[Immunochemical study of nuclear matrix proteins localization in the structure of perinucleolar chromatin].

Immunofluorescence labeling of proteins with molecular mass of 27, 38, 40, 50 and 65 kDa obtained from serum of patients with autoimmune disease demonstrated different patterns (small clusters or granules) in interphase nuclei of pig kidney cells. It was remarkable that there was no staining inside the nucleoli, but the proteins immunoreactivity was detected around them in the regions of perinucleolar chromatin. Moreover, expression of nucleolar proteins, such as fibrillarin and B23, was found only in nucleoli. After extraction of DNA, PNA and histones, the proteins with molecular mass 27 and 38 kDa were found in the periphery of residual nucleoli, and proteins with molecular mass 40, 50 and 65 kDa had similar localization and were also present in karyoplasm of cells as small clusters. According to our data, nucleolar protein, fibrillarin, was distributed regularly throughout the whole volume of residual nucleoli. At the same time, B23 protein was revealed only at their periphery, where perinucleolar chromatin had localized before treatment. Thus, it has been revealed that the proteins of nuclear matrix with molecular mass 27, 38, 40, 50 and 65 kDa, as well as nucleolar protein B23 are the parts of perinucleolar chromatin, which could be considered as special chromosomal domain associated with the functioning of the nucleolus.

[Electron microscope analysis of cardiomyocytes in the rat left ventricle under simulation of weightlessness effects and artificial gravitation].

Electron microscopic study of left ventricle cardiomyocytes and quantitative analysis of their mitochondriom was performed in rats exposed to tail-suspension, as a model of weightlessness effects, to artificial gravity produced by intermittent 2G centrifugation and a combination of these effects. It was found that the cardiomyocytes ultrastructure changed slightly after tail-suspension and after intermittent 2G influence, as well as under a combination of these effects. However, the number of intermitochondrial junctions increased significantly in the interfibrillar zone of cardiomyocytes under a combination of tail-suspension and intermittent 2G influence, which agrees with the cell hypertrophy described earlier.

[Non-histone skeleton of mitotic chromosome in situ].

Studying giant nuclei of Chironomus plumosus in situ (Makarov, Chentsov, 2010), we concluded that polythene chromosome structure appears after 2 M NaCl and DNase treatment in presence of 2 mM CuCl2. Cu2+ -ions may stabilize bonds between specific non-histone components, arranged into non-histone matrix of polythene chromosome. Here, we investigated the non-histone matrix of pig embryo mitotic chromosomes in situ, using 2 mM CuCl2-stabilization method. In 2 mM CuCl2-stabilized cells the residual chromosome body (non-histone matrix) could be visualized in every stage of mitosis. Mitotic chromosome non-histone matrix had the same reaction on preliminary hypotonic treatment as normal chromosome: different decondensation of non-histone material was observed. Topoisomerase IIalpha and SMC 1 had uniform localization inside chromosomal body and did not form any axial structures.

[Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization].

Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.

[The nuclear matrix proteins (mol. mass 38 and 50 kDa) are transported by chromosomes in mitosis].

It was shown by immunofluorescence method that serum M68 and serum K43 from patients with autoimmune disease stain interphase nuclei and periphery of mitotic chromosomes of pig kidney cells. Western blotting reveals the polypeptide with mol. mass of 50 kDa in serum M68, and the polypeptide with mol. mass of 38 kDa in serum K43. In the nuclear protein matrix, the antibodies to protein with mol. mass of 38 kDa stained only nucleolar periphery, while the antibodies to the protein with mol. mass of 50 kDa stained both the nucleolar periphery and all the interphase nucleus. It shows that among all components of nuclear protein matrix (lamina, internuclear network, residual nucleoli) only nucleolar periphery contains the 38 kDa protein, while the 50 kDa protein is a part of residual nucleolar periphery and takes part in nuclear protein network formation. In the interphase cells, both proteins were in situ localized in the nuclei, but one of them with mol. mass of 50 kDa was in the form of small clearly outlined granules, while the other (38 kDa) was in the form of small bright granules against the background of diffusely stained nuclei. Both proteins were also revealed as continuous ring around nucleolar periphery. During all mitotic stages, the 50 kDa protein was seen on the chromosomal periphery as a cover, and the 38 kDa protein formed separate fragments and granules around them. After nuclear and chromosome decondensation induced by hypotonic treatment, both antibodies stain interphase nuclei in diffuse manner, but in mitotic cells they stained the surface of the swollen chromosomes. The polypeptide with mol. mass of 50 kDa maintained strong connection with chromosome periphery both in norm and under condition of decondensation induced by hypotonic treatment and at subsequent recondensation in isotonic medium. In contrast, the protein with mol. mass of 38 kDa partially lost the contact with a chromosome during recondensation appearing also in the form of granules in cytoplasm. The data allow to consider, that nuclear matrix proteins can be transported as a part of peripheral chromosomal material, and that they can have connection of different stability with chromosomal periphery as well as the main nucleolar proteins (fibrillarin, B-23, nucleolin et al.) and some non-nucleolar components of nuclear protein matrix.

