The characteristics of antibodies of mice immunized by human unconventional myosin 1c.

Specific antibodies produced against a protein of interest are invaluable tools for monitoring the protein structure, intracellular location and biological activity. Inoculation of murine lymphoma cells into the peritoneal cavity of immunized mice provides generation of ascitic fluid containing a significant amount of antibody with desired antigen specificity. Here we demonstrated that the intraperitoneal administration of murine lymphoma NK/Ly cells in mice immunized with 48 kDa isoform of human blood serum unconventional myosin 1c leads to generation of ascitic fluid that contained specific IgG-antibodies. These antibodies were capable of binding of the unconventional myosin 1c isolated from blood serum of patients with multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosis, and could be used for diagnostics of several autoimmune diseases, the multiple sclerosis in particular.

[Genetic transformation of the moss Ceratodon purpureus by novel polycationic carriers of DNA].

There is a big progress in application of genetic engineering for improving the biological properties of different organisms. Viral and non-viral carriers are used for delivery of genetic material into target cells. Nanoscale polymeric materials of natural and synthetic origin are the most promising gene delivery agents. These polymers have demonstrated high efficiency of DNA delivery into the mammalian cells, although they were not very effective in plant cells. Here, the procedure for genetic transformation of Ceratodon purpureus (Hedw.) Brid. moss protoplasts is described. Method is based on the application of novel surface-active polymeric carriers of the polyDMAEM structure and controlled length and charge. It allows obtaining more transient and stable moss transformants per microgram of plasmid DNA when compared with known protocol based on using polyethyleneglycol. It is easier, more convenient, and cheaper than the "gene gun" method. Perspectives for further improvement of structure and functional characteristics of novel polymeric carriers are considered for delivery of genetic material into plant cells.

[Effect of free and polymer carrier encapsulated doxorubicin towards HCT116 cells of human colorectal carcinoma].

Development of novel nanoscale functionalized carriers is nowadays one of the most urgent problems in cancer treatment. The aim of our study was to compare the antineoplastic effect of free doxorubicin and its complex with a nanoscale polymeric carrier towards HTC116 colorectal carcinoma cells. It was established that application of the complex of poly(5-tret-butylperoxy)-5-methyl-1-hexene-3-in-co-glycydyl metacrylat)-graft-polyethyleneglycol (poly(VEP-GMA-PEG)-graft-PEG), where VEP--5-tret-butylperoxy)-5-methyl-1-hexene-3-in; GMA--glycydyl metacrylat; graft-PEG--graft-polyethyleneglycol accordingly, functionalized with phosphatidylcholine for doxorubicin delivery increased 10 times the efficiency of cytotoxic action of this drug, as compared wich such efficiency in case of the action of free doxorubicin. The encapsulated form of doxorubicin caused more intensive cleavage of the reparation enzyme PARP and longer delay in G2/M cell cycle arrest, compared to such effects of free doxorubicin. The developed carrier itself is non-toxic to the used mammalian cells and does not cause impairment in their cell cycle. A deletion in both alleles of p53 gene did not affect the antineoplastic action of doxorubicin that was immobilized on the nanoscale carrier. Thus, p53-dependent signaling pathways are not involved in the cytotoxic action of doxorubicin-carrier complex. It is suggested that novel nanoscale polymeric carrier poly(VEP-GMA-PEG)-graft-PEG functionalized with phosphatidylcholine could be a promising carrier for targeted delivery of anticancer drugs.

Changes in signaling pathways of cell proliferation and apoptosis during NK/Ly lymphoma aging.

Expression of specific proteins involved in regulation of cell proliferation and apoptosis was studied at the initial (7-8 days after tumor inoculation), median (13-14 days), and terminal (20-21 days) stages of murine NK/Ly lymphoma development. Western-blot analysis using antibodies to MEK-ERK signaling pathway, E2F-1/2 and c-Myc, pSTAT1, pSTAT3, pSTAT5, anti-apoptotic Bcl-XL and pro-apoptotic p53 and Rb proteins, as well as active cleaved forms of caspases-3, -6, -7, was carried out to investigate the growth and survival status of NK/Ly cells. There was a marked increase in the expression of E2F-1/2 transcription factors, MAPK signaling cascade and c-Myc, which suggests intensive proliferation of lymphoma cells at terminal stage of tumor development. However, cytomorphological investigation and electrophoretic study of DNA fragmentation have shown degeneration of NK/Ly lymphoma cells and increase in their death. No expression of p53 protein or cleaved forms of caspases-3, -6, -7 was found, which suggests a caspase-independent type of apoptosis in these cells. Ascitic fluid collected at a terminal stage of NK/Ly lymphoma development was significantly weaker in supporting tumor cell growth than ascitic fluid collected at the initial stage of tumor development. It is suggested that uncontrolled cell proliferation at terminal stage of the NK/Ly lymphoma development causes nutrient deprivation and deficiency of specific growth factors in the ascitic fluid, due to overexpression of MEK-ERK, E2F and c-Myc, thereby leading to the induction of apoptosis.

