization of protein synthesis oscillations and diluted cultures, in which synchronization is induced by phenylephrine or gangliosides (standard preparation of total gangliosides from the bovine brain). Preincubation of the diluted or dense cultures in the presence of 2 mM EGTA for 1-2 h with subsequent protein assay in a medium with EGTA did not affect the kinetics of protein synthesis: no rhythm was found in the diluted cultures, while it was preserved in the dense cultures. When the diluted cultures preincubated in the presence of EGTA were placed in a medium with EGTA and 2 microM phenylephrine for 2 min, the rhythm was visualized. The treatment of diluted cultures with 100 microM TMB-8 for 5 or 10 min with subsequent washing and incubation in a medium with 3 microM gangliosides led to visualization of the protein synthesis rhythm, i.e., to the synchronization of oscillations, while no rhythm was found in the standard cultures. Preincubation of the diluted cultures in a medium with 10, 15, or 20 microM BAPTA-AM did not affect the kinetics of protein synthesis. When, after such preincubation, the diluted cultures were placed in the medium with gangliosides, the rhythm was visualized. In the dense cultures, normally capable of self-synchronization, no rhythm of protein synthesis was found after their treatment with 10-20 microM BAPTA-AM for 1 h. The transfer of such cultures in the medium with gangliosides led to visualization of the rhythm. Thus, calcium affects the kinetics of protein synthesis: after the rise of Ca2+ in the cytoplasm was blocked, the rhythm of protein synthesis was not visualized due, supposedly, to disturbed mechanisms of medium conditioning. However, exogenous gangliosides in the dense or diluted cultures preincubated in the presence of BAPTA-AM ore TMB-8 allowed the rhythm visualization, i.e., synchronization may not depend on changes in the intracellular calcium concentration.

The circumhoralian rhythm of protein synthesis was determined in a monolayer culture of hepatocytes from rats at the age of 1 to 24 months and weighing from 45 to 480 g, respectively. The peptide lyvagen (Lys-Glu-Asp-Ala) obtained by directed chemical synthesis on the basis of amino acid analysis of the liver polypeptide preparations increased the level of protein synthesis in the hepatocytes from rats of different ages; the highest effect was observed in the cells of old animals. In old rats, lyvagen increased the amplitude of protein synthesis fluctuations. The peptide epitalon (Ala-Glu-Asp-Gly) constructed on the basis of analysis of the epiphysis peptides did not change the intensity of protein synthesis in the cultured hepatocytes.

We studied the effect of the alpha 1-adrenolytic prazosine on dense cultures of hepatocytes, which are normally characterized by the protein synthesis rhythm, and diluted cultures, in which such a rhythm is revealed after external synchronization. Exogenous gangliosides (a fraction of the total gangliosides of the bovine brain) then synchronize the rhythm in diluted cultures; this effect is also displayed in the presence of 10(-7) M prazosine. The synchronizing effect of the medium conditioned by dense cultures was also preserved in the presence of prazosine. In the dense cultures that don't normally require external synchronization, prazosine affected intensified the rhythmic patter of changes in the protein synthesis. After a total of 0.3 microM gangliosides were introduced in the medium with prazosine-pretreated dense cultures, the protein synthesis rhythm was visualized. We propose that, while blocking adrenoreceptors, prazosine does not prevent the action of exogenous synchronizing factors on the hepatocytes, but inhibits the release of such factors from the cell.

Circahoralian cellular rhythms have been revealed after the amendment of quantitative cytochemical methods initiated at the Department of Histology (Moscow State University) and Laboratory of Cytology (Institute of Animal Morphology, Russian Academy of Sciences). The first findings have been confirmed in many laboratories. Circumhoralian kinetics proved to be specific for various cell types (from bacteria to mammalian cells) and cellular functions. Independent physiological investigations found circahoralian rhythms for various tissue functions. Distribution of the rhythms as well as their possible nature and significance for tissue biology are reviewed.

Published data indicate that 1-3 microM adrenomimetic phenylephrine increases the concentration of calcium ions in the cytoplasm of cultured hepatocytes. We studied low-density cultures exhibiting no protein synthesis rhythm in the fresh medium and demonstrated that a 2 min action of 2 microM phenylephrine induces protein synthesis rhythm, i.e., synchronizes the synthesis oscillations in hepatocytes. A similar effect was observed for a selective inhibitor of the reticulum calcium pump, di-tert-butyl-benzohydroquinone, that increases the cytoplasm concentration of calcium ions by a receptor-independent mechanism. A calcium antagonist imipramine obviated the synchronization effect of phenylephrine. We propose that short-term changes in the cytoplasm concentration of calcium ions covering the whole cell population are among intracellular mechanisms of protein synthesis synchronization in hepatocytes in vitro.

