DEVELOPMENT OF NEW APPROACHES TO THE TREATMENT OF HEMANGIOMAS IN EXPERIMENT.

The aim of the study was to determine the endogenous factors regulating angiogenesis and develop new approaches to the treatment of uncontrolled growth of capillaries. The effect of a cell proliferation inhibiting thermostable protein complex (TPC), isolated from adult chicken liver, on an experimental model of Hemangioma (adolescent chicken comb) was studied. It has been proved that thermostable proteins isolated from the liver of adult chicken have the ability to inhibit the growth of adolescent bird comb. In particular, it has been shown that daily TPC injection (200 µg) into the comb of animals of the experimental group for 22 days causes visible changes in the color and size of the comb. To explain the changes in typical skin redness and the comb size, quantitative changes in capillaries in highly vascularized papillary layer of the dermis were assessed using histological assessment. Based on the results we can conclude that, the thermostable protein complex of adult chicken liver has the ability to inhibit capillary growth of adolescent chicken comb.

ISOLATION AND COMPERATIVE STUDY OF THE GROWTH INHIBITING THERMOSTABLE PROTEIN COMPLEX FROM THE BONE MARROW OF THE ADULT MICE.

The growth inhibiting thermostable protein complex (TPC) from the bone marrow cells of adult mice was isolated and partially characterized. A comparative analysis of TPC components was performed by polyacrylamide gel electrophoresis. It has been established that the complex isolated from the bone marrow of adult mice, like complexes obtained from other organs, contains two relatively high-molecular-weight and low-molecular-weight sub fractions of proteins. In addition, minor content of low molecular weight components was detected in bone marrow TPC. It has been established that the thermostable protein complex of adult mice bone marrow cells does not have the ability to inhibit the proliferation of homotypic cells. This may be due to the minor content of active component (low molecular weight subfraction) in the complex and the structural and functional properties of constantly renewable hematopoietic tissue, such as the nature of factors that regulate proliferation, their different cell penetration and the expression of membrane receptors.

PECULIARITIES OF ACTIVATION OF COMPENSATORY-ADAPTIVE PROCESSES IN ADULT RAT LIVER CAUSED BY UNILATERAL NEPHRECTOMY.

In order to determine the general patterns of activation of the inter organ compensatory-adaptive processes, the peculiarities of activation of compensatory processes caused by unilateral nephrectomy in adult rat liver at the initial stage of hepatopathy (first three days after the operation) have been studied. In particular, it has been established that only small but visible abnormalities in the spatial relationship of sinusoids and hepatic plates are revealed morphologically in rat liver histology. The increasing of the functional load caused by changes in histoarchitecture, at these times, does not stimulate hepatocyte proliferation. At the same time, it is revealed that at the initial stage of hepatopathy caused by unilateral nephrectomy, preference is given to polyploidy from compensatory-adaptive processes charachterised to liver. In particular, it has been shown that liver responses mainly by quantitative increases in binucleated cells (2cx2) to the functional load induced by unilateral nephrectomy at an early stage.

THE STUDY OF THE IMMUNE CORRECTIVE PROPERTIES OF GREEK WALNUT (JUGLANS REGIA L.) SEPTA ON THE EXPERIMENTAL MODEL OF LEUKOPENIA.

From the positive effects of the drugs prepared from various parts of walnut described for a wide variety of diseases, their antitumor effect is remarkable. This feature can be used for treatment of leukopenia caused by radiotherapy and/or chemotherapy. Therefore, to study the immunocorrective properties of Greek walnut, the walnut septa were selected, aqueous extract of which has been studied on experimental model of leukopenia in white mice caused by a single injection of cyclophosphamide. The material of the study were the blood and bone marrow smears from intact and tested adult mice stained by Giemsa's dye. The quantity of leukocytes in peripheral blood were determined by the counting chamber under the light microscope with standard protocol. Cloud-point extraction, HPLC analysis and UV-visible spectrophotometry were used to study the composition of the walnut septa extract. It has been established that the Greek walnut septa extract has the correction ability of suppressed myelopoiesis in white mice caused by injection of cyclophosphamide. The blood formula normalization process by the mentioned extract is provided by the fast increasing in number of immature (band neutrophil) and mature neutrophils in the peripheral blood. It was shown that walnut septa extract stimulates the division, differentiation and maturation of blast forms of myeloid as well as lymphoid line in the bone marrow of mice with leukopenia. Cloud-point extraction and liquid-chromatographic analysis revealed the presence of hydrophilic and hydrophobic components in the walnuts septum extract.

