[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 adhesion on the status of corneal tissue during in vitro culturing].

Newt and rat cornea with and without the corneal limbus was cultured under conditions of adhesion to a substrate (stationary culturing) and in the absence of adhesion (roller culturing). It was found that the contact of the tissue with the substrate play the key role in the transduction of the regulatory signal that ensures the maintenance of cornea viability and affects proliferation and migration of cornea cells.

[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.

[Role of a regulatory protein from the bovine cornea in activation of cell sources of corneal regeneration in vitro].

A highly purified regulatory protein isolated from the bovine cornea (RBC) was tested for the effect on the rat and newt corneas in vitro under different culture conditions. In the newt cornea, RBC stimulated limbus epithelial cells in roller cultures and cells in the basal layer of corneal epithelium in both roller and stationary cultures. In roller cultures of the rat cornea, RBC had no effect on weakly differentiated limbus cells but stimulated progenitor cells of the basal cornea layer to differentiation inbto definitive epithelial cells. In stationary cultures, RBC did not activate the involvement of cells into regeneration of the rat corneal tissue.

[A regulatory protein isolated from cattle milk].

A regulatory protein displaying biological activity at ultralow doses was identified in cow's milk. The isoelectric point for this protein falls into the pH range of 4.48-5.59, and the molecular weight does not exceed 10 kDa. A study of the secondary structure detected the predominant presence of beta-structures, especially antiparallel, in the molecule of this regulatory protein as well as the regions described in terms of a statistical globule. It was demonstrated that this protein is located extracellularly in the epithelium of mammary ducts, and that this regulatory protein is present in an active form in whole milk; however, it was detectable neither in dry milk nor in infant formula. The results obtained suggest that the milk regulatory protein characterized in this work was identical to low-molecular-weight serum glycoprotein, one of the proteins studied earlier, displaying biological activity at ultralow doses.

[New regulatory protein isolated from the bovine eye lens and its action on the cataract development in rat in vitro]. / Issledovanie vliianiia reguliatornogo belka, vydelennogo iz khrustalika glaza byka, na kataraktogenez u krys in vitro.

The regulatory protein was isolated from the eye lens extract by using an early designed scheme including by means of salting-out of proteins by ammonium sulphate, isoelectrofocusing in pH gradient and electrophoresis in PAAG. A high-purity fraction of the regulatory protein was obtained. The localization of the regulatory protein in the rat-eye lens was investigated by means of primary rabbit antibodies obtained within the case study and by FITS-marked secondary antibodies. Cataractogenesis was induced, in vitro, in Wistar rat lenses through adding, to the cultivation medium, hydrogen peroxide (0.5 mM) or calcium chloride (15 mM). The regulatory protein isolated from the bovine eye lens was added alongside with damaging antibodies to the nutrition medium, concentration 10(-12) mg/ml. The lenses were cultivated for as long as 8 days at 37 degrees C. The degree of opacification of lenses was evaluated visually with the help of a lined substrate as well as by spectrophotometry. The studied protein was shown immunohistochemically to be localized in the intercellular space of the lens epithelium in the region of the basic membrane. The cataractogenesis-related research of the regulatory protein was made on rabbit eye lenses, which were cultivated as a whole for as long as 8 days in vitro. Their transparency and morphology were preserved in them in full since they were cultivated in a serum-free nutrition without admixture of any destructive agents. Opacification of lenses was induced in vitro by changing the concentration of calcium ions in the cultivation medium or through adding hydrogen peroxide to the medium. The valuations of the lens opacity degree as observed in different research series and made by visual observation well correlate with the results of spectrophotometry of lenses made after their cultivation. It can be stated that the studied regulatory protein, when added to the cultivation medium, enhances about two-fold the lens transparency versus the lenses cultivated in the catactogenesis-containing medium. Finally, very small doses of the regulatory protein isolated from the bovine eye lens were found to prevent cataractogenesis in rats in vitro. Since the studied regulatory protein was localized by us in the region of epithelium, it can be suggested that its protective action is conditioned by its ability to contribute to regulating the main biological processes occurring in the lens capsule.

[Studies on the effect of the adhesion protein isolated from bovine eye on cell proliferation in the newt cornea].

