AIRR (International Association for Regeneration Research)

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Abstracts
of
Presentations and Posters of the
International AIRR Conference,
Oct. 1999,
in Tbilisi, Georgia
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 Bufo regularis, was examined by topical application of a concentrated solution of prolactin to the amputated limb stumps of newly metamorphosed toadlets. This treatment resulted in enhancement of regeneration; regenerative outgrowths of experimental toadlets were more frequent, larger in size and had improved morphology compared to their control counterparts. All of the regenerates were hypomorphic, consisting histologically of well-developed cartilaginous rods, caps or collars, surrounded by connective tissue and skin. Rare or no muscle development was detected. We conclude that prolactin topically administrated in concentrated dosages, exerts a restricted positive effect on the regenerative capacity of Bufo regularis.
The tail of the geckonid lizard, Bunopus tuberculatus was amputated at a proximal level and the animals were exposed to continuous darkness. The animals were intraperitoneally injected, daily for 30 days postamputation, with prolactin. The initiation of regeneration, growth rate, as well as mitotic index of blastema cells, were enhanced by prolactin administration. Time required for achievement of regeneration phases in prolactin-treated lizards decreased compared with control animals. It is concluded that prolactin may speed up the rate of tail regeneration in the geckos, and increases the mitotically active cell population during blastema formation.

EFFECT OF ENDOGENEOUS NUCLEOLAR TRANSCRIPTION FACTOR ON THE PROLIFERATING HEPATOCYTES.
T.Abramidze, I.Kakhidze, D.Dzidziguri
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University

During last years we have been studying white rat hepatocytes nucleolar transcription factors. These are thermostable proteins with opposite (stimulatory and inhibitory) effects on transcription process. At present, it’s known that regulation of cell growth and proliferation is realized by growth factors, receptors, kinases and transcription factors, therefore we are interested how nucleolar transcription factors influence on proliferating cells in homological tissue. In particular, the effect of nucleolar protein fraction on transcription activity of regenerating and physiological growth hepatocytes was studied in vitro. It was determined that indicated fraction stimulates RNA-synthesis in both types of growing cells. At the same time, stimulating effect of nucleolar fraction is more intensive in regenerating hepatocytes that is probably connected with different amount of active ribosomal genes in these cells. Also, the effect of nucleolar protein fraction on mitotic activity of hepatocytes was investigated. It was shown that on the early stage of postnatal development after injection of nucleolar fraction the mitotic index of hepatocytes was decreased (in comparison with control by 40%). This fact may be explained by effect of inhibitory component of nucleolar protein fraction.

GRAVITY INFLUENCES GROWTH AND DIFFERENTIATION IN ADULT NEWTS
Hermann Josef Anton*, Eleonora N. Grigoryan**, Victor I. Mitashov**, Katja Krupp-Beyerlein*, Heike Pitzer*
*Zoologisches Institut, Universität zu Köln, Weyertal 119, D-50923 Köln, Germany.
**Institute of Developmental Biology, Russian Academy of Sciences, 26, Vavilov str., 117808, Moscow, Russia

Life on earth has developed under the steady influence of gravity. All existing living systems up till now exist because of the very early gained ability to vanquish gravitation.
Since several years the opportunity exists to achieve microgravity (i.e. 10 – 5 to 10– 6 g) in space shuttles and stations. From our group experiments have been carried out first with adult newts (Pleurodeles Waltl) in space conditions. During the last 12 years regenerating newts lived in microgravity conditions aboard of the Russian unmanned bio-satellites for  time periods of 7 to 14 days and have been investigated histo- and cytologically immediately after return to earth and later. All experiments brought the amazing results that cell physiology is influenced by microgravity. This is expressed by increasing the rate of cell proliferation within mitotic active tissues. In connection with that an earlier redifferentiation during regeneration processes in all body parts which have been investigated till today  (eye lenses, neural retina, limb, tail).
This acceleration takes place not only during the stay in weightlessness, but seems to outlast after return from flight under the normal earth gravity conditions. Up till now we do not know too much about the triggering phenomenon. We only know that a 7 to 14 days stay in weightlessness is followed by growth acceleration.
We also have proved that simulation of weightlessness by clinorotation has comparable results. But we don’t know time and/or gravity threshold and the cytological switch on and channeling mechanisms too.
For more information look at  presentations No: 
ANALYSIS OF THE ACTIVITY OF KEY GLYCOGEN METABOLISM ENZYMES AFTER A PARTIAL HEPATECTOMY OF NORMAL AND CIRRHOTIC RAT LIVER
N.N. Bezborodkina, M.V. Kudryavtseva, B.N. Kudryavtsev Institute of Cytology, Russ. Acad.  Sci.., S.-Peterburg 194064, Russia

For a long time it has been known about the possibility of involution of pathological alterations in the liver. However, studies on the effects of the resection of the cirrhotic liver on the restoration of its functional activity remained poorly. One of the key tissue-specific functions of the liver is the capability for synthesizing and degradating glycogen by the parenchima cells. A study was made of the activity of key enzymes of glycogenolyses and glycogenesis. Activities of glucose-6-phosphatase (G6Pase), glycogen phosphorylase (GP) and glycogensynthase (GS) were determined in the rat liver in norm, in the liver cirrhosis (CCl4-induced cirrhosis) and after a partial hepatectomy (PH). The enzyme activity in the liver punctates was determined by methods modified for small tissue samples. We have shown that activities of G6Pase and GP fell after the 6-month-long CCl4 poisoning to 36 and 40% respectively of the normal levels of these enzymes. After the PH the activity of G6Pase remained low at 3 and 6 months to only slightly higher than that in the cirrhotic liver. The activity of GP remained effectively as low as in the cirrhotic liver at 3 months, while  further 3 months later it had reached the normal level. The activity of GS in the cirrhotic hepatic tissue did not differ from that in the normal liver, and no statistically significant changes in GS activity were revealed either during spontaneous recovery after the end of CCl4 poisoning or at 3 and 6 month after PH.

