Wound healing activity of aqueous dispersion of fullerene C60 produced by "green technology".

In addition to exhibited antioxidant and anti-inflammatory activity, fullerene C60 is a promising wound healing agent. An important stage in the production of fullerene-based ointments is the stability of the aqueous fullerene dispersion (AFD) with minimum size of colloidal fullerene aggregates and sufficiently high concentration. To achieve these parameters tangential flow filtration of fullerene C60 was used ("green technology"). As estimated by small-angle neutron scattering and dynamic light scattering purified AFDs with narrow-size distribution nanoclusters have a size of 6 nm and are assembled into agglomerates which reach a size of 150 nm. The ability of the AFD to exhibit regenerative activity was studied using the animal wound model. This study shows for the first time that the fullerene-based composition stimulates the healing of wounds of various origins. We assume that the mechanism of the AFD wound-healing activity is associated with the aryl hydrocarbon receptor and macrophages activity.

THE COMPARATIVE CHARACTERISTIC OF MORPHOLOGY AND PROLIFERATIVE ACTIVITY OF COELOMIC FLUID AND COELOMIC EPITHELIUM CELLS IN STARFISH ASTERIAS AMURENSIS AND STARFISH A. RUBENS.

Identification and characterization of cells responsible for the restoration of tissues in adult organisms is one of the main problems in regenerative biology. In this study, the comparative histological analysis of cellular suspensions in coelomic fluid (CF) and coelomic epithelium (CE) of two close species of Asteroidea has been done. Particular attention was paid to characteristics of small epithelial cells (SECs, diameter 4 mm) with high nuclear-cytoplasmic ratio more 0.9 and without visible signs of differentiation. Cells of this type constitute a significant proportion in CE in Asterias rubens, show proliferative activity and are probably the progenitor cells for the coelomocytes. Small cells with parameters identical to those of A. rubens SECs have been found both in CF and CE of A. amurensis. We have found subpopulation of weakly attached CE cells highly enriched with SECs-1. These cells were able to migrate from CE. Analysis of adhesion ability of CF and CE cells has revelaled the same patterns for these two closely releated starfish. Two-week primary cultures have demonstrated the speciality of A. amurensis CE cells consisting in the formation of «crystals¼, the potential centers of spiculogenesis that have not been revealed in A. rubens. Both small cells and larger cells with nuclear-cytoplasmic ration lower than 0.7 demonstrated proliferative activity in vivo and in vitro. Moreover, more high mitotic activity of coelomocytes has been found in A. amurensis. The hypotheses of coelomocytes origin are discussed.

[The characteristic of the coelomic fluid and coelomic epithelium cell populations of starfish Asterias rubens L., capable to attach and spread on various substrates].

Cultivation is one of the methods modeling processes occurring in vivo. The success of cultivation, in particular, is defined by a substratum choice. We studied the ability of coelomocytes and coelomic epithelial cells to attach and spread to fibronectin, laminin, polylysine, and glass. Qualitative composition of heterogeneous populations of coelomocytes and epithelial cells was determined after staining the cells with rhodamine-phalloidin and DAPI, and changes in the composition of populations evaluated in response to injury. Seven relative classes of coelomocytes has been identified, three of which has been shown to participate in the formation of clot during primary repair of wounds. There was a change in the proportion of these cells, attached to specific ligands in response to the injury. In coelomic epithelium 8 relative classes of cells has been identified, two of which are likely to be candidates for the role of progenitor cells for coelomocytes--coelomocyte-like and small epithelial cells with high nuclear-cytoplasmic ratio. The enrichment with the small cells in population of attached coelomic epithelium cells has been revealed when seeding on laminin. Continued viability of epithelial cells has been shown when cultured on laminin during 2 months.

[Cells of coelomic liquid and cells of different tissues of sea star Asterias rubens L. isolated from intact and post-traumatic animals: behaviour and proliferation under cultivation in vitro].

