Ammonium Salts Promote Functional Adaptation of Rat Erythrocytes on the Model of Forced Swimming.

Ammonium, an end-product of catabolism, in low doses can promote adaptation of metabolic pathways in erythrocytes under conditions of extreme physical exercise. We compared the effects of two ammonium salts, ammonium chloride and ammonium carbonate, in two doses on biochemical parameters of rat erythrocytes 1 day after extreme physical exercise in a 4-week cycle of forced swimming. Of 16 analyzed parameters, the maximum number of significant shifts from the control was revealed in the groups of rats receiving ammonium chloride in doses of 20 and 10 mg/kg, and the minimal number of differences was found in groups treated with ammonium carbonate in the same doses. The comparison of the levels of reduced glutathione and 2.3-bisphosphoglicerate and activities of 5'-nucleotidase and Ca2+- and Na/K-ATPases attested to more rigorous control of the mechanism of oxygen delivery to tissues by erythrocytes after administration of ammonium chloride in a dose of 20 mg/kg.

Ammonium Salts Increase Physical Performance and Reduce Blood Lactate Level in Rats in a Model of Forced Swimming.

We compared the effects of two doses of ammonium chloride and ammonium carbonate (10 and 20 mg/kg) on the duration of swimming and blood lactate level. Ammonium chloride in a dose of 20 mg/kg was more efficient than in a dose of 10 mg/kg. The efficiency of ammonium carbonate in a dose of 10 mg/kg was similar to that of ammonium chloride in a dose of 20 mg/kg. Increasing the dose of ammonium carbonate to 20 mg/kg led to a decrease in the duration of swimming. On the last day of the experiment, lactate level in 5 min after exhausting load was maximum in control rats, while in rats treated with 10 mg/kg ammonium carbonate and 20 mg/kg ammonium chloride it was lower by 27 and 33%, respectively. In the control group, the amplitude of the decrease in lactate concentration in 1 h after load was 2-fold greater than in the group receiving ammonium chloride in a dose of 20 mg/kg and 1.6-fold greater that in groups treated with ammonium carbonate in a dose of 10 mg/kg and ammonium chloride in a dose of 20 mg/kg.

Control of the Rate of Fibroblast Migration and Spreading by Defensin Oligopeptide Fragment.

The elaboration of modes for directed regulation of cell transition from the nonmotile fixed status to the motile state, and vice versa, is referred to the most important problems of practical medicine. Nowadays, the creation of biopreparations on the basis of naturally occurring compounds for the minimization or elimination of negative consequences at the cell malignization is an actual problem. The effect of synthetic peptide GERA (a fragment of antimicrobial polypeptides defensins) on the spreading and migration of embryonic fibroblasts was studied. The peptide was found to increase the number of spread cells in cell population compared to the control cells. In addition, the GERA peptide stimulates the directed migration of fibroblasts to wounded zone of cell monolayer (i.e., substrate areas free from cells). The most probable cell targets in spreading and migrating fibroblasts under peptide action are the structural and regulatory components of focal adhesions.

Hemoglobin deoxygenation and methemoglobinemia prevent regulatory volume decrease in crucian carp (Carassius carassius) red blood cells.

Fish red blood cells (RBCs) exhibit an oxygen-dependent regulatory volume decrease (RVD) in hypoosmotic environment. In higher vertebrates, membrane-associated hemoglobin is involved in the regulation of osmotic ion movements across the cellular membrane. However, whether the hemoglobin conformational state plays a role in the regulation of osmotic responses in fish red blood cells is still not fully understood. We found that changes in hemoglobin conformation influence the pattern of the regulatory volume decrease in Carassius carassius red blood cells. In oxygenated cells (96.4 ± 3.7% oxygenated hemoglobin), the volume recovery was completed within 125 min. Deoxygenation of hemoglobin (96.5 ± 2.7% of deoxygenated hemoglobin) inhibited the volume decrease in hyposmotically swollen red blood cells. Reoxygenation restored regulatory volume decrease in cells within 5 min. Induced methemoglobinemia (48.4 ± 1.8% of methemoglobin and 41.3 ± 2.3% of deoxygenated hemoglobin) blocked the process of volume recovery and significantly decreased osmotic stability of red blood cells.

[PHOSPHOLIPIDS, FATTY ACIDS AND HEMOGLOBIN OF ERYTHROCYTES IN RAT BLOOD UNDER STRESS CONDITIONS (SWIMMING AT A LOW TEMPERATURE)].

