M. S. Krasnov

Burn-Healing Effects of a Composition Containing Chitosan Gel and a Blood Serum Bioregulator

We studied the effect of combined preparation on the basis of chitosan containing a bioregulator isolated from cattle serum in a concentration of 10--10 mg/ml on healing of II-IIIa degree skin burns in mammals in vivo.

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 membranotropic 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.

Low-molecular-weight sclera protein biologically active at ultralow doses.

346 Myopia is one of the most prevalent ocular conditions, which leads to a partial sight and sometimes to complete blindness. The main cause of this pathology is the disturbed spatial functional organization of the extracellular scleral matrix [1]. High myopia results in scleral thinning because of abnormal spatial organization of collagen fibers: the thickness of the latter and their number in collagen bundles are reduced, which leads to degradation of the tissue. The search for and studying new endogenous substances capable of improving the state of the sclera in myopia is an urgent task of modern biochemistry. The subject of this study was to investigate the regulatory protein (RP) isolated from ox eye scleral tissue. In general, regulatory proteins exhibiting certain specific physical and chemical properties have been previously identified in various mammalian tissues including eye tissues [2–7]. These are low-molecularweight, highly glycosylated, ë‡ 2+ -binding proteins that are available in aqueous solutions as nanoparticles. Their secondary structure is mostly of unordered conformation and β -structures [4]. The biological activity of these RPs was recorded at ultralow doses; they displayed tissue specificity but not species specificity [4, 6]. Under conditions of organotypical cultivation of various tissues, the RPs studied were found to influence cell differentiation, proliferation, and migration and to support adhesive interactions and cell viability in tissues [4]. We assumed that similar RPs should be present in scleral tissue and have made an attempt to identify these compounds. Cattle scleral tissue was extracted with Ca 2+ -containing Ringer solution at 4 ° C for 2 h. At every stage of purification, the fractions were analyzed for the presence of the RP using the biotesting procedure that was developed earlier [8]. The procedure of extract fractionation was also described earlier; it included salting-out with saturated ammonium sulfate and isoelectric focusing (IEF) of supernatant [2, 4, 5, 7]. After fractionation by IEF, the fraction of acid scleral proteins that was biologically active at ultralow doses was isolated at pH < 3.0. This fraction (scleral RP) was analyzed by PAGE and shown to contain a low-molecular-weight protein with the apparent molecular weight lower than 14 kDa. Using the method of reversed-phase high-performance liquid chromatography (HPLC) in water–acetonitrile gradient (0–80%), the scleral RP fraction was divided into two components with the retention time 3.8 and 13.7 min (hydrophilic and hydrophobic components, respectively). Biotests showed that the first component was biologically active at ultralow doses. In aqueous Low-molecular-weight Sclera Protein Biologically Active at Ultralow Doses

Rotation Cultures of Isolated Newt Retina as a Tool for Obtaining Low-Differentiated Cells Proliferating in vitro

The cell source for the restoration of injured retina is being intensely studied [1–4]. In many studies, various models of retina traumas were developed and numerous morphological, immunochemical, and molecular methods were used to determine the changes in precursor cells and their proliferation and differentiation both in vitro and in vivo. These studies demonstrated the existence of stem cells in the fish retina and mammalian ciliary epithelium, multipotent cells in the growth region of amphibians, fishes, and birds, and differentiated cells (pigment epithelium and Müller’s glia) that are involved in the regeneration of the amphibian and bird retina. The newt retina is the most popular model for studying retinal regeneration, which is the most pronounced in this species. In the newt, differentiated pigment epithelium cells (PECs) and multipotent cells of the eye growth zone (the pars ciliaris–ora serrata complex) form the source for retina regeneration in situ [5]. In addition, the cells of Müller’s glia and microglia, as well as individual cells of the vitreal part of the inner nuclear layer were recently shown to synthesize DNA, whereas replaced bipolars with Landolt club share some morphological and biochemical features with photoreceptors and are assumed to be capable of substituting lost photoreceptors [1]. When the cell source for retina regeneration has been previously studied in newts, PEC cultures were grown in the eye cavity ( in oculi ) [6] and in vitro [7]. As a result, fragments of differentiated retina were obtained ( in oculi ), as well as individual types of neuronal populations ( in vitro ). In this study, we used a new method for culturing adult newt retina. The retina cleared from PECs was grown as a long-term in vitro organotypic culture under rotated on a mini-roller. This method is often used in neurotoxicological and physiological studies to maintain the embryonic brain tissue and mammalian retina [8] and to study retina regeneration in reaggregated suspensions of bird retinal cells [9]. The amphibian retina has never been cultivated by this method so far. Our approach showed that the retina of an adult newt Pl. waltl remains viable in vitro for a long time. Although some neurons died, retinal cells of the internal regeneration source proliferated, migrated, and populated the nuclear retina layers for four weeks. In addition, these blast cells sometimes aggregate in the cavities of spheroid formed by the retina.

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 specifi city 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.

On Mechanisms Underlying Regeneration and Reparation Processes in Tissues

Studies of the possibility of regulating the regenerative and reparative processes in pathologically modified tissues are reviewed. A ready cell system providing the realization of reparative and regenerative processes in all organs (cell sources of regeneration) exists in all organisms. The authors suggest that active bioregulators presented in this paper are involved in the mechanisms of tissue regeneration by modulating the cell sources of regeneration.

Experimental Models for Culturing of Eye Tissues of Pleurodeles Waltl for Evaluation of Specific Effects of Sclera Bioregulator in Ultra-Low Doses

Experiments on stationary culture of posterior eye and roll-bottle culture of the whole eye from adult water lizards Pleurodeles waltl showed that sclera bioregulator produces a stabilizing effects on adhesion interactions between the sclera, choroid, and pigment epithelium and on the maintenance of viability of sclera fi broblasts and pigment epithelium cells.

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.

In vitro organotypic cultivation of adult newt and rat retinas

Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiGFAP antibody dye demonstrated an increase in the parent Müller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the adult rat retinal spheroid developing in vitro; BrdU-positive cells and multiple mitoses were revealed in this fissue. However, the source of proliferation was not in the peripheral retina, and resident macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt inner retinas.

Modulators of activity of regulatory proteins acting at ultra low doses

New, previously unstudied bioregulators active at ultra low doses, 108 to 10−17 mg protein/ml, were isolated from vitreoretinal tissue. It was shown that these bioregulators contained regulatory peptides and modulators, represented by proteins with a molecular weight of 15–70 kDa, including blood serum albumin. The nanosize of the bioregulators correlated with their activity at ultra low doses.