[Variance analysis of Cd2+, Zn2+, and Mn2+ effect on membrane Na+,K(+)-ATPase activity of loach embryos].

The evaluation of influences Cd2+, Zn2+, Mn2+ in concentrations 10(-6), 10(-5), 10(-4) M (factor of dose) on the Na+, K(+)-ATP-ase activity in the early period of development (60-330 min.) of loach embryos (time factor) using one- and two-factor analysis of variance has been performed. It has been detected, that the changes of enzyme activity are mainly caused by action of the explored cations and do not depend on time of embryos development. The most influence on activity in the indicated period of embryos development of loach renders Cd2+ in concentration 10(-4) M, relative value of its influence being 95.7% (p = 0.01). Substantial concentration dependence of the Na+, K(+)-ATP-ase activity is exposed to the action of each of cations. The values of the influence of their concentration changes during the studied period of development differ insignificantly for all cations and make 76.2-77.5% (p < 0.01).

[Effect of heavy metal cations on the activity of membrane Na+, K+ -ATPase in embryos of loach (Misgurnus fossilis L.)]. / Aktyvnist' Na+, K+-ATP-azy membran zarodkiv v'iuna (Misgurnus fossilis L.) za diï kationiv vazhkykh metaliv.

The activity of membrane Na+, K+ -ATPase of embryos of loach (Misgurnus fossilis L.) at early stages of development in the normal conditions and under the influence of heavy metal cations has been investigated. It was established, that the influence of such heavy metal cations as Ni2+, Co2+, Sn2+, Zn2+, Mn2+ and Cd2+ in concentrations 10(-6) - 10(-4) M results in reduction of activity of membrane Na+, K+ -ATPase of loach embryos. It was shown, that the inhibition effect is more expressed with the increase of concentrations of heavy metal cations in the incubation medium. The definition of inhibition constants Io.5 has allowed to analyze the sensitivity of Na+, K+ -ATPase to influence of various cations of heavy metals at different stages of blastomer division. Possible mechanisms of influence of heavy metal cations on the activity of membrane Na+, K+ -ATPase of loach embryos have been considered.

[Factors of avian erythrocyte fusion: changes in the state of plasma membrane induced by dimethylsulfoxide and temperature]. / Faktory sliianiia éritrotsitov ptits: vliianie dimetilsul'foksida i temperatury na sostoianie plazmaticheskoi membrany.

Chicken erythrocytes cooled down to +2 degrees were incubated with 39% dimethysulfoxide (Me2SO) at 37 degrees, eventually resulting in a hemoglobin release. Incubation of erythrocytes with Me2SO at +2 degrees was also accompanied by hemoglobin release; the effect of Me2SO under these conditions was enhanced by Ca2+. Hypertonic concentrations of NaCl and sucrose suppressed this effect upon erythrocyte cooling. At the same time in the absence of Me2SO the level of cold hemolysis of erythrocytes increased as the tonicity of the medium rose. In this case low concentrations of Me2SO (8-15%) exerted a protective effect. The mechanisms of action of Me2SO and temperature on the structure of erythrocyte membrane are discussed.

[Effects of cryoprotectors on the properties of the mitochondrial membrane surface as determined by distribution in biphasic aqueous systems]. / Vliianie krioprotektorov na svoistva poverkhnosti membran mitokhondrii, opredeliaemykh po raspredeleniiu v dvukhfaznykh vodnykh sistemakh.

The effects of various cryoprotectors, e. g. dimethylsulfoxide (Me2SO), glycerol, ethylene glycol and polyethylene oxide (PEO, m. w. 400) on the distribution of rat liver mitochondria in a biphasic polymeric PEO 4000-Dextrane 500 system were studied. The type of criss-cross distribution of cryoprotector-treated mitochondria suggests that the isoelectric point for the organelles is shifted towards the acidic values of pH. The effects of these agents decrease in the following order: Me2SO greater PEO-400 greater than glycerol greater than ethylene glycol. The alteration of the distribution coefficient for the mitochondria after their incubation in aqueous polymeric systems in the presence of cryoprotectors is also indicative of a decrease of their surface charge.

[Effect of low temperatures on the phospholipase A2 activity of rat liver mitochondria]. / Bliianie nizkikh temperatur na aktivnost' fosfolipazy A2 mitokhondrii pecheni krys.

Phospholipase A2 is shown to be activated by freezing-thawing, possibly due to changes in the state of lipid bilayer under the effect of both the temperatures themselves and physicochemical factors formed in the low-temperature range.