SKELETAL MUSCLE REGENERATION IN EXTREMELY OLD RATS
B.M. Carlson, A.B. Borisov, E. Dedkov, and J.A. Faulkner
Department of Anatomy and Cell Biology and Institute of Gerontology, University of Michigan, Ann Arbor,  Michigan 48109, USA

Previous experimentation with cross-age muscle transplantation has shown that although extensor digitorum longus (EDL) muscles of 24-month old rats regenerate poorly in situ, they regenerate as well as young muscles if transplanted into the limbs of 4-month old host rats. Subsequent experiments showed that in the absence of nerve damage, muscles in old rats damaged by exposure to Marcaine regenerated to control levels of function. Recent experiments have examined the regenerative capacity of muscles in extremely old rats (32-35 months old) that are near death. Although control muscles in these animals are weak and show a number of morphological abnormalities, they nevertheless regenerated to control levels of function and higher than control mass 41 days after Marcaine injection. When EDL muscles from 32-month old rats were transplanted into legs of young host rats, they regenerated as well as young-into-young EDL muscle transplants. These experiments show that even at a very advanced age, when the muscles show numerous aging changes, rat muscles still retain sufficient biological reserve to be able to regenerate well as young muscles in vivo if they are placed in a favorable environment.
(Supported by grant PO1 AG-10821 from the NIH.)

THE CORRELATION OF NUCLEOLUS ORGANIZING REGIONS QUANTITATIVE AND STRUCTURAL PARAMETERS (NUMBER, VOLUME, 3D-ORGANIZATION) WITH CELL DIFFERENTIATION
*E.Cherkezia, **O.Zatsepina, ***M.Gabrichidze, ***Sh.Jinjolia, *P.Tchelidze
*Group of Computed 3D-Engineering of Biostructures and Tissues, Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University;
**Department of Electron Microscopy, A.Belozersky Laboratory of Physico-Chemical Biology, Moscow State University;
***Group of Computed 3D-Engineering of Biostructures and Tissues, Kutaisi State University;

The methods of ultrastructural computed tomography and Ag-NOR-protein staining have been used for the virtual 3D-reconstruction of metaphase nucleolus organizing regions (NORs) in intensively proliferating and differentiating erythroid cells, hepatocytes and endotheliocytes of 12-day old mouse embryos liver. It have been detected, that the process of differentiation is accompanied by the decreasing of active (Ag-positive) NORs number and sizes probably due to the reducing of transcribing ribosomal genes “dose”. Such changes of mitotic NORs correlate with the structural and quantitative alterations of interphase nucleoli during differentiation.
The material is presented in the form of computed animated film.

THE POSSIBLE ORIGIN OF THE STROMAL FIBROBLASTS IN THE PROCESS OF THE REGENERATION OF MOUSE CORNEA
K. Davitaia
Georgian Academy of Sciences, Institute of Zoology, Laboratory of Cytology, Tbilissi. Georgia

The origin of white adult unlinear mice eye corneal stromal cells after central perforating traumatisation of Cornea with sterile preparating needle was investigated.
Histological studies found out that the new formation of stromal cells takes its origin from corneal limb. These cells are moving to the area of stroma wound already by 3 hours after operation. The entire epithelisation wound of cornea occures only on 5th day, but stroma doused only on the 25-30th day after traumatisation.
The  autoradiographic  researches demonstrated, that regeneration of mechanically damaged mice eye corneal srtoma cells mainly occurs due to predecessor cells, intensively multiplied and migrated throughout the limbal trabeculi to the corneal stroma regeneration region.

With the help of immunofluorescence methods on the Xenogene chimere (rat-mouse) it was fixed, that regeneration of corneal stroma occurs by the fibroblasts, that take their origin from precursor cells, intensively proliferating beyond the borders of center of inflammation possibly in the hematopoietic organs, may be in bone marrow.

PROLIFERATIVE ACTIVITY AND THE DYNAMICS OF CELL DIVISIONS OF LYMPHOCYTES FROM PATIENTS WITH LUPUS AND RHEUMATOID
ARTHRITIS
N.A.Dvalishvili, N.N. Sigua, T.A. Lezhava Department of Genetics, Tbilisi State University, Georgia

Responses of peripheral blood lymphocytes to Phytohaemaglutinin (PHA) and cell rate divisions measured by  sister chromatid differential staining have been studied in patients with Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). In total 14 SLE and 12 RA patients were investigated on blasttransformative activities of lymphocytes in vitro. Lymphocyte cultures obtained from 10 phenotypically healthy individuals served as control. All samples were cultured for 72 hr under the same conditions in medium not containing serum. Despite the wide variations among SLE patients in the blast index they revealed the lowest values of indices. In 2 cases (chronic forms) cultures had the indices of response to the mitogen close to zero. The untreated RA patients investigated at an onset of desease showed significant increase of lymphocyte transformation patterns in vitro - in average of (84.6?0.81)% blasttransformed cells compared to (56.3?0.50)% in a control group, while the treated RA patients revealed dechne in response to PHA (an average index was (43.4?0.52%)).
Comparative evaluation of cell rate divisions revealed no significant differences in their values between the patients of the same groups. By the time of harvesting cell populations were asynchronous in all studied individuals. Character of the distribu-tion of cell divisions in case of RA was found to be considerably changed with a majority of the 2nd but with a rather high content of the 1st division cells (23.4% in comparison to 6.6% in control) suggesting a large population of cells with a delayed response to PHA. Dynamics of cell division for SLE patients did not differ from control patterns.