The phospholipid and fatty acid content of erythrocytes in rat blood was studied after exposure to stress - swimming up to drowning. This stress was found to increase the content of phospholipids cha- racteristic for the membrane outer layer (phosphatidilcholine by 13 % and sphingomyelin by 23 %). At the same time a decrease in the content of acid phospholipids belonging to membrane inner layer was ob- served (phosphatidylethanolamine by 16 %, phosphatidyl serine by 14 % and monophosphoinositide by 23 %). The data obtained indicate that erythrocyte membrane under stress conditions undergoes certain structural changes which seem to have an effect on its functional properties. At the same time it was fo- und that the content of saturated, unsaturated and ounsaturation index? of fatty acids does not undergo es- sential changes under stress exposure studied thereby, possibly, compensating maintenance of functional properties of the erythrocyte membrane under conditions of the impaired phospholipid structure. On the basis of examining absorption spectra of lipid extracts it was found that stress evokes a two-fold spectrum growth in the range of 390-410 nm appropriate to the hem. The fact of appearance of the hem in the ext- ract indicates to saponification of hemoglobin evoked by changed pH of the erythrocyte internal environ- ment. Actually, in the process of lipid extraction the hemoglobim passes into a disordered state due to the effects of not only temperature and environmental pH but also due to those of organic solvents possessing lower, than water, ability to form hydrogen bonds. Probably, transesterification of a minor portion of phospholipids occurs during their extraction from erythrocytes with the chloroform-methanol mixture.

A defensin fragment regulates CHO-K1 cell spreading and induces renewal of fatty acid composition in cell membrane phospholipids.

A tetrapeptide defensin fragment has been shown to stimulate the spreading of CHOK1 cells. The tetrapeptide investigated had virtually no effect on the composition of cell membrane phospholipids but participated in the regulation of the renewal of fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Incubation of cells with the peptide resulted in a change in the composition of the unsaturated fatty acid residues in the phospholipids investigated: specifically, the content of monoenoic and/or dienoic acids increased and that of polyenoic acids decreased. The possible role of the peptide investigated (1) in the regulation of the functional activity of integrin receptors, and (2) in changes in the packing density of the phospholipid acyl chains in cell membrane microdomains, which affects the rates of integrin clustering and adhesion complex formation, is discussed.

[PHOSPHOLIPIDS AND FATTY ACIDS IN ERYTHROCYTES OF THE LAMPREY LAMPETRA FLUVIATILIS DURING AUTUMN PRESPAWNING PERIOD AND THE ABSORPTION SPECTRUM OF THEIR LIPID EXTRACT].

The content of some classes of phospholipids and their fatty acid composition in erythrocytes of the lamprey Lampetrafluviatilis during the autumn period of its prespawning migration are investigated. It is found that the phospholipid spectrum of erythrocytes of the lamprey, the oldest representative of vertebrates, is similar to that of many mammals. A four-fold prevalence of phosphatidilcholine content over sphingomyelin content as well as prevalence of (ω3-acids over ω6-acids indicates the <> of lamprey's erythrocyte membranes - an important indicator of deformational ability of lamprey's erythrocytes. Phosphatidilethanol amine and its plasmalogenic form are the most unsaturated phospholipids (their unsaturation indices are 230 and 342, correspondingly). Phosphatidilcholine is the most saturated one (UI is 167). It is found that the basic acid indicators characterizing the fluidity of erythrocyte membranes remain unchanged during the whole period of prespawning migration of lampreys up to spawning. The blood contains several buffer systems, in particular, membrane phospholipids which neutralize acids and alkali incoming into the blood. In the process of organism life a change of pH inside erythrocytes occurs. One can suppose that the base of the system associated with buffer properties of the blood is water dissociation. Inside thin vessels of the circulatory system the hemoglobin attaches and returns molecules of oxygen due to interaction of the buffer systems with water. The property of water to dissociate as well as ion transfer produce in erythrocytes, lying within narrow vessels of the circulatory system, a local pH alteration allowing displacing/attaching the molecule of oxygen from hemoglobin.

[A current viewpoint on structure and evolution of collagens. II. The fibril-associated collagens with interrupted triple helices].

Fibril-associated collagens with interrupted triple helices (FACITs) form one of the subfamilies of collagen family. Being minor components of connective tissues in multicellular animals, FACITs play an important role in structurization of extracellular matrix whose peculiarities determine differences among tissues. FACITs take part in regulation of the sizes of banded collagen fibrils and are also a link between diverse components of extracellular matrix and cells in different tissues. The functional characteristics of FACIT molecules are determined by peculiarities of α-chain structure (interruptions in collagenous domains and module structure of N-terminal noncollagenous regions), trimeric molecules (trimerization domains), and supramolecular assemblies (mainly, association with banded fibrils and inability to form homopolymeric suprastructural aggregates). The evolution of FACITs is also discussed. A hypothetical model of structural changes leading to formation of FACIT subfamily is propounded.

