[Cytokine-inducing and anti-inflammatory activity of chitosan and its low-molecular derivative].

A low-molecular derivative of the polysaccharide (5 kDa) was obtained and its cytokine-inducing and anti-inflammatory activity was studied by free radical depolymerization of chitosan (110 kDa). It was shown that high-molecular chitosan in vitro inhibited the synthesis of anti-inflammatory cytokine, the tumor necrosis factor alpha induced by endotoxin. In the case of peroral introduction to experimental animals, high- and low-molecular chitosans stimulated synthesis of the anti-inflammatory cytokine IL-10 in the blood serum of mice; in this case, the activity of the high-molecular derivative was two times higher as compared with the initial polysaccharide. With peroral introduction, the initial polysaccharide (50 mg/kg) and its derivative inhibited the development of chemically induced inflammation of experimental animals' large intestines, which was manifested as a decrease in the affected area and the degree of damage to the large intestine wall, as well as a two-fold reduction of myeloperoxidase activity. According to morphological and biochemical characteristics, the effect of chitosans was similar to that of a hormone anti-inflammatory drug, prednisolone.

[Effect of chitosan on the cell ultrastructure and activity of hydrolases in tobacco leaves].

Effect of chitosan on the mesophyll cell ultrastucture and activity of hydrolases in leaves of tobacco cv. Samsun was studied. It was shown that, in many cells, chitosan treatment stimulated the protein-synthesizing apparatus (nucleolus dimension and amount of both mitochondria and rough endoplasmic reticulum membranes increased) and, at the same time, caused some activation of lytic compartment expressed in the stimulation of the formation of dictyosomes, smooth ER elements and cytoplasmic vacuoles, which are all prominent constituents of this compartment. In biochemical experiments, it was established that chitosan substantially enhanced activity of hydrolases (acid phosphatase, RNase, proteases) in the leaves as compared to untreated leaves. In some cells chitosan treatment caused considerable destructive changes (condensation of nuclear chromatin, collapse of cytoplasm and so on) that can be classified as a result of programmed cell death development.

[Electron microscopic study of the chitosan effect on the intracellular accumulation and the state of tobacco mosaic virus particles in tobacco leaves].

Influence of chitosan on the accumulation and state of tobacco mosaic virus (TMV) in the mesophyll cells of Nicotiana tabacum L. var Samsun leaves in early period of infection development (3 days after infection of leaves) has been studied. The virus accumulated in the cells of the leaves treated for 24 h before infection with chitosan to a lesser degree than in the control cells. The chitosan affected the formation of TMV-specific granular and tubular inclusions which are known to consist of the viral replicase components. Three days after infection of the leaves treated with the chitosan, a typical sign of the infection development was the predominant formation of granular inclusions which are known to appear at the early stages of TMV replication. The infected cells of the leaves untreated with chitosan contained mainly tubular inclusions which had been shown previously to be formed from granular ones at the last stages of the infection process. This indicates that chitosan treatment of the leaves leads to a delay of the development of infection. In phosphotungstic acid-stained suspensions obtained from the infected leaves, abnormal (swollen and "thin") TMV particles were observed along with normal ones. The appearance of abnormal virus particles seems to be caused by virus-induced activation of intracellular lytic processes. The most lytic activity in the infected cells as well as the highest number of abnormal viral particles was observed under the chitosan action. Therefore, it appears that chitosan-mediated stimulation of lytic processes causing destruction of TMV particles may be one of the protective mechanisms limiting virus accumulation in cells.

[Chitosan antiviral activity: dependence on structure and depolymerization method].

Enzymatic (the action of lysozyme) and chemical (hydrogen peroxide) hydrolysis of chitosans with various degree ofacetylation (DA)--25, 17, and 1.5%--was performed. Purification and fractioning of the hydrolysis products were performed using dialysis, ultrafiltration, and gel-penetrating chromatography Low-molecular (LM) derivatives of the polysaccharide with molecular masses from 17 to 2 kDa were obtained. The study of their antiviral activity against the tobacco mosaic virus (TMV) showed that these samples inhibited the formation of local necroses induced by the virus for 50-90%. The antiviral activity of the LM chitosans significantly increased with the lowering of their polymerization degree. Furthermore, the products of the enzymatic hydrolysis possessed higher activity than the chitosan samples obtained as a result of chemical hydrolysis. It was revealed that the exhibition of the antiviral activity weakly depended on the degree of acetylation of the samples.

