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.

[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.

[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.

[The 3-dimensional organization of the nucleolus and the nucleolus organizer regions in differentiated cells. II. The reticular, vacuolized and nucleolonemal nucleoli of hepatocytes from the intact mouse liver and of hepatocytes stimulated to proliferation as a result of partial hepatectomy]. / Trekhmernaia organizatsiia iadryshka i iadryshko-obrazuiushchikh raionov differentsirovannykh kletok. II. Retikuliarnye, vakuolizirovannye i nukleolonemnykh iadryshki gepatotsitov intaktnoi pecheni myshi i gepatotsitov, stimulirovannykh k proliferatsii v rezul'tate chastichnoi gepatéktomii.

By means of stereological and morphological analysis, the dynamics of nucleolar structural changes in stimulated mouse hepatocytes has been studied. Reticulated and vacuolized nucleoli typical for normally functioning hepatocytes were shown to convert into the nucleolonemal nucleoli, first noticed 18 hours following operation. The process of activation of mouse hepatocyte nucleoli involves two steps. At the first step (within 1-18 hours following operation) a progressive growth of nucleoli volume, due to a simultaneous reduction of vacuolar sizes and growth of RNP component occurs. Such changes were observed in both groups of nucleoli being present in the intact mouse liver, because at this particular step a considerable decrease in the number of vacuolized nucleoli took place. Besides, in the stimulated hepatocyte nucleoli the number and total volume of fibrillar centers increase. However, in spite of their considerable changes, the nucleoli preserve the marks characteristic of the reticulated type. The second step of activation, noted 18 hours after stimulation of hepatocytes, includes more fundamental structural reconstructions. As the result, reticulated nucleoli obtain the nucleolonemal structure. At the same time, on the background of a further decrease in the individual volume and an essential growth of the total volume in the fibrillar centres, a spasmodic increase in the mass of the dense fibrillar component, which is associated with the formation of the continuous strand of the nucleolonema. All this is responsible for the complete changes in structural organization of nucleoli commonly seen in mouse hepatocytes. It is suggested that the cause of such a structural reconstruction may be due to the changes in topography of transcriptionally active regions of rDNA.

[The 3-dimensional organization of the nucleolus and the nucleolus organizer regions in differentiated cells. III. Changes in the nature of the distribution of argentophilic proteins in the nucleoli of mouse hepatocytes with stimulation of the hepatocytes to proliferation]. / Trekhmernaia organizatsiia iadryshka i iadryshko-obrazuiushchikh raionov differentsirovannykh kletok. III. Izmenenie kharaktera raspredeleniia argentofil'nykh belkov iadryshek gepatotsitov myshi pri stimuliatsii gepatotsitov k proliferatsii.

The stereological and morphological analysis of Ag-positive zones revealed the principal difference in quantity and distribution of Ag-NOR proteins in reticulated and nucleolonemal nucleoli of mouse hepatocytes. It is shown that reticulated nucleoli of resting hepatocytes are characterized with the presence of discrete rounded Ag-positive zones, whose quantitative parameters correspond to the fibrillar centers and dense fibrillar component taken together. The analysis of the three-dimensional structure of Ag-positive zones of nucleolonemal nucleoli has revealed considerable changes in quantity and distribution of Ag-NOR proteins due to proliferative stimuli. 22 hours following operation, a continuous, strongly winding strand of nucleolonema is revealed in the nuclei. Taking into account that Ag-NOR proteins are associated with the transcriptionally active regions of rDNA it may be suggested that this structural conversion was due to decondensation and activation of inactive regions of r-chromatin. A considerable growth of Ag-positive zone volume during the conversion of reticulated nucleoli into the nucleolonemal type may be explained by the increase in Ag-NOR protein contents.

[Analysis of soluble nuclear fractions regulating RNA synthesis]. / Izuchenie faktorov iadernogo soka, reguliruiushchikh sintez RNK.

The influence of the rat liver nuclear extract on RNA synthesis in the system of isolated cell nuclei of the rat liver was investigated. Two factors were discovered with opposite influence on RNA synthesis: one stimulating, while the other inhibiting the synthesis. A fraction of high-polymeric RNA (7-9 S) and a fraction of the total protein were isolated from the extract. The influence of these fractions on the transcription process was studied both in isolated nuclei and in the system containing pure liver DNA in the presence of RNA polymerase of E. coli. In both the cases the total fraction of protein turned out to stimulate RNA synthesis, while the high-polymeric fraction of RNA inhibited it. The soluble fractions, detected in the cells and influencing RNA synthesis, interact with RNA polymerase either increasing or decreasing the activity of the enzyme.

Studies on the action of the nuclear factor promoting actinomycin D-binding capacity of chromatin.

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.