Effect of Bioactive Peptide Complex Isolated from Bovine Serum on Proliferation and Migration of Mesenchymal Stromal Cells In Vitro and Reparation of Bone Defects In Vivo.

We studied the effect of a bioactive peptide complex isolated from bovine serum on the proliferative potential and migration rate of mesenchymal stromal cells in vitro, as well as on the healing of modeled bone defects in rats. This bioregulatory preparation stimulated proliferation of mesenchymal stromal cells from deciduous tooth pulp in vitro, but did not affect the rate of their migration in two-dimensional cultures. In vivo experiments showed that application of this preparation in combination with hydroxyapatite and chitosan gel accelerated bone tissue regeneration, thus ensuring restoration of morphologically normal bone matrix. Thus, cattle blood serum is an available source for the production of bioregulatory preparations for medical purposes.

[Biologically Active Peptides of King Crab Hepatopancreas].

Substances of a peptide nature isolated from the hepatopancreas of the king crab Paralithodes camtschaticus exhibited physicochemical properties and membranotropic and specific activities similar to those of membranotropic homeostatic tissue-specific bioregulators previously found in different mammalian and plant tissues. Their biological effect on vertebrate tissues was demonstrated on a model of roller organotypic cultivation of Pleurodeles waltl newt liver tissue.

[Peptide-containing fraction from a culture medium of Fusarium sambucinum: composition and biological effects].

The culture fluid of the fungus Fusarium sambucinum was investigated for the presence of new peptide-containing bioregulators, previously identified in various mammalian and plant tissues. A fraction containing peptides with molecular weights from 1000 to 2000 Da, which exhibited specific membranotropic activity and a number of physical and chemical properties characteristic of this group of bioregulators, was obtained. The effects of this fraction on the model roller organotypic cultivation of liver tissue of the Pleurodeles waltl newt in vitro were investigated for the first time. This fraction caused the additional activation of pigmented liver cells of newt (analogues to Kupffer cells of the liver of mammals) and provided the maintenance of cell-cell adhesive interactions in tissues. The results show that a new group of peptide bioregulators was present in the culture medium of the fungus F. sambucinum.

[Studies on the structure of the bioregulator purified from the rat brain].

From the brain tissue of Wistar rats,we purified a bioregulator, which is active at ultralow doses. Using reversed-phase HPLC, we prepared a homogenous polypeptide with a molecular weight of 4749 +/- 2 Da, which is responsible for the biological activity of the bioregulator. Using the CD spectroscopy method, we calculated the percentage of canonical elements of the secondary polypeptide structure in a solution. Using the methods of proteomics, we revealed that the structure of the investigated polypeptide was similar to the N-terminal sequence of a fragment of guanine-nucleotide binding G0-protein subunit alpha-1.

[Identification of a new bioregulator acting in ultralow doses in bulb onion (Allium cepa L.)].

A bioregulator that has physicochemical and biological properties similar to a group of bioregulators isolated from various animal tissues has been found in the bulb onion (Allium cepa L.). It was determined that the biological action of the plant bioregulator is determined by a peptide with molecular weight of 4036 +/- 2 Da whose 18-C-terminal amino acid sequence consisted of 18 residues. On models of seed germination of some vegetable cultures, the ability of the bioregulator isolated from supernatant of onion extract in ultralow doses (10(-13) mg of protein/ml) to inhibit growth and development was demonstrated.

[MALDI-TOF mass spectrometric identification of novel intercellular space peptides].

We performed the matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF) analysis of the peptides entering into the composition of not yet explored bioregulators derived from the extracellular matrix of the tissues of the various organs of the mammals, and also plants and fungi. The study included 15 different mammalian tissues, 13 species of plants, and 2 species of fungi. Exploring the bioregulators derived from eye tissues, we demonstrated that their composition includes peptide components with the same values of the molecular weight. The composition of the bioregulators derived from the tissues of various organs of mammals or different species of plants and fungi includes the peptides with different values of molecular weight. Obtained data indicate the growing evidence of the assumptions about the major function of the bioregulators of this group--their involvement in the regulation of tissue-organ homeostasis in the biological systems.

[Study of a new group of bioregulators isolated from the greater plantain (Plantago major L.)].

Proteins with physicochemical properties and biological activity similar to those of membrano-tropic homeostatic tissue-specific bioregulators that had been found earlier in various animal tissues were discovered in leaves of the common plantain (Plantago major L.). To study the specific activity of these plant proteins, we developed an experimental model for organotypic roller cultivation of newt (Pleurodeles waltl) skin tissue in vitro. We showed that the plant proteins of interest exert the wound-healing effect, which is characteristic of this plant, on the skin of vertebrates both in vitro and in vivo.