[Dynamics of autoimmune myocarditis development in rats].

The development of autoimmune myocarditis in rats after a single hypodermic injection of rat myosin mixed with a complete Freund's adjuvant (CFA) (400 microg/kg in 200 microl) was studied. The rats from the control group were injected with only CFA. The titer of antibodies to myosin, infiltration of lymphocytes into the myocardium, ultrastructural damage of myofibrils, mitochondria, and nuclei of cardiomyocytes were maximally pronounced on days 14-21 after the immunization with myosin, which indicates a peak of the inflammatory reaction. The content of nitrites and nitrates in the blood serum and myocardium of immunized rats were also studied. A certain contribution to the development of the inflammation is made by CFA: in rats injected with only CFA, morphological signs of myocarditis were found, but to a much lesser degree than in the group immunized with myosin.

[The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L].

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.

[High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus].

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.

[Electron microscopy description of cardiomyocytes from the left ventricle of rat heart after apoptosis induction by isoproterenol].

Changes in cardiomyocytes from the left ventricle of rat heart were studied by light and electron microscopic and morphometric methods in the myocardial regions neighboring necrotic foci formed after the injection of 80 mg/kg beta adrenomimetic isoproterenol. TUNEL assay was used to detect apoptotic cardiomyocytes. Three types of cardiomyocytes (A, B, and C) differing by the ultrastructure of the nucleus and the degree of mitochondrial changes were identified at all studied stages of necrotic focus development (4-48 h). B and C type cardiomyocytes could represent cells at different stages of apoptosis. The apoptotic changes in cardiomyocytes proved to prevail in early lesion foci (4-18 h), while cardiomyocytes at later stages were prone to necrosis; cardiomyocytes can exhibit signs of apoptosis and necrosis at the same time.

[Experimental visualization of chromoneme as one of the higher levels of chromatin compactization in the mitotic chromosome].

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1-0.2 microm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoeneme structure of the mitotic chromosomes in the presence of Ca2+. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl2. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosome swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca2+ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl2, were treated with a photosensbilizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and their general pattern of packaging in in the chromatic was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures.

[Light optical and electron microscopic studies of the tumor cells in rats exposed to vinblastine against a background of increased activity of the anticoagulating system]. / Svetoopticheskoe i élektronno-mikroskopicheskoe issledovanie opukholevykh kletok krys pri deistvii vinblastina na fone povyshennoi aktivnosti protivosvertyvaiushchei sistemy.

It is shown that the damage of sarcoma 45 cells at different stages of cell life cycle occurs under the effect of vinblastine treatment against a background of higher activity of the blood anticoagulating system. A decrease in the mitotic activity, mitosis accumulation in prophase and especially in the metaphase as well as essential changes in the interphase cell ultrastructure are detected.

[Effect of vinblastine during high activity of the anticoagulating system on the growth of sarcoma 45 in rats and several blood indicators in animals with tumors]. / Deistvie vinblastina pri povyshennoi aktivnosti protivosvertyvaiushchei sistemy na rost sarkomy 45 krys i nekotorye pokazateli krovi zhivotnykh s opukhol'iu.

The strong depressive effect of vinblastine on the rat sarcoma 45 growth and intensification of this effect by the combination of vinblastine treatment with the activation of the blood anticoagulating system (BAS) were found. Vinblastine had no negative effect on the BAS activation process and induced no persistent changes in the qualitative content of leucocytes in the peripheral blood.

[Behavior of pericentromere heterochromatin regions under different conditions of chromosome decondensation in isolated nuclei]. / Povedenie raionov pritsentromernogo geterokhromatina v razlichnykh usloviiakh dekondensatsii khromatina vydelennykh iader.