The properties and possible origin of circahoralian intracellular rhythms are discussed. Irregularity of these oscillations, as expressed in different time scales, determination, and resistance to external influences, including changes in the environmental temperature, are noted. No pacemaker of circahoralian rhythms was found. These specific features and analysis of the structure of circahoralian rhythms (Herst equation) suggest their similarity to fractals, chaotic but determined forms with a certain internal structure. Determination of circahoralian rhythms in a cell-free system and their possible fractal nature characterizes the rhythms as an imminent property of the cytoplasm, that does not require special regulation. Modulations of the frequency and amplitude of oscillation under the influence of external influences are described. It is proposed that the circahoralian rhythms are a protective factor for the cell and organ functions and a mechanism of adaptations under varying life conditions.

We studied the kinetics of proteins synthesis in the cultures of hepatocytes on collagen-coated slides in medium 199 enriched with 0.2 mg/ml albumin and 0.5 microgram/ml insulin and devoid of bovine serum. Circahoral fluctuations of proteins synthesis intensity were found in the monolayer cultures and sparse cultures in a conditioned medium. No protein synthesis rhythm was expressed in a fresh medium after repeated washing of the cultures. Addition of micromolar concentrations of gangliosides GM1 or GD1a to the medium revealed the rhythm in washed cultures. Addition of GT1b or GM3 to the medium did not synchronize the oscillations: the kinetics of protein synthesis did not differ from that in the control space cultures in a fresh medium without exogenous gangliosides. Accumulation of gangliosides GM1 and GM3 in the hepatocytes in vitro upon conditioning of a serum-free culture medium was shown using the indirect immunocytochemical method. The effect was found in dense monolayer cultures and in cultures with separated cells in a conditioned medium. The protein synthesis rhythm was found in such cultures. Gangliosides are weakly expressed in most cells of a repeatedly washed 24-hour monolayer and washed sparse cultures. No protein synthesis rhythm was found in washed cultures. Similar changes in the dynamics of the culture medium conditioning, accumulation of gangliosides in cells and rhythmic activity of cells population confirm the concept of the synchronizing role of gangliosides.

The kinetics of protein synthesis was studied in the cultures of rat hepatocytes on slides covered with collagen or fibronectin and kept in a serum-free medium 199 complemented by albumin and insulin. No protein synthesis rhythm was found in the cultures with markedly separated cells obtained from a diluted suspension of hepatocytes ("sparse cultures"). After 0.3-0.4 microM total gangliosides were added to the medium with such cultures, the rhythm was found in all experiments. Such an effect was earlier found under the influence of a medium conditioned by a monolayer on the sparse cultures. The results obtained suggest the involvement of gangliosides in synchronization of oscillations of the protein synthesis intensity and call for studies of the conditioned medium properties.

In a culture of rat hepatocytes, kept in a serum-free medium 199, the number of attached cells was decreased and a monolayer was not formed within 24 h. Soon after the cells were attached to collagen, the intensity of protein synthesis in the serum-free medium was decreased due to the smaller number of cells in the culture and was calculated per cell. The properties of cultures, i.e., the number and shape of cells, external appearance of the culture, and intensity of protein synthesis, were similar in a serum-containing medium and in the serum-free medium 199 complemented with 0.2 mg/ml albumin and 5 micrograms/ml insulin. A monolayer typical for the medium with serum was formed in the medium with albumin and insulin within approximately 20 h after attachment of the hepatocytes to the substrate.

Newborn rats were grown in litters of 8 (control), 4, or 16 individuals up to the day 21 of postnatal development and then transferred to usual keeping and feeding conditions similar for all groups. Liver and cerebellum were removed for the study at days 18, 45, and 90. At day 28, the body weight of animals from experimental groups differed by approximately three-fold, weight of liver, almost five-fold, and weight of the cerebellum, by 1.4 times. The body weight of animals also showed significant differences at day 90, however, the weight of the liver and cerebellum differed from the control only in animals that received excessive feeding. We demonstrate that in the control and in experimental rats, the intensity of the protein synthesis during the period from day 18 to day 90 shows similar changes, and it is always higher in the liver as compared with the cerebellum. Differences between fasting animals and animals given excess food generally were apparent mainly at day 90. Thus, feeding disturbances during the first weeks after birth result in uncompensated changes of animal growth and of the studied animal organs. These data are discussed in connection with perspectives of using this model to study specific aspects of three-dimensional organization of the nucleolar complex of hepatocytes and cerebellar neurons.

Incorporation of 3H-leucine in proteins and radioactivity of free leucine have been studied in hepatocyte culture on collagen-coated glass. A high density culture obtained from a suspension of isolated rat hepatocytes at 5 x 10(6) cells/ml was compared with low density cultures diluted from the same suspension 10- to 20-fold. The incorporation pool ratio showing the efficiency of the precursor utilization was higher in the low density culture as compared with the high density monolayer. The effect was due to disproportionate low pool in the diluted cultures. The total radioactivity of proteins was lower in the diluted cultures as compared with the monolayer, although the incorporation was not in proportion with the cell density. Specific rate of protein synthesis (per cell) was higher in the diluted cultures than in the monolayer.