THE EFFECT OF EXTRACT OF GREEK WALNUT (JUGLANS REGIA L.) SEPTA ON SOME FUNCTIONAL CHARACTERISTICS OF ERYTHROCYTES.

Administration of plant extracts for the treatment of several different diseases is an important approach ofmodern medicine.The reason must be an easy way of application, low price and the complex action of herbal medicines. The aim of the work was to study the effect of extract of walnut (Juglans regia L.) septa on the functional characteristics of erythrocytes during administration of cytotoxic agent - cyclophosphamide (experimental model of leukopenia). The material for the study was blood of the intact and experimental white mice. Sorption capacity and resistance to lysis of erythrocyte membrane have been determined by the spectrophotometric methods. According to the gained results, administration of cyclophosphamide had an influence on sorption capacity of erythrocytes and the given characteristic was increased only on the 8th day of cyclophosphamide administration, compared to control. Sorption capacity of erythrocytes was increased more on 8th day after combined application of cyclophosphamide and exctract of walnut septa. Resistance of erythrocytes to lysis was also increased after administration of the cyclophosphamide and this characteristic was further increased in case of combined application of cyclophosphamide and exctract of walnut septa. Thus, the stimulative effects of extract of walnut septa have been established on sorption capacity and resistance to lysis of erythrocytes in case of nonspecific damage of the cells.

COMPARATIVE STUDY OF GROWTH INHIBITORY PROTEIN COMPLEX DERIVED FROM DIFFERENT FORMS OF CHILDREN HEMANGIOMA.

The comparative analysis of the thermostable protein complexes obtained from the capillary and the cavernous hemangioma was carried out. It is shown that the active component (with low molecular weight 12-14 kD) is in minor content in the complexes obtained from capillary, as well as from the cavernous hemangioma. The effect of protein complexes from cavernous hemangiomas on proliferation of the brain and pancreatic cells in growing rats was studied. It was established that the thermostable protein complex from cavernous hemangiomas does not have the ability to inhibit the proliferation of heterotypic cells due to the minor content of the active ingredient in protein complexes.

Pancreatic cell proteome - qualitative characterization and function.

The macromolecular protein complex form adult white rat pancreas has been obtained and partially characterized. The most important distinguishing characteristics of the complex is the thermostability of its components. Using polyacrylamid gel by electrophoresis and chromatography of hydrophobic interaction has been established, that the components with relatively high (45-60 kD) molecular weight, are hydrophobic, while the low molecular weight components (11-12 kD) corresponds to hydrophilic proteins according to its column retention time. Participation of TPC in the regulation of the homotypic cell growth was determined. It is shown that the pancreatic TPC through inhibition of transcription decreases cell mitotic activity in growing rats. It is also shown that this complex is involved in pancreas regeneration processes. On the basis of the qualitative characteristics and cell growth regulatory function of this macromolecular protein complex described by us, it can be considered as a pancreatic cell proteome.

Influence of halothane narcosis on a change of the number of GABA-positive cells in the hippocampus of adult rats with the midasolam premedication.

The most difficult questions in pediatric anesthesiology still remain relevant at surgeries to correct congenital maxilla-facial pathologies, accompanied by severe respiratory distress, speech and auditory sensation. For a long time the anesthetics were widely used among the neonatal and young children with extreme care due to their indicated suppressive effects on the immature organ systems. According to this fact, the choice of anesthesia is a matter of extremely importance. In addition, the study of molecular mechanisms indicating to the adverse effects induced by application of anesthesia will make it possible to develop therapeutic strategies for prevention of postoperative complications. The purpose of present study is to investigate a change of the quantity of GABA positive cells in the hippocampus of white adult rats under the conditions of halothane narcosis and with the premedication by midazolam. Subjects of a study - 30 adult white rats with the weight of 130-140 g. Animals were subdivided into three groups. I group - intact animals; II group - sham-operated rats under the conditions of halothane narcosis; III group - sham-operated animals, premedicated by midazolam, under the halothane narcosis. A change in the quantity of GABA positive cells was determined by the immunohistochemical method (Rabbit Anti-GABA). All data were processed by the method of standard variation statistics. The authenticity of results was evaluated according to the Student's criterion within limits of 95-99% reliability. The results of conducted by us studies revealed, that during the application of halothane narcosis causes an increase in the GABA positive cells in the fields CA1 on hippocampus of adult white rats. In 24 hours after sham operation, at the condition of application of halothane narcosis a quantity of GABA positive cells raises in the CA3 field of hippocampus in II group animals as well. Moreover, in the CA1 field correlation between GAD65/67 positive and GABA positive cells is revealed which was not observed at later stages. The study of this question is the purpose of our next investigations.

