Melatonin Stimulates Epithelium Migration in Wound Models In Vitro and In Vivo.

We studied the effect of bovine brain gangliosides, individual ganglioside GM1, and melatonin on the rate of wound closure under in vitro conditions and the effect of melatonin on the rate of wound healing under in vivo conditions. It was shown that bovine brain gangliosides and melatonin reliably increased cell migration in the experimental wound model. This effect was detected when the cell cultures were treated with the test preparations after wound infliction and when the cultures of human keratinocytes were pretreated before wounding. Analysis of the effect of melatonin on the rate of wound healing in vivo showed that melatonin accelerated this process, especially at the middle stages corresponding to the proliferation phase (days 3-6 after surgery). Histological analysis revealed intensification of epidermal cell proliferation at the edges of the wound starting from day 4 after surgery.

Biochemistry of Direct Cell-Cell Interactions. Signaling Factors Regulating Orchestration of Cell Populations.

Biochemical mechanisms for the orchestration of cell populations are discussed in view of direct cell-cell interactions and composition of the intercellular medium. In our works of the last 20 years, we used circahoralian (ultradian) rhythm of protein synthesis as a marker of cell interactions. Experiments in cell cultures are described; some influences on the organism native medium were performed. Information is presented on the signaling membrane factors that trigger a cascade of processes in the cytoplasm and lead to the orchestration of cell activity in vitro and in vivo. Among these factors are blood serum neurotransmitters, gangliosides, and some hormones. Studying protein synthesis kinetics allowed us to understand the importance of maintaining the constant levels of signaling factors in mammalian blood. The literature on protein phosphorylation as a key process of cell organization is reviewed. The persistence of the organizing signal for several days is described as a type of cell "memory". It seems promising to extend the area for application of direct cell-cell interactions (respiration of cells, proliferation, etc.) to study possibilities of epigenetic regulation. It is important to continue the studies on the mechanisms of biochemical action of the known drugs as signaling factors.

Glutamic Acid Signal Synchronizes Protein Synthesis Kinetics in Hepatocytes from Old Rats for the Following Several Days. Cell Metabolism Memory.

The kinetics of protein synthesis was investigated in primary cultures of hepatocytes from old rats in serum-free medium. The rats were fed mixed fodder supplemented with glutamic acid and then transferred to a regular mixed fodder. The amplitude of protein synthesis rhythm in hepatocytes isolated from these rats increased on average 2-fold in comparison with the rats not receiving glutamic acid supplement. Based on this indicator reflecting the degree of cell-cell interactions, the cells from old rats were not different from those of young rats. The effect was preserved for 3-4 days. These results are discussed in connection with our previous data on preservation of the effect of single administration of gangliosides, noradrenaline, serotonin, and other synchronizers on various cell populations. In contrast to the other investigated factors, glutamic acid is capable of penetrating the blood-brain barrier, which makes its effect possible not only in the case of hepatocytes and other non-brain cells, but also in neurons.

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Glutamic Acid - Amino Acid, Neurotransmitter, and Drug - Is Responsible for Protein Synthesis Rhythm in Hepatocyte Populations in vitro and in vivo.

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell-cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.

Circahoralian (ultradian) metabolic rhythms.

This review presents data concerning metabolic rhythms with periods close to one hour (20 to 120 min): their occurrence, biochemical organization, nature, and significance for adaptations and age-related changes of cells and organs. Circahoralian (ultradian) rhythms have been detected for cell mass and size, protein synthesis, enzyme activities, concentration of ATP and hormones, cell respiration, and cytoplasm pH. Rhythms have been observed in bacteria, yeasts, and protozoa, as well as in many cells of metazoans, including mammals, in vivo and in cell cultures. In cell populations, the rhythms are organized by direct cell-cell communication. The biochemical mechanism involves membrane signal factors and cytoplasmic processes resulting in synchronization of individual oscillations to a common rhythm. Phosphorylation of proteins is the key process of coordination of protein synthesis and enzyme activity kinetics. The fractal nature of circahoralian rhythms is discussed as well as the involvement of these rhythms in adaptations of the cells and organs. Senescent decrease in rhythm amplitudes and correspondingly in cell-cell communication has been observed. The possibility of remodeling these changes through the intercellular medium has been predicted and experimentally shown. Perspectives for studies of the organizers and disorganizers of cell-cell communication in the intercellular medium along with appropriate receptors are discussed with special emphasis on aging and pathology. One perspective can be more precise definition of the range of normal biochemical and physiological state with the goal of correction of cellular functions.

Melatonin modifies the rhythm of protein synthesis.

Melatonin (5 nM) added to medium with primary hepatocyte cultures shifted the phase of circahoralian rhythm of protein synthesis and hence, can be a factor synchronizing fluctuations in protein synthesis and rhythm organizer in the hepatocyte population. Blockade of melatonin receptors with luzindole (20 nM) arrested rhythm organization of protein synthesis by melatonin. Prospects of studying biochemical mechanisms of protein synthesis rhythm organization with other drugs (calcium agonists, similarly to melatonin) are discussed.