Using the radiochemical method, we found a circahoralian rhythm of protein synthesis in slices of rat liver two weeks after transection of the vagus nerve anterior stem or bilateral subphrenic vagotomy with simultaneous transection of the celiac plexus hepatic branches. The rhythm was similar to those found in slices of normal liver or in a monolayer culture of hepatocytes. The incorporation of leucine in proteins and the pool of free leucine were similar in the denervated and normal liver. The self-synchronization of individual cell oscillations in protein synthesis intensity was shown earlier in experiments performed on cell cultures. A study of protein synthesis in the denervated liver suggests that cell interactions are also the main mechanism underlying the synchronization of oscillating cellular functions in the organ in situ.

A part of suspension with isolated hepatocytes was cooled and then warmed to normal temperature. In cultures formed with such cells, antiphase oscillations of the protein synthesis rate were detected concerning control cultures from another part the same hepatocyte suspension. Oscillations of protein synthesis were observed in the mixed cultures formed with equal parts of antiphase subpopulations of hepatocytes. The experiments established cell interactions in vitro resulted in synchronization of protein synthesis oscillations in different cells the same population.

Variability of cells is analyzed using data obtained in studies on polyploidy in the heart muscle and on ontogenetic changes in the number of myocytes. In normal myocardium, variation in the number and ploidy of muscle cells is observed. The types of genomic variation are similar to those found in populations of organisms, i. e., to hereditary and modifying variation. In studies on modifications of the myocyte genome, heart growth reserves were revealed, which manifest themselves in pathological states associated with myocardial hypertrophy. These reserves depend on the level of myocardial polyploidy that is determined during early postnatal development.

The rate of rRNA synthesis increased in hepatocytes within 14 h after partial hepatectomy. The increase, 1.7-fold on the average, was similar in diploid, tetraploid and octaploid nuclei. Under these conditions, the content and area of argentophilic (Ag-) nucleolar proteins were increased, although they varied between individual animals and nuclei of the same cell population. The changes of transcription and Ag proteins were not proportional. Thus, the Ag stain may characterize only the trend in activity of the ribosomal genes but not their exact state in hepatocytes.

Changes in the activity and number of nucleoli has been studied in hepatocytes of the same rat before and after partial hepatectomy. In the latter case, the rate of ribosomal RNA (rRNA) synthesis was increased by a factor of 1.7, on the average. The synthesis was evaluated by incorporation of adenosine triphosphate in situ after alpha-amanitine treatment of squashed cells. In 2c, 4c and 8c nuclei, the number of labeled nucleoli was less than in nucleolonemic nucleoli stained with silver nitrate. These values were drawn together during stimulation of rRNA synthesis but the difference was often preserved especially for highly ploid nuclei. The lack of activity in part of nucleolonemic nucleoli was assumed. After stimulation of RNA synthesis, the number of nucleolonemic and satellite nucleoli decreased, while the size of preserved nucleoli was enlarged. The number of nucleoli did not correspond to the nuclear ploidy; their total size was proportional to the gene dosage. The mean level of rRNA synthesis was similar in nuclei of the same ploidy with two, three and four nucleoli. A great variability was detected in the rate of rRNA synthesis as well as in the number of nucleoloneme and satellite nucleoli in hepatocytes of the control and experimental rats. Some difference was observed in the label degree for nucleoli of the same nucleus.

Cerebellum of rat embryos at day 17 of gestation was transplanted into sensomotor cortex of adult syngenic rats. DNA content in granule and Purkinje cells of the transplant was determined cytophotometrically on day 30 after surgery. It is shown that granule cells are diploid; about 3% Purkinje cells contain hyperdiploid and tetraploid nuclei which corresponds to the content of such cells in the adult cerebellum. The conclusion is drawn that cell hyperploidy does not depend on the functional load but is essential property of the tissue.

Radioligand and immunohistochemical analysis was used for studying the expression of an unusual estrogen-binding protein (UEBP) in the regenerated liver of male rats poisoned with CCl4. As a result of CCl4 poisoning, most hepatocytes intensely producing UEBP and located in the central part of the liver lobules are destroyed (40 to 90% of the liver parenchymal cells). Dead cells are substituted by new hepatocytes formed due to proliferation of periportal hepatocytes in which UEBP is expressed at a low level. In regenerated liver, however, a high level of UEBP expression characteristic of the control animals is restored, as well as a gradient mode of distribution of UEBP-containing cells with the maximum UEBP concentration in hepatocytes surrounding the central vein. The results obtained with animals with either intact or removed testes were similar, except that the dynamics of liver regeneration was slower in the castrated rats. These data suggest the existence in all hepatocytes of the complete androgen-related program for UEBP expression. This program is inherited by daughter cells in the absence of an inducing hormone and is similar in all cells, independently of the extent of its phenotypic expression in hepatocytes with different localization within the liver lobule.