[Fatty acid composition of phospholipids of erythrocytes of lamprey, frog, rat, and absorption spectra of their lipid extracts].

The work deals with study of content and fatty acid composition of phospholipids as well as of absorption spectra of lipid extracts of blood erythrocytes poikilothermal and homoiothermal animals of different evolutionary levels. Objects of study were poikilothermal lamprey (Lampetra fluviatilis) consuming oxygen from water and the common frog (Rana temporatia) consuming it both from water and from air. Homoiothermal animals were white rats (Rattus rattus) inhabiting in the air medium. The animals were studied at the winter-spring periods. There was established the twofold predominance of the phospholipid content in the lamprey plasma as compared with erythrocytes. In frog and rat the reverse ratio was observed. Based on study of the fatty acid composition of erythrocyte phospholipids it is suggested the higher density of membranes of lamprey as compared with frog membranes. As to fatty acides of the rat blood erythrocytic fraction, they turned out to be less diverse, with almost twofold predominance of saturated over unsaturated acids and not containing the long-chained (C22) Ω3 acids. All this leads to the low unsaturation index and, accordingly, to a dense packing of fatty acids in membrane structures of rat erythrocytes. Mechanism of reversible binding of O2 molecules by hemoglobin in erythrocytes is discussed. The mechanism of interaction of O2 molecules with water molecules is likely to interfere with exchange interaction electrons of hemoglobin iron atoms and oxygen molecule. This confirms our obtained absorption spectra showing that in the lipid extract practically not containing water the heme isolated from erythrocytes is converted to hemin.

[The effect of the collagen tripeptide fragment (GER) on the adhesion and spreading of fibroblasts depends on the properties of adhesive surface].

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates--polystyrene plastic and immobilized on plastic poly-L-lysine, fibronectin or gelatin was studied. Tripeptide GER has been found to participate in the regulation of fibroblast adhesion and spreading. Therewith, the tripeptide effect value on cell response was dependent both on the mode of tripeptide addition to culture medium and on the type of used substrate. During coincubation of fibroblasts with the tripeptide the stimulation of cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin was observed. At the same time the tripeptide did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with the tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. In was shown that the extent of activation and inhibition of adhesive processes on fibronectin was higher than such ones on gelatin after tripeptide treating. The data obtained support the assumption about concerted action of tripeptide GER (activity of which was dependent both on the used concentration of the tripeptide and on the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets on which the GER peptide may affect during the formation of cell-substrate interactions are discussed.

[A modern viewpoint on structure and evolution of collagens. I. Fibrillar collagens].

This review summarizes current data on structure of the most representative group of the collagen family--fibrillar collagens. Attention has been focused on structural organization of individual domains and their functional role in the hierarchical stacking of alpha-chains of collagens. There is presented characteristics of the main stages of biosynthesis and of supramolecular processing of fibrillar collagens. Also considered are some aspects of evolution of fibrillar collagens. The role of duplication of genome and genes, intergene rearrangements, and exon shuffling in evolution of collagen genes is discussed.

[Effect of stress actions on some hematologic and biochemical parameters of rat blood and on energetic intermolecular interactions in lipid extract under effect of light radiation].