The positive effects of Midazolam on functional activity of white rat brain cells in conditions of halothane anesthesia.

For the elimination of postoperative complications, which are evident in infants with congenital palatine and upper lip cleft after halothane anesthesia during standard premedication (Atropine, Dimedrol) the usage of agents of benzodiazepine group with anti-hypoxic effects is advised. The latter may modulate the blocked ion channels by neuromediators, which are activated by GABA and GABA receptor function. The neuro-protective ability of halothane is demonstrated. To reveal the mechanisms of positive effects using benzodiazepine group for premedication we have investigated the effects of midazolam of brain cell functional activity of experimental animals (white rats) in conditions of halothane anesthesia. For the estimation of white rat brain cells functional activity the nuclear transcriptional activity was studied (based on the intensity of [14C]-UTF inclusion), also the glutamic acid decarboxilaze activity (GAD65/67) using immunohistochemistry. It is estimated that halothane inhibits the transcription in rat brain cells. During midazolam premedication the halothane inhibitory effect on RNA synthesis is not revealed. After an hour of pseudo-operation halothane also induces proved decrease of quantity of GAD65/67 positive cells in CA3 hippocampal field. At the same time the quantity of similar cells are increased in CA1 field. The increase of GAD65/67 positive cells in CA1 is more evident during midazolam premedication. Based on the data received we can conclude that the positive effect of midazolam results in increase of GAD65/67 positive cells in CA1 hippocampal field.

Biliary hypertension as the cell proliferation trigger in bile duct ligated rats.

The aim of the study was to investigate the relationship between biliary pressure dynamics and proliferative activity of cholangiocytes and hepatocytes in BDL setting. 30 white Wistar male rats, weighting 200-220 gr. were involved in the modelled biliary hypertension setting induced by common bile duct ligation (CBD). The pressure in CBD was measured before (I) and at 1st (II), 3rd (III), 72nd (IV), 96th (V), 120th (VI) and 144 th (VII) hours after its ligation, with following data registered respectively: 169.67+/-4.16 (I), 205.0+/-1.0 (II), 213.67+/-3.06 (III), 252.67+/-3.51 (IV), 270.67+/-2.52 (V), 277.33+/-3.21 (VI) and 285.67+/-2.52 (VII) mm H(2)O. Pressure in portal vein was measured before (I) and at 1st (II), 6th (III), 24th (IV), 72nd (V) and 144 th (VI) hours past the bile flow obstruction. The corresponding indices were: 103.67+/-3.51 (I), 107.33+/-4.16 (II), 123.33+/-2.08 (III), 58.00+/-1.73 (IV), 172.00+/-3.61 (V) and 190.67+/-4.93 (VI) mm H(2)O. Liver histology was studied on paraffin embedded and Hand E stained slides in every above mentioned terms of CBD ligation. Mitotic indices for proliferating cholangiocytes and hepatocytes were counted at 24th, 48th, 72nd, 96th and 144 th hours after CBD ligation. The outcomes revealed that ductular reaction appears just in the first hours after CBD ligation. However the mitoses of epitheliocytes reach their peak (18.0+/-1.3) at 3rd day but mitoses of hepatocytes (31.5+/-2.1) - at 4th day after bile flow obstruction. Consequently, the following conclusions were made: biliary and portal hypertension, induced by the first respectively, represent the proliferative triggers for biliary ductules, cholangiocytes and hepatocytes in common biliary duct occlusion setting; However, dynamics, as well as the mechanisms of this process require further comprehensive investigation, including not only reduced inter-experimental term intervals and refinement of morphological and/or molecular - biological techniques of the study, but also the development of new, different experimental models.