The effect of the adhesion protein isolated from the bovine cornea was studied on the model of mechanical injury (cross cutting of the cornea). In the concentration of 10(-12) mg/ml, the protein influenced the proliferation of corneal epithelial cells in newt Pleurodeles waltl in vivo. Experiments were conducted using autoradiography, and the nuclear labeling index (NLI) was determined at different times after surgery and in different corneal regions. This adhesion protein significantly induced proliferation of corneal epithelial cells relative to control groups with the injured eyes treated with the serum adhesion protein at the same concentration or water. The differences between the experimental and control animals were most pronounced 7 days after surgery. By day 14, they were less pronounced but still significant. On day 28, no significant differences in NLI were observed between the three groups, although these values remained higher than in intact animals. An increased pool of proliferating cells in the corneal epithelium was observed both in the affected and intact areas. The data obtained indicate that the biological activity of this protein is not species specific and that it can be a proliferation factor for corneal epithelial cells.

[A study of the adhesive glycoprotein-inactivating protein from mammalian blood serum]. / Issledovanie belka-inaktivatora adgezivnogo glikoproteina iz syvorotki krovi mlekopitaiushchikh.

A protein with a molecular weight of 70 kDa was isolated from bovine blood serum and purified to a homogenous state. This protein inhibited reversibly the adhesive serum glycoprotein with a molecular weight of 12 kDa, which displayed biological activity at ultralow doses. Amino acid analysis showed that the protein inactivator belongs to the group of prealbumins from vertebrate blood serum. The secondary structure of its molecule was characterized by a considerable number of alpha-helices. The conditions for inactivation of serum glycoprotein were studied. The interaction between the serum glycoprotein and the protein inactivator occurred over a long period of time (1 day). It should be emphasized that the presence of calcium ions was a necessary condition for the inactivation of the serum glycoprotein. The data suggest that inactivation of serum glycoprotein results from the formation of a molecular complex consisting of the protein inactivator and the glycoprotein, which is related to the carbon-protein interaction.

[The effect of extremely low doses of the novel regulatory plant proteins ]. / Deistvie novykh reguliatornykh belkov iz rastenii v sverkhmalykh dozakh.

Searching and study on regulatory proteins, which can keep under control the scope of important processes as like as cell adhesion, proliferation, differentiation and morphogenesis, is an actual aim of the current biochemistry. Recently we have identified S-100 proteins in plants of following species: plantain (Plantago major L.), aloe (Aloe arborescens L.), and bilberry (Vaccinum myrtillus L.). Extraction and purification of S-100 proteins gotten from these plants were performed by the method we developed earlier for adhesion proteins of animal tissues. Homogeneity of the studied plant proteins was evaluated and confirmed by HPLC and SDS-electrophoresis in PAAG. Both, plant and animal proteins have appeared to be biologically active at extremely low doses. The tests were performed by adhesiometrical method in short-term tissue culture of mouse's liver in vitro. As a result it was established that the plant proteins insert a membranotropic effect being added in extremely low doses, corresponding to 10(-10)-10(-13) mg/ml. Keeping in mind that the plantain is well known remedy for wound protection and healing, in several experiments we studied the biological effect of plant S-100 proteins on animal cells. It was found that S-100 proteins obtained from plantain influences proliferation of human fibroblasts in vitro. It was found that after the treatment with this protein in low doses the cell growth rate increases essentially.

[Regulatory proteins of vertebrate eye tissues]. / Reguliatornye belki tkanei glaza pozvonochnykh.

In our work the new proteins likely belonged to the microenvironment of pigmented epithelium cells and retinal neurons in mammalian eye were studied. We attempted to understand the role of these proteins in the maintenance of normal morphological and functional state of these eye tissues. Earlier for the first time we identified the adhesion molecules with physico-chemical and biological properties much different from other known cell adhesion molecules of bovine eye. Probably, they represent one family of low molecular weigh, highly glicosylated proteins, that express biological activity in extremely low doses--10(-10) mg/ml. The homogeneity of studying proteins is confirmed by HPLC and SDS-electrophoresis in PAAG. It is shown also that these proteins are N-glycosylated, because they contain mannose and N-acetilglucosamine residues. They demonstrate as well a high calcium-binding activity, with Kd corresponded to 10(-4)-10(-6) mg/ml. For a study of the biological effect of these glycoproteins in extremely low doses, a new experimental model was proposed and developed. It was the cultivation in vitro of the posterior part of the eye obtained from the newt Pleurodeles waltl. In short-time culture system it was demonstrated that the studied glycoproteins could stabilize pigment epithelium cell differentiation and cellular interactions in the neural retina in vitro. In addition, glycoproteins, obtained from the pigmented epithelium of bovine eye could decrease the rate of bipolar cell apoptosis in the neural retina. Therefore, the novel adhesion glycoproteins, expressing their biological activity in extremely low doses, pretend to be the regulatory molecules with vivid gomeostatic effects necessary for the delicate adjustment of cell behavior action and function in sensory tissues.