G2 POPULATION OF HEPATOCYTES AND LIVER REGENERATION IN WHITE RATS
D.V. Dzidziguri, Z.N. Kokrashvili, E.L. Mikadze, I. Modebadze, T.M. Shavlakadze
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University

After partial hepatectomy the remnant liver cells rapidly re-enter the cell cycle. Maximum mitotic activity appears by the 30th hour after the operation. Here we show that mitotic activity of hepatocytes is directly dependent on their RNA synthesis peak obtained 6 hours after the operation. Today it is well known, that during 4 hours after the partial hepatectomy expression of immediate early response genes takes place. Products of these genes, which represent transcription factors provide for the maximum level of the transcriptional activity of delayed early responce (ribosomal) genes 6 hours after the operation. Processes connected with reparative growth are considerably changed in the conditions of hormonal disbalance. In the previous studies we have shown that in the adrenalectomized rats the first peak of transctiptional activity is detected 3 hours after partial hepatectomy. Also it was demonstrated that 6 hours after the operation mitotic activity of the hepatocytes increases, which was detected neither 4 days after adrenalectomy nor 6 hours after pure hepatectomy. Our results indicate the existence of G2 population of hepatocytes in the liver tissues, as this is the only population to enter mitosis within 6 hours after the operation.
We suggest that adrenalectomy causes accumulation of tetraploid cells during 4 days. However in order for these cells to enter mitosis, signal molecules are required, which activate genes regulators of cell growth. In the conditions of hormonal dibalance, when the cytostatic effect of cortisol is lost, a rapid expression of these genes takes place. Therefore all processes that facilitate entrance of the tetraploid hepatocytes into mitosis are being accelerated.
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THE MECHANISM OF DAMAGE REPAIR AND IMMORTALITY OF MALIGNANT TUMOURS MAY INVOLVE THE EVOLUTIONARY MEMORY OF THE LIFE CYCLE OF UNICELLULAR ORGANISMS
Je. Erenpreisa*, M. Cragg**, T. Illidge**, Ivanov*
*Lab. Tum. Cell Biol., A. Kicherstein Institute of Microbiology and Virology, Rige, Latvia;
**Lymphoma Lab., Tenovus Institute, Southampon General Hospital, UK.

In multicellural organisms the cells are differentiated for somatic and germ ones, correspondingly mortal and immortal. Malignant tumours originated from mortal somatic tissues are immortal. Progression of tumours proceeding from benigh to malignant, which lasts for years, can be modelled by the response of the resistant p53 mutant tumours to damage, which is displayed in a few weeks. In our model system of p53 mutant human Burkitt’s lymphoma cell lines, treated by one dosage external beam irradiation 10Gy, the following set of responses was observed using DNA flow cytometry, radioautography, and cytology: (1) transent G2 arrest followed by a few aberrant mitoses ending in mitotic catastrophe; (2) polyploidisation during the 1st week followed by de-polyploidisation on the 2nd; (3) abundant apoptosis of 80-90% of polyploid cells; (4) restoration of diploid cell line during the 3rd week postirradiation. Subnuclear differentiation displaying different rates of DNA synthesis, as well as shedding of degraded chromatin from giant cells remaining alive themselves were found. The rest of the giant nucleus underwent a bouquet-like reconstruction strongly reminding a meiotic prophase. The polyploid bouquets then returned to interphase and radially splitted into secondary nuclei, which resumed mitotic cycle and constituated the recovered cell line. It is supposed that normally lethal dsDNAs, which arise from irradiation damage, are healed within giant cells in the meiotic-like prophase by recombination repair. The processes observed strongly remind the life cycle of heterokaryotic protists, however, pedokaryogamy was not observed, at least cytologically. The evolutionary mechanism, which involves a germ-like differentiation, provides both damage-resistance and immortality of tumours as theoretically predicted by Janis Erenpreiss (1992, 1993).

INFLUENCE OF PLAFERON LB ON THE TRANSCRIPTIONAL ACTIVITY OF REGENERATING LIVER AND KIDNEY CELLS
M.G.Gagua, D.V. Dzidziguri, V.I.Bakhutashvili
Tbilisi State University, Institute of Medical Biotechnology, Georgia

Intricate modulatory role of several growth factors, main hormones and their intracellular secondary messengers remains poorly defined. Plaferon LB is a compound of biologically active substances produced from human amnion. A wide range of the pharmacological effects of Plaferon LB has been well studied. Here we show that the compound has an ability to regulate the transcriptional activity of the genes. Administration of Plaferon LB to adult white rats during 5 days leads to the considerable increase of the transcriptional activity of genes expressed in hepatocyte nuclei under the normal physiological conditions. Influence of Plaferon LB on the transcription of genes regulators of cell proliferation was studied on the regenerating liver model. Plaferon LB was administered by intraperitoneal injection to the experimental animals during five days and partial hepatectomy was performed. We have found that Plaferon LB effects transcriptional activity of genes involved in delayed early response. Particularly, 6 hours after the operation the RNA synthesis activity of liver cells increases by 30 per cent. It was also shown that 3-fold administration of the compound (1 hour prior to the operation, immediately after  and 1 hour after the operation) results in the opposite effect. Six hours after the operation peak of transcriptional activity is inhibited by 46%. As it has well been established this peak of transcriptional activity in hepatocytes stimulated for proliferation is controlled by the expression products of immediate early genes and  is directly related to the mitotic activity of hepatocytes. Similar results were obtained on kidney epithelial cells after unilateral nephrectomy. The obtained results indicate that Plaferon LB contains an active substance which has an ability to suppress the immediate early genes.

PARTIAL SERUM REPLACEMENT BY ALKALOIDS IN CULTURED FIBROBLASTS
M.Gedevanishvili, N.Gogitidze, M.Lebanidze, N.Malazonia and L.Tsutsunava
Inst. Pharmacochemistry, 380059 Tbilisi, P.O.a/71, Georgia

Culture of the most cells requires the presence of serum. Presumably, serum contains mitogens generated during the coagulation process, e.g. PDGF and serotonin. Serotonin appeared to stimulate cell multiplication in vascular smooth muscle and certain fibroblast cell cultures by interacting with specific plasma membrane receptors [Gedevanishvili et al., 1978; Nemecek et al., 1986]. We have found also that alkaloids of Peganum harmala, Veratrum lobelianum and Vinca herbacea which mimic pharmacologycal effects of serotonin in isolated organ systems accelerate growth of the same fibroblast cultures (e.g. mouse and human embryo, L-929, L-197 and L41 cell lines).Moreover, in presence of serotonin or of these alkaloids it was possible to grow cells using 2.0-2.5 % of bovine serum instead of standard 10%. The cell multiplication promoting activity was apparent also when alcaloids were added to “starved” (quiescent) for 48 hr fibroblasts. In such cases effect of 0.5%-5.0 % serum plus alkaloids was equal to effect of 10% serum in resuming growth of cultures. Alkaloids, serotonin and serotoninergic receptor blocking agents - cyproheptadine, tipindole and inmecarb-iodine methylate were added to culture media at the moment of seeding, or 6 hr after seeding, and to 4-day cultures starved for 48 hr, in ?M (10-6-10-7 M) concentrations. Cell density and in  special series, incorporation of [3H] Thymidine were determined. It is noticeable, that growth-resuming effect of 10% serum in starved  cultures was completely abolished by inmecarb which is known to bind exclusively to cell surface. Also, serotonin (and, respectively, alcaloids) showed mitogenic activity at concentrations that are comparable to those found in human platelet-poor plasma. Finally, most investigators nowadays came to conclusion that an ideal culture system would contain only the defined components necessary for the survival, differentiation, or proliferation  of the cell line [Wolfe et al., 1980]. Thus, the interaction among the many undefined components of serum and the cells in culture which can alter the biochemical and kinetic properties being investigated could be eliminated.