Proposed sources of coelomocytes in Asteroidea after traumatic injures are coelomic epithelium, axial organ or Tidemann's bodies. To study the involvement of cell division in the process, proliferation of cells from different tissues of starfish Asterias rubens L. has been studied after bromdeoxyuridine incorporation in vivo. To study the differentiation of coelomocytes in vitro a method for isolation and cultivation of different tissue cells has been worked out and cell behaviour and proliferation in culture has been analyzed. The reliable BrdU incorporation has been found in coelomic epithelium cells in vivo. Coelomocytes and coelomic epithelium cells behaviour in culture dependent on the post-trauma period after which the cells were loaded into the culture whereas no difference was revealed for axial organ and Tidemann's bodies cells. Two-month cultivation of coelomic epithelium cells resulted in formation of colony-like accumulations of the cells with high nuclear-cytoplasm ratio which of colony-like accumulation of the cells with high nuclear-cytoplasm ratio which incorporated BrdU. Thus, coelomic epithelium cells seem to be more promising object for the study of A. rubens cell differentiation in vitro.

[The comparative analysis of subcellular fractionation methods for revealing of alpha-actinin 1 and alpha-actinin 4 in A431 cells].

Alpha-actinin 1 and alpha-actinin 4 belong to a family of actin-binding proteins with shared structural function and regulation of several processes in a cell. Based on previous data on different distribution of these proteins in the nucleus and cytoplasm, we have explored in detail the distribution of alpha-actinin 1 and alpha-actinin 4 in subcellular fractions in A431 cells spread on fibronectin. Several methods of subcellular fractionation were used. Complex approach allowed resuming that revealing of alpha-actinin isoforms in fractions depended on the composition of lysis buffer and preliminary low-temperature freezing of the cells. We have drawn a conclusion that alpha-actinin 4 can be found in all cytoplasmic and nuclear subfractions, while alpha-actinin 1 is characterized by cytoplasmic and membrane localization with specificity of its distribution tightly to the nuclear membrane.

[The analysis of cellular elements in coelomic fluid during early regeneration of of the starfish Asterias rubens L].

Three main cell types were found in the coelomic fluid (CF) of intact starfishes: agranulocytes (55-80%) varying in size and form (spherical and ovoid) and with occasional pseudopodia, granulocytes (15-45%), and small cells (up to 2 %) with a high nuclear-cytoplasmic ratio. The starfish response to injury depends on the degree of coelomic fluid loss. After a slight wounding, when only insignificant portion of CF is lost, the cellular composition of circulating fluid changed only slightly. Unlike, a significant injury resulted in rising the share of small cells, regarded presumably as young cells. Besides, after injury the functional characteristics of SF also changed: the proportion of cells with decondensed chromatin and stained nucleoli increased, and coelomocytes acquired ability to form nets at adhesion. Moreover, some new cell types can be found (fusiform cells), with granulocyte proportion in nets increasing. We suppose that after slight wounding circulating coelomocytes may restore from the existing store of differentiated cells beyond the circulation, whereas after significant injury young undifferentiated coelomocytes are involved in the process of restoration.

[Dynamic of DNA-binding activity of transcription factors in A431 cells during their spreading on immobilized ligands].

Dynamics of actin cytoskeleton in A431 cells and specific NF-kappaB SRF and AP-1 DNA-binding activities were studied during a 2 h spreading of these cells on fibronectin, laminin-2/4 or an antibody to epidermal growth factor receptor. Cell spreading was shown to be accompanied by sequential formation of actin cytoskeleton structures, whose spatial organization depends on the type of immobilized ligand. We have determined the time intervals, within which certain forms of cytoskeleton do not change qualitatively and are specific for dominant part of cell population. It has been shown that DNA-binding activities of the above transcription factors studied oscillate during cell spreading. The cycles of DNA-binding activity were found to be equel to 15-40 min. The character of oscillations depends on both transcription factor and ligand type. The temporal comparison of presses of actin cytoskeleton formation and DNA-binding activity of NF-kappaB and SRF times suggest that actin cytoskeleton reorganization may be presumably associated with activation of NF-KppaB and SRF.

[SRE, NF-kappaB, and AP-1 DNA-binding activities induced by A431 cell adhesion correlate with actin cytoskeleton reorganization].