Comparative study has been carried of effect of the three-day long starvation, running, and their combination on morphological parameters of rat blood, lipid metabolism, and activity of blood Na,K-ATPase. Different effect has been shown of these stress factors on the blood erythrocyte composition. Starvation is accompanied by the most pronounced release of stored erythrocyte into blood, which results in a significant decrease both of the total amount of reticulocytes and the complete absence of reticulocytes of the I stage of maturity (the youngest). The running on treadmill led to a significant increase of the total amount of blood reticulocytes and to multiple increase of immature reticulocytes (RC-I and RC-II), which can indicate some stress of the bone marrow erythroid stem line. The curve of acid resistance of blood reticulocytes has shown the animal to experience the greatest stress at a combination of starvation and running. Starvation and running produced different effects on blood lipid characteristics. The content of triacylglycerides (TAG) in blood rose by 40% at starvation and decreased by 30% at running, a similar tendency being found for index of atherogeneity. The fatty acid composition of blood phospholipids at running and its combination with starvation practically did not differ from control. A change of Na,K-ATPase, which is so characteristic of reaction to various kinds of stress, sharply fell at starvation (by 22%), but increased at running (by 13%) and decreased markedly at combination of these actions. Absorption spectra of lipid extracts of the whole blood of the rats submitted to various stress actions showed that extracted from blood (at different amount depending on the kind of action) is an organic substance with coupled bonds, which absorbs light in the diapason of 360-620 nm. The absorption of light in the diapason of 400-410 nm has been found to belong to the Soret band of ferroheme and ferriheme. The shift of the Soret band indicates electron transitions in the iron cation. By the change and disappearance of the Soret band, it is possible to judge about the processes occurring in the lipid extract. The disappearance of the Soret band in the lipid extract indicates formation in it of steady radicals as a result of the ferriheme disintegration due to accumulation of energy in porphyrin, which does not seem to occur in the blood cell membranes. The iron atom in the ferriheme molecule is known to accept electron and yields a part of energy probably to porphyrin. Then ferriheme yields electron and becomes ferriheme with excess of energy in porphyrin. Hence, at admission of the next electron to the iron atom the porphyrin molecule is to get rid of the energy obtained earlier to prevent its disintegration. The heme is possible to be an accumulator and distributor of energy in tissue.

[Bounds of change in unsaturation index of fatty acid composition of phospholipids at adaptation of molluscs to biogenic and abiogenic factors of external medium].

Comparative study of fatty acid composition of total phospholipids, as well as of phosphatidylcholine and phosphatidylethanolamine from hepatopancreas and leg muscle was performed on several representatives of gasteropods (Gastropoda) molluscs and bivalve (Bivalvia) mussel (Mytilus edulus). The objects of our study were marine litorins (Littorina saxsatilis) adapted to different temperature conditions of White Sea and Barents Sea, freshwater lymnaea (Lymnaea stagnalis) infested by Trematoda and mussels from White Sea and Black Sea. It was shown that depending on the existence conditions of studied tissue or lipid, the maximal change is observed in the percentage of saturated acids (4-83 %), the percentage of unsaturated acids was less expressed (1-14 %) and the changes in unsaturation index (UI) did not exceed 20 % on average. It was supposed that observed quantitative bounds of UI change under the action of different external factors is utmost for maintenance of membrane fluidity necessary for normal vital activity of cell, particularly in studied ectothermic molluscs.

[Effect of total hypothermia on the fatty acid composition of blood phospholipids of rats and sousliks and light irradiation on chemical processes in lipid extract].

Effect of hypothermia on the fatty acid composition of rat and souslik blood phospholipids is studied. Different reaction of these animals to cooling is revealed: in rats no changes were observed in the fatty acid composition of blood phospholipids, whereas in the hibernating there were significant changes in the content of individual fatty acids (FA). The content of monoenic acids in sousliks decreased almost by 50%, while the content of saturated acid (C18) and of polyenic acids C18 : 2omega6 and C20 : 4omega6 rose significantly. Such changes seem to be the mechanism that promotes maintenance of the organism viability under conditions of a decreased level of metabolism, heart rhythm, and body temperature and is evolutionarily acquired. At the same time, the observed changes in the content of individual FA do not lead to sharp changes in such integrative parameters as the total non-saturation of phospholipids, which determines liquid properties of chylomicrons and other lipolipoprotein transport particles of the souslik blood. There are studied absorption spectra of blood lipid extracts of rats and sousliks under effect of light as well as effect of light upon the FA composition of lipid extracts of these animals. The FA composition of lipid extracts has been established to remain practically constant, whereas the character of changes of spectra under action of light indicates the presence in the extracts of oxidation-reduction reactions. The obtained data allow suggesting that in the lipid extract there occurs cooperation both of the phospholipid molecules themselves and of them with other organic molecules, which makes it possible for fatty acids to participate in processes of transport both of electrons and of protons. This novel role of FA as a participant of the electron transfer might probably be extrapolated to chemical reactions (processes) occurring inside the membrane.

[Fatty acids of phospholipids of brain cell nuclei in rat ontogenesis].

Experiments were carried out on the 19-day old rat embryos, the 5- and 45-day old rat pups, and the 1.4-1.5-year old rats. Phospholipids and their fatty acid composition in brain cell nuclei were studied using methods of extraction, two-dimensional thin-layer chromatography, and spectrofluorimetry. In the course of ontogenesis, the percentage of different classes of nuclear phospholipids was changed; at the postembryonic period, the unsaturation index of lipids (phosphatidylcholine and phosphatidylethanolamine) and the content of unsaturated (especially polyenic) fatty acids decreased. Microviscosity of nuclear membranes increased; this changes were also shown earlier in phylogenesis of vertebrates. Thus, the facts revealed in the present work correspond to the recapitulation law. It is suggested that such change of lipid ratio and of composition of their fatty acids as well as of the membrane microviscosity serve for regulation of functions of membrane proteins and have adaptive character.