PHARMACOLOGY OF GRANULOCYTE RESTITUTION IN BONE MARROW-DEPRESSED HOST
M.Gedevanishvili, A.Grkikian, N.Mushkiashvili and Z. Tkabladze
Inst. Pharmacochemistry, 380059 Tbilisi, P.O.a/71, Georgia

The role of specific growth factors defined as colony-stimulating factors (CSF) in the multiplication and differentiation of granulocyte cells is well established [Bessemer et al., 1986; Fagg, 1989; Hansen, 1997]. However, it is not clear whether another separate group of steady-state regulators and long-range mediators of generalized hematopoietic response exists. In the present study effects of biogenic amines and their functional analogues were investigated.
Alkaloid  maidine, epinephrine and phenilephrine promote restitution of granulocytopoiesis in X-irradiated or myelotoxic drug treated animals. These substances promote also granulocyte colony formation on cellulose-acetate membranes inserted into the peritoneal cavity of irradiated host. Accelerated restitution of granulocytopoiesis was observed also under the influence of polyphenols, morphine, pilocarpine and Li-carbonate. Specific receptor-blocking agents which belong to opiate, cholinergic and alpha-adrenergic groups of pharmacological antagonists eliminated granulocytopoietic effects, indicating that granulocyte-restitution promoters are active at different anatomical levels. The hypothesis is advanced that cholinergic sympathetic vasolidator system and subsequently output of epinephrine is enhanced. According to this scheme polyphenols, morphine and lithium operate at hypothalamic and midbrain levels, pilocarpine activates cholinoceptors in adrenal medullary chromaffine (postganglionic) cell membranes, while epinephrine and alkaloid (maidine) stimulate directly the bone marrow hematopoietic elements by alpha-adrenergic mechanism. However, it is not clear whether epinephrine, and bioamine analogues explored, act upon granulocytic cells directly, or stimulate hematopoietic microenvironment to produce CSFs and/or related factors. If the cells of granul-cytopoietic lineage really possess adrenergic receptors, it would be of indubitable interest to investigate consequences of cooperative action of alpha-adrenergic substances and CSFs in promoting granulo-cytopoietic differentiation and multiplication in stress conditions.

THE USING OF NUCLEOLAR PARAMETERS IN ESTIMATION OF WHITE RAT MYOCARD FUNCTIONAL STATE AFTER THE TREATMENT WITH SPECIFIC GROWTH-INHIBITING CARDIOMYOCYTE FACTOR
N.Giorgobiani, L.Rusishvili, D.Dzidziguri, G.Tumanishvili
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University

There is no doubt for the present that the general morpho-functional characteristics of nucleolus are the reliable exponent of functional state of tissue reflecting the synthetical and proliferative potencies of cells. The nucleolus is highly sensitive to different kind of exo- and endogenious factors both of physical and chemical nature. It is also well known that the nucleoli of dividing cells differ by their functional morphology from those of nondividing cells. It has been determined that in the myocard of newborn animals the proliferating cells as well as differentiated cardiomyocytes are presented. For the present time there is no data on the "chalone"- type factors effect on the ribosomal genes transcription sites. Earlier we have extracted and described a tissue-specific, thermostable factor of white rat myocard ventricles, which inhibited the proliferation and DNA-synthesis in newborn rat cardiomyocytes and thus can be referred as "chalone"-type factor. According to all mentioned above the possibility arose to observe the effect of our mitosis-inhibiting factor by means of nucleolar parameters.
For purifying the obtained factor the electrophoresis of its thermostable fractions has been carried out and the eluates presented by low-molecular protein fraction (LPF) have been collected. The objectives of our experiments were:I) the studying of LBF effect on proliferative activity of  7-day  newborn rats cardiomyocytes by means of colchicine mitotic index (MI); II) the using of nucleolar parameters for estimation of 7-day rat myocard functional state. For this purpose both paraffin and semi-thin sections were used. It has been shown that MI of 7-day rats decreased by approximately 50% after the treatment with LPF. On the semi-thin sections the nuclei with active (3.6%), low active (21.2%) and inactive nucleoli (75.2%) were counted. After the treatment with LPF the number of cells with active and low active nucleoli decreased by approximately 40% and 47% correspondingly, while the number of cells with inactive nucleoli increased by 40%. The obtained results showed that during the inhibition of proliferation the number of cells with inactive nucleoli increased, while the number of cells with active nucleoli decreased.

THE EFFECT OF  GAMMA-IRRADIATION ON INTENSITY OF REGENERATION DURING CALLUSOGENESIS OF HIGH PLANT ORGANISMS
M.Gogebashvili
Georgian Scientific Research Institute ofAgricultural Radiology

The forming of radiobiological response during callusogenesis of high plant in many respects depends on regeneration intensity of meristemal tissues.
Thus, the dedifferentiation of tissues in injured callusogenesis zone is the basis of the realization of the first processes. In this work the possibility of direct influence on regeneration intensity by gamma-irradiation and by this means the regulation of formbuilding processes in plant tissues is shown.
This fact can be used in applied aspect during the plant tissues transplantation and graft forming and also during regulation of totipotention response under conditions of tissues cultivation in vitro.
The hypothesis of principle of hierarchical structural plant organization as basis of organisms general regeneration ability is advanced.