[Inhibitory effect of kappa/beta-carrageenan from red alga Tichocarpus crinitus on the development of a potato virus X infection in leaves of Datura stramonium L].

The effect of kappa/beta-carrageenan from red alda Tichocarpus crinitus on the development of a potato virus X (PVX) infection in the leaves of Datura stramonium L. has been studied. The treatment of leaves with carrageenan stimulates a protein synthesis in the cells, causing an increase in the size of nucleoli and in the number of mitochondria and membranes of the granular endoplasmic reticulum. At the same time, such treatment slightly stimulates lytic processes, causing an increase in the number of agranular endoplasmic reticulum cisterns, dictyosomes, and cytoplasmic vacuoles and the formation of cytoplasmic zones, transparent to electron microscopy. The carrageenan-induced stimulation of lytic processes results in the destruction of viral particles and can be considered as one of the defense mechanisms, preventing the intracellular accumulation of viruses. The carrageenan-stimulated formation of PVX-specific laminar structures, able to bind viral particles and, therefore, prevent their intracellular translocation and reproduction, represents another carrageenan-induced mechanism of the antiviral defense in plant cells.

[Influence of kappa/beta-carrageenan from red alga Tichocarpus crinitus on development of local infection induced by tobacco mosaic virus in Xanthi-nc tobacco leaves].

The influence of kappa/beta-carrageenan from red marine alga Tichocarpus crinitus on the development of tobacco mosaic virus (TMV) infection in Xanthi-nc tobacco leaves was studied. It was shown that the number of necrotic lesions on the leaves inoculated with the mixture of TMV (2 microg/ml) and carrageenan (1 mg/ml) was reduced by 87%, compared to the leaves inoculated with the virus only. The suppression of virus infection was also observed when leaves were treated with carrageenan 24 h before or 24 h after leaf inoculation with TMV; however, in these cases, suppression was less evident than after inoculation with the virus-polysaccharide mixture. It is supposed that the antiviral activity of carrageenan applied together with TMV may be explained by its action not only on the plant but also on the virus itself. The inhibitory effect of carrageenan pretreatment can be explained by its favorable effect on tissue resistance to infection. The suppression of this resistance by actinomycin D indicates that carrageenan functions via its action on the cell genome.

[Effect of carrageenan on non-specific resistance to LPS-induced endotoxemia in mice].

The study showed that kappa/lamda-carrageenan increase non-specific resistance to impact of Escherichia coli lypopolysaccharide (LPS)-induced endotoxinemia in mice. Carrageenan was administered intragastrically in dose 100 mg/kg/day during 5 days followed by intraperitoneal injection of LPS in dose 1 mg/kg. Carrageenan hampered the involution of thymus, hypertrophy of adrenal glands, the changes on level of thyroid hormones and corticosterone in serum, the activation of glycogenolysis, glycolysis, and peroxidation of lipids in liver.

Natural polysaccharide carrageenan inhibits toxic effect of gram-negative bacterial endotoxins.

The protective effect of polysaccharide carrageenan on the damaging effect of endotoxins of gram-negative bacteria was studied in vivo and in vitro. Carrageenan increased mouse resistance to the toxic effect of LPS. The degree of protection depended on polysaccharide concentration and administration time and route. Pretreatment of donor platelets with carrageenan reduced their aggregation activity caused by cooperative effect of LPS and ADP.

Modification of biological activity of lipopolysaccharide in the complex with chitosan.

In the complex with chitosan, lipopolysaccharide partially lost its ability to induce lymphokines tumor necrosis factor and interleukin-8, but retained immunostimulating properties and increased phagocytic function of macrophages by improving digestion of bacteria.

[Comparative study of the physico-chemical properties of chitosans with varying degree of polymerization in neutral aqueous solutions]. / Sravnitel'noe izuchenie fiziko-khimicheskikh svoistv khitozanov razlichnoi stepeni polimerizatsii v neitral'nykh vodnykh rastvorakh.

The physicochemical properties of chitosan samples with high (130 kD) and low (30 kD) molecular masses in neutral aqueous solutions (pH 6.0) were studied by the methods of high-speed and equilibrium sedimentation, viscosimetry, and NMR and UV spectroscopies. Differences in the hydrodynamic characteristics of the samples were revealed. It was found that low-molecular-weight chitosan represents flexible linear macromolecules which undergo conformational changes upon temperature increase. The high-molecular-weight chitosan forms more rigid asymmetric structures whose conformation does not vary significantly with temperature increase. It was found that the high-molecular-weight chitosan has a higher constant of binding to the anionic dye tropeoline 000-II, which can be explained by different conformations of their macromolecules in solution.