Ganglioside-mediated metabolic synchronization of protein synthesis activity in cultured hepatocytes.

An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3-0.5 microm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06-0.2 microm GM1 or 0.1-0.2 microm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24 h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.

The rhythm of protein synthesis does not depend on oscillations of ATP level.

A rhythm of the [3H]leucine incorporation rate with a period of about one hour (circahoralian rhythm) has been found in rat hepatocytes grown in vitro as a monolayer and in liver organ culture. The periodicity of the incorporation rate remained after correction for changes in leucine pool size. A similar periodicity of the leucine incorporation rate was detected in a cell-free system prepared from rat hepatocytes. We have also found circahoralian oscillations of the ATP level and similar oscillations of the leucine tRNA aminoacylation rate in a hepatocyte monolayer. The addition of 1 mM ADP to the culture resulted in a considerable increase in the ATP level in the cells, but the rhythm of protein synthesis was retained under these conditions. The conclusion that there is a flexible association between changes in the ATP and GTP levels on the one hand, and oscillations of the protein synthesis rate on the other, is also supported by experiments with a cell-free system, in which the rhythm of protein synthesis rate was observed in the presence of excess ATP and GTP. We propose an hypothesis to explain the fractal pattern of circahoralian metabolic rhythms.

Lack of proportionality between gene dosage and total muscle protein content in the rat heart.

Counting of isolated cardiomyocytes has demonstrated that their number was 16.8 +/- 0.6 10(6) in both ventricles of weanling rats (28 days after birth), growing in litters of four (fast-growing). In rats growing in litters of 16 (slow-growing), the myocyte number was 11.8 +/- 0.8 10(6). In the control group (8 sucklings per litter), there were 14.2 +/- 10(6) cardiomyocytes. The fast-growing rats had more octoploid cells than slow-growing ones. Considering ploidy and cell number, the total number of myocyte genomes in fast-growing animals was 45% higher than in slow-growing ones. The total content of contractile proteins in fast-growing weanling animals was higher by 28% while sarcoplasmic proteins were 8% higher. This lack of correspondence between the number of myocyte genomes and muscle protein content was even more pronounced at the age of 110 days. The results are compared with the cytophotometric data concerning the lack of correspondence between the total protein content in a myocyte and its DNA amount and chromosome number, i.e., total dosage of the myocyte genes.

Cell differentiation as assayed by the topography and number of ribosomal genes.

In situ hybridization, Ag-staining and electron microscopy were used to study the distribution of ribosomal genes in isolated nuclei of rat cerebellar cells and the correspondence of the ribosomal genome topography to the nucleolar structure. rDNA-DNA autoradiography revealed clusters of silver grains, as well as diffuse groups and rows. The cluster frequencies corresponded to the frequencies of nucleoli on Ag-stained slides. Competitive hybridization in situ using unlabelled rat rRNA and hybridization with a nonspacer rDNA fragment showed that the diffuse groups and rows of grains also correspond to the ribosomal genes. Spatial organization of the ribosomal genome in the Purkinje cells differs from that in the other cerebellar neurons and glial cells. A 1.5-fold redundancy of the ribosomal genes was found in some Purkinje cells, while most of these as well as microneurons contained the diploid value of the genes.

Genome multiplication in cardiomyocytes of fast- and slow-growing mice.

The number of myocytes and the percentage of cells with a high degree of ploidy increased in the heart ventricles of fast-growing mice compared with slow-growing ones. The mean incidence of octa- and hexadecaploid (by summary DNA content) myocytes was 7% in the slow-growing and 23% in the fast-growing, weaned mice. In these groups, the total myocyte number varied by 20%. There were 43% more myocyte genomes in the heart ventricles of the fast-growing mice than in those of the slow-growing mice. The same differences in cell number and ploidy persist in 90-day-old mice in spite of feeding ad libitum after weaning.

An attempt to influence DNA content in postmitotic Purkinje cells of the cerebellum.

Feulgen-DNA content has been measured cytophotometrically in granule and Purkinje cells of the rat and mouse cerebellum. The study has confirmed that under normal conditions a very small part of the P. cell population in rats (less than 3%) possesses a Feulgen-DNA surplus ranging from 2C to 4C. In mice, the hyperdiploid (H2C) P. cells are even more rare. The occurrence of H2C-P. cells in rats and probably also in mice has not been substantially changed in animals exposed to factors interfering with chromatin structure and/or its template activity; the H2C-P. cells seem to be slightly more frequent after injecting mice with corticoids (Urbason) or in animals suffering from ectromelia or hereditary Purkinje cell degeneration. The incidence of H2C-P. cells has neither been substantially affected by experimental conditions which are known to lead to functional and/or metabolic stimulation of the cerebellum. Functional changes in the number of H2C-P. cell nuclei may, however, be short-term or transient in character and therefore might have excaped detection in our models. The findings rule out an impact of some reasons suspected for the artefactual origin of Feulgen H2C DNA values as e.g. compactness of the chromatin.