Protein synthesis intensity in the tissue of various portions of the brain of rats pre-exposed to 10 min systemic circulation arrest was studied with the use of 3H-leucin, a protein synthesis labeled precursor, and compared with that in intact animals. Protein synthesis intensity in the brain of resuscitated animals was found to correlate with the degree of their neurologic status recovery. In rapidly recovering animals a significant intensification of protein synthesis is observed on day 4 after resuscitation in the sensorimotor cortex, hippocampus, and cerebellum. In the animals whose resuscitation was not so smooth this process develops only in the hippocampus and the intermedial and lateral portions of the cerebellum. In patients with disturbed neurologic status protein synthesis intensity was unchanged in all the examined portions of the brain. The authors suggest that protein synthesis activation phase is an obligatory stage in full-value neurologic recovery of resuscitated animals.

The intensity of protein synthesis (incorporation/pool) is usually low in normal stomach mucosa and enhanced in exacerbation of peptic ulcer; following the effective laser therapy it decreases only in one third of cases. Therefore the scarring of ulcer not always coincides with the normalization of protein synthesis. The results characterize also the community of reactions in stomach and duodenal mucose membranes. The changes in protein synthesis intensity following the laser irradiation depended neither on the initial size of ulcer nor on the age of patient and the total duration of disease.

DNA cytophotometry has been performed in ventricular cardiomyocytes of hypertrophic human hearts. In the cases of hypertrophy in adults (generalized atherosclerosis, postinfarct scars), polyploidy expression did not exceed the limits of normal variability developed during childhood. In the cases of hypertrophy caused by congenital heart defects, high polyploidy has been revealed (the mean level 20c and more, where c is haploid DNA content), which considerably exceeded the upper limit of normal variability (approximately 10c). Our hypothesis has confirmed that heart hypertrophy in adults proceeds in conditions of stable genome rather than due to redundant polyploidization of the ventricular myocytes. The same idea assumes enhanced polyploidization of the myocytes in childhood in humans with congenital heart diseases.

With general atherosclerosis, the ploidy of left ventricle myocytes in the hearts of patients that underwent infarction corresponds to the norm variation irrespective of the ventricle and heart weights. At heart diseases the myocyte nucleus ploidy is often much higher than the norm variability both in hypertrophied ventricles and in those with normal weight. An additional polyploidization is suggested that may occur at some natural ontogenetic periods of human development (in the childhood) during heart diseases both innate or spontaneously appearing at the particular time. Unlike, the myocardial hypertrophy in adults does not stimulate myocyte polyploidy.

Parameters of ideal circadian cycle were compared with those of a circadian cycle composed of near-hour fluctuations. The integral cycle was optimized by the algorithm of matrix random search studying the approximation of its parameters to the ideal cycle after the phase shift of the latter. Computer calculations revealed a low efficiency of the search by fluctuation amplitude. The search by phase and frequency was effective in a narrow range of changes and needed time. The average level of fluctuations tuned up practically immediately: ideal and optimized curves coincided in all points. Examples of interactions between day, near-hour and shorter cycles are cited.

Lack of proportionality between DNA and protein content has been revealed in the human cardiac myocytes. The proportion 2:4:8:16 was observed in DNA of di-, tetra-, octa- and hexadeca myocytes while the protein content of the same cells was 2:3.5:5.2:7.2 in the inner layer, 2:3.5:6.5:8.9 in the central layer and 2:3.1:5.6:9.1 in the outer layer of the normal left ventricle. The protein content of myocytes of the same ploidy was higher in the inner layer than in other ones.

Possibility of cytophotometry application was established for the acid nucleolar proteins reacting with silver nitrate. The increase in transcription in the Purkinje neurons results in the expansion of the Ag-proteins areas, whereas their amount may be not increased. The amount and area of the Ag-proteins double in hepatocytes through polyploidization, but the number of nucleoli does not correspond to the gene dosage. This lack of correspondence was also revealed in micronucleoli. The number of nucleoli is not similar in the nuclei of some binuclear hepatocytes, and this disproportionality increases through polyploidization.

The permeability of the blood-brain barrier of rabbits was measured in response to different methods of endaural pharmacophysical intervention and intravenous injection of radioactive DL (2,3-3H2) leucine. The concentration of labelled leucine was measured in the inner ear fluids. The experiments were performed using 28 rabbits. The maximum amount of the drug entered the labyrinth as a result of endaural superphonoelectrophoresis. In this case the positive effect was reached after a smaller number of therapeutic sessions. This method is recommended for clinical treatment of cochleovestibular lesions.