INFLUENCE OF ETHANOL INTOXICATION ON THE HYPOTHALAMUS DEVELOPMENT IN THE RATS’ OFFSPRING
Gvinadze N.N., Japaridze N.Dj., Svanidze I.K.
I.Beritashvili Institute of Physiology, Georgian Academy of Sciences, Tbilisi, 380060, L.Gotua 14. Georgia

Influence of 15% ethanol on the number of differentiating nervous cells was investigated in the rats hypothalamus preoptic, supraoptic, and nuclei of the mammilary bodies. Total of 17 nuclei were investigated on the 1st, 3rd, 7th, and 15th days of the postnatal ontogenesis. Statistically significant decrease of the nervous cells was found in the following nuclei: N.periventriculari hypothalamy (n.Pe), Area preoptica med. (MPO), Area lateralis hypothalamy (LPO), N.suprachiasmaticus (n.SCH), N.supraopticus hypothalamy (n.SO), N.paraventricularis hypothalamy (n.PA), N.mammilarius hypothalamy (n.MM), which shows  different sensitivity of the neurons to ethanol and probably is a result of depression of their precursor cells proliferation and migration.

STUDY OF MOLECULAR EVENTS WHICH TRIGGER LIVER REGENERATION
Z.N. Kokrashvili, T.M. Shavlakadze, I.Modebadze, I.G. Kakhidze, M.G. Gurushidze, D.V. Dzidziguri
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University Georgia

Previously we have shown that under the conditions of hormonal disbalance, the hepatocytes stimulated for proliferation exhibit accelerated expression of genes regulators of the cell growth. The first peak of transcriptional activity emerges three hours after the partial removal of the liver tissue and is further followed by early mitosis (6 hours after the operation). We suggested that in the destructive liver tissue the increase of mitotic index should be due to G2 (possibly tetraploid) hepatocyte population. Entrance of these cells into mitosis should have been a result of the accelerated expression of the immediate and delayed early response genes. It is now known that the expression products of immediate early genes (which include foc and jun proto-oncogenes) induce the subsequent activation of delayed early genes. To suppress the activation of early response genes we used  their well known inhibitor  Doxorubicin (DOX) and also Cortisol. DOX, Cortisol and DOX+Cortisol was administered to intact white rats prior to partial hepatectomy, immediately after the operations and 1 hour after the operation. Transcriptional activity of hepatocyte nuclei was studied 6 hours after the operation. It was shown that separate injections of DOX and Cortisol as well as their combined administration results in almost two fold inhibition of RNA synthesis activity in hepatocytes. The obtained results suggested that the suppression of immediate early genes by cortisol, which is similar to that by DOX causes inhibition of delayed early gene expression. In the following seria of the experiments we showed that in the conditions of hormonal disbalance the intensity of transcriptional activity of the hepatocyte nuclei stimulated for proliferation is directly related to the level of mytotic index. Higher the transcriptional activity is, higher the is the mytotic index of the hepatocytes 6 hours after the operation. These results proved our suggestion that transition of G2 hepatocytes into mytosis is due to the activation of delayed early genes three hours after the operation.

THE AUTOREGULATION OF THE NUMBER OF UNICELLULARS IN CULTURE
A.A.Kozlov
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University, Georgia
The hypothesis has been made that the sizes of organs in embryogenesis, postnatal development and during reparative growth is controled by the mechanism formed on the earliest stages of evolution on the level of unicellular organisms assemblies. The experiments on Colpoda sp. have shown, that such mechanism limiting the whole number of cells in culture independently from the volume of culturing does work in the culture of these protozoans.
It have been confirmed that the explanation of the observed effects requires the involvement of physical mechanism of the transmission of information on the whole number of cells in culture to every single cell.
The variant of such a physical mechanism is discussed. The comparison of experiment and theoretical data is presented.

GLYCOGEN-FORMING FUNCTION OF HEPATOCYTES AT REGENERATION AFTER A PARTIAL HEPATECTOMY OF NORMAL AND CIRRHOTIC LIVER
Kudryavtseva M.V., Bezborodkina N.N., Kudravtsev B.N.
Institute of Cytology, Russian Academy of Sciences, S.-Peterburg 194064, Russia

Regeneration is both actual biological and medical problem. Studies of the regeneration process was made primary on the normal liver. However, for medicine it means significant the possibility of involution of pathological alterations in the liver. Morphological and functional investigations of the mammalian liver regeneration found significant regenerating possibility of this organ, promoting of involution cirrhotic alterations. This fact may be of the experimental substantiation for use of resection in clinic. Experimentally, there are detailed studies on the effects of the resection of the cirrhoric liver on the recovery of the liver morphological structure. Meanwhile, the recovery of the functional activity of the liver parenchyma cells has so far remained poorly studied. Using cytofluorimetric method the content of total glycogen (TG) and its labile (LF) and stable (SF) fractions were determined in isolated hepatocytes in norm, in the liver cirrhosis and during regeneration after a partial hepatectomy (PH) of the normal and cirrhotic liver. The liver cirrhosis was shown to be characterised by a 2-3-fold increase in the total glycogen content and to a similar increase in the LF content. Even more pronounced (4.6 times) was the SF accumulation in the cirrhotic liver cells. By 6 month after the PH of the cirrhotic liver, the TG and its fractions had completely returned to normal.

DIFFERENT REACTION OF THE BIRDS EYE RUDIMENT CELLS

ON THE INFLUENCE OF Act-D UPON DEPENDENCE OF ITS STAGE OF DEVELOPMENT
G. Kvinikhidze
Georgian Academy of Sciences, Institute of Zoology, Laboratory of Cytology, Tbilisi, Georgia