In isolated mouse nuclei the chromocenters were shown to be the pericentromeric heterochromatin regions (PCHR). After the decreasing of bivalent ion concentration (0.1 mM Ca2+, 2 mM Mg2+) the main and peripheral parts of the chromatin remained on the contrary as the compact chromatin bodies. The additional ultrasound treatment of isolated nuclei in the presence of 0.1 mM Ca2+ with DNAase II and triton X-100 resulted in the species enriched by the condensed PCHR.

[Chromocenters of interphase nuclei in the mouse liver contain satellite DNA]. / Khromotsentry interfaznykh iader pecheni myshi soderzhat satellitnuiu DNK.

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.

[Morphometric study of nucleolus-organizing regions of chromosomes from swine embryo kidney cells during Go-, G2-period and mitosis]. / Morfometricheskoe izuchenie iadryshkoobrazuiushchikh raionov khromosom kletok SPEV v Go-periode, v G2-periode i v mitoze.

Quantitative characteristics of nucleolus-organizing regions of chromosomes (NORs, or fibrillar centers, FCs) and some other nucleolar components have been studied with the aid of complete series of ultrathin sections of PK-cells. It has been found that: 1) the number of FCs per cell in the G0-period, in the G2-period and at metaphase is equal to 7.0, 33.7 and 8.0, respectively; 2) volumes of individual FCs in the G0-period (0.033 micron 3), G2-period (0.014 micron 3) and at metaphase (0.025 micron 3) are different; 3) the total volume of FCs, calculated for a haploid set of chromosomes, do not differ in the G0 (0.105 micron 3) and G2 (0.107 micron 3) periods, but exceed twice the FCs volume at metaphase (0.04-0.05 micron 3). These data show that the activation and inactivation of ribosomal genes in interphase PK-cells are not accompanied by a change in the total volumes of FCs and are probably connected with the "fragmentation" and fusion of FCs. Complete inactivation of ribosomal genes at mitosis leads to a decrease in the total volumes of FCs; 4) the nucleolus volume is proportional to the volume of the dense fibrillar RNP-component; in the G2-period the nucleolus volume also correlates with the number of FCs (r = 0.99); 5) the volume of the dense fibrillar component within individual fibrillar complexes--the structures corresponding to one nucleolus-organizing region--is not constant. This is an indirect evidence for the differences in the functional activity of NORs of different chromosomes.

[Repair of damage to cells in an SPEV culture after exposure to mitomycin C]. / Izuchenie reparatsii povrezhdenii kletok kul'tury SPEV posle deistviia mitomitsina C.

The action of mitomycin C on a porcine embryo kidney culture (in dose of 0.5-1.5 mkg/ml in the course of 24-72 hours) is accompanied by significant changes in the ultrastructure and morphology of cells. Moreover, mitomycin C sharply inhibits DNA synthesis, mitotic activity and much more weakly suppresses protein and RNA syntheses. After a prolonged (48 hours) washing of the antibiotics only the mitochondrial ultrastructure is restored in the fresh cultural medium. DNA synthesis and mitotic activity remain suppressed, while protein and RNA syntheses increase sharply, which leads to protein accumulation in cells, and to the enlargement of nuclei, nucleoli and cells. Such changed cells are unable to keep on living and perish.

[Stability in association of the peripheral material with mitotic chromosomes]. / Stabil'nost' sviasi perifericheskogo materiala s mitoticheskimi khromosomami.

The localization of nucleolar proteins (fibrillarin and B-23), and of the protein of interphase nuclear matrix (NMP-65) was studied in the perichromosomal material (CM) after of short hypotonic treatment (15% solution of Henks medium) on cultured pig embryonic kidney cells, followed by restoration of isotonic conditions. It is shown that during hypotonic shock the mitotic chromosomes demonstrate reversible swelling, but their periphery is bounded with a rim of PCM, containing antibodies to fibrillarin and NMP-65, but not to B-23. After returning the cells to the initial isotonic medium, all the three proteins can be detected again on the periphery of chromosomes. It suggests the existence of different stability in the association of free proteins with chromosome bodies. Besides, B-23 and fibrillarin could be visualized in residual nucleoli after a complete extraction of histones and DNA from nuclei.

