Comparative analysis of behavior and proliferative activity in culture of cells of coelomic fluid and of cells of various tissues of the sea star <Emphasis Type="Italic">Asterias rubens</Emphasis> L. Isolated from normal and injured animals

The sources of coelomocytes in Asteroidea are suggested to be the coelomic epithelium, the axial organ, or Tiedemann’s bodies. To study the problem of whether the cells are replenished at the expense of divisions, we analyzed the incorporation of bromodeoxyuridine (BrdU) in vivo into cells from different tissues of the sea star Asterias rubens L. To study differentiation in vitro, methods of isolating and cultivating cells from various tissues were elaborated and an analysis of the behavior and incorporation of BrdU in culture was performed. The reproduced BrdU incorporation was detected in coelomic epithelial cells. The behavior of coelomocytes and the coelomic epithelial cells in culture depended on the time after the injury of the animals in which the cells were isolated, whereas, for the axial organ and Tiedemann’s bodies, no differences were revealed. After 2 months of cultivation, the formation of BrdU-incorporating, colony-like cells with high nuclear-cytoplasmic ratios was characteristic of coelomic epithelial cells. Thus, the most prospective object for studying the processes of A. rubens cell differentiation in vitro seems to be the coelomic epithelium.     

Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes of types I and IV

Basing on the natural affinity of skin keratinocytes toward extracellular matrix proteins, we have attempted to dissect the population of these cells by varying the time of their adhesion to substrates from fibronectin and collagen of types I and IV. After selection for 10, 20, and 30 min, the keratinocytes were cultivated for 24 h under standard conditions. The area of cell projection on the substrate and the spreading coefficient were measured. Statistically significant morphological differences between cells selected on different substrates were found. The size of cells growing on type-I collagen was twice as large as that of the cells cultivated on collagen type-IV or on fibronectin. Independent of the substratum, up to 60–65% of the cells had a round shape. Keratinocytes cultivated on collagens revealed heterogeneity both in the control and after selection in their adhesion times, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interactions with extracellular matrix proteins in the organism. Original Russian Text O.G. Spichkina, G.P. Pinaev, Y.P. Petrov, 2008, published in Tsitologiya, Vol. 50, No. 2, 2008.     

Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and keratin 19 to actin ratio

Keratin 19 is considered to be a marker for skin stem cells that assume a particular subpopulation of these cells in the keratinocyte population. It is also known that keratinocytes have a natural affinity for extracellular matrix proteins. The aim of this work was to identify subpopulations of keratinocytes by cultivation on various substrates (type-I collagen, laminin 2/4 and fibronectin) and by measuring keratin 19/actin ratio (G/R). The areas of cell projection on a substrate, perimeter, the spreading coefficient, and G/R have been assayed. Keratinocyte populations were morphologically homogeneous both on fibronectin and laminin 2/4. Large cells comprised 12 and 20% of the populations, respectively. No correlation between morphological parameters of cells and keratin 19/actin ratio was found in keratinocytes cultivated in fibronectin. The cells grown on collagen behaved as a heterogenous population with large cells comprising more than 50% of the population. On average, the size of the cells grown on collagen was twice as large as the cells cultivated on fibronectin. On the other hand, the correlation between cell size and G/R was revealed in cells grown on both collagen and laminin 2/4. The cells with lower G/R values display a larger size and higher spreading. We assume that this correlation is determined by the presence of α1 and α2 integrin subunits in these cells. Keratin 19 assay did not reveal keratinocyte subpopulations. Our results raised doubts regarding the presence of stem cells in cultures of humanskin keratinocytes.     

Cell cultivation on microspheres coupled with histones

We have studied the application of histones for the modification of a cell-culture surface. Experiments were conducted on 293 cell lines (human embryonic kidney cells transformed by Ad5 adenovirus) and BALB/3T3 clone A31 (spontaneously transformed mouse embryonic fibroblasts). The cell’s interactions with various types of histons placed on a hydrophobic glass surface or 1.0 μm dextran microspheres was assessed. It was found that all histones studied exhibited adhesive properties but their complexes, such as the total and core histones, were the most adhesive and had improved cell morphology. Cross-linked conjugates of histone complexes immobilized on microshperes facilitated cell network formation by cellular interactions and cell contacts with several microspheres. For the 293 line, unlike BALB/3T3 clone, the A31 cells significantly increased their proliferative activity on microspheres coated with cross-linked conjugates of histone complexes. The study showed that the substrate may be applied for 3D pore matrices designed for tissue-like cell structures in vitro.     

Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.     

Chromosome fractionation. II. Features of the composition and behavior of biphasic polymer systems of metaphase chromosomes obtained from HeLa cells at acidic and neutral pH values

Metaphase chromosomes (MCh) isolated at pH 2.1 and 6.5 slightly differ in their composition and contain in average: 17% of DNA, 9% of RNA and 74% of protein. The three-fold increase of protein content in MCh was not accompanied by the change in nonhistone protein composition. The behaviour of chromosomes isolated at pH 2.1 (acid) and 6.5 (neutral) in two-phase polymer system is shown to be very similar. The function of certain chromosome components in the determination of MCh behaviour in two-phase systems and possible usefulness of acid and neutral MCh for fractionation of total MCh are discussed.