Vertebrates eye is a convenient model to study the process of differentiation, as it consists of many different types and origin cells. In previous work we had shown, that the differentiation of eye rudiment cells (retina and lens cells) is realized in accurate temporal  and special interrelation between the processes of specific synthesis, proliferation, cytochemical and specific ultrastructural organization of cells. Moreover, the termination of these processes is realized only after visualization of cells ultrastructural and cytochemical peculiarities of retina and lens rudiment cells. Thus, we decided to study how much inhibition of cells transcriptional activity influence on the process of these rudiments cells differentiation. For that aim Actinomicin D (Act D) was endued (1? on 1g of weight) in different stage of development chick embryos. Material was fixed after 20m, 1, 3 and 24 hours after the enduetion of the inhibitor. Concentration of RNA and common protein in cells was measured by quantitatively cytophotometrical methods on the zond photometer “Zetopon” (Reichert) and on the “Microvideomat-2” (Option). The proliferation activity of the cells was examined by enduing labeled 3H-thymidine in the egg. The ultrastructure of the cells was studied in the transmittional electron microscope (IEM-100). Date figures were processed statistically. Results of investigation showed, that inhibition of the transcription of Act-D causes the rapid decrease of the concentration of RNA and common protein, especially considerable in the first days of incubation (1 - 12 days of inc.). The decrease of the concentration of RNA and common protein is shown in the cells ultrastructure. Simultaneously the proliferation activity of cells decreases. Together these changes on the early stage of development (1-2, 3-4 days of incubation) cause the inhibition of development and sometimes the embryos death. 24-hour incubation of 6 and 7 days embryos with Act-D causes delay formation of the second lens fibers and appearance of abnormal, vacuolated lens. The inhibition in formation of membrane disks of the outer segments of the cones (photoreceptors of the retina) of 10-11 days embryos at 24 hours incubation with the Act-D, is registered as well. The results show, that transcription of mRNA for the membrane disks protein (opsin) are occurring just from 10 to 11 days of incubation Before hatching (17-19 days of incubation) in inhibitory influence of Act-D is observed only in first hours of incubation with an inhibitor. These results of the undertaken experiment show that the inhibition of transcription by Act-D causes different reaction in rudiment retina and lens cells of chick embryos in dependence of stage of development and grade of cells differentiation. It was supposed that significant inhibition effect of Act-D occurs in eye rudiment cells in period of active transcription of mRNP for specific structure proteins (Lens erystallins and cone photoreceptors-opsin).

POSSIBLE ROLE OF APOPTOSIS IN THE PROCESS OF RAT LIVER REGENERATION
Mikadze E.L., Mamatsashvili T.G.
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University, Georgia

The liver of hepatectomized rats in norm and under the effect of chloramphenicol was studied at different period of time after the operation; the degree of inclusion of 3H- thymidine into the nuclei of hepatocytes on the 22 hour after the organ resection and the transcriptional activity of the nuclei of hepatocytes before hepatectomy were investigated as well.
Apoptotic cells in normal regenerative liver were observed on the 6-10 hour after the operation, but they were not observed in the hepatectomized rat liver under the effect of chloramphenicol. The inclusion of 3H-thymidine into the nuclei of hepatocytes of experimental rats was inhibited by 46% compared to control, whereas the indications of transcriptional activity of nuclei of hepatocytes in both groups before the operation were equal within the limits of mistake.
On the basis of the analysis of the obtained data we supposed that the origination of apoptotic cell population is due to a loss of cell mass as a result of 1/3 organ resection, and that just the existence of apoptotic cells contributes to normal liver regeneration. Absence of population of apoptotic cells in the rat liver under the effect of chloramphenicol, directly connected with the inhibition of mitochondrial function, leads to the decrease of inclusion of 3H-thymidine into the nuclei of hepatocytes, i.e. to a partial block of S-phase. Equal values of transcriptional activity of the nuclei of hepatocytes in both groups of rats at the moment of operation suggest that the activation of chloramphenicol  effect causing the inhibition of apoptosis and decreasing the degree of inclusion of 3H- thymidine into the nuclei of hepatocytes of experimental rats is caused by the intensification processes in the cells and tissue of liver immediately after the operation.
The obtained results led us to the suggestion that the apoptotic cell population should contain substances, which facilitate the undamaged hepatocytes to enter a new cell cycle. The obtained data to a certain extent are in agreement with our earlier suppositions according to which in the process of programmed cell death the cell produces substances that can enable it to survive, but at the irreversible DNA degradation these substances, no longer important for this cell, can stimulate neighboring cell division.
We suggest that “altruism” of apoptotic cell is in its capacity to affect intact cells of the tissue when dying as mitogenic or growth factor and thereby regulate the cell balance of tissue.

THE HYPOTHETICAL MODEL OF THE POLYPLOIDIZATION OF RAT LIVER HEPATOCYTES
Mikadze E.L., Berulava M.N. Tumanishvili G.D.
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University,Georgia

In the liver tissue both of embryonic and postnatal (in norm and after hepatectomy) rats the “bimitosis” - a main stage of the hepatocyte polyploidization model, dominating in the literature, was not observed, but in the pair of cells located quite close to each other synchronous mitosis were detected. On the grounds of our own  as well as literary data, the scheme of synchronous mitosis formation was drawn up. This scheme, in certain aspect, is able to reflect the stages of hepatocyte polyploidization as well. At the 1st stage the diploid hepatocyte, as a result of acytokinetical mitosis, conditioned by the defect of cytotomy, forms a binuclear hepatocyte. At the 2nd stage the nuclei of binuclear hepatocyte synchronously pass G1 and S phases, that results in a forming of binuclear tetraploid. At the 3rd stage the binuclear tetraploid, due to the apoptotic cells excrescences undergoes cytokinesis and forms two monotetraploids. At the 4th stage the cells start mitosis as a result of which, in our opinion, not 4 mononuclear, but 2 binuclear diploid cells are formed. Therefore, at each stage of the process of synchronous mitosis, during a certain period of time, hepatocytes of various classes of ploidity are present. Since synchronous mitosis, the amount of which increases with the age of embryo, are formed as a result of violation of normal flow of mitosis, in the light of conception of cell mortality and the limited number of cell duplications, we may assume that synchronous mitosis are indicating the stage of the cell, and their rise is connected with the certain critical number of cell duplications. Further cell duplications and conformable shortening of telomeric segments of chromosome, as is well known, threaten the cell with the death. Due to this reason we think that in a response to the next external proliferative stimulus, the cell changes its mitotic cycle and the process of formation of synchronous mitosis is being blocked at some stage. The blocked cells start to differentiate and then to function, enriching the liver tissue with the hepatocytes of different classes of ploidity. Proceeding from mentioned above, we consider that the importance of ploidity is mainly the conservation of the vitality of the cell, its survival and is directed towards the defense of the cells from further divisions, and possible duplication, at the same time is limited by the duplication of its genetic material.

