Neuroprotective and Antioxidant Activity of Arachidonoyl Choline, Its Bis-Quaternized Analogues and Other Acylcholines.

The antioxidant activity and protective effect in the toxicity model of H2O2 were studied for arachidonic (AA-CHOL), docosahexaenoic (DHA-CHOL), linoleic (Ln-CHOL), and oleic (Ol-CHOL) fatty acids, as well as arachidonoyl dicholine (AA-diCHOL) and O-arachidonoyl bistetramethylaminoisopropanol (ABTAP). AA-CHOL, DHA-CHOL and Ln-CHOL provided a 20% increase in cell survival. AA-CHOL, AA-diCHOL, Ol-CHOL, and ABTAP had a radical-scavenging effect in the ABTS test, approximately equal to the activity of a standard radical scavenger Trolox.

Neuroprotective Action of Amidic Neurolipins in Models of Neurotoxicity on the Culture of Human Neural-Like Cells SH-SY5Y.

It was established that in neurodegeneration models in the human neuron-like cell line SH-SY5Y, amide derivatives of arachidonic and docosahexaenoic acids were inactive in experiments with MPP+ and CoCl2 but protected from H2O2. The protective activity of neurolipins decreased in the series DHA-DA > AA-SER ≥ AA-GLY > AA-GABA ≥ AA-EA and was manifested starting from a concentration of 0.5 nM.

[The optimization of the nitric oxide quantitative analysis for its determination in the cultural medium of mammalian cell culture].

The protocol for the quantitative analysis of nitric oxide as nitrite-ion suitable for determination of its production by a mammalian cell culture was developed. The optimal results were obtained using microvolume-adjusted Griess method after the preliminary reduction of NO3- to NO2- with non-activated cadmium. The protocol was verified on a rat glioma C6 cell culture. The developed method may be used for the nitric oxide determination in 96-well and 48-well microplates; the detection limit is 2.1 ± 0.1 µM for NO2- and 2.9 ± 0.1 µM for NO3-.

[Nanocomplexes of recombinant proteins and polysialic acid: preparation, characteristics, and biological activity].

A preparation of nanocomplexes containing recombinant proteins (interferons alpha2b and beta1b, insulin, and human granulocyte colony stimulating factor) and natural polysialic acid (PSA) has been described. The incorporation of protein into the complex changes its electrophoretic mobility. Atomic force microscopy reveals the average size of 23-kD insulin complexes with PSA of 10-20 nm and demonstrates that more than 60% of glycopolymer molecules carry a single protein molecule. Experiments with cultured cells show that cytokines bound to polysialic acid retain their ability to regulate cell proliferation. Insulin bound to PSA has a prolonged hypoglycemic effect in vivo.

[Modification of recombinant proteins by covalent polysialation illustrated with the example of human insulin].

Methods of selective and nonselective covalent immobilization of genetically engineered proteins on molecules of natural polysialic acid are described by the example of human insulin. Such modification increases insulin lifetime in vivo.

[Neutral glycosphingolipids in Ehrlich ascites carcinoma cells]. / Neitral'nye glikosfingolipidy kletokk astsitnoi kartsinomy Erlikha.

The structure of the neutral glycosphingolipids of the Ehrlich ascite carcinoma (EAC) cells was studied. The main four components were identified as glycosylceramide, lastosylceramide, N-acetylgalactosyllactosylceramide and galactosyl-N-acetyllactosylceramide (asialo-GM1). The neutral glycolipid pattern of the cells was found to depend on their density. Dilution of the cell suspension resulted in an increased content of asia-lo-GM1, whereas the content of the other neutral glycolipids remained unchanged. The possible connection between these changes and the earlier disclosed cell density dependence of the gangliosides in EAC cells is discussed.

[Lipoxygenase oxidation of arachidonic acid in murine splenocytes and its modulation by the lactone ganglioside GM3]. / Lipoksigenaznoe okislenie arakhidonovoi kisloty v splenotsitakh myshei i ego modulirovanie laktonom gangliozida GM3.

The main arachidonic acid metabolites released into the medium by mouse splenocytes have been identified on the basis of chromatographic and spectral studies as well as by mass spectrometry of the derivatives. In the absence or presence of exogenous arachidonic acid mouse splenocytes produce mainly 12-hydroxy-5,8,10,14-eicosatetraenoic and 12,20-dihydroxy-5,8,10,14-eicosatetraenoic acids. Both products are constantly released by intact cells into surrounding media without stimulation by exogenous substrate or other modulators. For the first time it is shown that exogenously added ganglioside GM3 lactone as well as ganglioside GM3 itself can influence the arachidonic acid metabolism in splenocytes.

[Glycosphingolipids from human T-lymphoma MOLT-4 cells]. / Glikosfingolipidy kletok T-limfomy MOLT-4 cheloveka.

The glycosphingolipids of human lymphoma MOLT-4 cells were studied, using biochemical methods and specific antisera to gangliosides. The major neutral glycosphingolipids were found to be glucosyl- and lactosyl ceramides. GM3, GM2, GM1 and GD1a were identified as ganglioside components.

[Comparative study of gangliosides from murine B- and T-lymphomas]. / Sravintel'noe izuchenie gangliozidov B- i T-limfom myshei.