[Significance of electron interactions of fatty acids of phospholipid molecules in the organism adaptation to habitation temperature].

Data in the fatty acid composition of muscle tissue phospholipids of some representatives of gastropod molluscs (Gastropoda) have been presented for the first time. In the lake phytophagues Lymnaea stagnalis and Lymnaea ovata the long-chained C22-acid was not detected, whereas in the predator common whelk Buccinum undatum, C22:6omega3 was present. Comparison of absorption spectra (240-720 nm) of lipid extracts of the studied invertebrates and of rat has been performed. The obtained data are discussed from the point of view of participation of pi-electrons of phospholipid fatty acid molecules in adaptation of membranes to the habitation temperature, which arises owing to interelectron attraction and to the process of formation of Cooper's pairs.

[The role of the collagen tripeptide fragment GER in the adhesion activation and modification of fatty acid composition in membrane phospholipids of CHO-K1 cells].

It has been found that multiply repeated tripeptide fragment GER (Gly-Glu-Arg) from different collagen types stimulates nonspecific adhesion of CHO-K1 cells. Activation of cell adhesion is accompanied by modifications in fatty acid composition of cell membrane phospholipids. Cell incubation with the synthetic peptide increases the unsaturation indexes of phosphatidylcholin (PC), phosphatidylethanolamine (PEA) and phosphatidylinositol (PI). Arachidonic (C20:4omega6) acid is mainly contributed to the increased unsaturation index of PI. In the case of PC and PEA not only arachidonic acid but also other unsaturated fatty acids: docosatetraenoic (C22:4omega6), docosapentaenoic (C22:5omega3) and docosahexaenoic (C22:6omega3) acids are implicated in the index increasing. Besides, the elevation of relative content of molecules with polyenoic fatty acids in the group of PI molecules is accompanied by decrease in monoenoic fatty acids caused mainly by decrease in the oleic (C18:1) acid level. The role of the investigated peptide: 1) in the activation of cell adhesion as a regulator of active or non active state of integrin receptors: 2) in the alterations of fatty acid composition in main classes of phospholipids as modulator of fluidity level in annular lipid zones around these adhesive molecules is discussed.

[About paired correlation of pi-electrons tissue lipid extracts of animals of different evolutionary levels].

With aid of optical methods, the presence of the paired correlations of pi-electrons has been revealed in phospholipids as well as in triacylglyceride molecules. Used for analysis were lipid extracts of individual representatives of animals of various evolutionary levels--cartilaginous and bony fish and mammals differing by the content of unsaturated fatty acids in lipids. It has been established that the necessary condition for formation of electron pairs is interaction of lipid molecules with each other. An opinion is put forward that in the liquid crystal structure of the membrane monolayer there are two zones able to form electron pairs--the zone of location of ester bonds and the zone in the region of double bonds. Besides, the paired correlation in the phospholipid molecule electron system is accompanied by the absence of electric resistance of the membrane monolayer, which provides the monolayer superconductivity at low rates of movement of the "electron fluid". It is to be noted that the very fact of the presence of the electron pair implies transfer of energy by small portions, which does not allow excitation of individual phospholipid molecules in the monolayer and promotes stability of the native membrane. Our data agree with the known statements of A. Pulman and B. Pulman that the life dynamicity is determined by dynamicity of the electron cloud in coupled or partially coupled systems.

[Effects of synthetic fragments of defensins on aggregation and adhesion of epitheliolike cells].

Normal course of processes of regeneration and epitheliazation of damage tissues has been shown to be based on the capability of cells participating in these processes for selective adhesion. In the case of the complete or partial absence of this capability in the cells-participants of the wound healing process, the so-called non-healing wounds appear. In this connection, it remains actual to search for natural agents promoting healing of chronic non-healing wounds. In the present work, we studied effects of synthetic fragments of leukocytic antimicrobial peptides defensines--GER, FGER, and GERA--on aggregation and adhesion of epitheliolike cells of the CHO-K1 line. These peptides have been established to have aggregate-stimulating properties; besides, they enhance adhesion of the cells to the untreated plastic and inhibit fibronectinmediated cell adhesion. Possible pathways of regulation by peptides of processes of intercellular and cell-matrix interaction are discussed as well as ways of release of these compounds in an organism and their functional role in an organism.