[Effect of the method of extracting the pore-forming protein from Yersinia pseudotuberculosis on its macromolecular organization]. / Vliianie sposoba ékstrktsii poroobrazuiushchego belka iz Yersinia pseudotuberculosis na ego makromolekuliarnuiu organizatsiiu.

By means of physico-chemical methods, lipid bilayer reconstitution and immunoenzyme assay, macromolecular organization of porin oligomers from Yersinia pseudotuberculosis, isolated by two extraction methods, was studied. Use of SDS and high temperature in the course of the extraction led to a partial denaturation of porin trimers at the level of the tertiary structure, these conformational changes affecting the porin's pore-forming activity and antigenic structure. At the same time, the partially denatured trimers are as stable under the treatment of urea and guanidine hydrochloride as the native protein.

[Features of a protein component of the endotoxin from Yersinia pseudotuberculosis]. / Kharakteristika belkovogo komponenta éndotoksina iz Yersinia pseudotuberculosis.

The protein moiety of endotoxin from Yersinia pseudotuberculosis was found to consist of two polypeptides with apparent molecular masses 40 and 14.5 kDa (4:1 w/w). The major protein (40 kDa) was isolated from the endotoxin pretreated with sodium deoxy cholate by gel chromatography on the Sephadex G-200 column. Comparative study of this protein and oligomeric form of porin from the outer membrane of Y. pseudotuberculosis using SDS--PAGE, velocity sedimentation, lipid bilayer experiments, chemical and serological analyses revealed their identity. The deoxycholate treatment of the endotoxin does not affect complexes of the major protein and LPS.

[Study of aqueous solutions of the lipopolysaccharide-protein complex from Yersinia pseudotuberculosis using hydrodynamic methods]. / Issledovanie vodnykh rastvorov lipopolisakharid-belkovogo kompleksa iz Yersinia pseudotuberculosis gidrodinamicheskimi metodami.

Physical and chemical properties of LPPS in aqueous solutions were studied. Hydrodynamic characteristics of LPPS depend upon the input concentration and method of solution preparation. Equation parameters for the relationship between [eta] and M omega were determined, and it was found that LPPS behavior in solution can be determined by the equation: [eta] = 5.5.10(-4).M0.57. In aqueous solutions complex macromolecules form compact particles ranging from 8 to 14 nm in size.

[Study of a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis in aqueous solutions by light scattering]. / Issledovanie lipopolisakharid-belkovogo kompleksa iz Yersinia pseudotuberculosis v vodnykh rastvorakh metodom svetorasseianiia.

Lipopolysaccharide-protein complex isolated from Yersinia pseudotuberculosis forms aggregates in aqueous solutions. Dissociation of aggregates was found by light scattering method for 1 M sodium chloride, 1 M guanidine chloride, and urea solutions. Very large particles were detected also in these solutions and acid media. Little light scattering alterations have been observed for 0.27 g/l solution, and pronounced effect of temperature has been detected for more diluted solutions.

[Different forms of a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis]. / O razlichnykh formakh lipopolisakharid-belkovogo kompleksa iz Yersinia pseudotuberculosis.

Two forms of lipopolysaccharide-protein complex with buoyant densities of 1,43 and 1,40 g/cm3 were found in the Yersinia pseudotuberculosis cell wall. These forms have the similar monosaccharide, fatty acid and polypeptide compositions, but differ in the length of O-specific chains. The differences in density are stipulated by the different contents of the main components of the complex. Both forms contain the related antigenic determinants but have some differences in the antigenic structure. The ability of the two forms to produce a hybrid form with the intermediate density of 1,41 g/cm3 has been shown.

[Sedimentation properties of a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis]. / Sedimentatsionnye svoistva lipopolisakharid-belkovogo kompleksa iz Yersinia pseudotuberculosis.

It was shown that in water media lipopolysaccharide-protein complex (LPSPC-) forms compact units with molecular weight 1.29 X 10(6) inclined to self-association. Disaggregation of LPSPC to subunits with molecular weight 7 X 10(4) under the action of the detergent and EDTA reveals the substantial role of hydrophobic interaction of ions of divalent metals in the formation of high molecular weight aggregates.