[An organotypic culture of the newt retina together with other tissues of the posterior eye segment as a model for studying the effects of the cell adhesion glycoproteins]. / Model' organotipicheskogo kul'tivirovaniia setchatki vmeste s tkaniami zadnego sektora glaza tritona dlia izucheniia deistviia adgezivnykh glikoproteinov.

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins and the localization of acetylcholinesterase activity were found to appear during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.

[Low-molecular-weight glycoprotein from cattle blood serum: structure and properties]. / Nizkomolekuliarnyi glikoprotein iz syvorotki krovi krupnogo rogatogo skota: struktura i svoistva.

Amino acid composition, structure, and physicochemical properties of a low-molecular-weight glycoprotein from cattle blood serum (SGP) were studied. The content of carbohydrates (represented by mannose-rich oligosaccharides) amounted to 45-50 wt %. The value of specific partial heat of SGP, measured by differential scanning calorimetry (DSC), equaled 1.8 J/g.K, which is characteristic of unfolded proteins. Circular dichroic (CD) spectra of SGP led us to conclude that it is not highly structured and that it occurs in the shape of a statistical globule. The protein was deglycated using anhydrous trifluoromethane sulfonate (TFMS), after which its amino acid composition and the sequence of a fragment were determined. The results indicate that SGP is a protein not studied previously.

[Study of adhesive proteins from neural tissues of the 8-day old chicken embryonic eye]. / Issledovanie adgezivnykh belkov iz neiral'nykh tkanei glaza 8-dnevnykh kurinykh zarodyshei.

Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epithelium in vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10(-12) M solutions.

[Experimental therapy of perforating wounds of cornea with adhelon]. / Primenenie Adgelona v lechenii pronikaiushchikh ranenii rogovitsy v eksperimente.

The efficacy of Adhelon, a new stimulator of repair regeneration belonging to the group of adhesion factors, was experimentally assessed in the treatment of ocular injuries on a model of perforating untreated wound of the cornea. Clinical and histologic studies in two groups of rabbits persuasively demonstrated the benign effect of adhelon on the course of wound process: the period of inflammatory reaction shortened, infectious complications were prevented, the wound edges closed sooner, epithelialization and reconstruction of newly formed cicatricial tissue was accelerated. Application of adhelon led to the formation of more delicate, compact, and vessel-free cicatrices and accelerated the repair regeneration of the wounded cornea.

[The effect of macromolecular adhesion factors on hepatocyte proliferation in organ cultures of mouse embryonic liver]. / Vliianie makromolekuliarnykh adgezionnykh faktorov na proliferatsiiu gepatotsitov v organnykh kul'turakh émbrional'noi pecheni myshei.

The use of macromolecular adhesion factors (MAF) as a molecular tool made it possible to establish that at least two molecular mechanisms of cell adhesion are violated in the livers of mice genetically predisposed to spontaneous hepatoblastomogenesis in the late embryonic stages and during the early postnatal period. The biological effect of MAF on the adhesion properties of embryonic hepatocytes correlated with their influence on cell proliferation. It has been proposed that violation of the molecular mechanisms of cell adhesion is the key moment in spontaneous blastomogenesis.

[Disruption of molecular mechanisms of cell adhesion in murine liver in genetic predisposition to spontaneous blastomogenesis]. / Narushenie molekuliarnykh mekhanizmov kletochnoi adgezii v pecheni myshei pri geneticheskoi predraspolozhennosti k spontannomu blastomogenezu.

Macromolecular adhesion factors (MAFs) were obtained from the liver of mice with a genetic predisposition for spontaneous hepatoblastomogenesis (CBA) and of resistant mice (C57B1). The MAFs from the liver of both strains are similar in their chemical nature and represents a complex of phosphoglycopeptides with a molecular mass of 20-40 kDa and glycosaminoglycans. The discovered basic difference in the biological effect of MAFs of these strains on the adhesion of hepatocytes appears to be due to disturbances of MAF glycosylation in CBA mice, which cause changes in the spatial-functional organization of the adhesion site in the hepatocytes.