To investigate the stress response and physiological adaptations of goldfish (Carassius auratus) to critical salinity (CS) waters, we analyzed high-density lipoprotein (HDL) stoichiometry, stress markers (cortisol, glucose), and plasma osmotic properties (Na+ , osmolality, water content) using ichthyology, biochemistry, and proteomics approaches. After 21 days of exposure to CS, plasma concentrations of cortisol, glucose, and Na+ increased, indicating stress. Total plasma osmolality (Osmtotal ) and osmolality generated by inorganic (Osminorg ) and organic osmolytes (Osmorg ) also increased, the latter by ~2%. We associated the increase of Osmorg with (1) increased metabolite concentration (glucose), (2) dissociation of HDL particles resulting in increased HDL number per unit plasma volume (~1.5-2-fold) and (3) increased HDL osmotic activity. HDL remodeling may be the reason for the redistribution of bound and free water in plasma, which may contribute to water retention in plasma and, at the same time, to hemodynamic disturbances under CS conditions. The study's findings suggest that HDL remodeling is an important mechanism for maintaining osmotic homeostasis in fish, which is consistent with current capillary exchange models in vertebrates.

Abstract In goldfish Carassius auratus acclimated under critical salinity conditions (CS, 11.5 g kg -1 ), the concentrations of cortisol, glucose, Na + in the blood plasma increased significantly, also large β - HDL particles decomposed into small α- ones with a higher negative charge and a smaller number of apolipoproteins molecules in 1 particle composition. Particles remodeling led to ~1.5-2-fold increase in their number per unit plasma volume. Taking into account that under CS conditions the contribution of organic osmolytes to plasma osmolality increased by ~2%, and the content of the bound water fraction in plasma increased by ~4 times, we assumed that goldfish have a “reserve” mechanism for maintaining homeostasis: when the possibilities of increasing the concentration of Na + and organic metabolites in plasma are limited, remodeling of HDL particles and an increase in their amount in plasma can help to maintain its hyperosmotic status and additional influx of water into the body.

AIM:To evaluate the effect of hypernatremia on the organization of blood plasma high density lipoproteins (HDL) in goldfish; to compare the state of hypernatremia in fish and humans; to assess the possible risks and consequences of the effect of hypernatremia on human plasma lipoproteins.METHODS:The fish were acclimated for 20 days at a critical salinity of 11.5 g/L; after that the salt water was gradually "desalinated". The concentration of Na+ and the content of total water were determined in tissues, cells, and body fluids. The HDL organization was assessed by the number of apolipoprotein molecules per particle. The methods of flame spectrophotometry, electrophoresis and MALDI were used.RESULTS:In fresh water, the state of normonatremia was maintained in the fish body; at critical water salinity, the state of hypernatremia. Against the background of hypernatremia, the initial signs of muscle and erythrocyte dehydration appeared in fish, the total water content in the plasma did not change, and HDL disintegrated into small particles, which, upon restoration of normonatremia, were combined into the original large forms.CONCLUSION:In goldfish at the state of normonatremia, large forms of HDL are stable while at the state of hypernatremia, the small forms of HDL are stable. Under conditions of a hypertonic environment and plasma hypernatremia, the breakdown of HDL prevents the loss of water from the fish organism and reduces the threat of their dehydration. Human hypernatremia is characterized by plasma sodium levels comparable to that in goldfish, however accompanied by life-threatening metabolic changes. The results of this study may be useful for assessing the risks of HDL breakdown at hypernatremia and for the development of protocols for the treatment of pathological conditions in humans (Fig. 4, Ref. 45).

A hypothesis on the involvement of high-density lipoproteins (HDL) in maintaining osmotic homeostasis in fish was proposed. For its verification, structural reorganizations of serum HDL in the goldfish Carassius auratus L. were investigated under natural conditions (during the seasonal dynamics) and in experiments on the effect of critical salinity. A common algorithm of HDL rearrangements in wild freshwater fish and those acclimated experimentally to critical salinity was revealed. It consists in reversible dissociation and association of HDL particles. Under natural (freshwater) conditions, this algorithm matches to the dynamics of total protein distribution relative to the capillary wall, under experimental conditions to changes in water salinity. This finding implies the possibility of implementing multiple strategies of maintaining osmotic homeostasis with the involvement of HDL in higher teleosts whose blood, in contrast to mammals, lacks a specialized osmotically active protein albumin. The role of HDL as universal metabolic regulators and stabilizers is discussed.