Gangliosides from murine B-lymphomas (MOPC 21 and MOPC 406) and T-lymphoma EL-4 were studied by thin-layer chromatography, immunoprecipitation with specific antisera to gangliosides and by treatment with neuraminidase. It was found that the gangliosides of all three lymphomas differ in their interaction with antisera and neuraminidase although they are similar in their chromatographic behaviour.

[Neutral glycosphingolipids of murine lymphoma EL-4]. / Neitral'nye glikosfingolipidy limfomy EL-4 myshei.

Neutral glycosphingolipids of murine T-lymphoma EL-4 were studied. The major glycolipid components were identified as GlcCer, LacCer, GgOse3Cer and GgOse4Cer. It has been shown for the first time that not only gangliosides but also neutral glycolipids are shed from the cell surface into the outer medium.

[Structure of lymphoma gangliosides in EL-4 mice]. / Struktura gangliozidov limfomy EL-4 myshei.

The structure of the major gangliosides from murine lymphoma EL-4 was established by acid hydrolysis, methanolysis, methylation analysis, neuraminidase treatment and chromium trioxide oxidation. Two of these gangliosides were identified as the N-acetyl and N-glycoloyl forms of the GM2 ganglioside. The third ganglioside making up to 40% of the total ganglioside pool contained two sialic acid residues and was identified as a GD2 ganglioside which up to now had not been reported in extraneural tissues.

[Gangliosides modulate lipoxygenase oxidation in human lymphocytes]. / Gangliozidy moduliruiut lipoxsigenaznoe okislenie v limfotsitakh cheloveka.

Using reverse phase high performance chromatography with UV-detection, the arachidonic acid cascade in human peripheral blood lymphocytes (PBL) was studied. It was found that PBL oxidized arachidonic acid via the lipoxygenase pathway, 12-hydroxyeicosatetraenoic acid (12-HETE) being the major metabolite of endogenous arachidonic acid. Exogenous arachidonic acid added to human PBL suspensions increased 12-HETE synthesis 5-7 times. In another experimental series the effects of gangliosides (GD3, GM1 and GM3) on lipoxygenase-catalyzed oxidation of arachidonic acid in human lymphocytes were investigated. All the gangliosides tested stimulated PBL to secrete 12-HETE both from endogenous and exogenous arachidonic acid. In most cases the stimulating effect of GD3 was much more apparent that those of GM1 and GM3.

Role of gangliosides in reception of influenza virus.

The ganglioside composition of Ehrlich ascites carcinoma (EAC) cells and the role of the individual gangliosides in binding and penetration into the cell of influenza virus were determined. EAC gangliosides identical with or close to GM3, GM2, GM1, GT1a and GT1b were characterized by thin-layer chromarography, compositional analyses, methylation analysis and mass-spectrometry. The ganglioside uptake capacity of native and neuraminidase-treated EAC cells was studied with tritium-labeled gangliosides of definite structure and the binding of influenza virus to cells was determinated by using [3H]uridine-labeled virus and by hemagglutination studies. Treatment of the cells with Vibrio cholerae neuraminidase largely decreased binding of the virus. Exogenous gangliosides with a terminal galactose unit or a penultimate galactose masked by neuraminic acid were able to restore the virus-binding capacity of neuraminidase-treated cells, however, the main ganglioside of EAC cells, GM2, which carbohydrate chain is terminated by N-acetylgalactosamine, was completely ineffective. The common carbohydrate sequence of the gangliosides showing binding activity (formula; see text) is proposed to be the main recognition structure of the influenza virus receptor on the surface of EAC cells. Penetration of labeled influenza virus into the nuclei of EAC cells was evaluated by measuring the radioactivity of the nuclei of neuraminidase-treated ganglioside-loaded cells after exposition to the labeled virus. Of all gangliosides tested only trisialogangliosides of the GT1b type were able to induce increased entry of the virus into the cells and accumulation of its radioactive component into the nuclei. It is suggested that GT1b gangliosides react specifically with the virus protein responsible for membrane fusion (apparently the hemagglutinin HA2 subunit) and thus are involved in virus penetration and delivery of the virus genome to the nuclei.

Hydrolysis of anandamide and eicosapentaenoic acid ethanolamide in mouse splenocytes.

The hydrolysis of anandamide has been studied in mouse splenocytes using tritiated anandamide analogs labeled in the acyl- or ethanolamide parts of the molecule. [3H]Anandamide undergoes rapid (t(1/2) = 2.5 min) uptake and hydrolysis, yielding ethanolamine and arachidonic acid. The anandamide hydrolysis in splenocytes is sensitive to inhibition by phenylmethylsulfonyl fluoride, and it is assumed that the observed activity is due to fatty acid amide hydrolase, which inactivates anandamide in central and peripheral tissues. Eicosapentaenoic acid ethanolamide and the 15-hydroxy-derivative of anandamide are shown to be amidohydrolase substrates as well. The fatty acids derived from hydrolytic cleavage of acylethanolamines undergo rapid oxidation by splenocyte lipoxygenase, yielding the corresponding 12-hydroxy-derivatives. Oxygenated ethanolamide derivatives were not found. The data suggest that polyenoic fatty acid ethanolamides are metabolic precursors of eicosanoids in splenocytes and that amide bond hydrolysis is the key point in switching of biological activity spectra between endocannabinoids and oxylipins.