Cell adhesion to extracellular matrix proteins induces activation of different signal molecules and influences gene expression. As shown earlier, epidermoid carcinoma A431 cell adhesion to fibronectin, laminin-2/4 or antibodies to receptor EGF (ab EGFR) results in reorganization of specific cell shape and actin cytoskeleton in the majority of cells. This study resolves a question whether morphological changes are accompanied with some cell response at the level of gene expression in nuclei. We have shown that cell reattachment promotes a specific DNA-binding of nuclear extracts with consensus sequences SRE, NF-kappaB and AP-1, compared to the control. NF-kappaB and AP-1 activities were considerably reduced in spread cells, which did not show actin filament's structures typical for the ligands. SRE specific proteins demonstrated other peculiarities and depended on the type of immobilized ligands. Our results argue that actin cytoskeleton reorganization, induced by cell adhesion to immobilized ligands at the early period after cell reattachment, is correlated with a specific answer at the levels of DNA-binding activity of transcription factors SRE, NF-kappaB and AP-1.

Small cytoplasmic RNA associated with polyadenylated RNA is involved in the hormonal regulation of gene expression.

The fraction of small RNA (sacc-RNA) associated with cytoplasmic rat liver poly(A)+ RNA by non-covalent, possibly complementary, interactions has been isolated and studied. Fingerprint analysis and Northern blot hybridization data reveal that the specific changes occur in the population of sacc-RNA in response to glucocorticoid treatment. The close similarity of the oligonucleotide composition of sacc-RNA and RNA-component of small nuclear RNP-acceptor of glucocorticoid hormones has been found. The hypothesis of the involvement of the small RNA in the hormonal regulation of posttranscriptional stages of gene expression in the cytoplasm has been put forward.

Cortisone-induced small RNP tightly bound to chromatin.

The small nuclear RNP (alpha-RNP) tightly bound to chromatin has been isolated. alpha-RNP can be removed from chromatin together with the acid-soluble proteins. The RNA from this RNP has been isolated; its electrophoretic mobility is equal to that of 4 S RNA. The study of the resistance of alpha-RNA to RNases (A, T1 and S1) in salt solutions of various ionic strengths allows us to conclude that the alpha-RNA has a well-developed secondary structure. The alpha-RNA is tightly associated with the protein moiety of alpha-RNP and has developed secondary structure. The alpha-RNA is tightly associated with the protein moiety of alpha-RNP and has a high metabolic activity.

[Study of the double-stranded fragments in the poly(A)-containing cytoplasmic RNA of the rat liver]. / Issledovanie dvuspiral'nykh uchastkov v poli(A)-soderzhashchikh RNK tsitoplazmy kletok pecheni krys.

The double-stranded sequences in the poly(A)-containing cytoplasmic RNA (c-dsRNA) hybridize mainly with the repetitive DNA. 70-80% of double-stranded regions in the cytoplasmic poly (A)-RNA are identical to double-stranded regions of heterogeneous nuclear RNA in normal as well as in cortisone-treated rats. The thermal stability of cytoplasmic double-stranded regions is higher in the presence of polyadenylate sequences than in their absence. It is suggested that the double-stranded sequences in the poly (A) +RNA interact with poly (A) stretches and form higher order structures. The thermal stability of c-dsRNA isolated from cortisone-treated rats is higher than that from control rats.

[Changes in the composition of double-stranded sequences of poly(A)-containing RNA in cell cytoplasm during hormonal induction]. / Izmenenie sostava dvuspiral'nykh posledovatel'nostei poli(A)-soderzhashchikh RNK tsitoplazmy kletok pri gormonal'noi induktsii.

Double-stranded segments (c-ds) have been studied in the poly(A)+ cytoplasmic rat liver RNA. Duplexes about 40 base pairs long have been shown to be of intermolecular character and originate from the interaction between ss-RNA and complementary regions of the poly(A)-containing RNA molecules. Shorter ds-sequences are, mainly, of intramolecular nature. Double-stranded sequences of different length differ also in their oligonucleotide composition, according to fingerprint analysis data. Under the action of cortisone, only several kinds of double-stranded sequences have been demonstrated to increase in the population of cytoplasmic poly(A)+RNA. The function of ds-regions in the hormonal regulation of gene expression is suggested.

[Glucocorticoids specifically regulate the expression of dispersed repeating sequences in liver cells. Detection of small ID-like antisense RNA]. / Gliukokortikoidy spetsificheski reguliruiut ékspressiiu dispergirovannykh povtoriaiushchikhsia posledovatel'nostei v kletkakh pecheni. Vyiavlenie maloi ID-podobnoi antismyslovoi RNK.

