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.

[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.

[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.

[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.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. III. The DNA-binding activity of nuclear alpha-RNP. The Signal Transmission from Growth Factors Group of the Medical Faculty, F. Schiller University, Jena, Germany]. / Novyi klass malykh RNP (al'fa-RNP), soderzhashchikh antismyslovuiu RNK, v kletkakh linii K-562. III. DNK-sviazyvaiushchaia aktivnost' iadernykh al'fa-RNP. Gruppa "Peredacha signala s rostovykh faktorov" pri Meditsinskom fakul'tete Universiteta im. F. Shillera, Iena, Germaniia.

DNA-binding activity of small nuclear alpha-RNP identified in acid-soluble fraction of chromatin of human proerythroleukemic cell line K-562 was studied using the technique of gel retardation. We found that nuclear alpha-RNP isolated from K-562 cells through treatment with dimethylsulfoxide, an agent inducing differentiation, acquire a capacity to specific interaction with Alu repeats of DNA leading to the formation of alpha-RNP-Alu-DNA complexes; nuclear alpha-RNP from cells that were not treated with dimethylsulfoxide do not show such capacity, although they are tightly bound with chromatin in the cell. Thus, the capacity of nuclear alpha-RNP to direct interaction with DNA Alu repeats appearing after the induction of K-562 cells to differentiation along erythroid pathway is an inducible property. We discuss hypothesis about the involvement of nuclear alpha-RNP in the control of expression of inducible genes at the level of chromatin and interaction with DNA.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. IV. The coordinated regulation of the expression of Alu-containing mRNA and alpha-RNA during differentiation]. / Novyi klass malykh RNP (al'fa-RNP), soderzhashchikh antismyslovuiu RNK, v kletkakh linii K-562. IV. Koordinirovannaia reguliatsiia ékspressii Alu-soderzhashchikh mPHK i al'fa-RNK v khode differentsirovki.

Small alpha-RNP of K-562 cells contain a small RNA as an RNA component, this RNA is homologous to Alu-repeating sequences of human DNA. When cells are exposed to dimethylsulfoxide, an agent inducing cell differentiation along the erythroid pathway, the content of both high-molecular-weight (heterogeneous nuclear and messenger) RNA enriched with Alu repeats and low-molecular-weight specific RNA, small Alu-homologous alpha-RNA undergoes a coordinated decrease. Using the technique of northern blot hybridization, we have demonstrated nonuniform distribution of Alu repeats both in the fraction of total low-molecular-weight RNA of the cytoplasm as well as in the fraction of messenger RNA. It is proposed that alpha-RNA (alpha-RNP) participates in the control of expression of non-linked Alu-containing genes.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. I. Their characteristics and changes during erythroid differentiation]. / Novyi klass malykh RNP (al'fa-RNO), soderzhashchokh antismyslovuiu RNK, v kletkakh linii K-562. I. Kharakteristika i izmeneniia pri differentsirovke po éritroidnomu puti.

A new class of small RNP (alpha-RNP) has been detected and identified in nuclei and cytoplasm of A-562 erythroid leukemia cell line; these RNPs have a characteristic spectrum of proteins containing conservative and specific components and a special RNA component, which contains a small antisense component (alpha-RNA), a homolog of short dispersed Alu repeats. alpha-RNP is highly stable, tightly associated with chromatin in the nucleus, and is found in the free state in cytoplasm. The composition of nuclear and cytoplasmic alpha-RNP differ and have a specific pattern of changes in response to dimethylsulfoxide, an agent causing differentiation.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. II. The interaction of the RNA--the alpha-RNP component--with heterogeneous nuclear and messenger RNA in the normal state and under DMSO exposure]. / Novyi klass malykh RNP (al'fa-RNP), soderzhashchikh antismyslovuiu RNK, v kletkakh linii K-562. II. Vzaimodeistvie RNK--komponenta al'fa-RNP--s geterogennymi iadernymi i informatsionnymi RNK v norme i pod vozdeistviem DMSO.

Small antisense RNA (alpha-RNA), components of a new class of small nuclear and cytoplasmic RNP (alpha-RNP) identified in the cells of K-562 human proerythroleukemia cell line, are capable of hybridizing under stringent conditions with precursors of mRNA (heterogeneous nuclear RNA or mRNA) and with mRNA of these cells. We found that DMSO, an agent inducing differentiation in K-562 cells, is capable of regulating the composition of alpha-RNA population and concomitantly changes the content of mRNA that has regions homologous (complementary) to alpha-RNA. Specifically, it has been demonstrated that DMSO decreases the level of alpha-RNA, which hybridizes with the actin gene. Results of restriction mapping of regions of complementary interaction of alpha-RNA with the actin gene point out that alpha-RNA hybridizes with regions containing the promotor area and 3'-nontranslated area of the gene. It is proposed that small antisense alpha-RNA (alpha-RNP) participates in the control of gene expression at posttranscriptional level in cell cytoplasm.

[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.

[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.

[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.