[The bioenergetic, cytochemical, and staining properties of normal hepatocytes and under functional loads, adaptations to environmental changes and in nonspecific injuries]. / Bioénergeticheskie, tsitokhimicheskie i tinktorial'nye svoistva gepatotsitov v norme, pri funktsional'nykh nagruzkakh, adaptatsiiakh k izmeneniiam sredy i pri nespetsificheskikh povrezhdeniiakh.

Two groups of cells were found among mouse liver cells and among hepatocytes in culture after their fixation and staining: weakly stained cells ("light" cells-LC), and strongly stained cells ("dark" cells- DC), the latter making about 30% of population. DC and LC do not differ in their capacity for DNA, RNA and protein synthesis, in contents of proteins, carbohydrates, lipids etc., and also in their ultrastructure. DC differ from LC in lower acidity (pH 6.5) of live cell cytoplasm, and in higher level of mitochondrial fluorescence after staining with Rhodamin 123. The live culture hepatocytes were seen in two alternative states: DC are able to become LC, and vice versa. The amount of DC increases after a high functional cell activation (partial hepatectomy, phlebotomy and hormonal stimulation), and after cell adaptation to modification of cultural medium conditions (modification of pH, hypo- and hypertonia, hyperthermia). In these cases hepatocytes maintain the capacity to granule formation after vital staining with Neutral red. After nonspecific moderate alteration (6% ethanol) of cultured hepatocytes, the acidity of cytoplasm and a temporary elevation of mitochondrial fluorescence may occur, in addition to the increase in the total number of DC. It is suggested that such a cell activation, both in normal physiological conditions and in the case of cell adaptation to modified cultural media, represents a general stress reaction of the cell preceding the "preparanecrosis" state.

[Structure of the centriolar complex in the hepatocytes of intact and regenerating mouse live]. / Stroenie tsentrioliarnogo kompleksa v gepatotsitakh intaktnoi i regeneriruiushchei pecheni myshi.

The structure of the cellular center in polyploid hepatocytes of intact and regenerating liver of adult mice has been studied. It was shown that the structure of the centriolar complex depends on stages of the cellular cycle. No pericentriolar structures (such as satellites, appendages and others) and cytoplasmic microtubules were found in the centriolar complex within G0-period. The satellites and appendages are formed in the half of the centrioles within G1-period. The microtubules can branch off some satellites; the daughter centrioles begin to form within S-period; there are diplosomes in the cells within G2-period, some mother centrioles are surrounded with the fine fibrillar halo. It is concluded that the structure of the centriolar complex within G0-period is distinguished by that within G1-period. The structure of the centriolar complex in polyploid hepatocytes has the same feature of reorganization in certain interphase periods of the cell cycle as in diploid cells of some cultured cells and the thyroid epithelium.

[Localization of fibrillarin, 53 kDa protein and Ag-NOR proteins in the nuclei of giant antipodal cells of the wheat Triticum aestivum]. / Osobennosti lokalizatsii fibrillarina, belka c mol. massoi 53 kDa i Ag-NOR-belkov v gigantskikh iadrakh kletok antipod pshenitsy.

Distribution of nucleolar argentophylic proteins, fibrillarin and 53 kDa protein, in highly polyploid nuclei of antipodal cells of Triticum aestivum L. was studied at different stages of the embryo sac development. The main results are as follows. 1. Ag-NOR proteins and fibrillarin form clusters are distributed in the giant nucleoli, whereas 53 kDa protein is mainly localized on the nucleolar periphery. Ag-NOR proteins and fibrillarin are accumulated as globular nucleolar-like particles--micronucleoli. 2. Dynamics of Ag-NOR proteins, fibrillarin and 53 kDa protein depends on the proliferative activity of endosperm cells. In embryo sacs with non-dividing endosperm cells at interphase stages, Ag-NOR proteins and fibrillarin were observed only within nucleoli and micronucleoli. In embryo sacs with dividing endosperm cells, fibrillarin and 53 kDa protein formed heterogeneous globular bodies varying in size. Simultaneously, some argentophylic material was observed in giant chromosomes. This may be due, presumably, to a partial or complete disappearance of the nucleoli of antipods and transition of some nucleolar components into the peripheral material of giant polytene chromosomes. We suggest that giant nuclei of antipodal cells may undergo cyclic transformation similar to those in the nuclei of dividing cells.