3D-MODELLING OF THE STRUCTURE AND FUNCTIONAL CHANGES IN RENAL TUBULAR EPITHELIUM DURING ADAPTIVE GROWTH
E.Mishatkina, G.Tumanishvili, P.Tchelidze
The Group of Computed 3D-Engineering of  Biostructures and Tissues, Laboratory of Developmental Biology,  I.Javakhishvili Tbilisi State University, Georgia

Earlier the striking functional heterogeneity of mice nephron proximal epithelium have been demonstrated, though, the whole range of questions remain unknown: 1) whether the shown functional polymorphism of mouse kidney epithelium is characteristic for all the mammalians; 2) whether such a polymorphism presents in nephron distal region as well. The objectives of the present study was to find out the character of functionally active cells distribution within the proximal and distal regions of nephron. It have been shown that: 1) white rats are also characterized with the functional polymorphism; 2) such functional polymorphism with the specific features presents also in distal region of nephron. The obtained results let us to suggest the presence of mechanisms regulating the activation of cells throughout the nephron.

CHANGES IN PROTEIN KINASE C (PKC) ACTIVITY AND ISOFORMS EXPRESSION DURING REGENERATION OF EDL AND SOLEUS RAT MUSCLES
Moraczewski,J.,* Nowotniak,A,.* Castagna M.,**Gautron,J.,*** Martelly,I.*
*University of Warsaw, Faculty of Biology, Department of Cytology,
Krakowskie Przedmieocie 26/28, 00-927 Warsaw, Poland
**INSERM, Oncogenese Applique, 94807 Villejuif ,France
***University Paris XII, Faculty of Science and Technology, Laboratory CRRET, 94010 Creteil, France

An in vivo model of crush-induced EDL and Soleus muscle regeneration in rat was used to study changes in protein PKC during the regeneration process. In this model, EDL muscle regenerates more rapidly than Soleus muscle and that has been associated with the level of mRNA expression of PKC isoforms as determinated by PCR. PKC activity was reduced by half in both muscles during the first 3 days following crushing, in the myolysis step. Then this activity highly increased in EDL muscle at day 7, in the recovering step of muscle fibers, then down to the contralateral levels at day 14 of regeneration. In Soleus muscle PKC activity was increased only at day 14. Surprisingly, PKC activity was transiently increased in both contralateral muscles. As shown by Western blotting technique, PKC isoforms alfa, epsilon, theta, delta, then beta, eta and  finally zeta and iota/lambda were sequentially expressed in regenerating muscles. PKC isoforms alfa, theta, epsilon, zeta were immunolocalized in fibers as well as in surrounding tissue in regenerating muscles. These studies support the possibility that PKC isoforms fulfil different functions in muscle regeneration.

DIFFERENTIATION OF CELLS IN SPINAL CORD AND SKELETAL MUSCLE EXPLANTS DURING THEIR SIMULTANEOUS CULTIVATION
D.P. Museridze, I.K. Svanidze, Ts.S. Tsaishvili
I. Beritashvili Institute of Physiology, Georgian Academy of Sciences, Tbilisi 380060, L. Gotua, 14.Georgia

Processes of histogenesis in vitro proceeds most completely during concomitant cultivation of tissue forming a unitary functional system in vivo. We have studied ultrastructural peculiarities of the spinal cord neural cells and the somatic muscle cells of 14 days old chick embryos after simultaneous cultivation.
The data obtained indicate that adaptation processes are completed within the first days of cultivation. Further cultivation leads to the differentiation of neurons and intensive growth of motoneuron axons toward the muscle tissue.
The present work shows that during simultaneous cultivation of somatic muscle tissue and spinal cord explants the vasic stages of differentiation, necessary to restore functional contacts between motoneurons and muscle tissue in vivo, are remained.

HOW THE REPARATIVE GROWTH SCENARIO EVOLVES DURING LIVER DEVELOPMENT?
T.M. Shavlakadze, I.R. Modebadze, Z.N. Kokrashvili, E.J.Tavdishvili, M.G. Gurushidze, D.V.Dzidziguri
Laboratory of Developmental Biology, Department of Biology and Medicine, Tbilisi State University. Georgia

The adult rat liver  regeneration following the partial  hepatectomy is a physiologic response to a growth stimulus. Restoration of the removed portion of the tissue starts with a rapid activation of early response genes (proto-oncogenes) and thereby with the establishment of a transcriptional cascade. Not  much  is known about proto-oncogenes which  regulate hepatocyte growth in the developing liver. Mostly, regulatory role of c-jun and c-met and expression of a number of liver specific genes is revealed. Also, expression of growth and transcription factors during fetal and neonatal development is now intensively studied. However, the exact mechanisms by which the proliferative growth response to injury may be evolved in the developing liver are unclear.
We studied the transcriptional activity of hepatocyte nuclei  in normal one month old rats and after  partial hepatectomy. Rhythmic changes of RNA synthesis activity  of growing liver cell nuclei was observed in 10 hour period (from 10 am to 8 pm). Also we showed that during the first 10 hours after the partial hepatectomy transcriptional activity of hepatocyte nuclei peaks twice, after 2 and 5 hours. We suggested that the first peak of transcriptional activity should be due to the expression of immediately early response genes (e.g. c-jun). To reveal the mechanism involved in the transcriptional activation of one month old rat hepatocytes in response to the partial hepatectomy, we used suppressers of genes regulators of cell proliferation- Doxorubicin and Cortisol.
Based  on the obtained  results  we suggest a pivot role of genes independently suppressed by Doxorubicin and Cortisol  for the realisation of the reparative growth program in the developing  liver. After one month old rat hepatocytes receive stimulus for the reparative growth, transcriptional activity of these genes increases and peaks after 2 hours.

NEW STRUCTURES IN RENEWING NEWT TISSUES
Charles Taban
59b. ch.Falquets, CH-1223 Cologny.