A hypothesis on the involvement of high-density lipoproteins(HDL) in maintaining osmotic homeostasis in fish was proposed. Forits verification, structural reorganizations of serum HDL in thegoldfish Carassius auratus L.were investigated under natural conditions (during the seasonal dynamics)and in experiments on the effect of critical salinity. A commonalgorithm of HDL rearrangements in wild freshwater fish and thoseacclimated experimentally to critical salinity was established.It consists in reversible dissociation–association of HDL particles.In the natural (freshwater) habitat, this algorithm is congruentwith the dynamics of total protein distribution across the capillarywall, while under experimental conditions with changes in watersalinity. These findings imply the possibility of multiple strategiesof maintaining osmotic homeostasis with the involvement of HDL inhigher teleosts the blood of which, in contrast to mammals, lacksa specialized osmotically active protein albumin. The role of HDLas universal metabolic regulators and stabilizers is discussed.

Mammalian plasma proteins play a key role in maintaining tissue fluid balance because they are retained within capillaries and thus create colloid osmotic pressure. Likewise, fish plasma contain a considerable concentration oligomeric proteins which likely serve a similar role. To elucidate the functions of these oligomeric proteins, we analyzed blood serum (BS) and interstitial fluid (IF) complexes in goldfish from the wild and under experimental conditions using 2D electrophoresis and matrix-assisted laser desorption/ionization (MALDI). We detected protein compounds with MWs ranging from 50 to 155 kDa, organized as oligomeric complexes. The protein compounds consisted of apolipoproteins АроА-I and Аро-14 which are homological to mammalian АроА-I and АроА-II, respectively. The 155-kDa and 50–125-kDa oligomer complexes were located very low-density lipoproteins (LDL) and high-density lipoproteins (HDL) areas on the BS/IF proteomic maps, respectively. The latter resembled mammalian HDL plasma particles by size and contained lipids, so we considered them as HDL particle populations. Investigation of the uniform dissociation/association mechanism for HDL and LDL oligomers in goldfish, from the wild and under critical salinity conditions, showed the “125/110 → 85/60 kDa” reorganization. This was associated with overcoming physiological stress during spawning and under critical salinity conditions. Opposite reorganization “85/60 → 125/110 kDa” was associated with restoration of metabolic processes after stress. The association/dissociation reorganizations promoted equilibration of BS and IF osmolarities in all fish groups. We discuss the connection of these reorganizations with total protein distribution across the capillary wall and salinity, as well as the role of oligomeric apolipoproteins as universal metabolic regulators.

The effect of the GM6001 metalloproteinase inhibitor on the regeneration of ambulacral structures in Eupentacta fraudatrix has been investigated. Inhibition of proteinase activity exerts a marked effect on regeneration, being dependent on the time when GM6001 is injected. When administration of the inhibitor begins on day 3 post-injury, regeneration is completely abolished, and the animals die. This means that early activation of proteinases is crucial for triggering the regenerative process in holothurians. When GM6001 in first injected on day 7 post-injury, the regeneration rate decreases. However, this effect has proven to be reversible: when inhibition ceases, the regeneration resumes. The effect of the inhibitor is manifested as a retarded degradation of the extracellular matrix, the lack of cell dedifferentiation, and, probably, a slower cell migration. The gelatinase activity is detected in all the regenerating organs of E. fraudatrix. In the holothurian Cucumaria japonica, which is not capable of healing skin wounds and ambulacrum reparation, no gelatinase activity was observed at the site of damage. A suggestion is made that proteinases play an important role in regeneration in holothurians. The most probable morphogenesis regulators are matrix metalloproteinases with gelatinase activity.

Changes in visual pigments were studied in two marine fish species, the masked greenling Hexagrammos octograrnmus and the prickleback Pholidapus dybowskii. A microspectrophotometric (MSP) analysis showed that the rods and cones of the fish collected from the natural marine environment in summer or kept in a tank at a high illumination level predominantly contained porphyropsins based on chromophore A2. As a result, lambda(max) of the double cones significantly shifted to longer wavelengths, reaching 625 and 609 nm, respectively. After several weeks of dark adaptation, the spectra of all the photoreceptor types shifted to shorter wavelengths, as the A1:A2 ratio switched to A1. The MSP data from the fish kept under controlled light conditions were confirmed by chromatography (HPLC), which showed that the changes in the chromophore ratio were reversible and independent of the water temperature. After the preliminary deep dark adaptation, the first noticeable shift in the pigment ratio from A1 to A2 occurred within two weeks of exposure to bright light. A novel finding in this study was a reverse polarity of A1/A2 changes, unlike the case in most other fish species, where A2 chromophore predominated after the dark exposure. This demonstration of the unusual phenomenon of visual pigment transformation suggests a modification or a new way for the activation of specific biochemical mechanisms of A1:A2 conversion at both high and low illumination levels.