[Kidney protein complexes that inhibit gene expression in the nuclei of homotypic cells].

The comparative study of protein complexes (PCs) isolated by alcohol extraction from white rat and human nephrocytes (post-operational material) have been performed. It has been shown that PCs of white rat cells inhibit RNA synthesis in the nuclei of intact as well as nephrectomized (unilateral nephrectomy) animals. The same effect has been shown while studying the PCs of patients with hydronephrotic kidneys. At the same time the inhibition of transcription has not been detected in case of RCC kidney cancer cell PCs. The chromatography studies revealed that the active component of PCs (12 kDa protein factor) which had been detected earlier has not been shown up in cancer cells of human kidney.

[Investigation of properties of the thermostable protein complex obtained from the myocardium left ventricles of adult white rats].

A thermostable protein complex (TSPC) obtained from the myocardium ventricules of adult rats inhibits mitotic and transcriptional activity of cardiomyocytes. At the same time this complex is more active on early stages of the postnatal ontogenesis in rats, aged 1 and 5 days. Following its action on RNA synthesis, the TSPC reveals tissue specificity only in cells with terminal differentiation, and is determined by its nuclear membranes. We continue studies for identifying the molecular weight and chemical nature of the TSPC, and the role of its different fractions in regulation of proliferation processes. Besides, it is planned to produce antibodies against TSPC fractions with the purpose to block its inhibitory effect on myocyte regeneration in the damaged myocardium.

The age related changes in cell cycle of mice cardiomyocytes.

Although rapid progress is being made in many areas of molecular cardiology, issues pertaining to the origins of heart-forming cells, the mechanisms responsible for cardiogenic induction, and the pathways that regulate cardiomyocyte proliferation during embryonic and adult life remain unanswered. The present study was performed to investigate the age related changes in cardiomyocytes' cell cycle in white mice. 18 white mice were used. The animals were distributed in three age groups: juveniles (2 months old), adults (10 months old) and senescents (18 months old). The animals were killed under ether narcosis. The heart was removed. Number of diploid, tetraploid, polyploid (octaploid, 16 n and 32 n), apoptotic nuclei and nuclei in S phase were counted by a Becton Dickinson (Mountain View, CA) FACScan flow cytometer. Statistical analyses were performed using Student t test. The heart of white mice contain diploid, tetraploid, octaploid, hexadecaploid (16 n) and 32 n cells. The amount of cardiomyocytes of different ploidy classes varies from one age group to another. The intensity of apoptosis increases with age. In all age groups the cardiomyocytes retain some capacity to synthesize DNA, which is not influenced by aging. However, the fact of DNA synthesis per se seems to be insufficient to drive adult cardiomyocytes through cell division.

Possible role of endogenous growth inhibitors in regeneration of organs: searching for new approaches.

A hydrophilic protein component (12-17 kDa) of the thermostable protein complex (TSPC) of different organs (heart, liver, kidney, brain) of adult white rats has been identified using hydrophobic interaction chromatography (HIC). Chromatograms and spectra of the myocardial hydrophilic component of different animals (snail, pigeon, rat, pig) detected within the UV region (190-360 nm) have shown that in different organs of the same animal species as well as in the same organ (e.g. heart) of different species, TSPC contains identical hydrophilic components, i.e. there is clearly a phylogenetically conserved group of thermostable proteins that regulates proliferation processes.

[Comparative study of the influence of renal endogenic factors on the proliferation activity of epithelial cells].

The proliferation activity of monolayer culture of Madin Darby Canine Kidney (MDSK) cells is suppressed by a thermostable protein factor of renal tissue of white rats and of humans. Under the influence of renal factors (RF), a decrease in cell number, and suppression of DNA synthesis and mitotic activity in MDCK cells occur. The inhibition of proliferative activity of cultured cells under the influence of RF was substantiated also by MTT assay. It was established that the inhibitory influence of RF is stipulated by suppression of RNA synthesis. It follows that RF may inhibit division of MDCK cells via suppression of gene expression in G1-phase. Similar factors were obtained from renal cells of different systematic groups of organisms (snail, frog, fish, pigeon, guinea pig, swine).