Unexpected structures forming meshes and most probably constituted by cell glycocalices interconnections were detected in the newt by application to cryostat sections of gold markers (Taban, 95) recognizing specific binding sites to substance P. dophamin, nor-adrenalin and serotonin. These structures were restricted to newt limb regenerates and in adult tissues, which are rapidly and constantly reconstituted, such as epidermis. In this last the mesh appears at mid-distance between the basal cell layer and the external surface, that is at a completely different site than the adepidermal membrane. The limb amputation trauma destroys these structures which are reconstituted during the regenerating process. It has been shown by A.Schmidt that this trauma destroys similarly the glycocalices ordinance, sustaining the view that the meshes seen with the gold marker include glycocalices. These results bring 1) a new evidence that several different neurotransmitters can bind the tissues of the regenerate; 2) show that groups of growing and differentiating cells of the regenerate have the possibility to react simultaneously since they are correlated directly by cell extensions. Studies carried out by others and visualizing collagenes, fibronectin or laminin in the newt did not see the described meshes. Current studies attempting to define the chemical components of the meshes are in progress.

POU-DOMAIN OCTAMER-BINDING TRANSCRIPTION FACTORS. DIFFERENTIAL EXPRESSION OF THE OCT3/4 DURING MOUSE SPERMATOGENESIS.
A.N.Tomilin, *G.-E.Seralini, *M.A.Drosdowsky, V.I.Vorob’ev,
Institute of Cytology, Russian Academy of Sciences, 194064, St.Petersburg, Russia. *Université de Caen, 14032 Caen, France

Molecular cloning of mammalian transcription factors Pit1, Oct1, Oct2, and nematode developmental regulatory protein Unc-86, revealed that all they had a common sequence referred to as the POU-domain, which recognizes the DNA octamer sequence (ATGCAAAT). Octamer motif required for both ubiquitous and tissue-specific expression of various genes. Oct1 was present in all cell types tested, while Oct2 was detected only in immunogenic cells.
The POU-domain octamer-binding transcription factor Oct3/4 is thought to be involved in maintaining of pluripotency of embryonal cells and to play a central role in establishment and maintenance of the germline in mammals. Using in situ hybridization, RT-PCR, immunofluorescence and Southwestern approaches we have found recently that in addition to the early pluripotent cells, primordial germ cells, and oocytes, the Oct3/4 protein is expressed in a stage-specific manner during spermatogenesis, being predominant throughout meiotic phase. The data obtained allow to suggest that Oct3/4 may exert regulatory function in the control of meiotic events and terminal differentiation during spermatogenesis.
A series of data indicate that Oct3/4 protein can activate transcription only in cooperation with cellular coactivator molecules through protein-protein interactions, and that these hypothetical proteins are functionally related to the adenovirus E1A protein. Attempts to isolate putative Oct3/4 coactivators by affinity methods have failed. We have used a bacterially produced recombinant mouse Oct3/4 (rOct3/4), as a protein-binding probe, to detect by far-Western assay the Oct3/4-associating proteins (OTAPs). Nuclear proteins extracted from pachytene spermatocytes, from embryonal carcinoma (EC) cells F9 and from the same cells after retinoic acid-induced differentiation were separated by SDS-PAGE, transferred onto nitrocellulose, and filters was probed with labelled rOct3/4. Both common and cell specific OTAPs were shown to interact directly with Oct3/4. Several OTAPs detected in pachytene spermatocyte may represent germ cell-specific Oct3/4 coactivators. Differentiation of the EC cells results in disappearance of most of OTAPs, supporting their coactivator nature.  (Supported by the Russian Basic Research Foundation).

PROLIFERATION AND DIFFERNTIATION CAPACITY OF RAT LATERAL VENTRICLE CELLS IN EMBRIONAL AND EARLY POSNATAL STAGES AFTER THEIR TRANSPLANTATION INTO NEOCORTEX OF ADULT RATS.
P.D. Bukia, U.G. Kharebava, E.Y. Kalandarishvili, A.D. Taktakishvili; M.T. Davitashvili, N.Sh. Gelashvili, Mepisashvili I.A.
Problematic morpho-physiological laboratory of Tbilisi State University, Georgia

The aim of the present investigation is to study the development and growth dynamic of germinative cells of dorsolateral wall of lateral ventricle transplanted from donors with different age.
The study was performed on white rats. We used 16, 17, 18, 20 days old embrions and 1,2,3,4,5 days old small rats as donors. Peaces of matrix (1 -1,5 mm2) of dorsolateral wall were implanted into the neocortex of adult rats.Recepients were decapitated on on different time points post transplantation. In particular, the material was obtaind from 2 days to 6 months post transplantation. The obtained material was treated for neuro-histological investigation.
The direct dependency of transplant lodge, growth and differentiation on the age of donor has been established. The growth and development of the transplant is the most successful when it is obtained from embrio, especially from  16 - 17 days old embrional matrix. Thus,  2 weeks after transplantation,  17 days old embrional germinative cells actively proliferate, the transplant  grows significantly and consists of poorly differentiated, densly packed elements. Several structural specifities of the transplant makes it easy to identify it from the surrounding tissues of the  recepient neocortex. These are the chain of glial cells on the edge, sizes of the neural cells, disorientation of cell bodies and their cytoplasmic branche, absene of normal cytoarchitecture. The level of lodging and integration of newborn donors matrix with surrounding tissues and proliferation of cellular elements is  less pronounced than in transplants from embrional matrix at  the same postimplantational time points.
Thus, based on the obtained results we suggest that after implantation of newborn rat matrix, precursors of neurons lodge, grow, differentiate and establish contacts with neural tissue of recepient. However, this process is lesser pronounces then in case of implantation of embrional matrix.
ribosomal genes in these cells. Also, the effect of nucleolar protein fraction on mitotic activity of hepatocytes was investigated. It was shown that on the early stage of postnatal development after injection of nucleolar fraction the mitotic index of hepatocytes was decreased (in comparison with control by 40%). This fact may be explained by effect of inhibitory component of nucleolar protein fraction.

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U. K.:
M. C. Thorndyke, Department of Biology, Royal Holloway University of London, Egham Surrey TW20 OEX U.K.
Germany:
H. J. Anton, Zoological Institute, University of Cologne, Weyertal 119, D-50923 Köln Germany,

Fax.: +49 221 9352682, e-mail: hjanton@biolan.uni-koeln.de

last update: October 1 1999
AIRR (International Association for Regeneration Research) ; News