One of the most important functions of plasma proteins in vertebrates is their participation in osmotic homeostasis in the organism. Modern concepts about plasma proteins and their capillary filtration are based on a model of large monomeric proteins that are able to penetrate the interstitial space. At the same time, it was revealed that a considerable amount of oligomeric complexes are present in the low-molecular-weight (LM) protein fraction in the extracellular fluids of fishes. The functions of these complexes are unknown. In the present study, we investigated the LM-fraction proteins in the plasma and interstitial fluid (IF) of redfins of the genus Tribolodon. This fish alternatively spends parts of its life cycle in saline and fresh waters. We identified the protein Wap65, serpins and apolipoproteins in this fraction. By combining the methods of 2D-E under native and denaturing conditions with MALDI, we demonstrated that only apolipoproteins formed complexes. We showed that serum apolipoproteins (АроА-I, Аро-14) were present in the form of homooligomeric complexes that were dissociated with the release of monomeric forms of proteins in the course of capillary filtration to IF. Dissociation of homooligomers is not directly correlated with the change in salinity but is correlated with seasonal dynamics. We found that there was a significant decrease in the total protein concentration in IF relative to plasma. Therefore, we suggested that dissociation of homooligomeric complexes from various apolipoproteins supports the isoosmoticity of extracellular fluids relative to capillary wall stabilization through a fluid medium in fish.

The analgetic activity of docosahexaenoic acid (DHA, 22:6 n-3) was studied using a chronic constriction injury (CCI) model in rats, and the dynamics of iba-1 (+) microglia/macrophages in the dorsal root ganglia (DRG) were characterized. DHA reduced the intensity and duration of neurogenic pain. The application of DHA led to an earlier stabilization of weight bearing in the incapacitance test and prevented the development of cold allodynia and degenerative changes in tissues of the denervated limb. DHA treatment significantly reduced satellite glia reaction and expression of the pro-apoptotic p53 protein in the DRG. Thus, DHA's anti-pain effect may be a result of the modulation of microglia/macrophages activity and the development of neuroprotective effects at the level of the dorsal root ganglia.

A wide range of the osmotic gradient created by plasma and tissue fluid proteins in relation to the capillary wall in redfins of the genus Tribolodon during an annual cycle was revealed. The maximum gradient values are close to those in mammals. The plasma protein distribution between the intraand extravascular body fluids depends on the annual cycle period in the particular season (spawning and feeding completion) and the barrier properties of the capillary wall (peritoneum, brain, and white muscles). Among the osmotic reflection coefficient values characterizing the barrier properties of the capillary wall, those corresponding to its absolute impermeability were not found. This strategy can be explained by the fact that fish have to sustain the fluctuations in the intravascular fluid volume and its rapid recovery after hypovolemia, as well as by the features of protein metabolism in fish.

The organization of the low-molecular fraction (LMF) of proteins from fish plasma was studied in Far Eastern redfins of the genus Tribolodon and other representatives of the family Cyprinidae. The common principle of the organization of the plasma LMF was established. According to this principle, the LMF includes two subfractions, one of which consists of oligomeric and the other of monomeric proteins. During the pre-spawning period, a decrease in the apparent molecular weight of proteins as a result of changes in their oligomeric structure was observed in the former subfraction; a reduction of the heterogeneity of the proteins occurred in the latter one. The analysis of these rearrangements allows one to differentiate two main types of the LMF: "basic" and "plastic." The former type is characterized by a low level of metabolic processes; the latter one, by their activation during the pre-spawning period. By using MALDI mass spectrometry, polymeric forms of apolipoproteins, fetuin, and albumin-like protein were identified within the oligomeric subfraction; hemopexin and inhibitors of proteinases were found within the monomeric subfraction.