[Kinetic features of a white rat hepatocyte population under hormonal disbalance before and after partial hepatectomy].

Bilateral adrenalectomy, followed in 4 days by a partial hepatectomy, was performed using white rats weighing as much as 120-140 g. Under hormonal disbalance caused by bilateral adrenalectomy, the number of polyploid (4c, 4c x 2, and 8c) hepatocytes significantly increased, compared to that in non-operated control rats. Six hours after a partial hepatectomy, the share of highly ploid hepatocytes falls, being accompanied by a 9-fold increase in mitotic index. It is supposed that under hormonal disbalance condition, a partial hepatectomy may induce "early" mitoses in hepatocytes blocked in G2-phase of the cell cycle.

Increased functional load on mouse kidney proximal tubule epithelial cells causes changes in nucleolar 3-D architecture.

Ultrastructural 3-D analysis of nucleolar architecture and Ag-NOR protein distribution in mouse kidney-cortex proximal-tubule epithelium has been performed. A principal scheme of structural changes of the nucleolus and organization of its components during the intensification of pre-rRNA synthesis (dynamic model of a nucleolus) based on computer spatial modelling has been advanced. According to the nucleolar composition, three groups of cells, which differ from each other by rRNA synthesis, are defined in normal kidney. Most nephron proximal-section cells (about 52%) are characterized by lower activity of RNA synthesis. Such kind of cells are defined as group I (nucleolar diameter 0.7-1.5 microm) and always contain resting, ring-shaped or close to ring-shaped dense nucleoli, which have 2 or 3 fibrillar centers. Nucleoli of group II cells (about 37%, nucleolar diameter 1.5-2.5 microm) have a higher level of activity, contain 4-7 fibrillar centers, and their structural organization is close to reticulated forms due to the first indications of vacuolar network (identified as prereticulated nucleoli). The most active cells of group III (about 11%, nucleolar diameter 2.5-3.5 microm) include cells with typical reticulated nucleoli with a well expressed vacuolar network and numerous fibrillar centers (18-22). Increased functional load of the epithelium caused by unilateral nephrectomy and diuretic (4-chlor-H [2-furylmethyl] 5-sulphamyl-antranic acid) injection changed the proportion of the different cell groups: group I decreased (about 25%), whereas groups II and III increased (about 8% and 17%, respectively). The increase of nucleolar activity first causes a deformation of the individual fibrillar centers as well as complication and growth of their surface. Further, a progressive fragmentation of the fibrillar centers and the growth of their total volume is observed. The complication and growth of the total volume of Ag-positive zones is another indication of the nucleolar activation. The vacuolar system develops by a gradual fusion of small isolated cavities into a united vacuolar network. Nucleoli with 2-7 fibrillar centers are considered to be intermediate forms reflecting successive stages of its activation or inactivation: from the resting ring-shaped nucleolus via transient stages of increasing functional activity to the active reticulated nucleoli and vice versa. The observed differences in the nucleolar ultrastructure are regarded as evidence of the functional heterogeneity of cell populations within one functional segment of nephron.

[The dependence of the 3-dimensional organization and length of the nucleolonema in the hepatocyte nucleoli of normal and regenerating rat livers on the nucleosome structure of the intranucleolar chromatin]. / Zavisimost' trekhmernoi organizatsii i dliny nukleolonemy iadryshek gepatotsitov normal'noi i regeneriruiushchei pecheni krysy ot nukleosomnoi struktury vnutriiadryshkovogo khromatina.