Effect of bioregulators isolated from the liver, blood serum, and bile of mammals on the state of newt liver tissue in organotypic culture.

We developed models of in vitro organotypic culturing of newt liver tissue with and without adhesion to the substrate. The effects of bioregulators isolated from mammalian liver, blood serum, and bile were studied on the developed models and their specificity was demonstrated. The state of the liver was evaluated by the area of clusters of pigmented cells and by the number of mitoses in the connective tissue cells of the cortical layer. These bioregulators exhibited their biological effects only under conditions of roller organotypic culturing of newt liver tissue.

Structural-functional characteristics of a new bioregulator isolated from bovine pigmented epithelium tissue.

A new bioregulator operating in ultralow doses corresponding to 10(-17) mg/ml has been isolated from tissue of pigmented epithelium of bovine eyes. It has been established that the functional basis of this bioregulator is a complex of a low molecular weight regulatory peptide (4372 Da) and a modulator consisting of a mixture of proteins with molecular weights of 14.980-66.283 kDa. It has been shown that the regulatory peptide is responsible for membranotropic activity of the bioregulator, and the modulator proteins are responsible for biological action in ultralow doses. The data demonstrate an interrelation between nanocondition of the bioregulator and its ability to show activity in ultralow doses.

[Modulators of the regulatory protein activity acting at microdoses].

New, previously not studied bioregulators active in the ultra low doses corresponding of 10(-8) - 10(-17) mg/ml have been isolated from vitreoretinal tissue of eye. It has been shown that these bioregulators comprise some regulatory peptides-modulators represented by proteins with molecular weights 15-70 KDa one of which is bovine serum albumin. Correlation between the nanosize of bioregulators and their ability to show activity in ultra low doses is established.

[A serine protease inhibitor from the anemone Radianthus macrodactylus: isolation and physicochemical characteristics].

A serine protease inhibitor with a molecular mass of 6106 +/- 2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed alpha/beta or alpha + beta polypeptides. It was established that InhVJ is highly specific toward trypsin (Ki 2.49 x 10(-9) M) and alpha-chymotrypsin (Ki 2.17 x 10(-8) M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.

[Synthesis of artifical genome as the basis of synthetic biology].

Recent achievements in the whole-genome sequencing especially viral and bacterial ones, together with the development of methods of bioinformatics and molecular biology, have created preconditions for transition from synthesis of genes to assembly of the whole genomes from chemically synthesized blocks, oligonucleotides. The creation of artificial genomes and artificial cells, will undoubtedly render huge influence on a deepening of knowledge of mechanisms of functioning of living systems at a cellular level, on a way of origin and evolution of life, and also on biotechnology of the future, and will generate preconditions for the further development of synthetic biology and nanobiotechnology.

Proteinase inhibitors from the tropical sea anemone Radianthus macrodactylus: isolation and characteristic.

Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex Peptide 10/30 FPLC, and Nucleosil C(18) reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/alpha-chymotrypsin have been determined. The K(i) values of RmIn I and RmIn II for trypsin and alpha-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals.

[Primary structures of actinoporins from sea anemone Oulactis orientalis].

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with specific primers to the N-terminal regions of the 0. orientalis actinoporin sequences and to the C-terminal region, which is known to be highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were established by sequencing the cDNA clones obtained by the fast amplification of 3'-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain lower hemolytic activities of Or-A and Or-G than that of actinoporins from the tropical species.

[Actinoporins from the Sea of Japan anemone Oulactis orientalis: isolation and partial characterization].

Two cytolytic toxins (cytolysins Or-A and Or-G) were isolated from the Sea of Japan anemone Oulactis orientalis and characterized. Their purification scheme involved a hydrophobic chromatography on Polychrom 1, a gel filtration on Akrilex P-4, a cation-exchange chromatography on CM-32 cellulose, and a reversed-phase HPLC on a Nucleosil C18 column. The molecular masses of Or-A and Or-G were determined by SDS-PAGE in 14% PAG to be ca. 18 kDa. The absence of Cys residues and a high content of basic amino acid residues are characteristic of their amino acid compositions. The hemolytic activities of Or-A and Or-G were found to be 295.86 and 322.58 HU/mg, respectively; these are by three orders of magnitude lower than those of sphingomyelin-inhibitable cytolysins from the tropic sea anemones. The amino acid sequences of the N-terminal fragments of Or-A and Or-G were determined to be ATFRVLAK and GAIIAGAA, respectively. Action of the cytolysins on the erythrocyte membrane is inhibited by exogenous sphingomyelin. They form ion channels in bilayer lipid membranes with the conductivity of 16, 32, and 40 pSm in 0.1 M NaCl and 168, 240, and 320 pSm in 1 M NaCl at pH 7.2. Therefore, they were attributed to the group of actinoporins.