Among the potential bipolar analgesics the imidazoline receptor stimulants are currently under consideration, their effect probably simulates the one of certain endogenous agonist, the most likely candidate for the role of which is a polyamine agmatine. Methods. In the experiment on nonlinear male rats during intraperitoneal introduction the researchers examined the agmatine sulphate effect on the severity of pain sensitivity and nociceptive threshold parameters on the model of tonic inflammatory pain. After taking the animals out of the experience the researchers using the method of high-performance liquid chromatography determined the agmatine content in different parts of the brain and in the cerebrospinal fluid. Results. Introduction of agmatine sulphate in the dose of 50, 100 and 200 mg/kg has reduced the severity of pain, mainly, at the tonic pain stage. Introduction of agmatine to the intact animals did not change the initial threshold of the temperature pain sensitivity but increased activity of «analgesia caused by pain.» After injection the drug accumulates in the spinal cord, brain and cerebrospinal fluid. Conclusions. Agmatine anti-algetic effect, probably, is due to its effect on the activity of different signaling mechanisms of the central nervous system, particularly the system of nitric oxide synthesis. The presence of the drug after intraperitoneal introduction in different parts of the brain indicates its ability to break the blood-brain barrier. This fact, along with the results of testing the agmatine biological activity allows to consider agmatineergic system of the brain as a potential target of analgesics.

Обращают на себя внимание весьма близкие спек‐ тры поглощения экстрактов корней цикория, изго‐ товленные разными предприятиями и даже в разных странах (России и Индии), что служит показателем об‐ щности набора веществ в конечных продуктах. Вместе с тем, спектры поглощения водных растворов сухих экстрактов из корней и настоев на корнях цикория по характеру значительно различаются, у первых один максимум, а у вторых – два. Вероятно, в процессе изготовления сухих водорастворимых экстрактов из корней цикория их химический состав искажается, технологически теряются или подвергаются превра‐ щениям вещества, обеспечивающие наличие второго максимума, однако, это требует уточнения и более углубленных исследований. В заключение можно добавить, что набор фото‐ метрических параметров может служить показателем качества конечного продукта. Использованная срав‐ нительно простая спектрофотометрическая методика пригодна не только для решения задач фармации, но может быть принята на вооружение и в гигиене пита‐ ния для характеристики качества пищевых добавок. Литература 1. Инулин [сайт]. URL: http://www.inulin.ru/ob‐inuline (дата обращения 5.02.2014). 2. Колдаев В.М. Спектры поглощения экстрактов из лекарствен‐ ных растений Приморья. М.: Спутник+, 2013. 128 с. 3. Мазнев Н. И. Новейшая энциклопедия лекарственных расте‐ ний. М.: ДОМ. XXI век, 2009. 621 с. 4. Максимов О.Б., Кулеш Н.И., Горовой П.Г. Полифенолы даль‐ невосточных растений. Владивосток: Дальнаука, 2002. 332 с. 5. Минина С.А., Каухова И.Е. Химия и технология фитопрепа‐ ратов. М.: ГЭОТАР‐Медиа, 2009. 560 с. 6. Одуванчик лекарственный // Neboleem.net [Интернет‐портал]. URL: http://www.neboleem.net/oduvanchik‐lekarstvennyj.php (дата обращения 5.02.2014). 7. Топинамбур // Tumannyj.ru [сайт]. URL: http://tumannyj.ru/ p0303.htm (дата обращения 5.02.2014).

Experiments on white rats showed that sciatic nerve ligation induced the development of neuropathic pain syndrome: thermal pain threshold decreased, significant reduction in weight bearing of the injured limb, and degenerative changes in the foot tissues. Administration of docosahexaenoic acid reduces activity and duration neuropathic pain syndrome, promoted reversion of weight bearing asymmetry, and prevented the development of degenerative changes in the foot tissues.

The Wnt5 protein localization in holothurian Eupentacta fraudatrix tissues was examined in the norm and during regeneration. In healthy E. fraudatrix, Wnt5 was found in solitary cells of the hypodermis and in the radial nerve cords. During regeneration, the number of Wnt5 positive cells increased. They were observed in the connective tissue of the body wall and the pharyngeal bulb, in nervous system tissues, the coelomic epithelium, and amoebocytes. The Wnt5 protein may participate in regulating the regeneration in the holothurian E. fraudatrix; it probably modulates cell migration, extracellular matrix reorganization, and neurogenesis.

The experiment on white rats confirms that the sciatic nerve ligation causes neuropathic pain syndrome characterised by a decrease in the temperature pain threshold, uneven weight distribution on hind-limbs, and dystrophic changes in foot tissues. Depending on dose used to treat animals with sciatic nerve injury, the docosahexaenoic acid reduces the intensity and period of neuropathic pain syndrome and allows earlier stabilisation of weight distribution, thus hindering the dystrophic changes in foot tissues.