A biochemical and ultrastructural stereo-morphological analysis, with special reference to spatial organization and length of nucleonema and Ag-positive zones, was performed for various modifications of nucleolonemal type nucleoli in normal and regenerating (6 and 22 hours after partial hepatectomy) rat hepatocytes. To determine possible disorders on nucleosomal and supranucleosomal levels, chromatin DNA degradation was carried out during micrococcal nuclease hydrolysis, followed by analysis of electrophoretically separated particles. Functional characterization of intranucleolar chromatin was performed by testing the rate of DNA degradation after DNAase I treatment as well as by detection of free G-C pairs during titration with actinomycin D. Transcriptional activity of nucleoli was determined according to the intensity of [14C]-UTP uptake with isolated nucleoli. It is shown that the total chromatin from control nucleoli contains nucleosomal fibrils, although deprived of high compactization level. Nucleosomes themselves are strongly destabilized. In activated nucleoli structural differences of chromatin are more perceptible. In 6 hour preparations the bulk of chromatin fibrils (about 70%) undergo a further relaxation and lose the nucleosomal structure. Therefore at this point of experiment, the maximum length of nucleolonema and Ag-positive zones was registered in addition to the highest quantity of free G-C pairs, and sensibility to DNAase I transcriptional activity of isolated nucleoli. 22 hours after hepatectomy, the transcriptional activity and functional parameters of intranucleolar chromatin markedly decreased compared to the 6 hour period. Simultaneously, the share of chromatin restituting the nucleosomal structure increased, while the length of nucleolonema was shorter than in nucleoli 6 hours after hepatectomy. The main results could be resumed in the following way: the general composition of nucleolonemal type nucleolus variations described in our experimental conditions is in close relation with the with the compactization grade of ribosomal DNP-fibrils.

Transcriptional activity and ultrastructure of morphologically different types of nucleoli isolated from hepatocytes of normal and hepatectomized rats.

Morphologically different types of nucleoli were isolated from liver of normal and partially hepatectomized rats, to allow their ultrastructure and transcriptional activity to be precisely correlated. Transcriptional activity was estimated from the intensity of incorporation of [14C]-UTP during periods of maximal RNA-polymerase I activity. RNA synthesis in hepatocyte nucleoli was maximal at 6 and 22 h after partial hepatectomy. The changes in transcriptional activity coincided with changes in nucleolar ultrastructure. Pseudonucleolonemal nucleoli, in which the prominent nucleolonemal network has a large dense fibrillar component and a small granular part, were first seen 6 h after the operation and showed the highest transcriptional activity. After 22 h, a second peak of RNA synthesis was recorded. Nucleolar size had almost doubled, and most hepatocytes showed hypertrophy of the granular component, indicative of intensified pre-rRNA processing.

[The 3-dimensional organization of the nucleolus and nucleolus-organizer regions of differentiated cells. IV. The structural and functional heterogeneity of the nucleoli in the epithelium of the proximal nephron in the mouse]. / Trekhmernaia organizatsiia iadryshka i iadryshkoobrazuiushchikh raionov differentsirovannykh kletok. IV. Strukturnaia i funktsional'naia geterogennost' iadryshek épiteliia proksimal'nogo otdela nefrona myshi.

By means of stereological and morphometrical analysis, the ultrastructure of nucleoli in epitheliocytes of mouse kidney cortex proximal tubuli has been studied. In accordance to the nucleolar composition, three main groups of nephrocytes with different levels of rRNA and protein synthesis were defined. Functional heterogeneity of proximal tubuli epithelium was established by correlation between different variants of ultrastructural organization of nucleoli and the total RNA synthesis activity, determined by 3H-uridine incorporation intensity. It has been shown that a greater part of cells (about 52%) in the nephron proximal section, which is characterized by slow RNA synthesis, causing a low functional activity of these cells, presumably represents a reparative cellular reserve. Such cells, defined as the 1st group cells, have resting, ring-shaped nucleoli with one fibrillar centre, and nucleoli similar to the ring-shaped ones but containing 2-3 fibrillar centres. Nucleoli of the 2nd group of nephrocytes (about 37%), most actively incorporating labeled precursor, contain 4-6 fibrillar centres. Their structural organization is closer to the reticular type of nucleoli. The 3rd most actively labeled group of nephrocytes includes cells with typical reticulated nucleoli. The number of fibrillar centres in the reticulated nucleoli is much higher (18-22) than in the 1st and 2nd groups of nephrocytes. Structural and functional polymorphism of nephrocytes was revealed not only in the proximal part of one nephron. During the increase in functional activity of nephrocytes, caused by unilateral nephrectomy, the quantitative correlation between cells related to these different groups was seen to change. The number of cells of the 1st group decreased by 24%, whereas that in the 2nd and 3rd groups increased by 9 and 15%, respectively. Nucleoli with 2-3 fibrillar centres are considered as transitional forms between the inactive ring-shaped nucleoli and the active reticulated nucleoli. Differences in the ultrastructure of nucleoli may be considered as an evidence of functional heterogeneity of nephrocytes within the proximal segment of nephron.