Differential regulation of the expression of various families of the rat genome interspersed repetitive sequences by glucocorticoid hormones in liver has been demonstrated. Small ID-like RNA complementarily associated with high-molecular-weight cytoplasmic poly(A)-containing RNAs has been identified. The results obtained indicate that glucocorticoids hormones induce expression of the B2, ID, and L1 families in rat liver. The content of the transcripts of these repetitive elements is increased by the hormones in the molecules of heterogeneous nuclear RNA. In the molecules of high-molecular-mass poly(A)-containing cytoplasmic RNA glucocorticoids increase the content of the ID-homologous sequences. In the population of the small specific RNA the content of the ID-homologous transcripts is also increased. A suggestion is put forward about the involvement of the ID-like element in the hormonal posttranscriptional regulation of gene expression at the mRNA level.

[Participation of small RNAs, associated with poly(A)+RNA in cytoplasm in hormonal regulation of genome expression]. / Uchastie malykh RNK, assotsiirovannykh s poli(A)+-tsitoplazmy, v gormonal'noi reguliatsii ékspressii genoma.

The fraction of small RNA (sacc-RNA), associated with cytoplasmic rat liver poly(A)+RNA by non-covalent, possibly complementary interactions, has been isolated and studied. Fingerprint analysis data reveal that the specific changes occur in the population of sacc-RNA in response to glucocorticoid treatment. The close similarity of oligonucleotide composition of sacc-RNA and RNA-component of small nuclear RNP-acceptor of glucocorticoid hormones has been found. The tightly boynd peptides were observed in the fraction of sacc-RNA. The hypothesis of the involvement of the small RNA in the hormonal regulation at post-transcriptional stages of gene expression in the cytoplasm has been forward.

[Double-helical structures in the make up of poly(A)-containing RNA of rat liver cells. Effect of cortisone]. / Dvuspiral'nye struktury v sostave poli(A)-soderzhashchikh RNK tsi toplazmy kletok pecheni krys. Vliianie kortizona.

Cytoplasmic poly(A)-containing RNA from rat liver cells are shown to contain RNase-resistant double-stranded regions. Their content increases after cortisone injection. The isolated double-stranded (ds) regions in polyacrylamide gel electrophoresis are seen as discrete bands of three size classes. Their melting curves are typical for double-stranded nucleic acids. The ds-structure isolated from poly(A)+-RNA of cortisone-stimulated rat livers has higher thermostability, with that of control livers. After denaturation 90% of ds-regions become single-stranded. About 50% of denatured double-stranded fragments reassociate at the range of COT = 10(-3)-10(-2). The origin of ds-structures in cytoplasmic poly(A)+-RNA and their possible functions are discussed.

[A new class of RNP particles containing small RNA homologous to short dispersed DNA repetitive sequences]. / Novyi klass RNP chastits, soderzhashchikh malye RNK, gomologichnye korotkim dispergirovannym povtoriaiushchimsia posledovatel'nostiam DNK.

Here we show that small RNAs homologous to short interspersed repetitive DNA sequences: ID, B1, B2--in rat cells and Alu in human cells are complexed with specific proteins to form small nuclear and cytoplasmic RNP particles (alpha RNP) with common properties. alpha-RNP differ from other ribonucleoproteins by composition and properties. alpha RNA molecules are apparently transcribed by RNA polymerase III. alpha-RNAs are capable of stable antisense hybridization with specific messenger RNAs. Expression of alpha-RNA is specifically regulated by gene regulatory factors. The data obtained support the suggestion that alpha-RNA may belong to the group of regulatory eukaryotic RNAs and that alpha-RNP might be involved in the coordinative control of the expression of the sets of genes with SINE-homologous sequences in regulatory regions.

[Intracellular distribution of tyrosine-phosphorylated actin-binding proteins in A431 cells spread on different ligands]. / Vnutrikletochnoe raspredelenie aktinsviazyvaiushchikh belkov, fosforilirovannykh po tirozinu, pri rasplastyvanii kletok A431 na raznykh ligandakh.

Spreading A431 cells on extracellular matrix elements fibronectin, laminin 2/4 and antibody to EGF receptor (5A9 clone) leads to tyrosine phosphorylation of actin-binding proteins, which participate in focal adhesions formation. Tyrosine phosphorylation of the proteins is retained for 1 h of cell spreading. When cells interact with ligands, focal adhesion kinase (FAK) becomes tyrosine phosphorylated, and eventually phosphorylates the target proteins. The cooperative effect of integrins and EGF receptor in FAK autophosphorylation at cell spreading on antibody to EGF receptor is discussed.

[Morphology of epidermoid carcinoma A431 cells spread on immobilized ligands]. / Analiz morfologicheskikh osobennostei populiatsii kletok épidermoidnoi kartsinomy A431, rasplastannykh na immobilizovannykh ligandakh.

Cell interaction with extracellular matrix is a multi-step process characterized by cell attachment to substrata with subsequent cell spreading accompanied by actin cytoskeleton and cellular membrane receptor reorganization. It has been shown elsewhere that epidermoid carcinoma A431 cells, spread on solid substrata coated with fibronectin, laminin-2/4 or antibodies to EGF receptor, form specific actin filament structures typical for each particular ligand. Here quantitative analysis of heterogeneous A431 cell population spread on the above ligands has been reported. Cells were subdivided into morphological classes, according to their shape and actin filament structure, and the relationship among classes under various experimental conditions were quantitatively estimated for every ligand. We studied the influence of cell detachment pattern, short-term and long-term starvation, and cell incubation in suspended state in the medium before plating on the cell population composition. It was possible to recognize the modal morphological class of cells with typical actin cytoskeleton structure dominating for the ligand in the population. Long-term starvation and incubation in suspension before cell spreading are considered as the crucial experimental parameters leading to dramatic changes in cell population.

[The epidermal growth factor induces specific changes in the expression of small RNA and of the set of small RNP in A-431 cells]. / Epidermal'nyi faktor rosta vyzyvaet spetsificheskie izmeneniia ékspressii malykh RNK i nabora belkov malykh RNP v kletkakh A-431.

Specific small ribonucleoprotein (alpha-RNP) complexes have been identified and characterized in the human epidermal carcinoma A-431 cells. The alpha-RNP complexes contain Alu-homologous small RNA, along with other small antisense RNA species. The epidermal growth factor (EGF) has been shown to induce selective specific changes in the expression of the small alpha-RNAs, the expression of the Alu-like RNA being repressed. Specific changes in the protein composition of the alpha-RNP complexes have been detected under the influence of EGF.

[alpha-Actinin-4 and p65/RelA subunit of NF-kappaB transcription factor are co-localized and migrate together into the nucleus in EGF-stimulated A431 cell]. / alpha-Aktinin-4 i p65-sub"edinitsa transkriptsionnogo faktora NF-kappaB solokalizuiutsia i sovmestno migriruiut v iadro v kletkakh A431 pod deistviem épidermal'nogo faktora rosta.

The NF-kappaB/Rel family of transcription factors in mammalian cells regulates inducible transcription of a large number of genes in response to diverse stimuli. Despite a great number of publications on this subject, little is known about precise NF-kappaB localization in the cytoplasm. As previously demonstrated, in normal rat fibroblast and human epidermoid carcinoma A431 cells p65/RelA subunit of NF-kappaB is co-localized in the cytoplasm with actin structures. However, the mechanism of NF-kappaB interaction with actin remains unclear. We have investigated localization of p65/RelA subunit NFkappaB and alpha-actinin isoforms during cell activation by epidermal growth factor (EGF). Using confocal microscopy, we have shown that alpha-actinin-4 and p65/RelA subunit of NF-kappaB transcription factor are co-localized in A431 cells. Cell treatment with EGF leads to translocation of the proteins to membrane ruffles, and eventually to migration into the nucleus. Pretreatment of A431 cells with cytochalasin D or wortmannin prior to EGF treatment increases p65/RelA and alpha-actinin-4 accumulation in nuclear extracts. Co-localization of alpha-actinin-4 with p65/RelA subunit of NF-kappaB was found in nuclei isolated from stimulated cells. These results support the notion that actin cytoskeleton reorganization and alpha-actinin-4 are involved in NF-kappaB signaling.